Proteases from Entamoeba spp. and Pathogenic Free-Living ... · HindawiPublishingCorporation JournalofTropicalMedicine Volume2013,ArticleID890603,32pages ReviewArticle Proteases from

Hindawi Publishing CorporationJournal of Tropical MedicineVolume 2013 Article ID 890603 32 pageshttpdxdoiorg1011552013890603

Review ArticleProteases from Entamoeba spp and PathogenicFree-Living Amoebae as Virulence Factors

Jesuacutes Serrano-Luna Carolina Pintildea-Vaacutezquez Magda Reyes-LoacutepezGuillermo Ortiz-Estrada and Mireya de la Garza

Departamento de Biologıa Celular Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico NacionalAvenida Instituto Politecnico Nacional 2508 07360 Mexico DF Mexico

Correspondence should be addressed to Mireya de la Garza mireyadelagarzayahoocommx

Received 25 September 2012 Accepted 28 November 2012

Academic Editor Carlos E P Corbett

Copyright copy 2013 Jesus Serrano-Luna et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The standard reference for pathogenic and nonpathogenic amoebae is the human parasiteEntamoeba histolytica a direct correlationbetween virulence and protease expression has been demonstrated for this amoeba Traditionally proteases are considered virulencefactors including those that produce cytopathic effects in the host or that have been implicated in manipulating the immuneresponse Here we expand the scope to other amoebae including less-pathogenic Entamoeba species and highly pathogenic free-living amoebae In this paper proteases that affectmucin extracellularmatrix immune system components and diverse tissues andcells are included based on studies in amoebic cultures and animal models We also include proteases used by amoebae to degradeiron-containing proteins because iron scavenger capacity is currently considered a virulence factor for pathogens In additionproteases that have a role in adhesion and encystation which are essential for establishing and transmitting infection are discussedThe study of proteases and their specific inhibitors is relevant to the search for new therapeutic targets and to increase the powerof drugs used to treat the diseases caused by these complex microorganisms

1 Introduction

Amoeba is a general name that is used for protists thatform a large and diverse assemblage of eukaryotes that arecharacterized by various types of pseudopodia [1 2] Someamoebae are pathogenic and even parasitic to human andother vertebrate hosts The four amoebae that are dealtwith in this paper have been classified under two SuperGroups Amoebozoa and Excavata as follows (a)EntamoebaAcanthamoeba and Balamuthia are classified under theSuper Group Amoebozoa (b) Naegleria fowleri is classifiedunder Super Group Excavata [1 2] The genus Entamoebaincludes several species such as E histolytica which causesamoebiasis an infection in the gut characterized by invasionof the intestinal mucosa that occasionally spreads to otherorgans mainly the liver and E dispar and E moshkovskiiwhich are morphologically similar to E histolytica and havebeen recently recognized as separate species Although Edispar andEmoshkovskiihave no apparent invasive potential

they exhibit some pathogenicity [3 4] Molecular phylogenyanalysis places the genus Entamoeba on one of the lowermostbranches of the eukaryotic tree closest to Dictyostelium [5]Although Entamoeba originally thought to lack mitochon-dria nuclear-encoded mitochondrial genes and a remnantorganelle have now been identified The establishment ofE dispar and E moshkovskii as distinct but closely relatedspecies has profound implications for the epidemiology ofamoebiasis because most asymptomatic infections are nowattributed to these noninvasive amoebae [3]

Free-living amoebae belonging to the genera Acantha-moeba Balamuthia and Naegleria are responsible for oppor-tunistic and nonopportunistic infections in humans andother animals The distinction between parasitic and free-living protozoa is generally sharp with organisms fallingreadily into one or the other category Some of the free-livingamoebae are unusual in that they straddle the line separatingthe two groups of organisms and yet are as destructiveas any of the classic parasitic protozoa [52] Unlike their

2 Journal of Tropical Medicine

parasitic counterparts these free-living amoebae are not welladapted for parasitism Furthermore as free-living formswith a broad distribution they are not dependent upon a hostfor transmission and spread Unlike the parasite E histolyt-ica free-living amoebae are mitochondrion-bearing aerobicorganisms that can complete their respective life cycles inthe environment without a host thus these organisms havebeen called amphizoic amoebae in recognition of their abilityto live endozoically although they are capable of free-livingexistence [52]

All pathogenic amoeba species have in common thecapability to phagocytose bacteria erythrocytes and celldetritus The major virulence factors are adhesins toxinsamoebapores and proteases which lead to the lysis deathand destruction of a variety of cells and tissues in thehost Because of their relevance to amoebic pathogenesisproteolytic enzymes are of particular interest Proteases areimportant in tissue invasion migration and host pathologyThe main goal of this paper is to review the proteases of theEntamoeba genus and of free-living amoebae

2 Entamoeba histolytica

Amoebiasis is a human parasitic infection caused byEntamoeba histolytica an extracellular protozoan Cysts aretransmitted through the fecal-oral route by contaminatedwater or food Parasite destruction of host tissues appearsto be the basis of amoebiasis which leads to invasivedisease pathologies such as intestinal amoebiasis which ismainly characterized by lesions in the colon that producefever abdominal pain dysentery and ulcerative colitis withmucous and blood Amoebae can spread through the portalvein to other organs such as the liver lungs kidneys andbrain Hepatic amoebiasis is characterized by liver abscessesthat can be fatal [53ndash57]

Amoebiasis is the third leading cause of death dueto parasites after malaria and schistosomiasis Amoebiasispresents a high index of morbidity and mortality mainlyin developing countries According to the World HealthOrganization (WHO) 500 million people are infected withamoebae 10 of infected individuals have virulent E his-tolytica resulting in 40000ndash100000 deaths annually [56] Ehistolytica infects only humans The life cycle of E histolyticaconsists of two main stages the trophozoite (also calledamoeba) or invasive form and the cyst or infective formCysts can tolerate the stomach acidic pH and excyst in theterminal ileum Trophozoites colonize the large intestine andin later phases of amoebiasis they invade the epithelium andmucosa In response to unknown stimuli amoebae undergomorphological and biochemical changes that lead to theformation of new cysts which are eliminated in the fecescompleting the cycle [53 55 56]

The cytopathogenic effect caused by E histolytica tropho-zoites is multifactorial Intestinal flask-shaped ulcers a hall-mark of amoebic colitis are characterized by severe damageto enteric cells as well as migration to the lamina propriaand blood vessels [57 58] The contact between trophozoitesand target cells appears to be the first step for cell lysis and

phagocytosis Several molecules are involved in this inter-action including the 260 and 220 kDa lectins and 112 kDaadhesin which participates in the adherence to epithelial cellsand erythrocytes [8 59ndash63] It has been proposed that forthe initial amoeba contact or adhesion surface carbohydrateson the target cell are recognized by specific molecules fromthe parasite One of the better studied amoebic moleculesis the GalGalNAc lectin which mediates binding to hostcarbohydrate determinants that contain galactose andor N-acetyl-D-galactosamine (GalNAc) [64 65] Adherence tocolonic mucosa is conducive to the continued reproductionof parasites and tissue damage by the products secreted byamoebae such as the pore-forming peptide amoebapore [66]which permits a massive influx of extracellular Ca+2 that iscombined with the release of amoebic proteases at the site ofcontact with the subsequent degradation of substrates Oncethe targets are partially digested the amoeba internalizesthe cell debris and substrate fragments by phagocytosis [67]Other proteins also contribute to host cell binding on targetcells and destruction such as phospholipases [68 69]

21 Proteases of E histolytica and Their Role in VirulenceStudies of E histolytica proteinases (proteases) have mainlybeen performed in the strain HM-1IMSS from axenicallygrown trophozoites De la Torre et al [70] isolated this strainfrom cysts of a Mexican patient suffering from intestinalamoebiasis Most of the cellular and molecular studies ofE histolytica throughout the world including the genomicsequence have been performed with this strain It hasbeen cultured for years and passed through the liver ofSyrian golden hamsters an experimental model in whichhepatic abscesses are reproduced tomaintain and increase thevirulence of E histolytica

Cysteine proteases (CPs) are the predominant proteolyticactivity associated with pathogenicity in E histolytica basedon many studies in which the degradation of differentsubstrates has been investigated including purified pro-teins of the extracellular matrix (ECM) immunoglobulinscomplement and mucin Figures 1 and 2 show the roleof these proteases during intestinal amoebiasis and bloodvessel transit of trophozoites respectively Animal modelstudies have confirmed that CPs have an important role invirulence E histolytica mutants impaired in genes encodingCPs have a diminished ability to produce hepatic abscesses[71 72] Figure 3 shows the role of proteases during amoebicliver abscess EhCPs are expressed both intracellularly andextracellularly and are referred to as cathepsin-like enzymesbecause their structure is similar to that of cathepsin L how-ever their substrate specificity resembles that of cathepsinB [34 73ndash75] Some proteases have been characterized assurface localized hence they have the potential to contributeto host tissue breakdown in vivo [74] (Figure 4) E histolyticamost studied proteases are summarized in Table 1

211 Purification and Cloning of the Main Cysteine Proteasesfrom E histolytica Several genes encoding E histolytica CPshave been cloned In 1990 Eakin et al [76] amplified CPgene fragments from trophozoites by PCR from genomic

Journal of Tropical Medicine 3

Mucuslayer

Extracellularmatrix

Epithelialcell

Tightjunctionjunction

EhCP5

EhCP4EhCP2 EhCP5EhCP112

EhCP4sIgA

Plasma cells

EhCP LfNeutrophils

EhCP5EhCPLf

EhCP Lf

Eh

EhEh

EhEh

Eh

Eh

EhCP1

EhCP tight

CollagenFibronectin

Laminin-1

Lactoferrin fromneutrophils

Lactoferrin fromexocrine glands

SecretoryIgA Intestinal

microbiotapIL-1

IL-18

Figure 1 Proteases from E histolytica as virulence factors during intestinal amoebiasis

DNA using degenerate oligonucleotide primers designedbased upon amino acid sequences flanking the active site ofCPs conserved in eukaryotic cells The amplified DNA frag-ments were subcloned and sequence analysis and alignmentrevealed significant sequence similarity to other members ofthe eukaryotic CP family (45 identical to chicken cathepsinL) The cysteine histidine and asparagine residues that formthe catalytic triad are also conserved Subsequently [77]isolated three genes from the strain HM-1IMSS designatedas acp1 acp2 and acp3These genes have since been renamedand the prefix ldquoehrdquo has been added so that these genes are nowdenoted ehcp1 (acp3) ehcp2 (acp2) and ehcp3 (acp1) Nextwe will review studies of the purification and cloning of Ehistolytica CPs Most of these studies were published beforethe establishment of an amoebic protease nomenclature orinclude experiments that only demonstrate degradation ofthe substrate for these studies we will focus on proteolyticactivity and Mr

By screening a genomic library from E histolytica [35]identified six genes encoding prepro-forms of CPs (ehcp1ndashehcp6) The nucleotide sequences of these genes differedby 40ndash85 and three of the genes ehcp1 ehcp2 andehcp5 exhibited the highest levels of expression Amoebalysates were analyzed for CP activity against a synthetic sub-strate Enzyme purification revealed that EhCP1 EhCP2 andEhCP5 are the main proteases representing approximately

90 of CP expression Tillack et al [78] analyzed 79 of 86genes encoding putative proteases by microarray hybridiza-tion Of these 50 encode CPs of various families all of whichbelong to the clan CA Interestingly under standard cultureconditions the authors consistently observed the expressionof only 20 genes Very few differences were apparent amongthe isolates from asymptomatic and amoebic disease-affectedindividuals even though the isolates had different geographicorigins Again only three peptidase genes were expressedat high levels ehcp1 ehcp2 and ehcp5 Accordingly in theless pathogenic strain HK-9 the expression of EhCP5 wasdecreased by 23-fold However these E histolytica isolateswere grown under axenic conditions and the expressionprofiles of CPs have been shown to adapt to different stimuliThis adaptation was confirmed in a transcriptional analysisof trophozoites isolated from the colons of infected miceversus trophozoites cultured in vitro In contrast to what isobserved in axenic cultured trophozoites EhCP4 EhCP6and EhCP1 were the most upregulated CPs during invasionand colonization of mice [79]

EhCP1 (Amoebapain) A thiol-dependent protease was iden-tified and partially purified by means of ammonium sulfatefractionation gel filtration and isoelectric focusing TheCP had Mr of 21 plusmn 2 kDa by gel chromatography Themaximal activity occurred at pH 44 but the CP was active


EhCP3

EhCP4

EhCP3

EhN

EhCP4-5

C3

IgG

Transferrin

Erythrocytes

Hemoglobin

Lactoferrin

Ferritin

EhCP4

Figure 2 E histolytica proteases participating during trophozoite transit in blood vessels

(a)

Hemoglobin

Ferritin

Lactoferrin

EhEh

EhEh

EhNN

N

EEEEhhhhhhhhhhhhhEh

(b)

Figure 3 (a) Hamster amoebic liver abscess of eight days of infection (Photo kindly donated by G Dominguez) (b) Proteases involved inthe development of human amoebic liver abscess

at pHs of 34 and 85 [80] Its proteolytic potential towardnative substrates and bovine insulin B-chain was examined[81 82] The CP was purified by two gel chromatographysteps ion exchange chromatography on DEAE-cellulose andaffinity chromatography on organomercurial-SepharoseThepurified protease was monomeric with a Mr of 27 plusmn 2 kDa its activity required an arginine at the P2-position of thesubstrateTheproteasewas able to digest native collagen type-I with an initial attack at the alpha 2-chain [6] EhCP1 islocated on the cell surface [34]

By using an antiserum against the 27 kDa protease acDNA clone was obtained Northern blot analyses suggestedthat the CP is more expressed in pathogenic isolates thanin nonpathogenic isolates [83] Accordingly using a purifi-cation method similar to that described earlier cathepsin Bactivity was obtained from virulent strains (HM-1IMSS andRahman) which yielded significantly more activity per mil-ligram protein than less virulent strains (HK-9 Laredo andHuff) These results suggest a correlation between amoebiccathepsin B and the pathogenesis of amoebiasis [26]


Table 1 Cysteine proteases of Entamoeba histolytica that degrade host proteins and their implication in virulence

Tissue or protein Purified Name Mr (kDa) Comment localization ReferenceECM components

Collagen

Yes EhCP1 27 In vitro surface [6]Yes EhCP2 26 In vitro membrane-associated [7]Yes EhCP112 355 In vitro (zymogram) surface [8]

Yes EhCP5 30 In vitro (zymogram) and it is essential ex vivo forthe cleavage of the collagen network surface [9 10]

Yes mdash 56 In vitro secreted [11]No mdash 72 In vitro (zymogram) crude extracts and EDG [12]

Yes mdash 27ndash29 In vitro [13]

LamininYes EhCP4 26 In vitro secreted and nuclear [14]Yes mdash 56 In vitro secreted [11]


FibronectinYes EhCP112 355 In vitro (zymogram) surface [8]Yes mdash 56 In vitro secreted [11]


Gelatin No mdash 110 68 56 and 22 Induced by collagen conditioned medium [15]Immunoglobulins

IgA Yes EhCP4 26 In vitro secreted and nuclear [14]Yes mdash 70 In vitro [16]

IgG

Yes EhCP5 30 In vitro surface [9]Yes EhCP4 26 In vitro secreted and nuclear [14]Yes EhCP1 27 In vitro surface [17]Yes mdash 56 In vitro secreted [11]

Complement

C3Yes EhCP4 26 In vitro secreted and nuclear [14]Yes EhCP1 27 In vitro surface [17]Yes mdash 56 In vitro [18]

C3a and C5a Yes mdash 56 In vitro [19]Cytokines

proIL-18Yes EhCP4 26 In vitro secreted and nuclear [14]Yes EhCP5 30 In vitro surface [20]Yes EhCP1 27 In vitro surface [17]

proIL-1120573 Yes EhCP1 27 In vitro surface [21 22]EhCP2 26 In vitro membrane-associated [21 22]

Iron-containing proteins

Hemoglobin

Yes EhCP112 355 In vitro (zymogram) surface [8]Yes EhCP5 30 In vitro (zymogram) surface [9]Yes mdash 41 27 In vitro intracellular [23]No mdash 32 40 In vitro total extract [24]Yes mdash 22 In vitro [25]Yes mdash 16 In vitro [26]

No mdash 116 82 28 and 21 Human porcine and bovine Hb in vitro(zymogram)

[27]

Transferrin No mdash 130 43 20 and 6 Total extractin vitro (zymogram) [28]

No mdash 130 70 50 35and 30

Conditioned mediumin vitro (zymogram) [28]


Table 1 Continued

Tissue or protein Purified Name Mr (kDa) Comment localization Reference

Lactoferrin No mdash 250 100 40 and22

Total extractin vitro (zymogram) [29]

Ferritin No mdash 100 75 and 50 Total extractin vitro (zymogram) [30]

Other proteins or tissues

Mucin Yes EhCP5 30 In vitro (zymogram) although ex vivo is not neededto cross the mucus surface

[9 31]

Proteoglycan Yes EhCP2 26 In vitro membrane-associated [7]

Villin Yes EhCP1 27 In vitro surface [14]Yes EhCP4 26 Iin vitro secreted and nuclear [14]

Fibrinogen Yes EhCP5 30 In vitro (zymogram) surface [9]

BSA Yes EhCP5 30 In vitro (zymogram) surface [9]EDG electron-dense granules

Ly

EhCP4EhCP5

EhCP1

EhCP2

NucleusLy

EhCP112EhCP3

Ly = Lysosome

Figure 4 Localization of proteases in E histolytica

EhCP2 (Histolysain) A one-step method for the purificationto homogeneity of the histolysain of a soluble parasite extractby affinity chromatography to a synthetic substrate has beenreported The enzyme showed an apparent Mr of 26 kDa bySDS-PAGE and 29 kDa by gel chromatography Its optimumpH varied widely with synthetic substrates from 55 to 95EhCP2 did not degrade type-I collagen or elastin but wasactive against cartilage proteoglycan and kidney glomerularbasement-membrane collagen EhCP2 also detaches cellsfrom their substrates in vitro and could play a role in tissueinvasionThis EhCP is located on the cell surface as well as ininternal membranes [7]

EhCP3 (ACP1) EhCP3 (ACP1) was obtained by PCR am-plification of E histolytica genes using primers based onconserved structural motifs of eukaryotic CPs It was in-itially reported to be present only in E histolytica [77]

but a related homologue gene (edcp3) with 95 iden-tity has been detected in E dispar [35 84] EhCP3 (308amino acids) is synthesized as a preproenzyme in which 13amino acids form a signal peptide and 79 form a profrag-ment (httpwwwuniprotorguniprotP36184) As a (His) 6-tagged protein themature enzymemigrates with an apparentMr of 31 kDa [75] The enzyme was cloned and expressedin baculovirus and purified rEhCP3 was active in a broadpH range of 45ndash80 and had a neutral optimal pH EhCP3was sensitive to E-64 a specific CP inhibitor The sequenceof EhCP3 is most similar to that of cathepsin L while itssubstrate preference for positively charged amino acids inthe P2 position is consistent with cathepsin B [75] AlthoughEhCP3 is a relatively low-level transcript in HM-1IMSS afterEhCP2 EhCP5 and EhCP1 [85] its expression is 100-foldhigher in the Rahman strain [86]

Using confocal microscopy with monoclonal antibodies(mAbs) to EhCP3 EhCP3was localized primarily in the cyto-plasm Following erythrophagocytosis EhCP3 colocalizeswith phagocytic vesicles [75] The presence of a homologousgene inE dispar in addition to its localization inE histolyticaphagosomes has led to the hypothesis that the primaryfunction of EhCP3 may be associated with the digestion ofnutrients rather than virulence however further studies (ieknockdown experiments) are needed to clarify its function

EhCP4 EhCP4 was described as a positive clone in agenomic library screened with an oligonucleotide probederived from a sequence conserved between ehcp1-3 andehcp5 However by Northern blot the expression of ehcp4and ehcp6 was barely detected in amoebae grown in vitroAnalogous genes are present in E dispar but just as whatoccurs with E histolytica their expression is almost unde-tectable in vitro [35] In a recent transcriptional analysiscomparing E histolytica HM-1IMSS isolated from mouseintestine with cultured amoebae from the same strain bothEhCP4 and EhCP6 were upregulated in trophozoites inthe mouse gut (20ndash35 fold and 10-fold increased resp) infact ehcp4 (ehcp-a4) was the most upregulated CP geneduring invasion and colonization in a mouse cecal model


of amoebiasis [79] Upregulation of ehcp4 in vivo correlatedwith the finding that coculture of amoebae with mucin-producing T84 cells increased ehcp4 expression up to 6-foldEhCP4 was cloned including the prodomain and catalyticdomain for expression in bacteria EhCP4 was purified andunderwent autocatalytic activation at acidic pH but hadgreatest proteolytic activity at neutral pH The calculated Mrof the mature enzyme was 23 kDa but the apparent Mr was26 kDa as assessed by SDS-PAGE [14]

Sequence alignment and computer modeling confirmedthat EhCP4 is a member of Clan CA C1A subfamily with acathepsin L-like structure [73 75 87] similar to previouslycharacterized EhCPs However unlike previously character-ized EhCPs which have a preference for cathepsin B or Lsubstrates with arginine in the P2 position EhCP4 displaysa unique preference for valine and isoleucine at P2 [14]EhCP4 is localized in the peri- and intranuclear regionsas well as in variable-sized vesicles in the cytoplasm Inaxenic amoebae EhCP4 is primarily cytoplasmic but in thoseisolated from infected mouse ceca EhCP4 is found in boththe nuclear and cytoplasmic extracts suggesting a role forEhCP4 in the cell cycle or differentiation as is the case forhigher eukaryotic CPs with a nuclear localization [88ndash90]Following coculture with colonic cells EhCP4 appeared inacidic vesicles and was released extracellularly Indeed inthe infected murine cecum immunofluorescence stainingof EhCP4 yielded strong signals in vesicle-like structureslocated in discrete surface areas Thus EhCP4 is apparentlysecreted in vivo [14]

EhCP4 cleaves several host proteins in vitro althoughin agreement with the different substrate specificity thepredicted digestion patterns differ from those of EhCP1EhCP4 degrades C3 unlike EhCP1-mediated C3 processingwhich produces an active C3b molecule [17 18] EhCP4also degrades IgA IgG pro-IL-18 villin-1 and laminin-1 [14] Thus like EhCP1 EhCP4 could play an importantrole in destroying the integrity of tissues and evading theimmune system and may contribute to the inflammatoryresponse in amoebic lesions [91] Based on the substratepreference and homology modeling a specific vinyl sulfoneinhibitor WRR605 was synthesized which selectively inhib-ited rEhCP4 Treatment with WRR605 notably decreasedthe amoeba burden and intensity of cecal inflammation in amouse cecal model of amoebiasis [14] Together these resultssuggest that EhCP4 is an important virulent factor in Ehistolytica and a promising therapy target

EhCP5 A soluble protein of Mr 30 kDa with an affinity formembranes was purified from amoebic extracts N-terminalsequencing and subsequentmolecular cloning revealed that itwas a member of the CP family of E histolytica The enzymestrongly associates with the membrane retaining its prote-olytic activity and produces cytopathic effects on culturedmonolayersThe 3D structure of EhCP5 revealed the presenceof a hydrophobic patch that may account for membraneassociation potential of the protein Immunocytochemicallocalization of the enzyme at the amoeba surface suggests apotential role in tissue destructionThe gene encoding EhCP5is not expressed in the closely related but nonpathogenic

species E dispar [87 92] Trophozoites of the virulent straintransfected with an antisense gene encoding EhCP5 hadonly 10 activity but retained their cytopathic effect onmammalian cell monolayers and were incapable of inducingthe formation of liver lesions in hamsters However thisactivity cannot be attributed only to EhCP5 because EhCP5antisense mRNA also inhibited the expression of other CPswhichmay be due to a high degree of sequence homology andconservation of residues critical for protease function [71 93]

