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Immunology Letters 150 (2013) 12– 22

Contents lists available at SciVerse ScienceDirect

Immunology Letters

jou rn al h om epa ge: www.elsev ier .com/ locate / immlet

eview

taphylococcus aureus virulence factors in evasion from innate immune defensesn human and animal diseases

lfonso Zecconi ∗, Federico Scaliniversità degli Studi di Milano, Dip. Scienze Veterinarie e Sanità Pubblica, Via Celoria 10, 20133 Milano, Italy

r t i c l e i n f o

rticle history:eceived 5 November 2012eceived in revised form 9 December 2012ccepted 8 January 2013vailable online 31 January 2013

eywords:taphylococcus aureus

a b s t r a c t

In the last decades, Staphylococcus aureus acquired a dramatic relevance in human and veterinarymedicine for different reasons, one of them represented by the increasing prevalence of antibiotic resis-tant strains. However, antibiotic resistance is not the only weapon in the arsenal of S. aureus. Indeed,these bacteria have plenty of virulence factors, including a vast ability to evade host immune defenses.

The innate immune system represents the first line of defense against invading pathogens. This sys-tem consists of three major effector mechanisms: antimicrobial peptides and enzymes, the complementsystem and phagocytes. In this review, we focused on S. aureus virulence factors involved in the immune

nnate immunityLRdhesinsost defense peptides

evasion in the first phases of infection: TLR recognition avoidance, adhesins affecting immune responseand resistance to host defenses peptides and polypeptides.

Studies of innate immune defenses and their role against S. aureus are important in human and vet-erinary medicine given the problems related to S. aureus antimicrobial resistance. Moreover, due tothe pathogen ability to manipulate the immune response, these data are needed to develop efficacious

ainst

vaccines or molecules ag

. Introduction

Staphylococcus aureus has a marked ability to adapt to differentnvironments and represents a primary cause of infection both inumans and in several animal species [1–3]. In the last decades. aureus has acquired a dramatic relevance in human medicineor different reasons, one of them represented by the increasingrevalence of antibiotic resistant strains [4–6]. In United States andmong infective pathogens, S. aureus represents one of the majorauses of death. Indeed, it is involved in over 18,000 deceases pernnum; about 290,000 hospitalizations and almost 12 million ofedical visits and treatments [5–7]. In veterinary medicine the

everity of the diseases are much lower when compared to humanide, but several different pathologies were described in both petsnd food producing animals [8,9]. Particularly in dairy cows, a highrevalence of S. aureus udder infections is observed worldwide.hese infections have an impact on milk yield and quality, and theosts of this infection make S. aureus the most expensive contagious

athogen in dairy cattle, worldwide [10–12]. The importance of S.ureus as a pathogen for human and veterinary medicine increasedurthermore, with the increasing frequency of involvement of

ethicillin-resistant S. aureus (MRSA) in severe human disease

∗ Corresponding author. Tel.: +39 0250318073; fax: +39 0250318079.E-mail address: alfonso.zecconi@unimi.it (A. Zecconi).

165-2478/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.imlet.2013.01.004

S. aureus.© 2013 Elsevier B.V. All rights reserved.

cases. MRSA has been isolated since many years [13,14]. Initially,they seemed to be restricted to hospital environment (HA-MRSA)[15], but they were soon identified also in non-hospitalized patients(CA-MRSA) [16,17]. Recently, cows and pigs emerged as MRSAreservoirs for human infection (LA-MRSA) [4,18–21]. The problemof MRSA is outside the scope of this paper and this topic was cov-ered by excellent reviews [2,22–27]. However, MRSA problem inhuman and veterinary medicine is one of the best examples for theconcept of “one health” (http://www.onehealthinitiative.com) andsupports the interest in using animal models, other than mice, toincrease our knowledge in S. aureus pathogenesis.