EhCP5 has been confirmed to degrade a broad spec-trum of biological and synthetic substrates such as mucinfibrinogen collagen hemoglobin bovine serum albumingelatin IgG Z-Arg-Arg-pNA and Z-Ala-Arg-Arg-pNA butnot Z-Phe-Arg-pNA The identification of rEhCP5 as a CPwas determined by using specific inhibitors [9] EhCP5activity and mRNA levels were analyzed in E histolyticasamples isolated from patients presenting different clinicalprofiles The degree of virulence of the isolates determinedin hamster livers correlated well with the clinical form ofthe patients and with the culture conditions EhCP5 mRNAlevels were also determined in fresh samples of amoebicliver abscesses Differences were not observed in the levels ofEhCP5 mRNA and CP specific activity among the culturedsamples However different levels of EhCP5 mRNA wereobserved in amoebae freshly isolated from hepatic lesionsThese results emphasize the importance of EhCP5 for amoebavirulence and the need for additional studies to validate themechanisms involved in the pathogenesis of amoebiasis [94]

EhCP5 was expressed in the bacterium Escherichia coli asa proenzyme and purified to homogeneity under denaturingconditions in the presence of guanidine-HCl EhCP5 wasrenatured in buffer containing reduced and oxidized thiolswhich led to a soluble but enzymatically inactive proen-zyme Further processing and activation was achieved inthe presence of DTT and SDS The recombinant enzyme(rEhCP5) was indistinguishable from native EhCP5 purifiedfrom amoebic lysates Under reducing and nonreducing con-ditions rEhCP5 exhibited Mr of 27 and 29 kDa respectivelywith the same optimal pH and similar specific activity againstazocasein [9]

EhCP112 EhCP112 is a papain-like proteinase [95] It wasfirst identified by using a mAb recognizing a protein presentin wild-type trophozoites but missing in an adherence-deficient mutant The mAb inhibited adhesion of amoebaeto target cells and a 112 kDa protein was identified byWestern blot [59] The DNA encoding this molecule wasisolated from a genomic library revealing that the 112 kDaadhesin is formed by two polypeptides (49 kDa and 75 kDa)encoded by two different genes separated by 188 bp The49 kDa polypeptide is a CP (EhCP112) whereas the 75 kDaprotein has a domain involved in the adherence of amoebato target cells (EhADH112) both proteins form a complexcalled EhCPADH Within this complex the peptides couldbe joined by covalent or strong electrostatic forces and theirproximity in the genome suggests a coregulated expression[60]

EhCP112 (446 amino acids) is synthesized as a pre-proenzyme in which 19 amino acids form the signal peptide


and 112 amino acids form the profragment The matureprotease has a putativeMr of approximately 34 kDa EhCP112contains a transmembrane segment domain characterized bythe catalytic triad (C H andN) in addition to an RGDmotifwhich is an integrin attachment domain [95] A previousreport suggests that the majority of E histolytica CP genesincluding the ehcp112 gene are not expressed during in vitroculture [73] However Northern blot and RT-PCR assaysindicate that the ehcp112 gene is expressed in trophozoites[60 96] Different culture conditions or different pathogenic-ities of trophozoites could be responsible for the distinctresults

EhCPADH is located in cytoplasmic vesicles and theplasmamembrane and is also secreted EhCPADH is translo-cated from the plasma membrane to phagocytic vacuolesduring phagocytosis [60] This implies a function in nutri-ent acquisition However EhCPADH has been associatedwith amoebae virulence Antibodies against this complexinhibit adherence phagocytosis and destruction of MDCKmonolayers by live trophozoites and extracts Antibodiesalso greatly reduce the ability of amoebae to produce liverabscesses in hamsters [59 60 97ndash99]

To elucidate the role of EhCP112 in virulence itwas cloned expressed in bacteria and purified rEhCP112degrades collagen type I and fibronectin and destroys cellmonolayers (MDCK) rEhCP112 also binds to erythrocytesand digests human hemoglobin [8] rEhCP112 is active ina broad pH range (3ndash10) with the highest activity at pH70 for azocasein and pH 60 for hemoglobin EhCP112 wasenzymatically active from 5 to 60∘Cwith a maximum activityfor both substrates at 37∘C [95] Interestingly the EhCP112enzyme is immunogenic in patients with amoebiasis [8]

212 Proteases Involved in Trophozoite Adhesion Leish-manolysin (Gp63) is a metalloendopeptidase that is essen-tial for the virulence of Leishmania major Leishmanolysindegrades ECM proteins during tissue invasion and preventscomplement-mediated lysis of promastigotes [100 101] Twoleishmanolysin homologues are encoded in the E histolyticagenome but only one copy of the gene is present in theclosely related E dispar The E histolytica specific familymember EhMSP-1 is a functional metalloprotease (MP) thatis localized to the cell surface Silencing EhMSP-1 expressiondramatically increases amoebae adherence to live and deadcells reducing mobility in cell monolayers and increasingphagocytosis The knockdown of EhMSP-1 also reduces cellmonolayer destruction but has no effect on the lysis of Jurkatlymphocytes Thus one possibility is that this phenotypecould be the consequence of defective adherence becauseincreased adherence may reduce the ability to move whichin turn causes the amoebae to be less able to destroy cellmonolayers The ultimate mechanism by which EhMSP-1affects adherence remains unknown However it is knownthat EhMSP-1 knockdown does not affect steady-state sur-face abundance of previously identified adhesins such asthe GalNAc-specific lectin and the serine-rich E histolyticaprotein (SREHP) [102ndash104] The ability of the CP inhibitorE-64 to almost completely block cell monolayer destruction

suggests that EhMSP-1 silencing affects monolayer destruc-tion indirectly and that EhMSP-1 itself likely does not directlydegrade cell monolayers [91 102 105] Apparently EhMSP-1plays no role in in vitro resistance to complement in contrastto leishmanolysin

The rhomboid-like proteins are a large family of seven-pass transmembrane serine proteases (SPs) that were firstidentified in Drosophila melanogaster and whose active sitelies within the lipid bilayer allowing them to cleave trans-membrane proteins [106 107] and hence trigger signalingevents [108] Substrates of rhomboid proteases are largelysingle-pass transmembrane proteins with transmembranedomain containing helix-breaking residues [109] The Ehistolytica genome encodes four rhomboid-like genes ofwhich only a single gene EhROM1 contains the necessarycatalytic residues for proteolytic activity [110] In resting con-ditions EhROM1 is localized at the trophozoite surface andin internal punctuate structures upon erythrophagocytosisit relocalizes in internal vesicles and during surface receptorcapping in the base of the cap The heavy subunit of theGalGalNAc lectin (Hgl) is a substrate of EhROM1 in vitro[110] EhROM1 knockdown leads to defects in both adhesionand phagocytosis but not cap formation or complementresistance

Importantly the reduced phagocytosis and adhesion phe-notypes appear to be independent implying that EhROM1has distinct roles in both pathways Interestingly there wereno significant changes in the expression or localization of theonly known substrate the heavy subunit of the GalGalNAclectin in EhROM1 knockdown parasites Several scenarioshave been proposed for how EhROM1 regulates parasiteadhesion without affecting the expression or localizationof the heavy subunit of the GalGalNAc lectin EhROM1may process a different adhesin or a different substrate thatmasks the GalGalNAc lectin adhesion or it may play arole in signaling during the adhesion process by detachingthe signaling integrin-like motif present in the cytoplasmicdomain from the rest of the GalGalNAc lectin (Figure 5)Alternatively EhROM1 may have a noncatalytic role in theadhesion process as has been described for other rhomboidproteases [111 112]

Cell-adhesion processes and proteolytic mechanismsfunction in a coordinated manner to provide directed cellmigration and are critical at the molecular level adhesionreceptors (integrins) are displayed on the cell surface andbind to ECM proteins whereas proteases (eg MPs and CPs)modify or degrade ECM components However some pro-teases interfere with cell adhesion andor migration in a non-proteolytic fashion [113ndash116] In cell-adhesion proteins suchas fibronectin RGD motifs serve as ligand recognition sitesfor cell-surface receptors such as integrins An RGD motifhas been found in the pro-region of the lysosomal cathepsinX from higher eukaryotes and apparently this motif canmediate adhesion and migration processes via binding tointegrins [114] Only EhCP5 EhCP18 and EhCP112 presentthis motif [73] The RGD motif is present in the pro-formof EhCP5 (EhPCP5) the RGD motif binds to 120572(V)120573(3) inte-grin on Caco-2 colonic cells and stimulates NF120581B-mediatedproinflammatory responses Binding to this integrin triggers


EhROM1

EhROM1

LgI

IgIHgI

EhROM1

LgI

IgIHgI

(a) (b) (c)

Figure 5 Proposed scenarios for how EhROM1 regulates parasite adhesion EhROM1 may process an unknown adhesin (a) or a differentsubstrate that masks the GalGalNAc lectin adhesion (b) or EhROM1 may play a role in signaling during the adhesion process by detachingthe signaling integrin-like motif present in the cytoplasmic domain from the rest of the GalGalNAc lectin (c)

integrin-linked kinase- (ILK-) mediated phosphorylation ofAkt-473 which binds and induces the ubiquitination of theNF-120581B essential modulator (NEMO) (Figure 6) As NEMOis required for the activation of the IKK120572-IKK120573 complex andNF120581B signaling these events markedly up-regulate proin-flammatory mediator expression in vitro in Caco-2 cells andin vivo in colonic loop studies in wild-type and Muc2(minusminus)mice lacking an intact protective mucus barrierThese resultshave revealed that the EhPCP5 RGDmotif represents a novelmechanism by which trophozoites trigger an inflammatoryresponse in the pathogenesis of intestinal amoebiasis [117]

As mentioned earlier EhCPADH is a surface complexthat binds to target cells [59 60 99] The role of EhCP112 inadhesion could be limited to its association with the adhesinEhADH112 although EhCP112 contains the RGD domainsuggesting that it binds to cellular integrins [8 118] Theparasite may utilize surface-bound proteases to interact withintegrins via the RGD motif to tether and propel along theendothelium much like immune cells crawling along thevasculature to survey blood contents for pathogens [119]

213 Proteases That Degrade Mucin Mucous is the firstlayer that protects the colonic epithelium from potentiallypathogenic viruses bacteria and parasites [120]The ability ofamoebae to invade may be inhibited by the binding of mucinto lectin which inhibits lectin activity and by the physicalbarrier between the parasite and the intestinal epitheliumcreated bymucin [64]MUC2 is themajor gel-formingmucinsecreted by goblet cells of the small and large intestines and isthemain structural component of themucus gel [121 122] Togain access to the underlying epithelial cells amoebae mustfirst breach the protective mucous layer This phenomenonhas been observed in biopsies from patients with acuteamoebic colitis [58] and has been confirmed in an ex vivo

human colonic model of amoebiasis after just two hoursof incubation the mucus layer was no longer observablesuggesting that it had been removed by amoebae [123] Ehistolytica secreted CPs are responsible for degrading MUC2[124] cleavage at the major cleavage site is predicted todepolymerize the MUC2 polymers thereby disrupting theprotective mucus gel [125] In addition the parasite encodesglycosidases that degrade mucin oligosaccharides [126] Ehistolyticamay use the concerted actions of glycosidases andproteases to disassemble the mucin polymeric network

Trophozoites with reduced CP activity are ineffectivein degrading and crossing the protective mucus layer pro-duced by cell lines in culture [31 93] However contro-versy remains about the effector protease(s) responsiblefor mucin degradation Bioactive recombinant EhCP5 iscapable of degrading purified native mucin [9 31] Howeveran EhCP5amoebapore-silenced strain was able to crossthe mucus barrier in an ex vivo colonic model but wasunable to migrate within the mucosa This behavior suggeststhat other proteases produced by the EhCP5amoebapore-silenced strain appear to be involved in the removal ofthe colon mucin gel [123] Alternative effector proteasesinclude EhCP1 EhCP2 and EhCP4 because their expressionis increased after trophozoites are exposed to mucin [14 127]This suggests that the specific composition of mucin mayaffect the ability of E histolytica CPs to degrade the mucouslayer

214 Alterations in Microvilli and Tight Junctions Afterthe mucus the tight junction (TJ) complex constitutes thefirst barrier against the paracellular penetration of intesti-nal microorganisms This barrier is formed by the plasmamembrane-spanning proteins claudin and occludin whichare associated with the cytoplasmic proteins ZO-1 ZO-2 and


RGD

Aktt

Ub

ILKPI3K

Akt

P

Degradation

Host cell

P

P

+

P

Ub

P473

P473

IKK NEMO

IKKγNEMO

IKK IKK

IKKα IKKβ

I BI B

NF- B

NF- B

Nucleus

TNF-IL-1

EhPCP5

Figure 6 EhPCP5 stimulates NF120581B-mediated proinflammatory responses Attached to E histolytica surface or secreted EhPCP5 binds to120572(V)120573(3) integrin through the RGDmotif and triggers PI3K-mediated ILK activation ILK phosphorylates Akt-473 which binds and inducesthe ubiquitination of NEMO This activates the IKK120572-IKK120573 complex that phosphorylate I120581B120572 This phosphorylation event signals I120581B120572ubiquitin-mediated degradation and thereby the release of NF-120581B into the nucleus where it activates proinflammatory gene expression

ZO-3 E histolytica is able to disturb the TJ complex fromhuman colonic adenocarcinoma T84 and MDCK T23 cellsTrophozoites decrease the transepithelial electrical resistancein T84 cells and to a lesser extent in MDCK cells [128]The same group of researchers subsequently observed thedephosphorylation of ZO-2 loss of ZO-1 from ZO-2 anddegradation of ZO-1 when amoebae were cocultured withT84 cells [129] By contrast [130] analyzed the interactionbetween trophozoites and human Caco-2 cell layers andidentified a crucial role for amoebic CPs in the preventionof villin proteolysis and associated microvillar alterationsthrough the treatment of Caco-2 cell layers with inhibitorsthat completely block CP activity Moreover trophozoites ofstrains pSA8 and SAW760 which have strongly reduced CPactivity exhibited reduced proteolysis of villin in coculturewith enteric cells indicating that villin proteolysis is anearly event in the molecular cross-talk between enterocytesand amoebae that causes the disturbance of microvilliBoth EhCP1 and EhCP4 are capable of degrading humanvillin in vitro and thus they could play an important rolein destroying the integrity of microvilli in vivo (Che He2010) In addition [131] reported that TPCK and TLCKtwo SP and CP inhibitors can affect E histolytica pro-teases and prevent proteolysis of ZO-1 ZO-2 and villinin Caco-2 cells Taken together these results indicate thatamoebae use their proteinases to overcome microvilli andtight junction barriers during the invasion of enteric celllayers

215 Proteases That Degrade the Extracellular Matrix Cys-teine proteases have been demonstrated to act on a varietyof substrates in vitro In the host parasite proteases facilitatetissue penetration by dissolving the intestinal mucosa andextracellular matrix (ECM) and freeing epithelial cells [7 1127 125 130 132ndash134] More than 80 of patients sufferingfrom amoebiasis express antibodies to trophozoite CPs [135]Interestingly purified enzymes that degrade one of the ECMproteins have been found to cleave other ECM proteinsgenerally fibronectin collagen and laminin The interactionof amoebae with ECM components results in the proteolysisand destruction of the connective tissue

For 30 years proteases degrading native type-I and type-III collagen fibers have been studied in cultured E histolyticaalthough CPs were purified more recently Collagenase ismore active against type-I collagenThreemajor fragments of75 50 and 25 kDa were obtained from type-I collagen uponincubation with amoebae After 3 h of incubation smallerfragments of degraded collagen were observed possiblydue to the action of other proteolytic enzymes [136] Inother work FPLC-anion-exchange and chromatofocusingchromatography were used to purify the major neutralproteinase from secretions of the cultured E histolyticaHM-1IMSS strain This proteinase is a CP with a subunit Mrof approximately 56 kDa a neutral optimum pH and apI of 6 The importance of this enzyme in extraintestinalamoebiasis is suggested by its ability to degrade a modelof connective tissue ECM as well as purified fibronectin


laminin and type-I collagen The enzyme caused a loss ofadhesion of mammalian cells in culture most likely becauseof its ability to degrade anchoring proteins Experimentswith a peptide substrate and inhibitors indicated that theproteinase preferentially binds peptides with arginine at P-1It is also a plasminogen activator and could thus potentiatehost proteinase systems [11] A 56 kDa band was identifiedby polyacrylamide-gelatin gel electrophoresis in 10 of 10isolates from patients with colitis or amoebic liver abscessesbut in only 1 of 10 isolates from asymptomatic patientsPathogenic isolates appear capable of releasing significantlylarger quantities of this CP which is released during thecourse of invasive disease as demonstrated by the pres-ence of circulating antibodies detectable by enzyme-linkedimmunosorbent assay (ELISA) These studies support theimportance of the 56 kDa CP in the pathogenesis of invasiveamoebiasis [91]

The collagenase activity was mainly observed in electron-dense granules These granules were induced and secreted inresponse to the incubation of amoebae with collagen typeI in vitro A specific collagenase activity with Mr of 72 kDawas identified in crude extracts We observed this activityin electron-dense granules this activity could be related toactin cytoskeleton function because the cytoskeleton-alteredamoeba strain BG-3 derived from the HM-1IMSS strainexhibits lower collagenase activity [12 137] Incubation ofamoebae with type-I collagen not only promotes collagenaseactivity but also increases the secretion of other CPs [15] andtogether with Ca2+ type-I collagen can induce the activationof several amoebic genes related to virulence factors suchas amoebapore C and EhCP5 along with the stress-inducedprotein HSP70 and the ribosomal protein L27a [138] Inaddition a correlation between collagenolytic activity andvirulence was observed when the levels of activity of differentstrains (virulent HM-1IMSS and the less virulent 200-NIHand HK-9) [139ndash141] or of other virulence factors [142] werecompared suggesting that collagenase may play a role inthe pathogenesis of amoebiasis In a recent study Chavez-Munguia et al [143] demonstrated that electron-dense gran-ules contain multiple CP activities

Recently the behavior of wild-type and EhCP5-silencedE histolytica was compared on a 3D-collagen matrix andwithin human colon fragments for fibrillar collagen cleavageand migration Interstitial collagen fibers within the connec-tive tissue of the human colon visualized by multiphotonand second harmonic generation signal imaging presented adense scaffold at the subepithelial level and a loose meshworkwithin the chorion To penetrate the tissue wild-type Ehistolytica migrated on the dense scaffold that remainedintact reached the crypt of Lieberkhun migrated alongand then disorganized the loose scaffold to escape into themucosa However in vitro EhCP5 was not required forcollagenase activity and migration through the matrix butwas necessary within the tissue environment for collagenmeshwork remodeling and subsequent invasion The dataindicate that subsequent steps of the invasion relay with ECMdestruction require human components that are induced oractivated in the presence of EhCP5 [10]

Themajor 27ndash29 kDaCPhas been studied since 1989Thiscystatin-inhibitable CP degrades the ECM proteins collagentypes IV and V as well as laminin and fibronectin withdifferent velocities and specificities under native conditionsWhereas the degradation of fibronectin and laminin proceedsrapidly collagen breakdown occurs slowly and incompletelyThis CP represents by far the highest portion of solubleproteolytic activity in E histolytica and is sufficient to destroythe host ECM [13] Similar results have been reported for the27 kDa CP with respect to the binding specificity for immo-bilized laminin over collagen and fibronectin Inactivation ofthe CP with the active-site inhibitor E-64 abolishes lamininbinding by the enzyme and conversely laminin inhibitsthe cleavage of a fluorogenic dipeptide substrate of the CPsuggesting that the substrate binding pocket of the enzymebinds to laminin Furthermore the addition of laminin butnot fibronectin or collagen to amoebae significantly reducesamoebic liver abscess formation in severe combined immun-odeficient (SCID) mice further supporting the assumptionthat CPs play an important role in amoebic pathogenesis[21]

An in vitromodel was developed to analyze the adhesionto and cleavage of human fibronectin-covered surfaces Thismodel revealed the specificity of the binding and occur-rence of structural and biochemical events in amoebae thatparticipate in and promote adhesion to the substrate andits degradation Similar results were obtained with lamininand Matrigel [144 145] A putative amoebic fibronectinreceptor with Mr of 37 kDa was identified Adhesion tofibronectin triggers the release of proteases which facilitatesthe local degradation of the substrate [144 146] Some ofthe proteases may generate fragments with chemotactic andchemokinetic properties that are able to promote the bindingand locomotion of amoebae [147] In later studies ECMproteins have been shown to be cleaved in vitro by identifiedCPs collagen is degraded by EhCP1 [6] EhCP2 [7] andEhCP112 [8] fibronectin by EhCP112 [8] and laminin byEhCP4 [14]

216 Cytopathic Activity Several studies have shown a directcorrelation between E histolytica CPs and tissue damage asCP activity decreases tissue damage decreases This relation-ship has been tested using trophozoites with low levels ofCPs in an amoebic liver abscess hamster model [71] or inhuman colonic xenografts [22] The inhibitor of CP activityE-64 reduces abscess formation in SCID mice [148] andspecific inhibitors of EhCP4 reduce cecal inflammation [14]or specific inhibitors of EhCP1 in human colonic xenografts[17] In the opposite strategy overexpression of ehcp5 but notehcp1 or ehcp2 significantly increased abscess formation ingerbils [149]

The results of in vivo experiments reflect all of thepleiotropic effects of EhCPs in the pathogenic mechanismof amoebiasis Some research has sought to probe the directcytopathic effect of each EhCP but the resulting data arecontroversial Cytopathic activity as measured by in vitromonolayer disruption was increased in EhCP5- and EhCP2-overexpressing E histolytica [149 150] However silencing


of the major ehcp genes (ehcp1 ehcp-2 ehcp-5 and ehcp7)or a reduction of expression to only 10 of the regularlevels of CP activity did not influence trophozoite-inducedcell monolayer disruption suggesting that these peptidasesare dispensable for cytopathic activity [93 151] All of theseresults were obtained using live intact trophozoites howeverlysates of virulent E histolytica have also been shown todestroy monolayers [152] Lysates of amoeba with only 10of the total EhCP activity exhibited very poor activity againstmonolayers This result was confirmed using the generalCP inhibitor E-64 while CPs of viable trophozoites areresponsible for 50 of cytopathic activity this inhibitorcompletely blocks the cytopathic activity of E histolyticalysates [150] The effects of E-64 suggest that CPs are notthe main virulence factor involved in the destruction ofmonolayers by intact trophozoites but they must play somerole particularly whenever there is also some lysis of thetrophozoites [93]

Nevertheless these data should be interpreted cautiouslybecausemonolayer destructionwas assayed these assays can-not distinguish between monolayer releases reflecting ECMdegradation from actual cell death [57] The investigationof the response of cultures of a human liver sinusoidalendothelial cells (LSECs) line to incubation with virulentor virulence-attenuated E histolytica shed some light onthis controversy The data obtained suggest that amoebainterference of the integrin-focal adhesion signaling pathwayplays a role in the induction of human cell retraction anddeath Using silenced strains the authors reported that thisphenomenon occurred independently of the cytolytic amoe-bapore but required galactose-inhibitable parasite adhesionlikely involving amoebic GalGalNAc lectin and active Ehistolytica CPs [153] This is in agreement with the findingthat secretory CPs of E histolytica are capable of inducinganoikis in MDA-MB-231 cells Anoikis is a specialized typeof apoptosis that mammalian epithelial endothelial andvarious other cell types experience upon their detachmentfrom the extracellular matrix (ECM) Based on the results ofthese two studies a model of pathogenesis of amoebiasis maybe proposed involving CP-mediated degradation of ECM-cell and cell-cell attachments thereby inducing anoikis ofhepatocytes and enterocytes which could lead to liver abscessand colonic ulcer formation However further investigationsare needed to elucidate the role of anoikis in amoebiasis[154]