1.1. S. aureus infections in humans

S. aureus has large array of virulence factors and the differentcombinations of these factors, the site of infection and the variabil-ity of host immune response explain the wide range of outcomesrelated to these infections. Indeed, the bacteria may have a negli-gible impact on health, acting as a commensal (on skin or anteriornasal mucosa), or it may cause moderate local infections, but alsosevere and invasive infections [28]. Some authors estimated thatroughly 20% of human beings carries the bacteria in the anterior

nasal mucosa for their entire life and 60% are recurrent carrierswithout any consequences [29]. Indeed, S. aureus can infect boththe upper and the lower respiratory tract, and it is responsible forthe 26% of community-acquired pneumonia in USA, and a serious

A. Zecconi, F. Scali / Immunology Letters 150 (2013) 12– 22 13

Table 1aS. aureus virulence factors: toxins.

Name it Abbreviation Function References

� toxin Hlb Sphingomyelinase with cytolytic activity [52,53]� toxin Hla Cytolytic pore-forming toxin [52,53]Leukocidins D, E and M LukD/E/M Kill leukocytes; bi-component pore-forming leukotoxins [53,54]PSM peptides PSMs Pore-forming toxins or detergent activity [55]Exfoliative toxins A, B and D ETA/B/D Exotoxins with superantigen activity; gluamate-specific serine proteases that digest desmoglein 1 [52,56]Enterotoxins SEs Gastroenteric toxicity; immunomodulation via superantigen activity [52,57]SE-like proteins SEls Unknown/No gastroenteric toxicity; immunomodulation via superantigen activity [57,58]Toxic shock syndrome toxin-1 TSST1 Endothelial toxicity (direct and cytokine-mediated); superantigen activity [52]

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oxins with activity related to immune evasion are reported in Tables 2a–d.

hreat to the survival of the patients affected by severe pneumonia29].

.2. S. aureus mastitis in dairy cows

S. aureus mastitis intramammary infections are reported in allhe countries with prevalence from 5% up to 70% of cows and 90%f herds. It does appear that these levels are decreasing due to themprovement of health management of dairy cows, but eradications far to be achieved. The cost of S. aureus intramammary infections

as estimated in 100–200$/cows [11]. As for human diseases, theathogenesis and spread of S. aureus are dependent on the bacte-ia virulence factors [12,30–33], but also the environment [34,35]nd the host play an important role in the frequency and sever-ty of these infections. In bovine mastitis pathogenesis, a peculiarspect is represented by the pivotal importance of innate immu-ity against invading pathogens [36–39]. This is supported by thevidence of a poor preventive activity of mastitis vaccines undereld conditions [40–44]. Indeed, several vaccines against S. aureusave been developed both experimentally and commercially, butone of them up to now showed consistent results as reviewed byefs. [45–47].

. S. aureus virulence factors

S. aureus is well known for the high frequency of antibiotic-esistant strains, but antibiotic resistance is not the only weapon in

able 1b. aureus virulence factors involved in adhesion to host cells.

Name Abbreviation Function

Fibronectin binding proteins A, B FnBPA,B Adhesins for fibrinoCollagen-binding adhesin Cna Adhesin for collagenSerin-aspartate repeat proteins C, D and E Sdrs Adhesins

Bone sialoprotein-binding protein Bbp Adhesin for bone siaElastin-binding protein EbpS Transmembrane adhPolysaccharide intercellular adhesin PIA Adhesin for aggregaIron-regulated surface determinant A IsdA Binds fibronectin, fib

(expressed in iron-rIron-regulated surface determinant B IsdB Binds hemoglobin aIron-regulated surface determinant C IsdC Binds hemin

Iron-regulated surface determinant H IsdH Binds haptoglobulinS. aureus surface proteins Sass Bind to the extracellS. aureus surface protein G SasG Binds to the extraceSerine-rich surface protein SraP Binds platelets