217 Proteins of the Immune System AreCleaved by Cysteine Proteases

Immunoglobulins Secretory IgA (sIgA) Abs are considered afirst line of specific defense against natural infections in thevast area occupied by mucosal surfaces [155] sIgA functionsin vivo by reducing mucosal colonization by pathogens andneutralizing diverse toxins and enzymes [156] E histolyticaelicits a local immune response in which an increase inspecific IgA is detectable in several compartments associatedwith themucosa [157 158] IgA inhibits the in vitro adherenceof amoebae to epithelial cell monolayers by recognizing

several membrane antigens [157 159] reduces proteolyticactivity [160] and has amoebicidal action [161] Several linesof evidence indicate that sIgA protects against E histolyticainfection [157] Children with stool IgA specific for lectinappear to be protected from intestinal infection [162] andGal-lectin heavy subunit-specific intestinal IgA is sufficientto provide immunity against E histolytica intestinal infectionin a baboon model [163]

Interestingly several lines of in vitro evidence supportthat E histolytica strikes back by using its surface andsecreted CPs to degrade host sIgAWhen serum and sIgA areexposed to viable axenic trophozoites (strain HM-1IMSS) aparasite sonicate or conditionedmedium by incubation withlive amoebae sIgA is completely degraded and proteinaseactivity ismaximal at a neutral pH and is completely inhibitedby E-64 [16] Serum and sIgA are susceptible to degradationby amoeba surface-associated CPs both sIgA1 and sIgA2 aredegraded in a similar fashion by surface amoeba proteasesHowever sIgA2 is functionally more resistant to proteolysisthan sIgA1 [156] The CP identity was not determined but itcould be a 70 kDa protease [16] EhCP4was recently shown todegrade IgA in vitro [14] however it is unclear if it is the sameprotease or if E histolytica has more than one IgA-degradingprotease In fact Garcia-Nieto identified this activity as asurface protease and EhCP5 unlike EhCP4 is found inthe amoeba plasma membrane [87] leading the authors tosuggest that this enzyme could be the main protease involvedin sIgA degradation [156]

Specific antiamoeba IgG responses are developed ingt95of patients with amoebiasis or even in individuals with Ehistolytica asymptomatic colonization [164] However it isdifficult to ascribe a protective role to IgG because the levelof Ab response correlates with the length of disease not withthe clinical response to infection [164] and mixed resultshave been obtained with animal models [165 166] This maybe because IgG is cleaved by amoebic CPs [34] A 56 kDaproteinase purified from E histolytica cleaved polyclonalhuman and monoclonal murine IgG in a dose-dependentmanner Intact trophozoites also cleave IgG The resultingcleaved mAb bound to trophozoites with lower affinity thanthe uncleaved antibody indicating a decrease in affinity andlimiting the effectiveness of the host humoral response tothe parasite [11 167] The identity of the CP in this study isalso unknown although later in vitro studies with purifiedrecombinant enzymes have demonstrated that EhCP1 [17]EhCP4 [14] and EhCP5 [9] degrade IgG

Immunoglobulin cleavage in amoebiasis could decreasethe affinity of the antibody for antigen or if the Fc portionwas removed trophozoites could evade the immune systemby coating their surface molecules in Fab fragments [133]This would prevent the activation of complement by theclassical pathway and attack from immune cells bearingcorresponding Fc receptors [34 119]

Complement Complement (C) is activated when zymogensin serum are cleaved and produces an enzyme cascade thatresults in nonspecific binding of C components to pathogensurfaces The two primary functions of C are to directlylyse foreign cells by the membrane attack complex C5b-9


and to opsonize pathogens with C molecules [119] C3 isthe central molecule of the alternative pathway hydrolysisof C3 generates its active fragments C3a and C3b Covalentdeposition of C3b on nearby surfaces triggers a cascade ofevents that end in membrane attack complex-mediated lysis[168]

The interaction of amoebae with C seems to be quitecomplex In a comparative in vitro analysis of several amoebicstrains a tight correlation between C resistance and thedegree of amoebic virulence was observed [169] Howeverduring liver abscess development in hypocomplementemicanimals tissue damage and parasite survival increase [170]E histolytica can resist lysis mediated by the C systemthrough extracellular C activation through the action ofEhCPs The 56 kDa secreted neutral CP activates the alter-native pathway of C by cleaving C3 in the fluid phaseCP action is similar to C-derived C3 convertases and nofurther degradation occursTherefore the C3b-like moleculeproduced is hemolytically active as demonstrated by itsability to accelerate activation of the alternative pathway of Cin rabbit erythrocytes [18] Only C-sensitive nonpathogenicE histolytica are lysed by this fluid phase-activated C3b-like molecule while pathogenic strains are resistant [171]Pathogenic amoebae most likely achieve this by inhibitionof surface deposition of the membrane attack complex [119172] The protease involved has not been identified howeverEhCP1 has been shown to cleave C3 in a manner identical tothat of the secreted 56 kDa native proteinase [18] to generatethe 1205721015840 subunit and possibly form an active C3bmolecule [17]Likewise EhCP4 cleaves C3 and produces a fragment with asize similar to that of C3b however unlike EhCP1 EhCP4degrades C3b-like ones [14] Further studies are needed todetermine whether C3 is actually activated in vivo by EhCP1or by an as-yet unidentified protease or if it is actuallydegraded by EhCP4

C3a and C5a the small cleavage fragments released byC activation are potent mediators of inflammation and ana-phylatoxins [173]The same extracellular proteinase (56 kDa)of pathogenic E histolytica is capable of limiting a potentialhost defense mechanism by degrading C3a and C5a [19] Theanaphylatoxin blockade decreases immune detection in theblood and reduces inflammation in amoebic lesions partiallyexplaining the lack of severe inflammation in advanced liverand intestinal lesions [119]

Molecules Involved in Inflammation In a SCIDmouse-humanintestinal xenograft model infection with E histolyticatrophozoites elicits a robust inflammatory response fromthe grafted tissue characterized by strong IL-1120573 and IL-8expression an early neutrophil influx and extensive damageto the intestinal graft [174] Trophozoites with reduced CPactivity fail to induce intestinal epithelial cell production ofthe inflammatory cytokines IL-1120573 and IL-8 and cause signif-icantly less gut inflammation and damage to the intestinalpermeability barrier [22] Purified amoebic CPs possess IL-1120573 converting enzyme (ICE or caspase-1) activity in vitrocleaving recombinant human pIL-1120573 into a biologically activeform of IL-1120573 [22] These purified proteases are most likelyEhCP1 and EhCP2 [21 22] In this work the authors postulate

that amoebae first bind to intestinal epithelial cells and thenlyse those cells through the action of amoebapore [175 176]The lysed cells may release pIL-1120573 which could then beactivated by extracellular amoebic CPs with ICE activity andfurther amplify the inflammatory process in amoebic colitis[22]

IL-18 is expressed in intestinal epithelial cells [177] andis a coinducer of the Th1 response The resulting stimulationof IFN120574 then activates macrophages the major cell capableof killing E histolytica trophozoites [178] IL-18 and IL-1120573maturation requires cleavage by caspase-1 However in con-trast to the activation of proIL-1120573 by amoebic lysates purifiedrEhCP5 rEhCP1 and rEhCP4 cleave proIL-18 and matureIL-18 to biologically inactive fragments [14 17 20] Thesecontradictory findings in which EhCPs have opposite effectsin two proinflammatory cytokines have not been explainedthe balance between these EhCPs is likely controlled by acomplex interplay of parasite and host molecules

218 Proteins Containing Iron Are Degraded by AmoebicProteases for Use as Iron Sources for Growth Iron is a vitalelement for the survival of almost all organisms Howeverunder physiological conditions Fe3+ is not soluble and Fe2+is soluble but toxic and readily oxidizes to Fe3+ To increasesolubility avoid toxicity and keep iron away from intrudersthis element is normally complexed to proteins thus the freeiron concentration is far too low to sustain the growth ofintruders However successful pathogens are able to scavengeiron from host proteins [179ndash183] E histolytica as wellas other amitochondriate protists (eg Tritrichomonas Tri-chomonas and Giardia) requires particularly high amountsof extracellular iron in vitro (sim100 120583M) surpassing that of themajority of both eukaryotic and prokaryotic cells (04ndash4120583M)[184] This high iron requirement is attributable to the heavyreliance of their energy metabolism on Fe-S proteins [185ndash187]

We have reported that E histolytica trophozoites are ableto use four of the human iron-containing proteins as ironsources for the parasitersquos growth in axenic culture mediumin which ferric ammonium citrate was substituted by theferrous- or ferric-protein under investigation These proteinsare hemoglobin transferrin lactoferrin and ferritin [185] Inall cases amoebae were able to endocytose and cleave theprotein to obtain the needed iron (Figure 7) The use of theseproteases by trophozoites could be considered a virulencefactor because the pathogens seek out host iron to survive inthe hostile host environment [179 182 183 188 189]

Hemoglobin Hemoglobin (Hb) is a globular protein that ispresent at high concentrations in erythrocytes or red bloodcells (RBCs) The function of Hb is to trap oxygen in thelungs and transport it through the blood to tissues and cellsIn adultmammals Hb is composed of two alpha and two betachains each containing one hemeprosthetic group thereforethere are four Fe2+ atoms in the Hb molecule which has Mrof 645 kDa [190] Hb uptake by E histolytica trophozoitesoccurs by disrupting the RBC cytoplasmic membrane withsurface hemolysins and phospholipases The major amoebic


CPs

Ly

Clathrin-coated vesicles

EE

LE

Ly

Internalizationunknown

Erythrocyte

Amoebic clathrin

h-Hololactoferrin

h-Lactoferrin binding protein

Hemoglobin binding protein

Ferritin binding protein

h-Transferrin binding protein

Haemoglobin

Ferritin

Lipid raft-dependent

h-Holotransferrin

Nucleus

Vesicles with Hb sucked

Ph

Ly

LyLL EEL

Fluid phase

ADH2

Acetaldehydealcohol dehydrogenase2

EE = early endosomeLE = late endosome

Ly = lysosome

Ph = phagocytic vacuole

Figure 7 Host proteins containing iron are internalized and degraded by amoebic proteases for use as iron sources for growth

hemolytic activity has been characterized in rat RBCs thisactivitywas detected in a vesicular fraction [191 192] An alka-line phospholipase has also been associated with virulence[193]

In E histolytica there is little information regardinghow iron is obtained from Hb This parasitic protist isextremely active as a phagocytic cell once phagocytosedhuman RBCs are broken down by amoebae Chevez et al[194] described complete RBC digestion in sim6ndash8 h by Perlrsquosstaining experiments Quantitative digestion assays usingdiaminobenzidine staining to visualize RBCs revealed thatafter 9 h of RBC phagocytosis Hb was thoroughly degraded[194ndash196] Several researchers have studied the role of amoe-bic hemoglobinases in the cleavage of different types of HbThe degradation of native bovine Hb at pH 76 by extractedproteinases from different monoxenic strains was observed[197] Thirty-five years ago two proteinases against nativebovine Hb were purified [23] one of 41 kDa with optimal

activity at pH35 and another of 27 kDawith optimal activityat pH 60 Subsequently a cytotoxin of 22 kDa with strongproteolytic activity against denatured Hb at an optimal pH of45 was described and a cathepsin B of 16 kDa that was activeagainst native and denatured Hb was purified [24ndash26] Perez-Montfort et al [24] identified two proteins of 32 and 40 kDathat were able to degrade denatured Hb

Our group has described three proteases of 21 82and 116 kDa in extracts of E histolytica HM-1IMSS Theseproteases were able to degrade human bovine and porcineHbs mainly at pH 70 and were inhibited by PHMB E-64NEM and IA all of which are specific CP inhibitors [27]Becker et al [198] reported a 30 kDa protease in vacuoles thatpreviously contained phagocytosed RBCs electrophoreticanalysis revealed the incorporation of Hb monomers intotrophozoites In parallel to the decrease in human Hb duringRBC digestion X-ray analysis revealed a loss of iron content[198 199] In vitro assays have demonstrated that purified


recombinant EhCP112 and EhCP5 are able to degrade Hb[8 9]

Transferrin In mammals iron is mainly transported bytransferrin (Tf) a protein found in serum and lymph thatdelivers iron to all sites mainly to tissues with active celldivision and bonemarrow erythroid cells synthesizingHb Tfis part of a family that also contains lactoferrin Tf has a119870

119889for

Fe3+ of 10minus22M and is extremely stable against degradationwhen saturated Tf is a glycoprotein of 80 kDa with two lobeseach containing one binding site with differing affinity forFe3+ TfR1 is a dimeric glycoprotein of approximately 90 kDaper subunit that is expressed in nearly all cells [200 201]

Interestingly one of the amoebic receptors for holoTfis the acetaldehydealcohol dehydrogenase-2 an enzymethat requires iron HoloTf is endocytosed through clathrin-coated vesicles and transported to lysosomes likely losing thefirst iron in early endosomes and the second in lysosomesdue to the acidic pH [28 202] To determine whethertrophozoites possess cytoplasmic or secreted proteases thatcan degrade holoTf total extracts and culture supernatants(SN) of medium with ferric citrate or in the absence of ironwere analyzed for holoTf cleavage Four bands of holoTfdegradation corresponding to 130 43 20 and 6 kDa wereobserved in the extracts In contrast five bands of 130 70 5035 and 30 kDa were observed in the SN All of the proteolyticactivities were of the cysteine type Secreted CPs could play akey role in cleaving Tf when amoebae travel by the portal veinto the liver and when upon remaining in the liver producehepatic abscesses [28]

Lactoferrin Lactoferrin (Lf) is a glycoprotein from the innateimmune system that is secreted to mucosae it is abundant incolostrum andmilk Lf is secretedwithout iron (apoLf) by thesecondary granules of neutrophils at the infection site thus itis amarker of inflammatory bowel diseases (IBDs) [203] Oneof the functions of Lf is to chelate iron to make it unavailableto intruders Lf is a single polypeptide chain that is folded intotwo lobes like Tf each lobe can bind one Fe3+ The degreeof Lf glycosylation determines its resistance to proteases andto very acidic conditions Apo-Lf has a higher avidity foriron than apo-Tf HoloLf releases iron only in very acidicenvironments (eg pH lt 4) and its conformation changesaccording to the saturation state When saturated Lf is morestable and resistant to proteolysis [204ndash206]

HoloLf can be used as a sole iron source for in vitrogrowth by E histolytica trophozoites in a similar fashionto that observed for ferric citrate HoloLf was recognizedby two proteins (45 and 90 kDa) located in the amoebicmembrane and its binding was specific [29] HoloLf entersthe amoeba by a clathrin-independent via (possibly caveolae-like structures) Following endocytosis holoLf is found invesicles similar to early endosomes and is then deliveredto late endosomes and lysosomes Delivery of holoLf tolysosomes may be required for its digestion by proteases andiron release which only occurs in a very acidic milieu CPsof 250 100 40 and 22 kDa from amoebic extracts cleavedholoLf at pH7 however the activity increased considerably atpH 4 [29] In acidic lysosomes the iron from holoLf is likely

released and the protein is degraded by CPs Culture SN didnot contain proteolytic activity against holoLf Whether Ehistolytica contains a reductase capable of changing the ironoxidation state remains unknown This mechanism seems tobe shared by other parasites such as Tritrichomonas foetus[207] As amoebae develop in the intestinal mucosa where Lfis found this protein could be the iron source for the parasiteat the beginning of infection in addition to iron-containingbacterial proteins

FerritinDue to the toxicity of iron all life forms must have amechanism to storescavenge excess iron Human ferritin isa major cytosolic protein with the capacity to capture up to4500 iron atoms When the intracellular iron level increasesferritin sequesters iron inside its cavity to detoxify the celland prevent damage Ferritin is abundant in the liver whichstores sim50 of the bodyrsquos total iron reservesThemammalianferritin family generally consists in spherical proteins Each474 kDamolecule consists of 24 subunits that are either heavy(H) or light (L) with a molecular mass of sim21 and sim19 kDarespectively [208ndash211]

Ferritin uptake by amoeba may be mediated by a bindingprotein because it is concentration and time dependenthighly specific and saturable at 46 nM ferritin E histolyticacan cleave ferritin into several fragments Three neutral CPs(100 75 and 50 kDa) were observed to degrade ferritin inculture extracts Ferritin entrance is constrained by inhibitorsof clathrin-coated pits and after 30min of incubation ferritincolocalized with an anti-rat LAMP-2 Ab in lysosomes [30]The liver invasion by E histolytica is poorly understood Onceliver cells are destroyed by amoebic enzymes ferritin can bereleased andmay be endocytosed by trophozoites and used asa source of iron andnutrients to formhepatic abscesses In theliver amoebae may also use Hb as an iron source howeverferritin can provide up to 1000-fold more iron than Hb Thecapacity of E histolytica to utilize ferritin as iron source maywell explain its high pathogenic potential in the liver

3 Entamoeba histolytica Related Strains

Virulence is a complex phenomenon that depends on twogeneral properties the invasiveness or ability of microorgan-isms tomultiply and to cause localized tissue destruction andtoxigenicity or the ability to produce and secrete substancesthat can cause distant lesions However the virulence ofE histolytica related strains likely depends mainly on thetissue-damaging potential of individual trophozoites and thenumber of invasive amoebae in the infected host [212]

The role of amoebic proteases as responsible for tissuedestruction in amoebiasis has been discussed in the previoussection [7 13 91] Amoebic proteases have been shownto degrade several tissue components such as collagenfibronectin and laminin [13 91] The pathogenicity anddegree of virulence of various amoebic strains are determinednot only by their level of protease activity but also bythe nature of their corresponding enzyme proteins Themost studied proteases from E histolytica related strains aresummarized in Table 2


Table 2 Enzymes from E histolytica related species that degrade host proteins and their implication in virulence

Parasite Tissue or protein Name Mr (kDa) Catalytic type Comment ReferenceE moshkovskii

ECM components mdash asymp45 CP Zymogram and activity of proteasessecreted in azocasein [32]

FIC strain Gelatin mdash 90 CP Zymogram [33]Laredo strain Gelatin mdash 90 CP Zymogram [33]

E dispar

ECM components EdCP3 asymp30 CP Sequence homology with E histolyticaLocated in cytoplasmic granules [34 35]

E invadensPZ strain Gelatin 99 90 CP Zymogram [33]

IP101 strain Gelatin 99 90 45 CP Zymogram [33]

IP-1 strain Gelatin 56 58ndash66 44ndash54 43 CP Zymogram [36]Gelatin 45 SP Zymogram [37]Gelatin 130ndash230 55 35 CP MP Zymogram [38]Gelatin 130ndash230 60 CP MP Zymogram [39]

Azocasein 28 CP Zymogram [40]

31 Entamoeba moshkovskii or Entamoeba histolytica LaredoType The Entamoeba histolytica Laredo strain was isolated50 years ago from a resident of Laredo (USA) who sufferedfrom diarrhea weight loss and epigastric pain [213] Fur-ther molecular studies and biological features revealed thatthe E histolytica Laredo strain is identical to Entamoebamoshkovskii [214] a species with a worldwide distributionthat is considered a nonpathogenic free-living amoebaRecently some studies have reported the possibility that Emoshkovskii could be a pathogenic species with the capacityto infect humans or be associated with gastrointestinalsymptoms [215]

E moshkovskii is frequently found in regions whereamoebiasis is highly prevalent It has been isolated fromwastewater freshwater from rivers and lakes brackish waterand human feces samples this last findingwould seem to sug-gest that Emoshkovskii could be pathogenic Both the Laredostrain and E moshkovskii grow at room temperature as wellas at body temperature (37∘C) They present trophozoite andcystic forms and their size varies depending on the strain Inculture trophozoites are able to complete cycles of divisionencystation and excystation Usually one to four nuclei areobserved but there are formswithmore nuclei which tend tooccupy a central position in the mature cysts [213 215 216]

When observed by light microscopy E moshkovskii doesnot have morphological features that permit its distinctionfrom E histolytica and E dispar [217] E histolytica cangrow at temperatures ranging from 27 to 365∘C whileE moshkovskii can grow at temperatures from 4 to 40∘Cand in the presence of low amounts of nutrients that arenot suitable for the growth of other Entamoeba species Emoshkovskii can also adapt to extremely hypotonic culturesas it develops a contractile vacuole a characteristic that isnot present under normal growth conditions [215] An assaywas recently reported for the differential detection of E

histolyticaE dispar andEmoshkovskii Thismethod consistsof a single PCR step and is highly sensitive and potentiallyquantitative It is possible to detect 02 pg E histolytica and2 pg E dispar or E moshkovskii DNA [218]

Homogenates of the Laredo strain or E moshkovskii weretested against RBCs from different mammalian species Thehomogenates had significant activity against hamster RBCsbut human RBCs were highly resistant to lysis [212] Acomparison of the acid and neutral protease activities of theLaredo strain with other less virulent strains revealed thatthe Laredo strain had significantly less activity [25] Thisstrain was tested for its capacity to digest native radioactivetype-I collagen gels and produce liver abscesses in newbornhamsters Laredo strain did not show collagenolytic activityand failed to produce lesions Thus the susceptibility toinvasive infection may depend on the characteristics of theextracellular components of host tissues and the potentialvirulence of the parasites [141] Additionally this amoeba wasunable to produce secreted electron-dense granules whichare associated with an increase in collagenolytic activity inE histolytica [137] Amoebic proteases and host leukocyteswere studied in a model of acute experimental amoebiasisthat was produced by the intratesticular injection of axenictrophozoites in ratsThe degree of inflammation and necrosisproduced by E moshkovskii was indistinguishable from thecontrol without lesions and this was correlated with theprotease activity measured against azocasein In this casethe protease patterns of E histolytica type Laredo and Emoshkovskii were different because E histolytica type Laredodisplayed high protease activity and caused minimal tissuedamage

The current evidence proposes that the risk factors foracquiring infections by E moshkovskii are similar to thosedescribed for E histolytica and E dispar However thecapacity of E moshkovskii to grow in the environment and


adapt to adverse conditions suggests that the risk of infectionis even greater than that forE histolytica orE dispar infection[213 215 218]

32 Entamoeba dispar E dispar is a nonpathogenic (com-mensal) amoeba with a morphology genetic backgroundcell biology and host range similar to that of E histolyticaTogether E histolytica and E dispar infect approximately10 of the worldrsquos population but E dispar is much morecommon Studies of isoenzymatic patterns antigenicity andgenetics have led theWHO to declare thatE histolytica andEdispar should be classified as distinct species based on geneticdifferences inmultiple genes [133 219] RecentlyE disparwasreported to present pathogenic behavior depending on thestrain and culture conditions [220] E histolytica equivalentgenes for virulence have been detected in E dispar Theimportance of CPs in the pathogenesis of invasive amoebiasisis indisputable CPs are important virulence factors and themain proteolytic enzymes in E histolytica [34 133]

In the beginning of the 1990s studies of E dispar strainsgrown in xenic cultures revealed important differences inthe proteolytic activity on albumin and gelatin compared toE histolytica axenic and xenic cultures [221] As discussedearlier within the total sequence of the E histolytica genomemore than 40 genes encoding CPs have been identified [7173 222] only 8 of these CPs are moderately expressed inculture [73] The majority of the protease activity detectedin E histolytica lysates is the result of the ehcp1 ehcp2and ehcp5 genes E dispar encodes 4 genes (edcp1 to edcp4)homologous to those of E histolytica [73 133] Functionalgenes homologous to ehcp1 and ehcp5 are absent in thisnonpathogenic species [35 92]

The characterization of proteases in E dispar has revealedimportant insights for the understanding of invasive amoe-biasis [34 133] CP genes that are associated with virulencewere thought to be expressed in E histolytica but not Edispar such as acp1 (ehcp3) [77] a related homologue with95 identity was identified in E dispar (edcp3) [84 223]Thesame phenomenon was observed for the ehcp2 gene whichwas subsequently detected in clinical strains of E dispar[35 73 77] The strategy was changed and E histolytica pro-teases were expressed in E dispar EhCP2 was overexpressedin E dispar by episomal transfection yielding almost thesame amount of total CP activity as E histolytica EhCP2overexpression produced increasedmonolayer destruction incultured mammalian CHO cells but was unable to produceliver abscesses in an animal model The same result wasobserved when E dispar wasmodified to overexpress EhCP5a CP that is not present in this amoeba in vitro [71 149]EhCP5 activity increased almost 30-fold leading to greatermonolayer destruction [87]

In an effort to characterize the molecular basis for thefailure to express a CP5-analogous enzyme in E dispar theehcp5-containing genomic regions from E histolytica and Edispar were compared The gene corresponding to ehcp5 ispresent and the location is conserved inE dispar but the geneis highly degenerate and does not contain any open readingframe suggesting that the gene has been nonfunctional for

a considerable period of time during the evolution of thenonpathogenic amoeba species [87] Degeneration of theedcp5 gene constitutes a loss of a specific and functionalaccessory molecule important for processing CP5 [87 92224] Recent advances in DNA-mediated gene transfer in Ehistolyticamay help to prove or disprove this hypothesis

In comparison with E histolytica the expression of CPsin E dispar is lower (CP activity in E histolytica is 10ndash1000-fold higher than in E dispar) which could explain thedifferences in pathogenicity between these species [35 73]However it is not clear if the ability of E histolytica to invadeis due to the synthesis of more CPs or because these enzymesdiffer in activity [75] Thus the systematic comparison ofE histolytica and E dispar constitutes an important area ofresearch to identify and analyze factors thatmay be importantfor amoebic pathogenicity

After phagocytosis cytoplasmic (EhCP3) and mem-brane-associated (EhCP2) proteases are released into phago-cytic vesicles in E histolytica and E dispar [75] A compar-ative analysis of maturation of phagolysosomes by acidifica-tion recruitment of hydrolases and degradation of phago-somal content between the two species was performed thissequence of events is crucial for the ability to ingest and killmicroorganisms and host cells Phagosomes of E histolyticaaremuch larger and contain a greater number of bacteria thanthose of E dispar and have more efficient acidificationThesedifferences reflect the nature of proton pumping across thephagosomal membrane and membrane trafficking leadingto phagosome maturation Acidification is essential for theactivation of hydrolytic enzymes and degradative proteinssuch as EhCP1 EhCP2 EhCP4 and EhCP5 phospholipasesand lysozyme Degradation in E histolytica phagosomesoccurs more rapidly than in E dispar phagosomes inagreement with the previous findings that CP activity in Ehistolytica is 10ndash1000-fold higher than in E dispar and thatspecific inhibitors do not prevent degradation in E disparphagosomes possibly because the CPs from E dispar arerelativelymore resistant to inhibitors or these enzymes are notessential for digestion in phagosomes These differences inphagosomedegradation and the enzymes involved in host celldegradation could determine the difference in the outcome ofinfection with these two species [225]

Recently the capacity of the E dispar ICB-ADO strainto cause liver damage and destroy cell culture lines inthe presence of common intestinal bacteria was reportedThis amoeba which was isolated from a patient who wassymptomatic but not dysenteric was able to induce livernecrosis in hamsters in an even more severe form than thatproduced by E histolytica and exhibited high CP activity[220] Thus E dispar could exhibit pathogenic behaviorwith the potential to have an invasive role depending onthe strain and host conditions The authors also discussedwhether E dispar is a commensal or pathogenic parasitethey present evidence that bacteria are able to contribute tothe regulation of factors such as proteases which could havean important impact on E dispar virulence Liver lesionswith a large number of amoebae are in close contact withhepatocytes and induce an inflammatory reaction ultimatelyresulting in an extensive necrotic area with a limited number


of trophozoites compared to E histolytica HM-1IMSS Bycontrast the damage produced to MDCK cells was lessextensive Accordingly the proteolytic activity of an E disparpolyxenic strain was increased by almost 75 in comparisonwith a monoxenic strain and by 50 in comparison with Ehistolytica [220]

Research on E histolytica and E dispar has primarilyfocused on the large family of CPs which has been firmlylinked to amoebic virulence but numerous proteases areencoded in the E histolytica genome Only one copy of thegene is present in E dispar this gene is most closely relatedto ehmsp-2 The ehmsp-1 gene functions in the regulation ofadherence and also affects motility tissue culture monolayerdestruction and phagocytosis as described previously [226]

33 Entamoeba invadens E invadens is a protozoan thatcauses invasive disease in reptiles and has the same two stagesof life cycle and pathogenic potential as E histolytica Thetrophozoite is the stage that lives within the infected hostwhile the transmissible stage is the quadrinucleated cystIn some of its hosts E invadens causes liver and intestinaldamage with similar pathology to that of E histolytica-infected individuals This amoeba is studied as a model ofencystation and excystation in the Entamoeba genera sinceE histolytica in vitro encystation has not been achieved[227 228] Proteases and in particular CPs are importantfor several cellular processes such as differentiation or hostcell invasion In contrast to E histolytica very little is knownabout the E invadens genome [227]

To study proteases and their role in virulence in Enta-moeba spp amoebic cytopathogenicitywas compared amongseveral axenized strains Each amoeba species possessesdistinctive virulence defined by the rate of tissue destructionwhich in turn can be correlated with the presence orabsence of hydrolases To identify specific cytopathic effectshemolytic profiles of homogenates of two E invadens strainswere compared in RBCs of different origins The E invadensstrain PZ has no activity on RBCs while the strain IP-1 hasvery high specific hemolytic activity against hamster andmouse RBCs [212] A subsequent study revealed that theenzymatic activity is similar in E histolytica and E invadensProteases from these species were stable under denaturedconditions and were inhibited efficiently by NEM and IAindicating that they are CPs [229] The importance of Einvadens CPs became apparent upon the observation thatthe major CP degrades azocasein at optimal pH of 48 andis similar to the cathepsin B-like CP of E histolytica Inaddition E invadens proteases are activated by DTT andinhibited by typical CP inhibitors (E-64 IA and PHMB)Further studies demonstrated that CPs may be critical forthe survival of E invadens as the specific inhibition of theseproteases may ultimately interrupt parasite transmission[40] Interestingly in the presence of SDS theE invadensCPsseem to be processed for autoproteolysis [33]

A model of acute experimental amoebiasis in rat testiswas used to study the role of proteases In this work theseverity of testicular lesions was correlated with the level ofprotease activity observed in E histolytica strains and in E

moshkovskii The exception was E invadens which displayedhigh protease activity but produced minimal testicular dam-age suggesting that both the pathogenicity and degree ofvirulence of various amoebic strains are determined by thelevel of protease activity and by the type of enzyme [32]Several proteaseswith homology to the ehcp3 and ehcp2 genesof E histolytica have since been reported in E invadensPurified E invadens CPs exhibit substrate specificity similarto that of E histolytica CPs and are inhibited by specificinhibitors E invadens possesses CPs MPs and SPs CPsclearly play a role in encystation because the use of inhibitorsreduces the degree of encystation but this effect seems to beproduced by decreased amoebic viability [38] In addition toCPs other proteins that are differentially expressed duringencystation have been described such as chitinases that aredirected to the cell surface inside small secretory vesicles[230 231]

Proteases are involved in the differentiation processin E invadens The proteasome system is very importantduring the conversion of trophozoites into infectious cystsas evidenced by the disruption of the encystation processby proteasome inhibitors while CPs inhibitors only delaythe process [232] In the cyst the most abundant proteasesare serine-type proteases which could be involved in thesynthesis of chitin most likely by activating the chitin-synthase proenzyme because AEBSF a specific inhibitor ofSPs prevented the formation of viable mature cysts Thuslife cycle and transmission can be interrupted by specificinhibitors of SPs [39] Therefore both chitinase and SPs areessential for cyst wall destruction during the excystationprocess [37] The participation of CPs in encystation andexcystation of E invadens was confirmed by using E-64 [36]

Presently several CP genes have been identified in Einvadens IP-1 based on their homology with genes in Ehistolytica (eicp-a3 -a5 -a9 -a11 -b7 -b9 -b10 and -c2)Twenty of these genes are expressed during axenic parasitecultivation whereas the remaining are not expressed orexpressed at very low levels The expression patterns ofeight of the identified E invadens CP genes were evaluatedduring the encystation or excystation processes eicp-b9 isthe major gene expressed during encystation that is notinvolved in Entamoeba autophagy but its specific functionduring encystation is unknown [227] The MPs encoded byehmsp-1 and ehmsp-2 inLeishmaniamajorwere obtained inEhistolytica and E dispar but were absent in E invadens Thisimplies that ehmsp-1 and ehmsp-2 are the result of geneduplication just previous to the divergence of E histolyticaand E invadens Thus E histolytica is more closely related toE dispar than to E invadens but in some time E dispar hadboth gene copies before losing edmsp-1 [226]

4 Pathogenic Free-Living Amoeba Species

Free-living amoebae are protist organisms distributed world-wide A small number of species have been implicated inhuman disease Naegleria fowleri Acanthamoeba spp andBalamuthia mandrillaris Some of these infections are oppor-tunistic and mainly occur in immunocompromised hosts


while others affect healthy people only under certain con-ditions Although the number of infections caused by theseprotozoa is low research is needed due to the difficulty indiagnosing these diseases and the lack of specific treatmentswhich results in high mortality mainly by encephalitisUnfortunately molecular studies of free-living amoebae havenot been performed However some virulence factors ofthese parasites have been purified and characterized Themost studied proteases from pathogenic free-living amoebaspecies are summarized in Table 3

41 Acanthamoeba spp Acanthamoeba is the most commonfree-living amoeba genus and is ubiquitously distributedAcanthamoeba species are opportunistic amphizoic proto-zoa that are found in soil air and water although theyhave also been isolated from vegetables and some animalsThese amoebae cause several diseases in humans such asgranulomatous amoebic encephalitis (GAE) in immune-compromised patients and keratitis in contact lens wear-ers Protozoa are resistant to diverse environments becausethey can tolerate a wide range of osmolarity salinity pHand temperature Acanthamoeba species that cause humaninfections are A castellanii A polyphaga A culbertsoniA hatchetti A healyi A astronyxis A lugdunensis and Adivionensis All of these species have a simple life cycle withtwo phases a vegetative stage or trophozoite (8ndash40120583m) anda resistant stage or cyst (8ndash29 120583m) The name Acanthamoebacomes from the presence of fine spine-like structures or acan-thopodia projecting outward from the surface of the body[233]

Typical forms of infection byAcanthamoeba spp are GAEand nasopharyngeal or cutaneous invasion amoebae spreadby the hematogenous via GAE is rather rare approximately150 cases have beendescribedworldwideDue to the difficultyin diagnosis it is possible that other cases of GAE have beenmisdiagnosed By contrast more than 30 cases of keratitisdue toAcanthamoebawere reported in Chicago (USA) aloneAs of August 2006 more than 5000 cases of keratitis due toAcanthamoeba are estimated to have occurred in the UnitedStates but the actual number of infections around the worldis unknown Large numbers of cases have also been reportedin the United Kingdom and India [52]

The adherence of Acanthamoeba trophozoites to targetcells or tissues is an important step in host invasion Amoebaecan adhere to human and animal corneal epithelial cellsOmana-Molina et al [234] reported the early adhesionsteps of A castellanii and A polyphaga trophozoites tohamster cornea After adherence to the epithelial surfacethe trophozoites form clumps and migrate to cell borderscausing separation of adjacent cells and cytopathic damagein distal cells Amoebic adhesion may be mediated by a130 kDamannose-binding protein (MBP) which is a surface-expressed protein [235] Other adhesins include a laminin-binding protein of 28 kDa [236] and a 55 kDa protein thatbinds to the laminin of the pathogenic strain A culbertsoni[237] Interestingly A polyphaga binds to the ECM proteinscollagen type IV laminin and fibronectin [238] and calciumenhances this binding [239] In these interactions amoebae

exhibited a strong attachment to the basal membrane com-ponents laminin and collagen IV The adherence to thesemolecules leads to secondary responses such as phagocytosisand toxin production that result in host cell death via thephosphatidylinositol 3-kinase (PI3-K) pathway [240]

In Acanthamoeba several groups have shown that adher-ence to the corneal epithelium results in the productionof diverse proteases [41 241] The action of the proteasesincludes damage to the collagen shield and degradation ofglycoproteins such as plasminogen fibrinogen laminin andhemoglobin [241] In additionAcanthamoeba species displayplasminogen activator activity which can trigger host matrixMPs leading to degradation of basement membranes Inanother study Alizadeh et al [241] demonstrated that theaddition of mannosylated proteins to the cornea of Chinesehamsters induced the expression of a protease of 133 kDa(MIP133) that mediated apoptosis of corneal epithelial cellsfacilitated corneal invasion and degraded the corneal stromaAcanthamoeba spp also possess hydrolytic enzymes such aselastases [242] phospho-lipases [243] SPs [47 244ndash247] CPs[47 244ndash247] and contact MPs [248]

In the last twelve years several proteases from distinctspecies of A polyphaga have been purified and character-ized Proteases have been obtained from both total crudeextracts and conditioned culture medium and tested withnatural and synthetic substrates Alfieri et al [244] uti-lized gelatin-containing gels and azocasein to demonstrateazocasein hydrolysis by cell lysates at an optimal pH of40-50 this hydrolysis was predominantly associated withCP activity By contrast culture SN contained significantazocasein hydrolyzing activity corresponding to SPs We [47]performed a partial biochemical characterization of proteasesin total crude extracts and conditioned culture mediumfrom A castellanii and A polyphaga strains by using gelatin-containing gels and azocoll assays (both denatured type-I col-lagen) Interestingly 17 proteolytic bands distributed betweenboth Acanthamoeba strains were observed The Mr of thesebands ranged from 30 to 180 kDa in A castellanii and 34 to144 kDa in A polyphaga Incubation with protease inhibitorsrevealed that the proteolytic activities mainly belonged to theSP type followed by CPs in both total crude extracts andconditioned culture medium

Numerous proteases inAcanthamoeba are able to degradeECM components He [249] described the presence of acollagenolytic enzyme from A castellanii culture mediumthat partially digested collagen shields after 4 h of incu-bation with complete degradation by 8 h Similar resultswere obtained with shields incubated in purified collagenasesolutions More importantly when naıve Lewis rats weretreated with Acanthamoeba-conditioned cultured mediumcorneal lesions were produced that were clinically similar tothose found in biopsy specimens of human patients diag-nosed with acanthamoebic keratitis The use of nonspecificprotease inhibitors and EDTA-Na in the Acanthamoeba-cultured medium completely blocked the degradation ofcollagen shields and the use of EDTA-Na in vivo also blockedamoebic collagenase activity The authors demonstrated thatthe parasite-derived culture medium most likely containedconsiderable amounts of collagenase and low concentrations


Table 3 Enzymes from free-living amoebaea that degrade host proteins and their implication in virulence

Pathogen Tissue or protein Purified Name Mr (kDa) Catalytictype Localization Reference

Acanthamoeba spp

Elastin No 70ndash130 SP Secreted [41]Collagen fibronectin andlaminin Yes 12 SP Secreted [42]

Corneal stroma (ex vivo)Collagen I and IV Yes MIP-133 133 SP Secreted [43]

Acanthamoebacastellanii

Corneal stroma (ex vivo)Collagen Yes MIP-133 133 SP Secreted [44]

Collagen I and IIIElastin No 130 SP Secreted [45]

Collagen I and III No 150 MMP Secreted [45]Collagen I and IVFibronectin hemoglobinalbumin IgA and IgG

Yes 33 SP Secreted [46]

Gelatin No

178 144123 11078 75 7234 30

SP Crude extracts [47]

Gelatin No 188 15753 34 SP Conditioned medium [47]

Acanthamoebapolyphaga

Gelatin No144 10578 7245 40 34



Acanthamoeba healyiCollagen I and IVFibronectin hemoglobinalbumin IgA and IgG


Acanthamoebalugdunensis

Collagen I and IVFibronectin hemoglobinalbumin IgA and IgG


Naegleria fowleri

Collagen I and elastin Yes 30 CP Secreted and cytoplasmic [48]Bovine mucin No 37 CP Crude extracts [49]

Gelatin No 130 10073 62 CP Crude extracts [50]

Gelatin No 310 178164 147 CP Conditioned medium [50]

Balamuthiamandrillaris

Collagen I and III elastinand plasminogen No 40ndash50 MMP Crude extracts [51]

Collagen I and III elastinand plasminogen No 73

40ndash50 MMP Conditioned medium [51]aAll results have been performed in in vitro assays MIP mannose induced protease SP serine protease CP cysteine protease MMP matrix metalloprotease

of other proteolytic enzymes Thus the authors speculatedthat in the initial infection Acanthamoeba-derived collage-nase acts as a priming agent to produce collagenolysis edemaand neutrophils infiltration in human keratitis

Excretory and secretory products of A polyphaga culturehave been described by [247] who observed major bandsof proteolytic activity in nondenatured gelatin substrategels with Mr of 36 49 and 66 kDa in homogenate andexcretedsecreted products These proteases showed a widepH range of activity with an optimumat pHs 7ndash9The authors

also described the collagenolytic activity of A polyphagaculture medium on the substrates azocoll and gelatin andnative type-I collagen They concluded that A polyphagasecretes multiple SPs CPs and MPs and that all of theseproteases contribute to the collagenolytic effect RecentlyFerreira et al [250] characterized elastase activities in theculture medium of A polyphaga These activities are inthe range of 70ndash130 kDa with an optimal pH of 75 inaddition these activities are inhibited by PMSF antipainchymostatin and 110-phenanthroline and partially reduced


by elastinal andEDTAThis study demonstrated that amoebictrophozoites secrete elastase activities and suggested high-molecular-weight SPs as possible elastase candidates

A secreted SP of A healyi was purified by Kong et al[251] The protease has a molecular weight of 33 kDa and anoptimum pH of 80 interestingly the optimum temperaturewas 40∘C This protease degrades type-I and -IV collagenand fibronectin The protease activity is inhibited by PMSFand DIFP both SP inhibitors Na [42] purified a secreted SPfrom A castellanii with an approximate Mr of 12 kDa Thismolecule is a chymotrypsin-like protease that can degradevarious substrates such as collagen fibronectin lamininsIgA IgG plasminogen fibrinogen hemoglobin and rabbitcorneal protein The purified protein was also used to testcytopathogenicity toward HEp2 cells which resulted in aloss of viability within 12 h The cytopathogenic events werecompletely inhibited when the protease was pretreated withPMSF before addition to the HEp2 cells

A 33 kDa SP secreted by A lugdunensis was purified byKim et al [46] The protease showed a pH optimum of 85and a temperature optimum of 37∘C This protease is ableto degrade collagen types I and IV fibronectin fibrinogenhemoglobin albumin IgG and IgA PMSF inhibited nearlyall of the protease activity Also the same authors [252]reported that this 33 kDa protease could be purified fromother Acanthamoeba strains with different degrees of viru-lence

Sissons et al [45] identified two proteases of 130 and150 kDa from an Acanthamoeba isolate capable of inducingGAE The 130 kDa protease was inhibited by PMSF sug-gesting that it is an SP whereas the 150 kDa protease wasinhibited by 110-phenanthroline suggesting that it is an MPBoth proteases exhibited maximal activity at neutral pHand over a range of temperatures These proteases degradeECM components such as collagen types I and III elastinand plasminogen as well as casein and hemoglobin Finally[41] described the partial biochemical characterization ofproteolytic enzymes secreted by Acanthamoeba spp tropho-zoites isolated from the corneal tissues of different patientsDifferent enzymatic patterns of proteases were observed thatvaried between single and multiple protease activities Low-molecular-weight SPs were secreted by trophozoites andwereassociated with a more severe clinical course of keratitisProtein extracts of Acanthamoeba trophozoites were assayedfor specific activity against type-I collagen Collagenases wereobserved in amoebic extracts of different patients These iso-lates produced a single band of approximately 36 kDa All ofthese patients suffered from severe infections ConsequentlyAcanthamoeba proteolytic enzymes could play a role in thedegree of virulence and clinical manifestations of disease inhuman keratitis

Pathogenic Acanthamoeba species are also known toinfect the central nervous system (CNS) resulting in fatalGAE disease The pathophysiological complications of thisdisease include induction of proinflammatory responsesinvasion of the blood-brain barrier and connective tissue andneuronal damage Intranasal intrapulmonary and intrac-ardiac inoculations of trophozoites lead to invasion of theCNS suggesting that amoebae can enter by different routes

including the blood-brain barrier [253] Several lines ofevidence suggest thatAcanthamoeba entry into the CNSmostlikely occurs at the cerebral capillary endothelium includingthe observation that lesions are more frequent in the brainparenchyma of GAE lesions [253ndash255] Some studies havefocused on Acanthamoeba interactions with primary humanbrain microvascular endothelial cells (HBMECs) and havedemonstrated that amoebae produce HMBEC dysfunction[256] Proteases appear to play an important role inweakenedtight junctions in HBMECs cultures [257] Acanthamoebaprotease-mediated disruption of HBMECs can be inhibitedby the SP-inhibitor PMSF implicating SPs in blood-brain-barrier perturbation [45 257] Furthermore recent studieshave shown that Acanthamoeba proteases target ZO-1 andoccludin proteins [258] More studies are needed to compre-hend the importance of Acanthamoeba spp proteases in thediseases that caused by these species

42 Naegleria spp Naegleria spp are amoeboflagellates thatare foundworldwide in warm freshwater and that feedmostlyon bacteria These amoebae transform from trophozoitesto the flagellate form if nutrients are limited in order tomove toward other rich nutrient niches Amoebae can alsotransform into cysts to survive adverse conditions Speciesof Naegleria have been known for over a century [259] butit was only approximately 40 years ago that one speciescalledNaegleria fowleriwas found to cause primary amoebicmeningoencephalitis (PAM) in humans [260] There is astrong indication that the pathogenicN fowleri evolved fromthe nonpathogenic species N lovaniensis [259] Only 235PAM cases have been reported worldwide so the diseaseis rare PAM is an acute fulminant and often fatal diseasethat occurs mainly in apparently healthy children or youngpeople with a recent history of swimming among which onlyaround 5 of patients survive [259] Infection is acquired byexposure towater in ponds pools or lakes contaminatedwithN fowleri [259 260]

Other researchers and our group have found that Nfowleri trophozoites gain access to the CNS by crossing theolfactory bulbs in experimental animals [261 262] Oncein the CNS amoebae divide rapidly causing inflammationassociated with tissue destruction leading to death in a fewdays The mechanisms involved in the tissue invasion anddestruction are poorly understood However various in vitrostudies suggest the presence of numerous virulence factorsThese factors include adhesins [263] pore-forming proteins[264 265] phospholipases [266] contact-dependent lysis[267] elastase [242] and secreted proteolytic enzymes withcytopathic effects [48 50]

There are few reports concerning the adherence of Nfowleri to ECMproteins An integrin-likemolecule that bindsto immobilized fibronectin has been reported [263] Thisprotein was described as an 120572-integrin subunit with a role incytotoxicity Shibayama et al [268] described the interactionof N fowleri with human type-I collagen Recently adhesionto collagen and fibronectin by the pathogenic strain Nfowleri and the nonpathogenic N lovaniensis was com-pared revealing greater adherence ofN fowleri to fibronectin


[269 270] N fowleri presents higher levels of surface gly-coconjugates that contain 120572-D-glucose and terminal 120572-L-fucose residues than N gruberi Cytosolic and membraneglycoconjugates were more highly expressed in N fowlerithan in N gruberi These differences could be related to theadherence to different substrates and therefore they couldalso be related to the pathogenesis of N fowleri