Extracellular matrix protein-binding protein Emp Binds extracellular mExtracellular adherence protein Eap/Map Impairs angiogenesiEmp homologue Ebh Binds extracellular mPlasmin-sensitive protein Pls Binds lipids of host cSecond immunoglobulin-binding protein Sbi Binds Fc domain of i

C3-C3b conversionVon Willebrand factor binding protein vWbp Binds and activates

Extracellular adherence protein Eap/Map MHC-II analog protebiofilm formation

dhesion factors with activity related to immune evasion are reported in Tables 2a–d.

or [53]

the arsenal of these bacteria. Indeed, these bacteria have plenty ofvirulence factors, which are the basis for its pathogenicity but alsogave them a vast ability to evade host immune defenses [48–51].Previous reviews reported the characteristics of S. aureus virulencefactors [52–54]. Therefore, the general description of these factorswill be not covered in this review, but an updated list of knownvirulence factors is reported (Tables 1a–c). These tables report onlyvirulence factors apparently not involved in innate immune eva-sion, while the factors involved in these latter mechanisms arelisted in Tables 2a–d.

3. Host response to S. aureus: innate immune defenses

The innate immune system represents a first line of defenseagainst invading pathogens [78]. This system consists of threemajor effector mechanisms: antimicrobial peptides and enzymes,complement system and phagocytes [49,78]. The importance andefficiency of these three effector mechanisms is different depend-ing on the site of infection and on bacteria characteristics [78,79].

An animal model where innate immune defenses are pivotal incontrolling S. aureus infection is represented by S. aureus mastitisin dairy cows. In the past, leukocytes were considered the mainsource of molecules involved in inflammatory and immunological

responses. It has been shown that other cells could be an importantsource of these mediators. Indeed, lung epithelial cells could mod-ulate the inflammatory response in the airways and modulate cellrecruitment through producing chemokines, cytokines, receptors

References

gen (FnBPA only), fibronectin and elastin [52,53,59,60] (type I and IV) [52,53,59,60]

[52,60]loprotein (SdrE allelic variant), binds fibrinogen [52,59,61,62]esin for elastin and tropoelastin [52,60]

tion; involved in biofilm formation [61]rinogen, transferrin, hemoglobin, hemin and fetuin

estricted environments)[53,59,60]

nd hemin [60][60]

and haptoglobulin–hemoglobin complex [60]ular matrix [53,60]llular matrix; involved in biofilm formation [60,63]

[60]atrix of host cells; involved in biofilm formation [60,64]

s and wound healing; stimulates production of TNF� and IL-6 [65–70]atrix of host cells [60,71]

ells; adhesion to nasal epithelial cells [60]mmunoglobulin; binds complement protein C3 and promotes [72,73]

prothrombin; binds fibrinogen and vW factor [74]in; adhesion to S. aureus cells and host cells; involved in [52,64]

14 A. Zecconi, F. Scali / Immunology Letters 150 (2013) 12– 22

Table 1cS. aureus virulence factors: enzymes and other proteins.

Name Abbreviation Function References

Coagulase Coa Binds and activates prothrombin; promotes conversion of fibrinogen to fibrin [52,74]V8 protease – Serine protease [52,53]Glycerol ester hydrolases lip, geh, beh, Triacylglycerols degradation [53]Fatty acid-modifying enzyme FAME Fatty acids modificationO-acetyltransferase OatA Peptidoglycan O-acetylation [53]PtdIns-phospholipase C Plc Phosphotidylinositol-specific lipase activity [53]Enolase Eno Catalyzes phosphor-glycerate to phosphoenol-pyruvate; binds to laminin [53]Arginine catabolic mobile element (3 types) ACME I/-II/-III Unclear role (aids colonization); seems to contain several enzyme and proteins

(arginine deaminase system, oligopeptide permease, zinc-containing alcoholdehydrogenase, spermine/spermidine acetyltransferase and others)

[75–77]

FPR-like 1 inhibitory protein FLIPr Binds formyl peptide receptor [53]

Enzymes and other proteins with activity related to immune evasion are reported in Tables 2a–d.

Table 2aVirulence factors enabling S. aureus to evade innate immune defenses related to leukocyte migration and phagocytic activity.

Effector mechanism S. aureus evasion factor Abbreviation Function References

Neutrophil migration Staphylococcal superantigen-like 5 SSL5 Specific binding to P-selectin glycoprotein ligand-1blocking PMN rolling

[93,197]

Staphylococcal superantigen-like 11 SSL11 [198]Staphylococcal superantigen-like 1 SSL10 Binds to chemokine receptors [199]Chemotaxis inhibitory protein CHIPS Blocks C5a receptor and formyl peptide receptors [53,142]Staphylococcal superantigen-like 7 SSL7 Binds to the Fc region of IgA and block recognition by

neutrophils[93]

Neutrophils lysis � toxin Hlg Bicomponent leukocidin; hemolysis [52,53]� toxin Hld Cytolytic toxin; binds neutrophils and monocytes [52,53]Panton-Valentine leukocidin PVL Bicomponent leukocidin; pore-forming toxin; kills

leukocytes[52,76]

Leukocidins A and B (alt. names H and G) LukAB/-HG Bi-component pore-forming leukotoxin that kills PMNs [54,200]

Resistance to oxidative burst Staphyloxanthin – Carotenoid (protects against ROS) [53]Catalase and alkylhydroxide reductase CatA, AhpC Inactivate hydrogen peroxide; pivotal for nasal

colonization[53]

Thioredoxin and thioredoxin reductase – Inactivates ROS [53]

Table 2bVirulence factors enabling S. aureus to evade innate immune defenses related to complement.

Effector mechanism S. aureus evasion factor Abbreviation Function References

Complement inactivation Capsular polysaccharides CPSs Alter C3 (CPS5 and 8) or C3b (CPS1) deposition [53,75]Staphylokinase Sak Plasminogen activator (plasminogen-serine

protease plasmin conversion)[53,73]

Staphylococcal complement inhibitor SCIN Inhibits convertase [53,142]Extracellular complement-binding protein Ecb Inhibits convertase [142]Clamp factor A ClfA Platelets adhesion (fibrin-mediated); binds

complement regulator factor I[52,53,59,60,74]

drE

fb

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TV

Staphylococcus aureus surface protein E SExtracellular fibrinogen-binding protein E

nd adhesion molecules [80,81]. Similarly, mammary gland epithe-

ial cells could produce both cytokines and immunomodulating

olecules [36,82–84]. Changes of innate immune response cane observed during lactation. Indeed, recently, Ref. [84] showedhat several soluble factors (lactoferrin, lysozyme, NAGase, and IgG)

able 2cirulence factors enabling S. aureus to evade innate immune defenses related to immuno

Effector mechanism S. aureus evasion factor Abbreviation

Degradation of immunoglobulins Protein A SpA

Staphylokinase Sak

Cloaking of opsonins Serotypes 5,8 PNAGClamp factor A

ClfA

Clamp factor B ClfB

Binds complement regulator factor H [142]Binds fibrinogen; inhibits C3 and C5convertases; binds complement C3

[52,73,142]

have a different pattern during lactation, in absence of intramam-

mary infections.

Among the different innate defenses, lysozyme showed a pecu-liar behavior when S. aureus intramammary infection occurred.Indeed, lysozyme activity in milk showed to be decreased in

globulins and opsonization.

Function References

Binds Fc domain of immunoglobulin, vonWillibrand factor and TNFR-1; Bindscomplement protein C3 and promotes C3–C3bconversion

[52,60,137]

Plasminogen activator (plasminogen-serineprotease plasmin conversion)

[53,73]

Platelets adhesion (fibrin-mediated); bindscomplement regulator factor I

[52,53,59,60,74]

Platelets adhesion (fibrin-mediated); bindscytokeratin 10.