Aldape et al [48] partially purified a secreted proteaseof 30 kDa consisting of two proteins from N fowleri Thebiochemical properties of the two forms ofN fowleri proteaseactivity were indistinguishable suggesting that they may beposttranslationally modified isoforms of the same gene prod-uct This activity was abolished by E-64 and leupeptin bothCP inhibitors Trophozoites or secreted protease activitieswere able to degrade mainly collagen and elastin this effectwas inhibited by Z-FA-FMK a specific CP inhibitor Wehave described proteolytic activities from N fowleri and Ngruberi that are able to degrade azure and azocoll at 37∘CThese activities were mainly inhibited by the CP inhibitorsPHMB and E-64 which indicates that the main proteaseactivities in N fowleri are thiol-proteases although there arealso lesser quantities of SP activity [50] More studies areneeded to elucidatewhether specific proteases fromN fowlerican degrade ECM proteins such as type-I and -IV collagenfibronectin elastin and laminin

We recently evaluated the role of mucins in the naturalimmune response and the role of the mucinolytic activityof trophozoites of N fowleri [49] A CP with Mr of 37 kDawith mucinolytic activity was identified This protease couldbe an important molecule in mucin degradation and evasionof the host innate immune response The study of Naegleriavirulence factors is still limited therefore much future workis needed particularly with respect to the role of proteases inthe invasion of the CNS

43 Balamuthia mandrillaris Balamuthia mandrillaris isthe causative agent of Balamuthia granulomatous amoebicencephalitis a life-threatening brain infection [271] Firstisolated from the brain tissue of a mandrill baboon [272] Bmandrillaris is naturally found in soil [273] and occasionallyinvades humans to cause an insidious infection that canoccur in either immunocompetent or immunosuppressedpatients To date there have been approximately 120 reportedcases of Balamuthia encephalitis worldwide with only twosurvivors [274] The incubation period is long and usuallyresults in drastic neurological damage that includes singleand multiple space-occupying brain lesions The amoebicentry routes may be the nasal mucosa or skin injuriesresulting from traumas [275] Studies in mouse models haveshown that B mandrillaris is able to migrate through theolfactory nerve pathway and access the brain in this form[276 277] Until now there have been few in vitro studiesin B mandrillaris This amoeba has cytotoxic activity againstmonkey kidney cells [278 279] and HBMECs [280] butthe cellular mechanisms of cytopathogenesis are unknownRocha-Azevedo et al [281] described the specific binding ofB mandrillaris to three ECM proteins in vitro collagen-Ifibronectin and laminin-1 Binding of amoeba to lamininwas

greater than that to collagen and fibronectin The authorsfound that binding was inhibited when the amoebae werepretreated with sialic acid

There is little information about protease activities in Bmandrillaris Matin et al [51] used isolates of B mandrillaris(from human and baboon) to describe protease activitiesin zymographic assays that revealed major protease bandswith approximate Mr of 40ndash50 kDa The protease bandswere inhibited by 110-phenantroline suggesting that theseactivities belong to the MP group These activities wereobserved over a pH range of 5ndash11 with maximum activityat neutral pH and 42∘C The B mandrillaris proteases coulddegrade type-I and -III collagen elastin and plasminogenThe authors also demonstrated that these proteases do notparticipate in the cytotoxic effects on HBMECs

A complete understanding of the pathogenic molecularmechanisms of this disease is needed and may lead to theidentification of potential targets for therapeutic manage-ment and an accurate diagnosis

5 Concluding Remarks

The occurrence of tissue damage during infection is acomplex phenomenon that is mediated by virulence factorsfrom the parasite as well as exacerbated responses fromthe host The numbers of amoebic surface and secretedproteases suggest that they are intended not only for nutrientacquisition but also as virulence factors It is well establishedthat there is a correlation between virulence and the level ofprotease expression in virulent amoebae such as E histolyticaorAcanthamoeba but not in other species such asE invadensfor which the quality and not the quantity of expressioncould be important This could also be true for proteasesof other species previously considered nonpathogenic amoe-bae such as E dispar Parasite tissue destruction mayrequire the participation of many types of protozoan parasiteproteases CPs SPs and MPs The predominant type ofprotease varies depending on the species for example Ehistolytica uses mostly CPs while Acanthamoeba spp prefersSPs

There are a great variety of protease targets such asmucinECM components tight junction proteins immunoglobu-lins complement and cytokines Iron-containing proteinscan also be included as protease targets because iron scav-enger capacity is currently considered a virulence factor forpathogens pathogensmust overcome hostmultiple strategiesthat limit iron availability as an innate host defense mech-anism In addition some proteases have a role in adhesionor encystation which are essential for establishing andtransmitting infection Thus the lack of proteolytic functionof some proteases demonstrates the multipurpose potentialof these valuable virulence factors

Amoebic proteases appear to be nonspecific becausethey degrade a wide variety of substrates in vitro howeverto correctly assess the role of each protease in infectionit is necessary to evaluate parasite protease activities withmodels in vitro models of complex substrates such as 3Dsynthetic ECM and ex vivo assays to test the proteolytic


activity under conditions similar to that of an actual infectionThe participation of host cells and bacteria in the invasionof parasites is also relevant because these cells can greatlyinfluence the amoeba protease profile as has been reportedfor E histolytica and E dispar the protease profiles whichchange extensively after contact with host cells or bacteriaAlthough a lot of valuable information has been obtainedstudying axenically grown trophozoites this informationshould be carefully extrapolated to what occurs duringinfection

Previously it was thought that amoebic proteases werenot able to exert cytopathic damage to cells however excitingnew evidence suggests that by degrading the anchoragepoints of host cells such as the ECM E histolytica proteaseslead to a special type of apoptosis called anoikis in hostcells In addition in Acanthamoeba a protease has beendescribed that mediates apoptosis of corneal epitheliumcells

For many of these parasites the identities of the proteasesare unknown because the reports refer only to proteolyticactivities of certain molecular mass ranges with the excep-tion of the extensively studied E histolytica Therefore itis important to identify each gene responsible for theseproteolytic activities to obtain a better understanding ofparasite pathogenesis Once the gene has been identified itis important to use parasites in which protease genes aredeleted or overexpressed Such studies will be of great valueto elucidate the actual roles of parasite proteases as virulencefactors This type of study has only been performed in Ehistolytica and E dispar with restrictions In E histolyticathe only available strategy requires the use of the G3 strain inwhich the amoebapore is silenced as a genetic background toperform the knockdown of the protease gene This strategyhas the limitation that a protease is assessed as a virulencefactor with an already attenuated trophozoite In additionsilencing strategies have proven to have some degree ofnonspecificity so it has been difficult to assign a function toa specific protease

Finally most patients suffering from amoebiasis expressantibodies to trophozoite CP making these proteases anattractive potential vaccine target or a potential tool toimprove the early diagnosis of human parasitic diseasesBecause transmission invasion and life cycle could beinterrupted by using specific parasite proteases inhibitors thestudy of proteases and their specific inhibitors is relevant tothe search for new therapeutic targets or to increase the powerof drugs currently used to treat these diseases such as in thereduction of tissue damage when specific inhibitors are usedin animal models

Abbreviations of Inhibitors andActivators of Proteases

AEBSF 4-(2-Aminoethyl) benzenesulfonyl fluorideDIFP Diisopropyl fluorophosphatesDTT DithiothreitolEDTA Ethylenediaminetetraacetic acid

E64 Trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane

IA IodoacetamideNEM N-ethylmaleimidePHMB p-HydroxymercuribenzoatePMSF PhenylmethylsulfonylfluorideTLCK Tosyl-lysyl-chloromethylketoneTPCK 1-Chloro-3-tosylamido-4-phenyl-2-butanoneZ-FA-FMK Benzyloxycarbonyl-Phe-Ala-fluoromethyl

ketone

Authorsrsquo Contributions

J Serrano-Luna and C Pina-Vazquez are contributed equallyto this paper

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgment

The authors thank CONACyT Mexico for supporting thiswork Projects no 60102 and 179251

References

[1] J Pawlowski and F Burki ldquoUntangling the phylogeny ofamoeboid protistsrdquo Journal of Eukaryotic Microbiology vol 56no 1 pp 16ndash25 2009

[2] A J Roger and L A Hug ldquoThe origin and diversificationof eukaryotes problems with molecular phylogenetics andmolecular clock estimationrdquo Philosophical Transactions of theRoyal Society B vol 361 no 1470 pp 1039ndash1054 2006

[3] T F H G Jackson ldquoEntamoeba histolytica and Entamoeba dis-par are distinct species clinical epidemiological and serologicalevidencerdquo International Journal for Parasitology vol 28 no 1pp 181ndash186 1998

[4] S A Tengku and M Norhayati ldquoPublic health and clinicalimportance of amoebiasis in Malaysia a reviewrdquo TropicalBiomedicine vol 28 no 2 pp 194ndash222 2011

[5] M Romeralo R Escalante and S L Baldauf ldquoEvolution anddiversity of dictyostelid social amoebaerdquo Protist vol 163 no 3pp 327ndash343 2012

[6] H Scholze and W Schulte ldquoOn the specificity of a cysteineproteinase from Entamoeba histolyticardquo Biomedica BiochimicaActa vol 47 no 2 pp 115ndash123 1988

[7] A L Luaces and A J Barrett ldquoAffinity purification andbiochemical characterization of histolysin the major cysteineproteinase of Entamoeba histolyticardquo Biochemical Journal vol250 no 3 pp 903ndash909 1988

[8] R Ocadiz E Orozco E Carrillo et al ldquoEhCP112 is anEntamoeba histolytica secreted cysteine protease that may beinvolved in the parasite-virulencerdquo Cellular Microbiology vol7 no 2 pp 221ndash232 2005

[9] A Hellberg N Nowak M Leippe E Tannich and I Bruch-haus ldquoRecombinant expression and purification of an enzy-matically active cysteine proteinase of the protozoan parasiteEntamoeba histolyticardquo Protein Expression and Purification vol24 no 1 pp 131ndash137 2002


[10] R Thibeaux A Dufour P Roux M Bernier A C Baglin andP Frileux ldquoNewly visualized fibrillar collagen scaffolds dictateEntamoeba histolytica invasion route in the human colonrdquoCellular Microbiology vol 14 no 5 pp 609ndash621 2012

[11] W E Keene M G Petitt S Allen and J H McKerrow ldquoThemajor neutral proteinase of Entamoeba histolyticardquo Journal ofExperimental Medicine vol 163 no 3 pp 536ndash549 1986

[12] J De Jesus Serrano M De La Garza M A Moreno etal ldquoEntamoeba histolytica electron-dense granule secretioncollagenase activity and virulence are altered in the cytoskeletonmutant BG-3rdquo Molecular Microbiology vol 11 no 4 pp 787ndash792 1994

[13] W Schulte and H Scholze ldquoAction of the major protease fromEntamoeba histolytica on proteins of the extracellular matrixrdquoJournal of Protozoology vol 36 no 6 pp 538ndash543 1989

[14] C He G P Nora E L Schneider et al ldquoA novel Entamoebahistolytica cysteine proteinase EhCP4 is key for invasive ame-biasis and a therapeutic targetrdquo Journal of Biological Chemistryvol 285 no 24 pp 18516ndash18527 2010

[15] J De Jesus Serrano M De La Garza M Reyes G LeonR Tovar and M De Lourdes Munoz ldquoEntamoeba histolyticaproteinase secretion induced by collagen type I is dependent oncytoskeleton integrityrdquo Parasitology Research vol 82 no 3 pp200ndash205 1996

[16] B L Kelsall and J I Ravdin ldquoDegradation of human IgA byEntamoeba histolyticardquo Journal of Infectious Diseases vol 168no 5 pp 1319ndash1322 1993

[17] S G Melendez-Lopez S Herdman K Hirata et al ldquoUseof recombinant Entamoeba histolytica cysteine proteinase 1 toidentify a potent inhibitor of amebic invasion in a humancolonic modelrdquo Eukaryotic Cell vol 6 no 7 pp 1130ndash1136 2007

[18] S L Reed W E Keene J H McKerrow and I Gigli ldquoCleavageof C3 by a neutral cysteine proteinase of Entamoeba histolyticardquoJournal of Immunology vol 143 no 1 pp 189ndash195 1989

[19] S L Reed J A Ember D S Herdman R G DiScipioT E Hugli and I Gigli ldquoThe extracellular neutral cysteineproteinase of Entamoeba histolytica degrades anaphylatoxinsC3a and C5ardquo Journal of Immunology vol 155 no 1 pp 266ndash274 1995

[20] X Que S H Kim M Sajid et al ldquoA surface amebic cysteineproteinase inactivates interleukin-18rdquo Infection and Immunityvol 71 no 3 pp 1274ndash1280 2003

[21] E Li W G Yang T Zhang and S L Stanley ldquoInteraction oflaminin with Entamoeba histolytica cysteine proteinases and itseffect on amebic pathogenesisrdquo Infection and Immunity vol 63no 10 pp 4150ndash4153 1995

[22] Z Zhang L Wang K B Seydel et al ldquoEntamoeba histolyticacysteine proteinases with interleukin-1 beta converting enzyme(ICE) activity cause intestinal inflammation and tissue damagein amoebiasisrdquo Molecular Microbiology vol 37 no 3 pp 542ndash548 2000

[23] J McLaughlin and G Faubert ldquoPartial purification and someproperties of a neutral sulfhydryl and an acid proteinase fromEntamoeba histolyticardquo Canadian Journal of Microbiology vol23 no 4 pp 420ndash425 1977

[24] R Perez-Montfort P Ostoa-Saloma L Velazquez-Medina IMontfort and I Becker ldquoCatalytic classes of proteinases ofEntamoeba histolyticardquoMolecular and Biochemical Parasitologyvol 26 no 1-2 pp 87ndash97 1987

[25] W B Lushbaugh A F Hofbauer and A A Kairalla ldquoRelation-ship of cytotoxins of axenically cultivated Entamoeba histolytica

to virulencerdquo Gastroenterology vol 86 no 6 pp 1488ndash14951984

[26] W B Lushbaugh A F Hofbauer and F E Pittman ldquoEntamoebahistolytica purification of cathepsin Brdquo Experimental Parasitol-ogy vol 59 no 3 pp 328ndash336 1985

[27] J De Jesus Serrano-Luna E Negrete M Reyes and M DeLa Garza ldquoEntamoeba histolytica HM1IMSS hemoglobin-degrading neutral cysteine proteasesrdquo Experimental Parasitol-ogy vol 89 no 1 pp 71ndash77 1998

[28] M Reyes-Lopez R M Bermudez-Cruz E E Avila and M DeLa Garza ldquoAcetaldehydealcohol dehydrogenase-2 (EhADH2)and clathrin are involved in internalization of human transfer-rin by Entamoeba histolyticardquo Microbiology vol 157 no 1 pp209ndash219 2011

[29] N Leon-Sicairos M Reyes-Lopez A Canizalez-Roman et alldquoHuman hololactoferrin endocytosis and use as an iron sourceby the parasite Entamoeba histolyticardquoMicrobiology vol 151 no12 pp 3859ndash3871 2005

[30] F Lopez-Soto A Gonzalez-Robles L Salazar-Villatoro et alldquoEntamoeba histolytica uses ferritin as an iron source andinternalises this protein by means of clathrin-coated vesiclesrdquoInternational Journal for Parasitology vol 39 no 4 pp 417ndash4262009

[31] D Moncada K Keller S Ankri D Mirelman and K ChadeeldquoAntisense inhibition of Entamoeba histolytica cysteine pro-teases inhibits colonic mucus degradationrdquo Gastroenterologyvol 130 no 3 pp 721ndash730 2006

[32] R Perez-Tamayo I Becker I Montfort P Ostoa-Saloma andR Perez-Montfort ldquoRole of leukocytes and amebic proteinasesin experimental rat testicular necrosis produced by Entamoebahistolyticardquo Parasitology Research vol 77 no 3 pp 192ndash1961991

[33] R Lopez-Revilla and M Baez-Camargo ldquoImmediate autopro-teolysis and new proteinases in Entamoeba invadens and Enta-moeba moshkovskii trophozoitesrdquo Archives of Medical Researchvol 23 no 2 pp 95ndash97 1992

[34] V Kissoon-Singh L Mortimer and K Chadee ldquoEntamoebahistolytica cathepsin-like enzymes interactions with the hostgutrdquo Advances in Experimental Medicine and Biology vol 712pp 62ndash83 2011

[35] I Bruchhaus T Jacobs M Leippe and E Tannich ldquoEntamoebahistolytica and Entamoeba dispar differences in numbers andexpression of cysteine proteinase genesrdquo Molecular Microbiol-ogy vol 22 no 2 pp 255ndash263 1996

[36] AMakioka M Kumagai S Kobayashi and T Takeuchi ldquoEnta-moeba invadens cysteine protease inhibitors block excystationand metacystic developmentrdquo Experimental Parasitology vol109 no 1 pp 27ndash32 2005

[37] A Makioka M Kumagai S Kobayashi and T TakeuchildquoInvolvement of serine proteases in the excystation andmetacystic development of Entamoeba invadensrdquo ParasitologyResearch vol 105 no 4 pp 977ndash987 2009

[38] M Sharma K Hirata S Herdman and S Reed ldquoEntamoebainvadens characterization of cysteine proteinasesrdquo Experimen-tal Parasitology vol 84 no 1 pp 84ndash91 1996

[39] Y Riahi and S Ankri ldquoInvolvement of serine proteinasesduring encystation of Entamoeba invadensrdquo Archives of MedicalResearch vol 31 no 4 pp S187ndashS189 2000

[40] H Scholze and W Schulte ldquoPurification and partial char-acterization of the major cysteine protease from Entamoebainvadensrdquo Biomedica Biochimica Acta vol 49 no 6 pp 455ndash463 1990


[41] F R De Souza Carvalho L C Carrijo-Carvalho A MChudzinski-Tavassi A S Foronda and D de FreitasldquoSerine-like proteolytic enzymes correlated with differentialpathogenicity in patients with acute Acanthamoeba keratitisrdquoClinical Microbiology and Infection vol 17 no 4 pp 603ndash6092011

[42] B K Na J C Kim and C Y Song ldquoCharacterization andpathogenetic role of proteinase fromAcanthamoeba castellaniirdquoMicrobial Pathogenesis vol 30 no 1 pp 39ndash48 2001

[43] M Hurt S Neelam J Niederkorn and H AlizadehldquoPathogenic Acanthamoeba spp secrete a mannose-inducedcytolytic protein that correlates with the ability to causediseaserdquo Infection and Immunity vol 71 no 11 pp 6243ndash62552003

[44] MHurt JNiederkorn andHAlizadeh ldquoEffects ofmannose onAcanthamoeba castellanii proliferation and cytolytic ability tocorneal epithelial cellsrdquo Investigative Ophthalmology and VisualScience vol 44 no 8 pp 3424ndash3431 2003

[45] J Sissons S Alsam G Goldsworthy M Lightfoot E L JarrollandNAKhan ldquoIdentification andproperties of proteases froman Acanthamoeba isolate capable of producing granulomatousencephalitisrdquo BMCMicrobiology vol 6 article 42 2006

[46] H K Kim Y R Ha H S Yu H H Kong and D I ChungldquoPurification and characterization of a 33 kDa serine proteasefrom Acanthamoeba lugdunensis KAE2 isolated from a Koreankeratitis patientrdquoThe Korean Journal of Parasitology vol 41 no4 pp 189ndash196 2003

[47] J D J Serrano-Luna I Cervantes-Sandoval J Calderon FNavarro-Garcıa V Tsutsumi and M Shibayama ldquoProteaseactivities ofAcanthamoeba polyphaga andAcanthamoeba castel-laniirdquoCanadian Journal ofMicrobiology vol 52 no 1 pp 16ndash232006

[48] K Aldape H Huizinga J Bouvier and J McKerrow ldquoNaeg-leria fowleri characterization of a secreted histolytic cysteineproteaserdquo Experimental Parasitology vol 78 no 2 pp 230ndash2411994

[49] I Cervantes-Sandoval J D J Serrano-Luna E Garcıa-LatorreV Tsutsumi and M Shibayama ldquoMucins in the host defenceagainst Naegleria fowleri and mucinolytic activity as a possiblemeans of evasionrdquoMicrobiology vol 154 no 12 pp 3895ndash39042008

[50] J Serrano-Luna I Cervantes-Sandoval V Tsutsumi and MShibayama ldquoA biochemical comparison of proteases frompathogenic Naegleria fowleri and non-pathogenic Naegleriagruberirdquo Journal of Eukaryotic Microbiology vol 54 no 5 pp411ndash417 2007

[51] A Matin M Stins K S Kim and N A Khan ldquoBala-muthia mandrillaris exhibits metalloprotease activitiesrdquo FEMSImmunology and Medical Microbiology vol 47 no 1 pp 83ndash912006

[52] G S Visvesvara H Moura and F L Schuster ldquoPathogenic andopportunistic free-living amoebae Acanthamoeba spp Bala-muthia mandrillaris Naegleria fowleri and Sappinia diploideardquoFEMS Immunology and Medical Microbiology vol 50 no 1 pp1ndash26 2007

[53] A Martinez-Palomo ldquoThe pathogenesis of amoebiasisrdquo Para-sitology Today vol 3 no 4 pp 111ndash118 1987

[54] CXimenez ldquoEpidemiology of amebiasis inMexico amolecularapproachrdquo Archives of Medical Research vol 37 no 2 pp 263ndash265 2006

[55] M Espinosa-Cantellano and A Martınez-Palomo ldquoPathogen-esis of intestinal amebiasis from molecules to diseaserdquo ClinicalMicrobiology Reviews vol 13 no 2 pp 318ndash331 2000

[56] S L Stanley Jr ldquoAmoebiasisrdquo Lancet vol 361 no 9362 pp1025ndash1034 2003

[57] K S Ralston and W A Petri Jr ldquoTissue destruction andinvasion by Entamoeba histolyticardquo Trends in Parasitology vol27 no 6 pp 254ndash263 2011

[58] K Prathap and R Gilman ldquoThe histopathology of acuteintestinal amebiasis A rectal biopsy studyrdquo American Journalof Pathology vol 60 no 2 pp 229ndash246 1970

[59] R Arroyo and E Orozoco ldquoLocalization and identification ofan Entamoeba histolytica adhesinrdquo Molecular and BiochemicalParasitology vol 23 no 2 pp 151ndash158 1987

[60] GGarcıa-RiveraMA Rodrıguez ROcadiz et al ldquoEntamoebahistolytica a novel cysteine protease and an adhesin form the 112kDa surface proteinrdquoMolecular Microbiology vol 33 no 3 pp556ndash568 1999

[61] J I Ravdin ldquoEntamoeba histolytica from adherence toenteropathyrdquo Journal of Infectious Diseases vol 159 no 3 pp420ndash429 1989

[62] WA Petri Jr and J I Ravdin ldquoCytopathogenicity ofEntamoebahistolytica the role of amebic adherence and contact-dependentcytolysis in pathogenesisrdquo European Journal of Epidemiologyvol 3 no 2 pp 123ndash136 1987

[63] J L Rosales-Encina I Meza A Lopex-De-Leon P Talamas-Rohana and M Rojkind ldquoIsolation of a 220-kilodalton proteinwith lectin properties from a virulent strain of Entamoebahistolyticardquo Journal of InfectiousDiseases vol 156 no 5 pp 790ndash797 1987

[64] W A Petri Jr R Haque and B J Mann ldquoThe bittersweetinterface of parasite and host lectin-carbohydrate interactionsduring human invasion by the parasite Entamoeba histolyticardquoAnnual Review of Microbiology vol 56 pp 39ndash64 2002

[65] J J McCoy B J Mann and W A Petri Jr ldquoAdherence andcytotoxicity of Entamoeba histolytica or how lectins let parasitesstick aroundrdquo Infection and Immunity vol 62 no 8 pp 3045ndash3050 1994

[66] M Leippe and H J Muller-Eberhard ldquoThe pore-formingpeptide ofEntamoeba histolytica the protozoan parasite causinghuman amoebiasisrdquo Toxicology vol 87 no 1ndash3 pp 5ndash18 1994