[52,59,60,74,134]

A. Zecconi, F. Scali / Immunology Letters 150 (2013) 12– 22 15

Table 2dVirulence factors enabling S. aureus to evade innate immune defenses related to antimicrobial peptides.

Effector mechanism S. aureus evasion factor Abbreviation Function References

Alteration of cell wall components Antimicrobial peptide sensor ApsS/R/X Binds and impairs antimicrobial peptides [182,183]Gra regulatory system GraR/S Impairs phagocytosis and LL-37 [186]Multiple peptide resistance factor F MprF Inserts lysine into teichoic acids [53]Dlt operon DltA/B/C/D Inserts d-alanine into teichoic acids [53]

Lysozyme resistance OatA gene OatA Changes in N-acetylmuramic acid [189]

Cathelicidin resistance Staphylokinase Sak A-defensine binding [192]Aureolysin Aul LL-37 clivage [194]

Mec

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Methicillin resistance

. aureus infected quarters [85]. The decrease may be the result ofn early exhaustion of lysozyme activity and was associated with aeduction in neutrophils respiratory burst activity [86,87], but alsoe related to the different expression of virulence factors [30,44].oreover, recent investigations suggest that lysozyme release from

ells is dose-dependent and at least 100.000 CFU/ml in milk areeeded to trigger its production from mammary gland epithelialells (Zecconi, unpublished data).

Thus, the role of mammary gland epithelial cells as a sourcef innate immunity component cannot be ignored, notably whenhe interactions between bacteria and host are of interest. Defense

olecules released by mammary gland epithelial cells seem to play major role in preventing mastitis, particularly when the level ofilk leucocytes is relatively low (<100.000 cells/ml), as suggested

y Refs. [36,38,88].

. Pathogenesis of S. aureus infections and innate immuneesponse: an evolving fight

The invasion of host tissues and organs triggers innate immu-ity, which is characterized by a quick response against theathogen. The timing of the immune response is strictly dependentn the first indispensable step: the recognition of pathogen-ssociated molecular patterns (PAMPs) [48,78,89]. Additionally,. aureus should adhere to host cell to multiply and, sometimes,nvade host cells [90], while the host replies by the immediateelease of antimicrobial components and of chemoattractants, thusecruiting immune cells for a more efficient response.

However, bacterial pathogens use very efficient strategies tovade host defenses in order to colonize and invade human andnimal tissues [90]. S. aureus has particular characteristics, and inhe initial steps of infections both in human and animals, whendhesion to epithelial cells is required, innate immunity represents

pivotal line of defense [12,48,91,92]. Whereas specific acquiredmmunity seems to play a major role in the further steps in theathogenic process, avoiding the diffusion of the bacteria and theevelopment of bacteremia and sepsis [91,92].

S. aureus virulence factors repertoire has plenty of mecha-isms to evade host innate immunity, including modificationsf structural component and secretion of a large array of spe-ific immune-modulating proteins acting in concert to counteractnnate immune defenses, and to create a microenvironment thatnables even better survival [48,49].

S. aureus has the capability to avoid TLR recognition; theirdhesins are quite efficient in reducing the effectiveness of com-lement system and phagocytes, and they developed resistanceechanism against host defense peptides (HDPs), similarly to the

ne developed against conventional antimicrobials.

Tables 2a–d report a list of the known factors involved in S.

ureus immune evasion classified by target: neutrophils and phago-ytosis, complement system, immunoglobulins and opsonins,DPs.

Correlation between methicillin resistance andcathelicin resistance, mechanism to beidentified

[195,196]

It is impossible to cover all the virulence factors and their rolein pathogenesis of S. aureus infection in a single review. There-fore, we focused this review on the S. aureus factors enabling theevasion from immune defenses expressed during the first steps ofthe pathogenic processes (TLR evasion, adhesins and HDPs). Fac-tors involved in avoiding cells’ recruitment and related immuneresponse were covered by other excellent reviews [49,79,93].

4.1. TLR family recognition

S. aureus invasion triggers a complex mechanism through theactivation of specific receptors for PAMPs, eventually startinginnate immunity response [79]. Toll-like receptors (TLRs) were thefirst receptors for PAMPs to be discovered, but in recent years, otherclasses of receptor have been identified namely NOD-like receptors,C-type lectin receptors and RIG-I-like receptors [94].

TLR-2 was one of the most investigated toll-like receptors due toits ability to identify Gram-negative bacteria LPS [95,96] and Gram-positive specific molecular patterns [97,98].

TLR-2 is located on the cell surface forming and heterodimericcomplex with TLR-1 or TLR-6. TLR-2/TLR-1 complex recognizesGram-negative’s triacylated groups, while TLR-2/TLR-6 complexidentifies Gram-positive’s diacylated groups [99]. The lipoteichoicacid and certain lipopeptides of S. aureus trigger TLR-2/TLR-6.TLR-2/TLR-6 complex leads to production and secretion of severalcytokines trough TIRAP and MyD88 pathways. In addition, TLR-2activation seems to be directly involved in the maturation of thephagosome [100,101].

TLR-2 showed to play an important role in the immunity againstS. aureus in different infection models; mutant mice without TLR-2appear more susceptible to cutaneous, intranasal or corneal infec-tion than wild ones; furthermore, these mice seem to be moredisposed to severe infection and eventually death [102–104].

TLR-4 has a central role in the innate immunity againstGram-negative bacteria because of its ability to recognizelipopolysaccharides (LPS) [94,105–107]. Nevertheless, TLR-4 seemsto have a relevant role in S. aureus infections too. In a brain abscessmodel, TLR-4−/− mice have shown more severe lesions, prolongedinfection and higher mortality than the control mice. These resultssuggest the role of TLR-4 for a complete recovery of the diseasedanimal [108]. TLR-4 also seems to be activated by S. aureus leuko-cidins, this activation leads to maturation of dendritic cells in thebone marrow [109].

The other receptors in toll-like family apparently do not seemto have a specific relevance in innate immune response againstS. aureus. However, a recent study highlighted the TLR-9 abil-ity to induce production of IL-12 after S. aureus exposure andthe relevance of TLR-9/TLR-2 crosstalk for the regulation of this

cytokines family. Furthermore, TLR-9 seems to mediate the classI IFN signaling in dendritic cells, role that may be detrimental inMRSA pneumonia due to the allegedly negative effects of IFN� andIFN� in these pneumonias [110].

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The NLRs (nucleotide oligomerization domain-like receptor orOD-like) represent a family of receptors involved in the regulationf innate immune response, they are typically located in the cytosolf cells with phagocytic activity. Nevertheless, NLRs can be foundedn various other cell types [111]. NLRs are a large family, over 20LRs were discovered in human cells, a number that probably will

ncrease considering that in mice were founded more than 30 NLRsnd in some invertebrate, like echinoderms, over 200 [111,112].

The role of NLRs still needs to be clarified. However, two mem-ers of the family, nucleotide-binding oligomerization domains 1nd 2 (NOD1 and NOD2), are widely studied, and they seem to have

role in innate immunity against S. aureus. Meso-diaminopimeliccid (m-DAP) triggers NOD1 [113,114] while muramyl dipeptideMDP) activates NOD2 [115,116]. Both m-DAP and MDP are com-onents of peptidoglycan. However, MDP is founded in almost allacterial cell walls whereas, m-DAP appears to be more character-

stic of Gram-negative bacteria. Nevertheless, some Gram-positiveacteria seem to be able to activate NOD1 [117,118].