[67] J I Ravdin F Moreau J A Sullivan W A Petri Jr andG L Mandell ldquoRelationship of free intracellular calcium tothe cytolytic activity of Entamoeba histolyticardquo Infection andImmunity vol 56 no 6 pp 1505ndash1512 1988

[68] M Leippe S Ebel O L Schoenberger R D Horstmann andH J Muller-Eberhard ldquoPore-forming peptide of pathogenicEntamoeba histolyticardquo Proceedings of the National Academy ofSciences of the United States of America vol 88 no 17 pp 7659ndash7663 1991

[69] S A Long-Krug K J Fischer R M Hysmith and J IRavdin ldquoPhospholipase A enzymes of Entamoeba histolyticadescription and subcellular localizationrdquo Journal of InfectiousDiseases vol 152 no 3 pp 536ndash541 1985

[70] M de la Torre R de la Hoz-Couturier L Landa and B Sepul-veda ldquoCultivos axenicos de Entamoeba histolyticardquo Archivos deInvestigacion Medica vol 2 supplement 1 pp 165ndash172 1971

[71] S Ankri T Stolarsky R Bracha F Padilla-Vaca and DMirelman ldquoAntisense inhibition of expression of cysteine pro-teinases affects Entamoeba histolytica-induced formation ofliver abscess in hamstersrdquo Infection and Immunity vol 67 no1 pp 421ndash422 1999


[72] R Bracha Y Nuchamowitz M Anbar and D MirelmanldquoTranscriptional silencing of multiple genes in trophozoites ofEntamoeba histolyticardquo PLoS Pathogens vol 2 no 5 article e482006

[73] I Bruchhaus B J Loftus N Hall and E Tannich ldquoTheintestinal protozoan parasite Entamoeba histolytica contains20 cysteine protease genes of which only a small subset isexpressed during in vitro cultivationrdquo Eukaryotic Cell vol 2 no3 pp 501ndash509 2003

[74] J H McKerrow E Sun P J Rosenthal and J Bouvier ldquoTheproteases and pathogenicity of parasitic protozoardquo AnnualReview of Microbiology vol 47 pp 821ndash853 1993

[75] X Que L S Brinen P Perkins et al ldquoCysteine proteinasesfrom distinct cellular compartments are recruited to phagocyticvesicles by Entamoeba histolyticardquo Molecular and BiochemicalParasitology vol 119 no 1 pp 23ndash32 2002

[76] A E Eakin J Bouvier J A Sakanari C S Craik andJ H McKerrow ldquoAmplification and sequencing of genomicDNA fragments encoding cysteine proteases from protozoanparasitesrdquo Molecular and Biochemical Parasitology vol 39 no1 pp 1ndash8 1990

[77] S Reed J Bouvier A S Pollack et al ldquoCloning of a virulencefactor of Entamoeba histolytica Pathogenic strains possess aunique cysteine proteinase generdquo Journal of Clinical Investiga-tion vol 91 no 4 pp 1532ndash1540 1993

[78] M Tillack L Biller H Irmer et al ldquoThe Entamoeba histolyt-ica genome primary structure and expression of proteolyticenzymesrdquo BMC Genomics vol 8 article 170 2007

[79] C A Gilchrist E Houpt N Trapaidze et al ldquoImpact of intesti-nal colonization and invasion on the Entamoeba histolyticatranscriptomerdquo Molecular and Biochemical Parasitology vol147 no 2 pp 163ndash176 2006

[80] H Scholze and EWerries ldquoA weakly acidic protease has a pow-erful proteolytic activity in Entamoeba histolyticardquo Molecularand Biochemical Parasitology vol 11 pp 293ndash300 1984

[81] H Scholze J Otte and E Werries ldquoCysteine proteinase ofEntamoeba histolyticamdashII Identification of the major splitposition in bovine insulin B-chainrdquoMolecular and BiochemicalParasitology vol 18 no 1 pp 113ndash121 1986

[82] H Scholze and E Werries ldquoCysteine proteinase of Enta-moeba histolyticamdashI Partial purification and action on differentenzymesrdquoMolecular and Biochemical Parasitology vol 18 no 1pp 103ndash112 1986

[83] E Tannich H Scholze R Nickel and R D HorstmannldquoHomologous cysteine proteinases of pathogenic and non-pathogenic Entamoeba histolytica differences in structure andexpressionrdquo Journal of Biological Chemistry vol 266 no 8 pp4798ndash4803 1991

[84] D Mirelman Y Nuchamowitz B Bohm-Gloning and BWalderich ldquoA homologue of the cysteine proteinase gene (ACP1or Eh-CPp3) of pathogenic Entamoeba histolytica is present innon-pathogenic E dispar strainsrdquo Molecular and BiochemicalParasitology vol 78 no 1-2 pp 47ndash54 1996

[85] K K Hirata X Que S G Melendez-Lopez et al ldquoA phago-cytosis mutant of Entamoeba histolytica is less virulent dueto deficient proteinase expression and releaserdquo ExperimentalParasitology vol 115 no 2 pp 192ndash199 2007

[86] P H Davis J Schulze and S L Stanley ldquoTranscriptomiccomparison of two Entamoeba histolytica strains with definedvirulence phenotypes identifies new virulence factor candidatesand key differences in the expression patterns of cysteine

proteases lectin light chains and calmodulinrdquo Molecular andBiochemical Parasitology vol 151 no 1 pp 118ndash128 2007

[87] T Jacobs I Bruchhaus T Dandekar E Tannich andM LeippeldquoIsolation and molecular characterization of a surface-boundproteinase of Entamoeba histolyticardquo Molecular Microbiologyvol 27 no 2 pp 269ndash276 1998

[88] E M Duncan T L Muratore-Schroeder R G Cook et alldquoCathepsin L proteolytically processes histoneH3duringmouseembryonic stem cell differentiationrdquo Cell vol 135 no 2 pp284ndash294 2008

[89] B Goulet A Baruch N S Moon et al ldquoA cathepsin L isoformthat is devoid of a signal peptide localizes to the nucleusin S phase and processes the CDPCux transcription factorrdquoMolecular Cell vol 14 no 2 pp 207ndash219 2004

[90] G Maubach M C C Lim and L Zhuo ldquoNuclear cathepsinF regulates activation markers in rat hepatic stellate cellsrdquoMolecular Biology of the Cell vol 19 no 10 pp 4238ndash4248 2008

[91] S L Reed W E Keene and J H McKerrow ldquoThiol proteinaseexpression and pathogenicity of Entamoeba histolyticardquo Journalof Clinical Microbiology vol 27 no 12 pp 2772ndash2777 1989

[92] U Willhoeft L Hamann and E Tannich ldquoA DNA sequencecorresponding to the gene encoding cysteine proteinase 5 inEntamoeba histolytica is present and positionally conservedbut highly degenerated in Entamoeba disparrdquo Infection andImmunity vol 67 no 11 pp 5925ndash5929 1999

[93] S Ankri T Stolarsky and D Mirelman ldquoAntisense inhibitionof expression of cysteine proteinases does not affect Entamoebahistolytica cytopathic or haemolytic activity but inhibits phago-cytosisrdquo Molecular Microbiology vol 28 no 4 pp 777ndash7851998

[94] M A R Freitas H C Fernandes V C Calixto et al ldquoEnta-moeba histolytica cysteine proteinase activity and virulenceFocus on cysteine proteinase 5 expression levelsrdquo ExperimentalParasitology vol 122 no 4 pp 306ndash309 2009

[95] L I Quintas-Granados E Orozco L G Brieba R Arroyo andJ Ortega-Lopez ldquoPurification refolding and autoactivation ofthe recombinant cysteine proteinase EhCP112 from EntamoebahistolyticardquoProtein Expression and Purification vol 63 no 1 pp26ndash32 2009

[96] S Carpeniseanu K Hirata X Que E Orozco and S LReed ldquoL6 a proteinase- and phagocytosis-deficient mutant ofEntamoeba histolyticardquo Archives of Medical Research vol 31 no4 pp S237ndashS238 2000

[97] X Madriz M B Martınez M A Rodrıguez et al ldquoExpressionin fibroblasts and in live animals of Entamoeba histolyticapolypeptides EhCP112 and EhADH112rdquo Microbiology vol 150no 5 pp 1251ndash1260 2004

[98] C Martınez-Lopez E Orozco T Sanchez R M Garcıa-Perez F Hernandez-Hernandez and M A Rodrıguez ldquoTheEhADH112 recombinant polypeptide inhibits cell destructionand liver abscess formation by Entamoeba histolytica tropho-zoitesrdquo Cellular Microbiology vol 6 no 4 pp 367ndash376 2004

[99] M A Rodriguez F Hernandez L Santos A Valdez andE Orozco ldquoEntamoeba histolytica molecules involved in thetarget cell-parasite relationshiprdquo Molecular and BiochemicalParasitology vol 37 no 1 pp 87ndash100 1989

[100] A Brittingham C J MorrisonW R McMaster B S McGwireK P Chang and D M Mosser ldquoRole of the Leishmania surfaceprotease gp63 in complement fixation cell adhesion and resis-tance to complement-mediated lysisrdquo Journal of Immunologyvol 155 no 6 pp 3102ndash3111 1995


[101] D M Mosser and A Brittingham ldquoLeishmania macrophagesand complement a tale of subversion and exploitationrdquo Para-sitology vol 115 supplement pp S9ndashS23 1997

[102] W A Petri Jr R D Smith P H Schlesinger C F Murphyand J I Ravdin ldquoIsolation of the galactose-binding lectinthat mediates the in vitro adherence of Entamoeba histolyticardquoJournal of Clinical Investigation vol 80 no 5 pp 1238ndash12441987

[103] S L Stanley K Tian J P Koester and E Li ldquoThe serine-richEntamoeba histolytica protein is a phosphorylated membraneprotein containing O-linked terminal N-acetylglucosamineresiduesrdquo Journal of Biological Chemistry vol 270 no 8 pp4121ndash4126 1995

[104] J E Teixeira and C D Huston ldquoParticipation of the serine-rich Entamoeba histolytica protein in amebic phagocytosis ofapoptotic host cellsrdquo Infection and Immunity vol 76 no 3 pp959ndash966 2008

[105] W E Keene M E Hidalgo E Orozco and J H McKerrowldquoEntamoeba histolytica correlation of the cytopathic effect ofvirulent trophozoites with secretion of a cysteine proteinaserdquoExperimental Parasitology vol 71 no 2 pp 199ndash206 1990

[106] E Bier L Y Jan and Y N Jan ldquorhomboid a gene requiredfor dorsoventral axis establishment and peripheral nervoussystem development in Drosophila melanogasterrdquo Genes andDevelopment vol 4 no 2 pp 190ndash203 1990

[107] U Mayer and C Nusslein-Volhard ldquoA group of genes requiredfor pattern formation in the ventral ectoderm of theDrosophilaembryordquoGenesampDevelopment vol 2 no 11 pp 1496ndash1511 1988

[108] JM Santos A Graindorge andD Soldati-Favre ldquoNew insightsinto parasite rhomboid proteasesrdquo Molecular and BiochemicalParasitology vol 182 no 1-2 pp 27ndash36 2012

[109] S Urban and M Freeman ldquoSubstrate specificity of Rhom-boid intramembrane proteases is governed by helix-breakingresidues in the substrate transmembrane domainrdquo MolecularCell vol 11 no 6 pp 1425ndash1434 2003

[110] L A Baxt R P Baker U Singh and S Urban ldquoAn Entamoebahistolytica rhomboid protease with atypical specificity cleaves asurface lectin involved in phagocytosis and immune evasionrdquoGenes and Development vol 22 no 12 pp 1636ndash1646 2008

[111] L A Baxt E Rastew R Bracha D Mirelman and U SinghldquoDownregulation of an Entamoeba histolytica rhomboid pro-tease reveals roles in regulating parasite adhesion and phago-cytosisrdquo Eukaryotic Cell vol 9 no 8 pp 1283ndash1293 2010

[112] K Karakasis D Taylor and K Ko ldquoUncovering a link betweena plastid translocon component and rhomboid proteases usingyeastmitochondria-based assaysrdquoPlant andCell Physiology vol48 no 4 pp 655ndash661 2007

[113] P C Brooks S Stromblad L C Sanders et al ldquoLocalization ofmatrixmetalloproteinaseMMP-2 to the surface of invasive cellsby interaction with integrin 120572v1205733rdquo Cell vol 85 no 5 pp 683ndash693 1996

[114] A M Lechner I Assfalg-Machleidt S Zahler et al ldquoRGD-dependent binding of procathepsinX to integrin120572 v1205733mediatescell-adhesive propertiesrdquo Journal of Biological Chemistry vol281 no 51 pp 39588ndash39597 2006

[115] A Mondino and F Blasi ldquouPA and uPAR in fibrinolysisimmunity and pathologyrdquo Trends in Immunology vol 25 no8 pp 450ndash455 2004

[116] M E Papaconstantinou C J Carrell A O Pineda et alldquoThrombin functions through its RGD sequence in a non-canonical conformationrdquo Journal of Biological Chemistry vol280 no 33 pp 29393ndash29396 2005

[117] Y Hou L Mortimer and K Chadee ldquoEntamoeba histolyticacysteine proteinase 5 binds integrin on colonic cells and stim-ulates NF120581B-mediated pro-inflammatory responsesrdquo Journal ofBiological Chemistry vol 285 no 46 pp 35497ndash35504 2010

[118] A F Horwitz ldquoIntegrins and healthrdquo Scientific American vol276 no 5 pp 68ndash75 1997

[119] L Mortimer and K Chadee ldquoThe immunopathogenesis ofEntamoeba histolyticardquo Experimental Parasitology vol 126 no3 pp 366ndash380 2010

[120] A Belley K Keller M Goettke and K Chadee ldquoIntestinalmucins in colonization and host defense against pathogensrdquoAmerican Journal of Tropical Medicine and Hygiene vol 60 no4 pp 10ndash15 1999

[121] N Asker M A B Axelsson S O Olofsson and G C HanssonldquoDimerization of the human MUC2 mucin in the endoplasmicreticulum is followed by a N-glycosylation-dependent transferof the mono- and dimers to the Golgi apparatusrdquo Journal ofBiological Chemistry vol 273 no 30 pp 18857ndash18863 1998

[122] A Herrmann J R Davies G Lindell et al ldquoStudies on thersquoinsolublersquo glycoprotein complex from human colon identifica-tion of reduction-insensitive MUC2 oligomers and C-terminalcleavagerdquo Journal of Biological Chemistry vol 274 no 22 pp15828ndash15836 1999

[123] D Bansal P Ave S Kerneis et al ldquoAn ex vivo human intestinalmodel to study Entamoeba histolytica pathogenesisrdquo PLoSNeglected Tropical Diseases vol 3 no 11 article e551 2009

[124] D Moncada K Keller and K Chadee ldquoEntamoeba histolyticacysteine proteinases disrupt the polymeric structure of colonicmucin and alter its protective functionrdquo Infection and Immunityvol 71 no 2 pp 838ndash844 2003

[125] M E Lidell D M Moncada K Chadee and G C HanssonldquoEntamoeba histolytica cysteine protease cleave the MUC2mucin in its C-terminal domain and dissolve the protectivecolonic mucus gelrdquo Proceedings of the National Academy ofSciences of the United States of America vol 103 no 24 pp9298ndash9303 2006

[126] D Moncada K Keller and K Chadee ldquoEntamoeba histolytica-secreted products degrade colonic mucin oligosaccharidesrdquoInfection and Immunity vol 73 no 6 pp 3790ndash3793 2005

[127] A Debnath J S Tashker M Sajid and J H McKerrow ldquoTran-scriptional and secretory responses of Entamoeba histolytica tomucins epithelial cells and bacteriardquo International Journal forParasitology vol 37 no 8-9 pp 897ndash906 2007

[128] A Leroy T Lauwaet G De Bruyne M Cornelissen andM Mareel ldquoEntamoeba histolytica disturbs the tight junctioncomplex in human enteric T84 cell layersrdquo FASEB Journal vol14 no 9 pp 1139ndash1146 2000

[129] A Leroy T Lauwaet M J Oliveira et al ldquoDisturbance oftight junctions by Entamoeba histolytica resistant vertebratecell types and incompetent trophozoitesrdquo Archives of MedicalResearch vol 31 no 4 pp S218ndashS220 2000

[130] T Lauwaet M J Oliveira B Callewaert et al ldquoProteolysis ofenteric cell villin by Entamoeba histolytica cysteine proteinasesrdquoJournal of Biological Chemistry vol 278 no 25 pp 22650ndash22656 2003

[131] T Lauwaet M J Oliveira B Callewaert G De BruyneM Mareel and A Leroy ldquoProteinase inhibitors TPCK andTLCK prevent Entamoeba histolytica induced disturbance oftight junctions and microvilli in enteric cell layers in vitrordquoInternational Journal for Parasitology vol 34 no 7 pp 785ndash7942004


[132] C Gitler E Calef and I Rosenberg ldquoCytopathogenicity ofEntamoeba histolyticardquo Philosophical Transactions of the RoyalSociety B vol 307 no 1131 pp 73ndash85 1984

[133] X Que and S L Reed ldquoCysteine proteinases and the pathogen-esis of amebiasisrdquo Clinical Microbiology Reviews vol 13 no 2pp 196ndash206 2000

[134] S Spinella E Levavasseur F Petek and M C RigothierldquoPurification and biochemical characterization of a novel cys-teine protease of Entamoeba histolyticardquo European Journal ofBiochemistry vol 266 no 1 pp 170ndash180 1999

[135] S L Reed J G Curd and I Gigli ldquoActivation of complement bypathogenic and nonpathogenic Entamoeba histolyticardquo Journalof Immunology vol 136 no 6 pp 2265ndash2270 1986

[136] M D L Munoz J Calderon and M Rojkind ldquoThe collagenaseofEntamoeba histolyticardquo Journal of ExperimentalMedicine vol155 no 1 pp 42ndash51 1982

[137] M De Lourdes Munoz E Lamoyi G Leon et al ldquoAntigens inelectron-dense granules from Entamoeba histolytica as possiblemarkers for pathogenicityrdquo Journal of Clinical Microbiology vol28 no 11 pp 2418ndash2424 1990

[138] A Debnath M A Akbar A Mazumder S Kumar and PDas ldquoEntamoeba histolytica characterization of human colla-gen type I and Ca2+ activated differentially expressed genesrdquoExperimental Parasitology vol 110 no 3 pp 214ndash219 2005

[139] H Gadasi and E Kessler ldquoCorrelation of virulence and col-lagenolytic activity in Entamoeba histolyticardquo Infection andImmunity vol 39 no 2 pp 528ndash531 1983

[140] M A Magos M De La Torre and M I Munoz ldquoCollagenaseactivity in clinical isolates of Entamoeba histolyticamaintainedin xenic culturesrdquo Archives of Medical Research vol 23 no 2pp 115ndash118 1992

[141] M De Lourdes Munoz M Rojkind and J Calderon ldquoEnta-moeba histolytica collagenolytic activity and virulencerdquo Journalof Protozoology vol 31 no 3 pp 468ndash470 1984

[142] V Tsutsumi A Ramirez-Rosales H Lanz-Mendoza et alldquoEntamoeba histolytica erythrophagocytosis collagenolysisand liver abscess production as virulencemarkersrdquoTransactionsof the Royal Society of TropicalMedicine andHygiene vol 86 no2 pp 170ndash172 1992

[143] B Chavez-Munguia G Castanon V Hernandez-RamirezM Gonzalez-Lazaro P Talamas-Rohana and A Martinez-Palomo ldquoEntamoeba histolytica electrondense granules secre-tion in vitro and in vivo ultrastructural studyrdquo MicroscopyResearch and Technique vol 75 no 2 pp 189ndash196 2012

[144] P Talamas-Rohana and I Meza ldquoInteraction betweenpathogenic amebas and fibronectin substrate degradation andchanges in cytoskeleton organizationrdquo Journal of Cell Biologyvol 106 no 5 pp 1787ndash1794 1988

[145] I Meza ldquoExtracellular matrix-induced signaling in Entamoebahistolytica its role in invasivenessrdquo Parasitology Today vol 16no 1 pp 23ndash28 2000

[146] J Vazquez-Prado and I Meza ldquoFibronectin rsquoreceptorrsquo inEntamoeba histolytica purification and association with thecytoskeletonrdquo Archives of Medical Research vol 23 no 2 pp125ndash128 1992

[147] E Franco J Vazquez-Prado and I Meza ldquoFibronectin-derivedfragments as inducers of adhesion and chemotaxis of Enta-moeba histolytica trophozoitesrdquo Journal of Infectious Diseasesvol 176 no 6 pp 1597ndash1602 1997

[148] S L Stanley Jr T Zhang D Rubin and E Li ldquoRole ofthe Entamoeba histolytica cysteine proteinase in amebic liver

abscess formation in severe combined immunodeficient micerdquoInfection and Immunity vol 63 no 4 pp 1587ndash1590 1995

[149] M Tillack N Nowak H Lotter et al ldquoIncreased expression ofthe major cysteine proteinases by stable episomal transfectionunderlines the important role of EhCP5 for the pathogenicity ofEntamoeba histolyticardquoMolecular and Biochemical Parasitologyvol 149 no 1 pp 58ndash64 2006

[150] A Hellberg R Nickel H Lotter E Tannich and I BruchhausldquoOverexpression of cysteine proteinase 2 in Entamoeba histolyt-ica or Entamoeba dispar increases amoeba-induced monolayerdestruction in vitro but does not augment amoebic liver abscessformation in gerbilsrdquo Cellular Microbiology vol 3 no 1 pp 13ndash20 2001

[151] H Irmer M Tillack L Biller et al ldquoMajor cysteine peptidasesof Entamoeba histolytica are required for aggregation anddigestion of erythrocytes but are dispensable for phagocytosisand cytopathogenicityrdquo Molecular Microbiology vol 72 no 3pp 658ndash667 2009

[152] K McGowan C F Deneke G M Thorne and S L GorbachldquoEntamoeba histolytica cytotoxin purification characteriza-tion strain virulence and protease activityrdquo Journal of InfectiousDiseases vol 146 no 5 pp 616ndash625 1982

[153] D M Faust J M Markiewicz A Danckaert G Soubigouand N Guillen ldquoHuman liver sinusoidal endothelial cellsrespond to interaction with Entamoeba histolytica by changesin morphology integrin signalling and cell deathrdquo CellularMicrobiology vol 13 no 7 pp 1091ndash1106 2011

[154] S Kumar R Banerjee N Nandi A H Sardar and P DasldquoAnoikis potential of Entameba histolytica secretory cysteineproteases evidence of contact independent host cell deathrdquoMicrobial Pathogenesis vol 52 no 1 pp 69ndash76 2012

[155] J MWoof andM A Ken ldquoThe function of immunoglobulin Ain immunityrdquo Journal of Pathology vol 208 no 2 pp 270ndash2822006

[156] R M Garcia-Nieto R Rico-Mata S Arias-Negrete and EE Avila ldquoDegradation of human secretory IgA1 and IgA2 byEntamoeba histolytica surface-associated proteolytic activityrdquoParasitology International vol 57 no 4 pp 417ndash423 2008

[157] J C Carrero C Cervantes-Rebolledo H Aguilar-Dıaz M YDıaz-Gallardo J P Laclette and JMorales-Montor ldquoThe role ofthe secretory immune response in the infection by Entamoebahistolyticardquo Parasite Immunology vol 29 no 7 pp 331ndash3382007

[158] B L Kelsall T G F H Jackson V Gathiram et al ldquoSecre-tory immunoglobulin A antibodies to the galactose-inhibitableadherence protein in the saliva of patients with amebic liverdiseaserdquo American Journal of Tropical Medicine and Hygienevol 51 no 4 pp 454ndash459 1994

[159] J C Carrero M Y Diaz M Viveros B Espinoza E Acostaand L Ortiz-Ortiz ldquoHuman secretory immunoglobulin A anti-Entamoeba histolytica antibodies inhibit adherence of amebaeto MDCK cellsrdquo Infection and Immunity vol 62 no 2 pp 764ndash767 1994

[160] G G Guerrero-Manrıquez F Sanchez-Ibarra and E E AvilaldquoInhibition of Entamoeba histolytica proteolytic activity byhuman salivary IgA antibodiesrdquo Acta Pathologica Microbiolog-ica et Immunologica Scandinavica vol 106 no 11 pp 1088ndash10941998

[161] N Leon-Sicairos F Lopez-Soto M Reyes-Lopez D Godınez-Vargas C Ordaz-Pichardo and M De La Garza ldquoAmoebicidalactivity of milk apo-lactoferrin slgA and lysozymerdquo ClinicalMedicine and Research vol 4 no 2 pp 106ndash113 2006


[162] R Haque D Mondal P Duggal et al ldquoEntamoeba histolyticainfection in children and protection from subsequent amebia-sisrdquo Infection and Immunity vol 74 no 2 pp 904ndash909 2006

[163] M D Abd Alla R Wolf G L White S D Kosanke DCary and J J Verweij ldquoEfficacy of a Gal-lectin subunit vaccineagainst experimental Entamoeba histolytica infection and colitisin baboons (Papio sp)rdquo Vaccine vol 30 no 20 pp 3068ndash30752012

[164] T F H G Jackson and V Gathiram ldquoSeroepidemiologicalstudy of antibody responses to the zymodemes of Entamoebahistolyticardquo Lancet vol 1 no 8431 pp 716ndash719 1985

[165] J C Carrero A Contreras-Rojas B Sanchez-Hernandez etal ldquoProtection against murine intestinal amoebiasis inducedby oral immunization with the 29kDa antigen of Entamoebahistolytica and cholera toxinrdquo Experimental Parasitology vol126 no 3 pp 359ndash365 2010

[166] D M Meneses-Ruiz J P Laclette H Aguilar-Diaz JHernandez-Ruiz A Luz-Madrigal and A Sampieri ldquoMucosaldelivery of ACNPV baculovirus driving expression of the Gal-lectin LC3 fragment confers protection against amoebic liverabscess in hamsterrdquo International Journal of Biological Sciencesvol 7 no 9 pp 1345ndash1356 2011

[167] V Q Tran D S Herdman B E Torian and S L Reed ldquoTheneutral cysteine proteinase of Entamoeba histolytica degradesIgG and prevents its bindingrdquo Journal of Infectious Diseases vol177 no 2 pp 508ndash511 1998

[168] D Ricklin G Hajishengallis K Yang and J D LambrisldquoComplement a key system for immune surveillance andhomeostasisrdquo Nature Immunology vol 11 no 9 pp 785ndash7972010

[169] S L Reed P G Sargeaunt and A I Braude ldquoResistance tolysis by human serum of pathogenic Entamoeba histolyticardquoTransactions of the Royal Society of Tropical Medicine andHygiene vol 77 no 2 pp 248ndash253 1983

[170] R Capin N R Capin M Carmona and L Ortız-Ortız ldquoEffectof complement depletion on the induction of amebic liverabscess in the hamsterrdquo Archivos de Investigacion Medica vol11 no 1 pp 173ndash180 1980

[171] S L Reed and I Gigli ldquoLysis of complement-sensitive Enta-moeba histolytica by activated terminal complement compo-nents Initiation of complement activation by an extracellularneutral cysteine proteinaserdquo Journal of Clinical Investigationvol 86 no 6 pp 1815ndash1822 1990

[172] A Olivos-Garcia E Saavedra E Ramos-Martinez M Nequizand R Perez-Tamayo ldquoMolecular nature of virulence in Enta-moeba histolyticardquo Infection Genetics and Evolution vol 9 no6 pp 1033ndash1037 2009

[173] Q Peng K Li S H Sacks and W Zhou ldquoThe role ofanaphylatoxins C3a and C5a in regulating innate and adaptiveimmune responsesrdquo Inflammation and Allergy vol 8 no 3 pp236ndash246 2009

[174] K B Seydel E Li P E Swanson and S L Stanley ldquoHumanintestinal epithelial cells produce proinflammatory cytokinesin response to infection in a SCID mouse-human intestinalxenograft model of amebiasisrdquo Infection and Immunity vol 65no 5 pp 1631ndash1639 1997

[175] R Bracha Y Nuchamowitz M Leippe and D MirelmanldquoAntisense inhibition of amoebapore expression in Entamoebahistolytica causes a decrease in amoebic virulencerdquo MolecularMicrobiology vol 34 no 3 pp 463ndash472 1999

[176] M Leippe ldquoAmoebaporesrdquo Parasitology Today vol 13 no 5 pp178ndash183 1997

[177] C A Dinarello ldquoInterleukin-18rdquoMethods vol 19 no 1 pp 121ndash132 1999

[178] R Seguin B J Mann K Keller and K Chadee ldquoThetumor necrosis factor alpha-stimulating region of galactose-inhibitable lectin of Entamoeba histolytica activates gammainterferon- primed macrophages for amebicidal activity medi-ated by nitric oxiderdquo Infection and Immunity vol 65 no 7 pp2522ndash2527 1997

[179] R L Jurado ldquoIron infections and anemia of inflammationrdquoClinical Infectious Diseases vol 25 no 4 pp 888ndash895 1997

[180] B R Otto A M J J Verweij-Van Vught and D MMacLaren ldquoTransferrins and heme-compounds as iron sourcesfor pathogenic bacteriardquo Critical Reviews in Microbiology vol18 no 3 pp 217ndash233 1992

[181] E D Weinberg ldquoInfection and iron metabolismrdquo AmericanJournal of Clinical Nutrition vol 30 no 9 pp 1485ndash1490 1977

[182] E D Weinberg ldquoIron and infectionrdquo Microbiological Reviewsvol 42 no 1 pp 45ndash66 1978

[183] E D Weinberg and J Miklossy ldquoIron withholding a defenseagainst diseaserdquo Journal of Alzheimerrsquos Disease vol 13 no 4 pp451ndash463 2008

[184] E D Weinberg ldquoIron and susceptibility to infectious diseaserdquoScience vol 184 no 4140 pp 952ndash956 1974

[185] F Lopez-Soto N Leon-Sicairos M Reyes-Lopez et al ldquoUseand endocytosis of iron-containing proteins by Entamoebahistolytica trophozoitesrdquo Infection Genetics and Evolution vol9 no 6 pp 1038ndash1050 2009

[186] J Tachezy andMWilson ldquoMore on iron acquisition by parasiticprotozoardquo Parasitology Today vol 15 no 5 p 207 1999

[187] S Vanacova D Rasoloson J Razga I Hrdy J Kulda andJ Tachezy ldquoIron-induced changes in pyruvate metabolism ofTritrichomonas foetus and involvement of iron in expression ofhydrogenosomal proteinsrdquoMicrobiology vol 147 no 1 pp 53ndash62 2001

[188] R A Finkelstein C V Sciortino and M A McIntosh ldquoRoleof iron in microbe-host interactionsrdquo Reviews of InfectiousDiseases vol 5 supplement pp S759ndashS777 1983

[189] E D Weinberg ldquoThe role of iron in protozoan and fungalinfectious diseasesrdquo Journal of Eukaryotic Microbiology vol 46no 3 pp 231ndash238 1999

[190] A Jacobs andMWorwood Iron in Biochemistry and MedicineAcademic Press London UK 1980

[191] S Said-Fernandez and R Lopez-Revilla ldquoSubcellular distribu-tion and stability of the major hemolytic activity of Entamoebahistolytica trophozoitesrdquo Zeitschrift fur Parasitenkunde vol 67no 3 pp 249ndash254 1982

[192] V Tsutsumi A Martinez-Palomo and K Tanikawa ldquoScanningelectron microscopy of erythrophagocytosis by Entamoebahistolytica trophozoitesrdquo Archives of Medical Research vol 23no 2 pp 173ndash175 1992

[193] J Vargas-Villarreal H Martinez-Rodriguez J Castro-GarzaB D Mata-Cardenas M T Gonzalez-Garza and S Said-Fernandez ldquoIdentification of Entamoeba histolytica intracel-lular phospholipase A and lysophospholipase L1 activitiesrdquoParasitology Research vol 81 no 4 pp 320ndash323 1995

[194] A Chevez I Iturbe-Alessio M Segura and D CoronaldquoPhagocytosis of human erythrocytes byEntamoeba histolyticardquoArchivos de Investigacion Medica vol 2 pp 2ndash286 1972

[195] J Mora-Galindo and F Anaya-Velazquez ldquoIntracellular diges-tion of human erythrocytes by Entamoeba histolytica a kineticstudy in vitrordquo Archives of Medical Research vol 24 no 4 pp347ndash351 1993


[196] J Mora-Galindo M Gutierrez-Lozano and F Anaya-Velazquez ldquoEntamoeba histolytica kinetics of hemolyticactivity erythrophagocytosis and digestion of erythrocytesrdquoArchives of Medical Research vol 28 pp 200ndash201 1997

[197] R Jarumilinta and B G Maegraith ldquoThe patterns of some pro-teolytic enzymes of Entamoeba histolytica and AcanthamoebaspmdashIThe action of E histolytica andAcanthamoeba sp on pro-tein substratesrdquo Annals of Tropical Medicine and Parasitologyvol 55 pp 505ndash517 1961

[198] I Becker R Perez-Montfort A Perez-Torres A Rondan-Zarate I Montfort and R Perez-Tamayo ldquoEntamoeba his-tolytica localization of a 30-kDa cysteine proteinase using amonoclonal antibodyrdquo Experimental Parasitology vol 82 no 2pp 171ndash181 1996

[199] J Mora-Galindo F Anaya-Velazquez S Ramırez-Romo and AGonzalez-Robles ldquoEntamoeba histolytica correlation of assess-ment methods to measure erythrocyte digestion and effect ofcysteine proteinases inhibitors in HM-1IMSS and HK-9NIHstrainsrdquo Experimental Parasitology vol 108 no 3-4 pp 89ndash1002004

[200] A Bezkorovainy ldquoBiochemistry of nonheme iron in manmdashIIron proteins and cellular iron metabolismrdquo Clinical Physiologyand Biochemistry vol 7 no 1 pp 1ndash17 1989

[201] J A Fernandez-Pol and D J Klos ldquoIsolation and characteriza-tion of Normal Rat Kidney cell membrane proteins with affinityfor transferrinrdquoBiochemistry vol 19 no 17 pp 3904ndash3912 1980

[202] M Reyes-Lopez J D J Serrano-Luna E Negrete-Abascal NLeon-Sicairos A L Guerrero-Barrera and M De la GarzaldquoEntamoeba histolytica transferrin binding proteinsrdquo Experi-mental Parasitology vol 99 no 3 pp 132ndash140 2001

[203] J Langhorst and J Boone ldquoFecal lactoferrin as a noninvasivebiomarker in inflammatory bowel diseasesrdquo Drugs Today vol48 no 2 pp 149ndash161 2012

[204] R D Brines and J H Brock ldquoThe effect of trypsin andchymotrypsin on the in vitro antimicrobial and iron-bindingproperties of lactoferrin in human milk and bovine colostrumUnusual resistance of human apolactoferrin to proteolyticdigestionrdquo Biochimica et Biophysica Acta vol 759 no 3 pp229ndash235 1983

[205] P Aisen and A Leibman ldquoLactoferrin and transferrin acomparative studyrdquo Biochimica et Biophysica Acta vol 257 no2 pp 314ndash323 1972

[206] E N Baker and H M Baker ldquoMolecular structure bindingproperties and dynamics of lactoferrinrdquo Cellular and MolecularLife Sciences vol 62 no 22 pp 2531ndash2539 2005

[207] J A Talbot K Nielsen and L B Corbeil ldquoCleavage ofproteins of reproductive secretions by extracellular proteinasesofTritrichomonas foetusrdquoCanadian Journal ofMicrobiology vol37 no 5 pp 384ndash390 1991

[208] P Arosio and S Levi ldquoFerritin iron homeostasis and oxidativedamagerdquo Free Radical Biology and Medicine vol 33 no 4 pp457ndash463 2002

[209] P M Harrison and P Arosio ldquoThe ferritins molecular proper-ties iron storage function and cellular regulationrdquo Biochimicaet Biophysica Acta vol 1275 no 3 pp 161ndash203 1996

[210] A M Koorts and M Viljoen ldquoFerritin and ferritin isoformsI structure-function relationships synthesis degradation andsecretionrdquo Archives of Physiology and Biochemistry vol 113 no1 pp 30ndash54 2007

[211] X Liu and E C Theil ldquoFerritins dynamic management ofbiological iron and oxygen chemistryrdquo Accounts of ChemicalResearch vol 38 no 3 pp 167ndash175 2005

[212] R Lopez-Revilla and S Said-Fernandez ldquoCytopathogenicityof Entamoeba histolytica hemolytic activity of trophozoitehomogenatesrdquo American Journal of Tropical Medicine andHygiene vol 29 no 2 pp 209ndash212 1980

[213] C ShimokawaM KabirM Taniuchi DMondal S Kobayashiand I K M Ali ldquoEntamoeba moshkovskii is associated withdiarrhea in infants and causes diarrhea and colitis in micerdquoJournal of Infectious Diseases vol 206 no 5 pp 744ndash751 2012

[214] C G Clark and L S Diamond ldquoThe Laredo strain andother Entamoeba histolytica-like amoebae are Entamoebamoshkovskiirdquo Molecular and Biochemical Parasitology vol 46no 1 pp 11ndash18 1991

[215] R D Heredia J A Fonseca and M C Lopez ldquoEntamoebamoshkovskii perspectives of a new agent to be considered in thediagnosis of amebiasisrdquoActa Tropica vol 123 no 3 pp 139ndash1452012

[216] M Goldman ldquoEntamoeba histolytica-like amoebae ocurring inmanrdquo Bulletin of the World Health Organization vol 40 pp355ndash364 1969

[217] I K M Ali C G Clark and W A Petri Jr ldquoMolecularepidemiology of amebiasisrdquo Infection Genetics and Evolutionvol 8 no 5 pp 698ndash707 2008

[218] Z Hamzah S Petmitr M Mungthin S Leelayoova and PChavalitshewinkoon-Petmitr ldquoDevelopment of multiplex real-time polymerase chain reaction for detection of Entamoebahistolytica Entamoeba dispar and Entamoeba moshkovskii inclinical specimensrdquo American Journal of Tropical Medicine andHygiene vol 83 no 4 pp 909ndash913 2010

[219] WHO ldquoldquoAmoebiasisrdquo World Health OrganizationPan Amer-ica Health Organization Expert Consultation on AmoebiasisrdquoWHOWeekly Epidemiological Record vol 72 pp 97ndash100 1997

[220] S S Dolabella J Serrano-Luna F Navarro-Garcıa et al etal ldquoAmoebic liver abscess production by Entamoeba disparrdquoAnnals of Hepatology vol 11 no 1 pp 107ndash117 2012

[221] W M Spice and J P Ackers ldquoThe effect of axenic versus xenicculture conditions on the total and secreted proteolytic activityof Entamoeba histolytica strainsrdquo Archives of Medical Researchvol 23 no 2 pp 91ndash93 1992

[222] B Loftus I Anderson R Davies et al ldquoThe genome of theprotist parasite Entamoeba histolyticardquo Nature vol 433 no7028 pp 865ndash868 2005

[223] I Bruchhaus and E Tannich ldquoA gene highly homologous toACP1 encoding cysteine proteinase 3 in Entamoeba histolyticais present and expressed in E disparrdquo Parasitology Research vol82 no 2 pp 189ndash192 1996

[224] E Tannich ldquoRecent advances in DNA-mediated gene transferof Entamoeba histolyticardquo Parasitology Today vol 12 no 5 pp198ndash200 1996

[225] B N Mitra T Yasuda S Kobayashi Y Saito-Nakano and TNozaki ldquoDifferences in morphology of phagosomes and kinet-ics of acidification and degradation in phagosomes betweenthe pathogenic Entamoeba histolytica and the non-pathogenicEntamoeba disparrdquo Cell Motility and the Cytoskeleton vol 62no 2 pp 84ndash99 2005

[226] J E Teixeira A Sateriale K E Bessoff and C D HustonldquoControl of Entamoeba histolytica adherence involves metallo-surface protease 1 an M8 family surface metalloprotease withhomology to leishmanolysinrdquo Infection and Immunity vol 80no 6 pp 2165ndash2176 2012

[227] F Ebert A Bachmann K Nakada-Tsukui et al ldquoAn Entamoebacysteine peptidase specifically expressed during encystationrdquoParasitology International vol 57 no 4 pp 521ndash524 2008


[228] G Jeelani D Sato A Husain A Escueta-de Cadiz MSugimoto and T Soga ldquoMetabolic profiling of the protozoanparasite Entamoeba invadens revealed activation of unpredictedpathway during encystationrdquo PLoS ONE vol 7 no 5 Article IDe37740 2012

[229] E E Avila M Sanchez-Garza and J Calderon ldquoEntamoebahistolytica and E invadens sulfhydryl-dependent proteolyticactivity1rdquo Journal of Eukaryotic Microbiology vol 32 no 1 pp163ndash166 1985

[230] S K Ghosh J Field M Frisardi et al ldquoChitinase secretionby encysting Entamoeba invadens and transfected Entamoebahistolytica trophozoites localization of secretory vesicles endo-plasmic reticulum and Golgi apparatusrdquo Infection and Immu-nity vol 67 no 6 pp 3073ndash3081 1999

[231] K L Van Dellen A Chatterjee D M Ratner et al ldquoUniqueposttranslational modifications of chitin-binding lectins ofEntamoeba invadens cyst wallsrdquo Eukaryotic Cell vol 5 no 5pp 836ndash848 2006

[232] J Gonzalez G Bai U Frevert E J Corey and D EichingerldquoProteasome-dependent cyst formation and stage-specific ubiq-uitin mRNA accumulation in Entamoeba invadensrdquo EuropeanJournal of Biochemistry vol 264 no 3 pp 897ndash904 1999

[233] H Trabelsi F Dendana A Sellami H Sellami FCheikhrouhou and S Neji ldquoPathogenic fre-living amoebaepidemiology and clinical reviewrdquo Pathologie Biologie vol 60no 6 pp 399ndash405 2012

[234] M Omana-Molina F Navarro-Garcıa A Gonzalez-RoblesJ d Serrano-Luna R Campos-Rodrıguez and A Martınez-Palomo ldquoInduction of morphological and electrophysiologicalchanges in hamster cornea after in vitro interaction withtrophozoites of Acanthamoeba spprdquo Infection and Immunityvol 72 no 6 pp 3245ndash3251 2004

[235] M Garate Z Cao E Bateman and N Panjwani ldquoCloningand characterization of a novel mannose-binding protein ofAcanthamoebardquo Journal of Biological Chemistry vol 279 no 28pp 29849ndash29856 2004

[236] Y C HongWM Lee H H Kong H J Jeong andD I ChungldquoMolecular cloning and characterization of a cDNA encoding alaminin-binding protein (AhLBP) from Acanthamoeba healyirdquoExperimental Parasitology vol 106 no 3-4 pp 95ndash102 2004

[237] B Da Rocha-Azevedo M Jamerson G A Cabral F C Silva-Filho and FMarciano-Cabral ldquoAcanthamoeba interactionwithextracellular matrix glycoproteins biological and biochemicalcharacterization and role in cytotoxicity and invasivenessrdquoJournal of Eukaryotic Microbiology vol 56 no 3 pp 270ndash2782009

[238] V R Gordon E K AsemMH Vodkin andG LMcLaughlinldquoAcanthamoeba binds to extracellular matrix proteins in vitrordquoInvestigative Ophthalmology and Visual Science vol 34 no 3pp 658ndash662 1993

[239] LWang E K Asem andG LMcLaughlin ldquoCalcium enhancesAcanthamoeba polyphaga binding to extracellular matrix pro-teinsrdquo Investigative Ophthalmology and Visual Science vol 35no 5 pp 2421ndash2426 1994

[240] J Sissons S K Kwang M Stins S Jayasekera S Alsam andN A Khan ldquoAcanthamoeba castellanii induces host cell deathvia a phosphatidylinositol 3-kinase-dependent mechanismrdquoInfection and Immunity vol 73 no 5 pp 2704ndash2708 2005

[241] H Alizadeh S Neelam M Hurt and J Y Niederkorn ldquoRoleof contact lens wear bacterial flora and mannose-inducedpathogenic protease in the pathogenesis of amoebic keratitisrdquoInfection and Immunity vol 73 no 2 pp 1061ndash1068 2005

[242] A Ferrante and E J Bates ldquoElastase in the pathogenic free-living amoebaeNaegleria andAcanthamoeba spprdquo Infection andImmunity vol 56 no 12 pp 3320ndash3321 1988

[243] PNMortazavi E Keisary L N Loh S Y Jung andNA KhanldquoPossible roles of phospholipase A2 in the biological activities ofAcanthamoeba castellanii (T4 Genotype)rdquo Protist vol 162 no 1pp 168ndash176 2011

[244] S C Alfieri C E B Correia S A Motegi and E M FPral ldquoProteinase activities in total extracts and in mediumconditioned byAcanthamoeba polyphaga trophozoitesrdquo Journalof Parasitology vol 86 no 2 pp 220ndash227 2000

[245] E Hadas and T Mazur ldquoProteolytic enzymes of pathogenicand non-pathogenic strains of Acanthamoeba spprdquo TropicalMedicine and Parasitology vol 44 no 3 pp 197ndash200 1993

[246] M M Mitra H Alizadeh R D Gerard and J Y NiederkornldquoCharacterization of a plasminogen activator produced byAcanthamoeba castellaniirdquoMolecular and Biochemical Parasitol-ogy vol 73 no 1-2 pp 157ndash164 1995

[247] K Mitro A Bhagavathiammai O M Zhou et al ldquoPartialcharacterization of the proteolytic secretions of Acanthamoebapolyphagardquo Experimental Parasitology vol 78 no 4 pp 377ndash385 1994

[248] N A Khan E L Jarroll N Panjwani Z Cao and T A PagetldquoProteases as markers for differentiation of pathogenic andnonpathogenic species of Acanthamoebardquo Journal of ClinicalMicrobiology vol 38 no 8 pp 2858ndash2861 2000

[249] Y He J Y Niederkorn J P McCulley et al ldquoIn vivo andin vitro collagenolytic activity of Acanthamoeba castellaniirdquoInvestigative Ophthalmology and Visual Science vol 31 no 11pp 2235ndash2240 1990

[250] G A Ferreira A CMMagliano EM F Pral and S C AlfierildquoElastase secretion in Acanthamoeba polyphagardquo Acta Tropicavol 112 no 2 pp 156ndash163 2009

[251] H H Kong T H Kim andD I Chung ldquoPurification and char-acterization of a secretory serine proteinase of Acanthamoebahealyi isolated from GAErdquo Journal of Parasitology vol 86 no 1pp 12ndash17 2000

[252] W T Kim H H Kong Y R Ha et al ldquoComparison ofspecific activity and cytopathic effects of purified 33 kDa serineproteinase from Acanthamoeba strains with different degree ofvirulencerdquoTheKorean Journal of Parasitology vol 44 no 4 pp321ndash330 2006

[253] A J Martinez S MMarkowitz and R J Duma ldquoExperimentalpneumonitis and encephalitis caused byAcanthamoeba inmicepathogenesis and ultrastructural featuresrdquo Journal of InfectiousDiseases vol 31 no 6 pp 692ndash699 1975

[254] A Martınez ldquoFree-living amoeba natural history preventiondiagnostic pathology and treatment of diseaserdquo p 156 CRCPress Boca Raton Fla USA 1985

[255] A J Martinez ldquoInfection of the central nervous system due toAcanthamoebardquo Reviews of Infectious Diseases vol 13 no 5 ppS399ndashS402 1991

[256] N A Khan ldquoAcanthamoeba invasion of the central nervoussystemrdquo International Journal for Parasitology vol 37 no 2 pp131ndash138 2007

[257] S Alsam J Sissons S Jayasekera and N A Khan ldquoExtracel-lular proteases of Acanthamoeba castellanii (encephalitis isolatebelonging to T1 genotype) contribute to increased permeabilityin an in vitromodel of the human blood-brain barrierrdquo Journalof Infection vol 51 no 2 pp 150ndash156 2005


[258] N A Khan and R Siddiqui ldquoAcanthamoeba affects the integrityof human brain microvascular endothelial cells and degradesthe tight junction proteinsrdquo International Journal for Parasitol-ogy vol 39 no 14 pp 1611ndash1616 2009

[259] J F De Jonckheere ldquoOrigin and evolution of the worldwide dis-tributed pathogenic amoeboflagellate Naegleria fowlerirdquo Infec-tion Genetics and Evolution vol 11 no 7 pp 1520ndash1528 2011

[260] F Marciano-Cabral ldquoBiology of Naegleria spprdquoMicrobiologicalReviews vol 52 no 1 pp 114ndash133 1988

[261] I Cervantes-Sandoval J D J Serrano-Luna E Garcıa-LatorreV Tsutsumi and M Shibayama ldquoCharacterization of braininflammation during primary amoebic meningoencephalitisrdquoParasitology International vol 57 no 3 pp 307ndash313 2008

[262] K L Jarolim J K McCosh M J Howard and D T John ldquoAlight microscopy study of the migration of Naegleria fowlerifrom the nasal submucosa to the central nervous system duringthe early stage of primary amebicmeningoencephalitis inmicerdquoJournal of Parasitology vol 86 no 1 pp 50ndash55 2000

[263] K L Han H J Lee H S Myeong H J Shin K I Imand S J Park ldquoThe involvement of an integrin-like proteinand protein kinase C in amoebic adhesion to fibronectin andamoebic cytotoxicityrdquo Parasitology Research vol 94 no 1 pp53ndash60 2004

[264] R Herbst C Ott T Jacobs T Marti F Marciano-Cabraland M Leippe ldquoPore-forming polypeptides of the pathogenicprotozoon Naegleria fowlerirdquo Journal of Biological Chemistryvol 277 no 25 pp 22353ndash22360 2002

[265] J D E Young and D M Lowrey ldquoBiochemical and functionalcharacterization of a membrane-associated pore-forming pro-tein from the pathogenic ameboflagellate Naegleria fowlerirdquoJournal of Biological Chemistry vol 264 no 2 pp 1077ndash10831989

[266] S E Barbour and F Marciano-Cabral ldquoNaegleria fowleriamoebae express a membrane-associated calcium-independentphospholipase A2rdquo Biochimica et Biophysica Acta vol 1530 no2-3 pp 123ndash133 2001

[267] D E Fulford and F Marciano-Cabral ldquoCytolytic activity ofNaegleria fowleri cell-free extractrdquo Journal of Protozoology vol33 no 4 pp 498ndash502 1986

[268] M Shibayama J D J Serrano-Luna S Rojas-Hernandez RCampos-Rodrıguez and V Tsutsumi ldquoInteraction of secretoryimmunoglobulin A antibodies with Naegleria fowleri tropho-zoites and collagen type Irdquo Canadian Journal of Microbiologyvol 49 no 3 pp 164ndash170 2003

[269] I Cervantes-Sandoval J Jesus Serrano-Luna J Pacheco-YepezA Silva-Olivares V Tsutsumi and M Shibayama ldquoDifferencesbetween Naegleria fowleri and Naegleria gruberi in expressionof mannose and fucose glycoconjugatesrdquo Parasitology Researchvol 106 no 3 pp 695ndash701 2010

[270] M Jamerson B da Rocha-Azevedo G A Cabral and FMarciano-Cabral ldquoPathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesionto and invasion of extracellular matrix proteinsrdquoMicrobiologyvol 158 pp 791ndash803 2012

[271] F L Schuster and G S Visvesvara ldquoFree-living amoebae asopportunistic and non-opportunistic pathogens of humans andanimalsrdquo International Journal for Parasitology vol 34 no 9 pp1001ndash1027 2004

[272] B A Rideout C H Gardiner I H Stalis J R Zuba T Hadfieldand G S Visvesvara ldquoFatal infections with Balamuthia man-drillaris (a free-living amoeba) in gorillas and other old worldprimatesrdquo Veterinary Pathology vol 34 no 1 pp 15ndash22 1997

[273] T H Dunnebacke F L Schuster S Yagi and G C BootonldquoBalamuthia mandrillaris from soil samplesrdquoMicrobiology vol150 no 9 pp 2837ndash2842 2004

[274] A Matin and N A Khan ldquoDemonstration and partial char-acterization of ecto-ATPase in Balamuthia mandrillaris and itspossible role in the host-cell interactionsrdquo Letters in AppliedMicrobiology vol 47 no 4 pp 348ndash354 2008

[275] A Martınez and G S Visvesvara ldquoBalamuthia mandrillarisinfectionrdquo Journal of Medical Microbiology vol 50 no 3 pp205ndash207 2001

[276] K Janitschke A J Martınez G S Visvesvara and F Schus-ter ldquoAnimal model Balamuthia mandrillaris CNS infectioncontrast and comparison in immunodeficient and immuno-competent mice a murine model of ldquogranulomatousrdquo amebicencephalitisrdquo Journal of Neuropathology and Experimental Neu-rology vol 55 no 7 pp 815ndash821 1996

[277] A F Kiderlen and U Laube ldquoBalamuthia mandrillaris anopportunistic agent of granulomatous amebic encephalitisinfects the brain via the olfactory nerve pathwayrdquo ParasitologyResearch vol 94 no 1 pp 49ndash52 2004

[278] F L Schuster T H Dunnebacke G C Booton et al ldquoEnvi-ronmental isolation of Balamuthia mandrillaris associated witha case of amebic encephalitisrdquo Journal of Clinical Microbiologyvol 41 no 7 pp 3175ndash3180 2003

[279] F L Schuster and G S Visvesvara ldquoAxenic growth and drugsensitivity studies of Balamuthia mandrillaris an agent ofamebic meningoencephalitis in humans and other animalsrdquoJournal of ClinicalMicrobiology vol 34 no 2 pp 385ndash388 1996

[280] S Jayasekera A Matin J Sissons A H Maghsood andN A Khan ldquoBalamuthia mandrillaris stimulates interleukin-6 release in primary human brain microvascular endothelialcells via a phosphatidylinositol 3-kinase-dependent pathwayrdquoMicrobes and Infection vol 7 no 13 pp 1345ndash1351 2005

[281] B Rocha-Azevedo M Jamerson G A Cabral F C Silva-Filhoand F Marciano-Cabral ldquoThe interaction between the amoebaBalamuthia mandrillaris and extracellular matrix glycoproteinsin vitrordquo Parasitology vol 134 no 1 pp 51ndash58 2007


parasitic counterparts these free-living amoebae are not welladapted for parasitism Furthermore as free-living formswith a broad distribution they are not dependent upon a hostfor transmission and spread Unlike the parasite E histolyt-ica free-living amoebae are mitochondrion-bearing aerobicorganisms that can complete their respective life cycles inthe environment without a host thus these organisms havebeen called amphizoic amoebae in recognition of their abilityto live endozoically although they are capable of free-livingexistence [52]

All pathogenic amoeba species have in common thecapability to phagocytose bacteria erythrocytes and celldetritus The major virulence factors are adhesins toxinsamoebapores and proteases which lead to the lysis deathand destruction of a variety of cells and tissues in thehost Because of their relevance to amoebic pathogenesisproteolytic enzymes are of particular interest Proteases areimportant in tissue invasion migration and host pathologyThe main goal of this paper is to review the proteases of theEntamoeba genus and of free-living amoebae

2 Entamoeba histolytica

Amoebiasis is a human parasitic infection caused byEntamoeba histolytica an extracellular protozoan Cysts aretransmitted through the fecal-oral route by contaminatedwater or food Parasite destruction of host tissues appearsto be the basis of amoebiasis which leads to invasivedisease pathologies such as intestinal amoebiasis which ismainly characterized by lesions in the colon that producefever abdominal pain dysentery and ulcerative colitis withmucous and blood Amoebae can spread through the portalvein to other organs such as the liver lungs kidneys andbrain Hepatic amoebiasis is characterized by liver abscessesthat can be fatal [53ndash57]

Amoebiasis is the third leading cause of death dueto parasites after malaria and schistosomiasis Amoebiasispresents a high index of morbidity and mortality mainlyin developing countries According to the World HealthOrganization (WHO) 500 million people are infected withamoebae 10 of infected individuals have virulent E his-tolytica resulting in 40000ndash100000 deaths annually [56] Ehistolytica infects only humans The life cycle of E histolyticaconsists of two main stages the trophozoite (also calledamoeba) or invasive form and the cyst or infective formCysts can tolerate the stomach acidic pH and excyst in theterminal ileum Trophozoites colonize the large intestine andin later phases of amoebiasis they invade the epithelium andmucosa In response to unknown stimuli amoebae undergomorphological and biochemical changes that lead to theformation of new cysts which are eliminated in the fecescompleting the cycle [53 55 56]

The cytopathogenic effect caused by E histolytica tropho-zoites is multifactorial Intestinal flask-shaped ulcers a hall-mark of amoebic colitis are characterized by severe damageto enteric cells as well as migration to the lamina propriaand blood vessels [57 58] The contact between trophozoitesand target cells appears to be the first step for cell lysis and

phagocytosis Several molecules are involved in this inter-action including the 260 and 220 kDa lectins and 112 kDaadhesin which participates in the adherence to epithelial cellsand erythrocytes [8 59ndash63] It has been proposed that forthe initial amoeba contact or adhesion surface carbohydrateson the target cell are recognized by specific molecules fromthe parasite One of the better studied amoebic moleculesis the GalGalNAc lectin which mediates binding to hostcarbohydrate determinants that contain galactose andor N-acetyl-D-galactosamine (GalNAc) [64 65] Adherence tocolonic mucosa is conducive to the continued reproductionof parasites and tissue damage by the products secreted byamoebae such as the pore-forming peptide amoebapore [66]which permits a massive influx of extracellular Ca+2 that iscombined with the release of amoebic proteases at the site ofcontact with the subsequent degradation of substrates Oncethe targets are partially digested the amoeba internalizesthe cell debris and substrate fragments by phagocytosis [67]Other proteins also contribute to host cell binding on targetcells and destruction such as phospholipases [68 69]

21 Proteases of E histolytica and Their Role in VirulenceStudies of E histolytica proteinases (proteases) have mainlybeen performed in the strain HM-1IMSS from axenicallygrown trophozoites De la Torre et al [70] isolated this strainfrom cysts of a Mexican patient suffering from intestinalamoebiasis Most of the cellular and molecular studies ofE histolytica throughout the world including the genomicsequence have been performed with this strain It hasbeen cultured for years and passed through the liver ofSyrian golden hamsters an experimental model in whichhepatic abscesses are reproduced tomaintain and increase thevirulence of E histolytica

Cysteine proteases (CPs) are the predominant proteolyticactivity associated with pathogenicity in E histolytica basedon many studies in which the degradation of differentsubstrates has been investigated including purified pro-teins of the extracellular matrix (ECM) immunoglobulinscomplement and mucin Figures 1 and 2 show the roleof these proteases during intestinal amoebiasis and bloodvessel transit of trophozoites respectively Animal modelstudies have confirmed that CPs have an important role invirulence E histolytica mutants impaired in genes encodingCPs have a diminished ability to produce hepatic abscesses[71 72] Figure 3 shows the role of proteases during amoebicliver abscess EhCPs are expressed both intracellularly andextracellularly and are referred to as cathepsin-like enzymesbecause their structure is similar to that of cathepsin L how-ever their substrate specificity resembles that of cathepsinB [34 73ndash75] Some proteases have been characterized assurface localized hence they have the potential to contributeto host tissue breakdown in vivo [74] (Figure 4) E histolyticamost studied proteases are summarized in Table 1

211 Purification and Cloning of the Main Cysteine Proteasesfrom E histolytica Several genes encoding E histolytica CPshave been cloned In 1990 Eakin et al [76] amplified CPgene fragments from trophozoites by PCR from genomic


Mucuslayer

Extracellularmatrix

Epithelialcell


EhCP5


EhCP4sIgA

Plasma cells

EhCP LfNeutrophils

EhCP5EhCPLf

EhCP Lf

Eh

EhEh

EhEh

Eh

Eh

EhCP1

EhCP tight

CollagenFibronectin

Laminin-1




microbiotapIL-1

IL-18







EhCP3

EhCP4

EhCP3

EhN

EhCP4-5

C3

IgG

Transferrin

Erythrocytes

Hemoglobin

Lactoferrin

Ferritin

EhCP4


(a)

Hemoglobin

Ferritin

Lactoferrin

EhEh

EhEh

EhNN

N

EEEEhhhhhhhhhhhhhEh

(b)







Collagen











IgG


Complement






Hemoglobin



[27]


No mdash 130 70 50 35and 30



Table 1 Continued







[9 31]





Ly

EhCP4EhCP5

EhCP1

EhCP2

NucleusLy

EhCP112EhCP3

Ly = Lysosome



























EhROM1

EhROM1

LgI

IgIHgI

EhROM1

LgI

IgIHgI

(a) (b) (c)









RGD

Aktt

Ub

ILKPI3K

Akt

P

Degradation

Host cell

P

P

+

P

Ub

P473

P473

IKK NEMO

IKKγNEMO

IKK IKK

IKKα IKKβ

I BI B

NF- B

NF- B

Nucleus

TNF-IL-1

EhPCP5


































CPs

Ly


EE

LE

Ly


Erythrocyte

Amoebic clathrin

h-Hololactoferrin





Haemoglobin

Ferritin


h-Holotransferrin

Nucleus


Ph

Ly

LyLL EEL

Fluid phase

ADH2



Ly = lysosome










119889for
















E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References







































































































































































































































































































Mucuslayer

Extracellularmatrix

Epithelialcell


EhCP5


EhCP4sIgA

Plasma cells

EhCP LfNeutrophils

EhCP5EhCPLf

EhCP Lf

Eh

EhEh

EhEh

Eh

Eh

EhCP1

EhCP tight

CollagenFibronectin

Laminin-1




microbiotapIL-1

IL-18







EhCP3

EhCP4

EhCP3

EhN

EhCP4-5

C3

IgG

Transferrin

Erythrocytes

Hemoglobin

Lactoferrin

Ferritin

EhCP4


(a)

Hemoglobin

Ferritin

Lactoferrin

EhEh

EhEh

EhNN

N

EEEEhhhhhhhhhhhhhEh

(b)







Collagen











IgG


Complement






Hemoglobin



[27]


No mdash 130 70 50 35and 30



Table 1 Continued







[9 31]





Ly

EhCP4EhCP5

EhCP1

EhCP2

NucleusLy

EhCP112EhCP3

Ly = Lysosome



























EhROM1

EhROM1

LgI

IgIHgI

EhROM1

LgI

IgIHgI

(a) (b) (c)









RGD

Aktt

Ub

ILKPI3K

Akt

P

Degradation

Host cell

P

P

+

P

Ub

P473

P473

IKK NEMO

IKKγNEMO

IKK IKK

IKKα IKKβ

I BI B

NF- B

NF- B

Nucleus

TNF-IL-1

EhPCP5


































CPs

Ly


EE

LE

Ly


Erythrocyte

Amoebic clathrin

h-Hololactoferrin





Haemoglobin

Ferritin


h-Holotransferrin

Nucleus


Ph

Ly

LyLL EEL

Fluid phase

ADH2



Ly = lysosome










119889for
















E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References







































































































































































































































































































EhCP3

EhCP4

EhCP3

EhN

EhCP4-5

C3

IgG

Transferrin

Erythrocytes

Hemoglobin

Lactoferrin

Ferritin

EhCP4


(a)

Hemoglobin

Ferritin

Lactoferrin

EhEh

EhEh

EhNN

N

EEEEhhhhhhhhhhhhhEh

(b)







Collagen











IgG


Complement






Hemoglobin



[27]


No mdash 130 70 50 35and 30



Table 1 Continued







[9 31]





Ly

EhCP4EhCP5

EhCP1

EhCP2

NucleusLy

EhCP112EhCP3

Ly = Lysosome



























EhROM1

EhROM1

LgI

IgIHgI

EhROM1

LgI

IgIHgI

(a) (b) (c)









RGD

Aktt

Ub

ILKPI3K

Akt

P

Degradation

Host cell

P

P

+

P

Ub

P473

P473

IKK NEMO

IKKγNEMO

IKK IKK

IKKα IKKβ

I BI B

NF- B

NF- B

Nucleus

TNF-IL-1

EhPCP5


































CPs

Ly


EE

LE

Ly


Erythrocyte

Amoebic clathrin

h-Hololactoferrin





Haemoglobin

Ferritin


h-Holotransferrin

Nucleus


Ph

Ly

LyLL EEL

Fluid phase

ADH2



Ly = lysosome










119889for
















E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References









































































































































































































































































































Collagen











IgG


Complement






Hemoglobin



[27]


No mdash 130 70 50 35and 30



Table 1 Continued







[9 31]





Ly

EhCP4EhCP5

EhCP1

EhCP2

NucleusLy

EhCP112EhCP3

Ly = Lysosome



























EhROM1

EhROM1

LgI

IgIHgI

EhROM1

LgI

IgIHgI

(a) (b) (c)









RGD

Aktt

Ub

ILKPI3K

Akt

P

Degradation

Host cell

P

P

+

P

Ub

P473

P473

IKK NEMO

IKKγNEMO

IKK IKK

IKKα IKKβ

I BI B

NF- B

NF- B

Nucleus

TNF-IL-1

EhPCP5


































CPs

Ly


EE

LE

Ly


Erythrocyte

Amoebic clathrin

h-Hololactoferrin





Haemoglobin

Ferritin


h-Holotransferrin

Nucleus


Ph

Ly

LyLL EEL

Fluid phase

ADH2



Ly = lysosome










119889for
















E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References







































































































































































































































































































Table 1 Continued







[9 31]





Ly

EhCP4EhCP5

EhCP1

EhCP2

NucleusLy

EhCP112EhCP3

Ly = Lysosome



























EhROM1

EhROM1

LgI

IgIHgI

EhROM1

LgI

IgIHgI

(a) (b) (c)









RGD

Aktt

Ub

ILKPI3K

Akt

P

Degradation

Host cell

P

P

+

P

Ub

P473

P473

IKK NEMO

IKKγNEMO

IKK IKK

IKKα IKKβ

I BI B

NF- B

NF- B

Nucleus

TNF-IL-1

EhPCP5


































CPs

Ly


EE

LE

Ly


Erythrocyte

Amoebic clathrin

h-Hololactoferrin





Haemoglobin

Ferritin


h-Holotransferrin

Nucleus


Ph

Ly

LyLL EEL

Fluid phase

ADH2



Ly = lysosome










119889for
















E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References


























































































































































































































































































































EhROM1

EhROM1

LgI

IgIHgI

EhROM1

LgI

IgIHgI

(a) (b) (c)









RGD

Aktt

Ub

ILKPI3K

Akt

P

Degradation

Host cell

P

P

+

P

Ub

P473

P473

IKK NEMO

IKKγNEMO

IKK IKK

IKKα IKKβ

I BI B

NF- B

NF- B

Nucleus

TNF-IL-1

EhPCP5


































CPs

Ly


EE

LE

Ly


Erythrocyte

Amoebic clathrin

h-Hololactoferrin





Haemoglobin

Ferritin


h-Holotransferrin

Nucleus


Ph

Ly

LyLL EEL

Fluid phase

ADH2



Ly = lysosome










119889for
















E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References
















































































































































































































































































































EhROM1

EhROM1

LgI

IgIHgI

EhROM1

LgI

IgIHgI

(a) (b) (c)









RGD

Aktt

Ub

ILKPI3K

Akt

P

Degradation

Host cell

P

P

+

P

Ub

P473

P473

IKK NEMO

IKKγNEMO

IKK IKK

IKKα IKKβ

I BI B

NF- B

NF- B

Nucleus

TNF-IL-1

EhPCP5


































CPs

Ly


EE

LE

Ly


Erythrocyte

Amoebic clathrin

h-Hololactoferrin





Haemoglobin

Ferritin


h-Holotransferrin

Nucleus


Ph

Ly

LyLL EEL

Fluid phase

ADH2



Ly = lysosome










119889for
















E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References







































































































































































































































































































EhROM1

EhROM1

LgI

IgIHgI

EhROM1

LgI

IgIHgI

(a) (b) (c)









RGD

Aktt

Ub

ILKPI3K

Akt

P

Degradation

Host cell

P

P

+

P

Ub

P473

P473

IKK NEMO

IKKγNEMO

IKK IKK

IKKα IKKβ

I BI B

NF- B

NF- B

Nucleus

TNF-IL-1

EhPCP5


































CPs

Ly


EE

LE

Ly


Erythrocyte

Amoebic clathrin

h-Hololactoferrin





Haemoglobin

Ferritin


h-Holotransferrin

Nucleus


Ph

Ly

LyLL EEL

Fluid phase

ADH2



Ly = lysosome










119889for
















E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References







































































































































































































































































































RGD

Aktt

Ub

ILKPI3K

Akt

P

Degradation

Host cell

P

P

+

P

Ub

P473

P473

IKK NEMO

IKKγNEMO

IKK IKK

IKKα IKKβ

I BI B

NF- B

NF- B

Nucleus

TNF-IL-1

EhPCP5


































CPs

Ly


EE

LE

Ly


Erythrocyte

Amoebic clathrin

h-Hololactoferrin





Haemoglobin

Ferritin


h-Holotransferrin

Nucleus


Ph

Ly

LyLL EEL

Fluid phase

ADH2



Ly = lysosome










119889for
















E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References



































































































































































































































































































































CPs

Ly


EE

LE

Ly


Erythrocyte

Amoebic clathrin

h-Hololactoferrin





Haemoglobin

Ferritin


h-Holotransferrin

Nucleus


Ph

Ly

LyLL EEL

Fluid phase

ADH2



Ly = lysosome










119889for
















E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References



























































































































































































































































































































CPs

Ly


EE

LE

Ly


Erythrocyte

Amoebic clathrin

h-Hololactoferrin





Haemoglobin

Ferritin


h-Holotransferrin

Nucleus


Ph

Ly

LyLL EEL

Fluid phase

ADH2



Ly = lysosome










119889for
















E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References

















































































































































































































































































































CPs

Ly


EE

LE

Ly


Erythrocyte

Amoebic clathrin

h-Hololactoferrin





Haemoglobin

Ferritin


h-Holotransferrin

Nucleus


Ph

Ly

LyLL EEL

Fluid phase

ADH2



Ly = lysosome










119889for
















E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References







































































































































































































































































































CPs

Ly


EE

LE

Ly


Erythrocyte

Amoebic clathrin

h-Hololactoferrin





Haemoglobin

Ferritin


h-Holotransferrin

Nucleus


Ph

Ly

LyLL EEL

Fluid phase

ADH2



Ly = lysosome










119889for
















E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References









































































































































































































































































































119889for
















E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References











































































































































































































































































































E dispar













































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References







































































































































































































































































































































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References





























































































































































































































































































































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References


















































































































































































































































































































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References









































































































































































































































































































Acanthamoeba spp








Gelatin No

178 144123 11078 75 7234 30




Gelatin No144 10578 7245 40 34








Naegleria fowleri










































ketone





Acknowledgment


References





































































































































































































































































































































ketone





Acknowledgment


References



























































































































































































































































































































ketone





Acknowledgment


References















































































































































































































































































































ketone





Acknowledgment


References






















































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































Documents

Proteases from Entamoeba spp. and Pathogenic Free-Living ... · HindawiPublishingCorporation JournalofTropicalMedicine Volume2013,ArticleID890603,32pages ReviewArticle Proteases from