The exact role of NOD2 in the innate response against S. aureuss still unclear. Indeed, preliminary studies on murine modelseported inconsistent results. In a systemic infection model, NOD2layed a critical role in the innate response. NOD2−/− mutant miceere more susceptible to infection, developed more severe diseases

nd had a larger production of some cytokines (TNF�, IFN� and IL-) than wild mice. Furthermore, NOD2−/− mice showed a higherortality in comparison with TLR-2−/− mice [119]. In a cutaneous

nfection model NOD2−/− mice developed more severe lesions thanild mice; however, NOD2−/− presented an inferior production of

NF� and IL-6 in comparison with wild mice [120].The role of NOD1 in S. aureus infection is even less well-known

han NOD2. Recent studies suggest that NOD1 may be relevant in. aureus killing by neutrophils. Indeed, neutrophils activity against. aureus in NOD1+/+ mice was higher than NOD1−/− ones whenre-stimulated with a Gram-negative bacteria [121].

.2. Evasion from TLRs recognition

The evasion from TLR recognition is a topic which is receivingn increasing interest, even if the studies available are relativelyew. Recently, a novel mechanism for staphylococci to escape theost immune system via interference with recognition by immuneells has been identified [122]. Staphylococcal superantigens likeSSL) proteins play an important role in this mechanism. Theseroteins are in a family of molecules able to interfere with mul-iple components of host immunity, including humoral immunity,psonization, and trafficking of leukocytes [167]. Therefore, theyay be involved in evasion both from innate and adaptive immu-

ity. Indeed, among the 14 members of the SSL family, SSL7 binds togA and C5; SSL5 binds to PSGL-1, chemoattractant receptors, and

MP-9, and SSL10 binds to chemokine receptor and human IgG123]. Among these families, SSL3 is the first-described bacterialrotein that blocks TLR-2 activation through direct extracellular

nteraction with the receptor [124]. Particularly, SSL3 binds to TLR- via its extracellular domain and inhibits tumor necrosis factorlpha (TNF�) production from mouse macrophages in responseo heat-killed S. aureus, peptidoglycan (PGN), or lipopeptide TLR-

ligands. In addition, SSL3 showed to inhibit IL-8 production inEK cells expressing TLR-1/2 and TLR-2/6 dimers through bindingf the extracellular TLR-2 domain. The SSL3-TLR-2 interaction isartially glycan dependent as binding of SSL3 to TLR-2 is reducedpon removal of sialic acid residues. Furthermore, the SSL3 (R308A)utant, lacking glycan-binding properties, shows lower TLR-2 inhi-

ition. An SSL3 mutant, lacking the N-terminal 126 amino acids, stilletains full TLR-2 inhibiting activity. Among the other SSLs tested,nly SSL4, which shares the highest homology with SSL3, blocksLR-2 activation. This unique function of SSL3 adds to the arsenal

Letters 150 (2013) 12– 22

of immune evasive molecules that S. aureus can employ to subvertboth innate and adaptive immunity [123].

The new insights into the SSL-TLR interactions will increase ourunderstanding not only of the bacterial infections but also in all thediseases related to TLR malfunction such as atherosclerosis [125].

4.3. Adhesion

Host-adhesion represents a pivotal phase of pathogenesis of S.aureus, and it developed a wide variety of molecules that aid thisprocess (Tables 1a–c) named adhesins. These molecules mediatethe adhesion to different substrates of the host: extracellular matrixproteins (ECMs), plasma proteins, epithelial cells and endothelialcells [59,60]. Furthermore, some of these adhesins interfere withhost immunity, as described later in this paper. Adhesins can beanchored on the surface of the bacteria, or they can be secreted.Surface adhesins are called microbial surface recognizing adhesivematrix molecules (MSCRAMMs) whereas secretable adhesins arecalled secretable expanded repertoire adhesive molecules (SER-AMs) [53,59]. Both MSCRAMMs and SERAMs are proteins, even ifother molecules, such as polysaccharide intercellular adhesin (PIA)and teichoic acids, can act as adhesins too.

The adhesion is a pre-requisite to have colonization and, eventu-ally, infection of the host. The most important sites of colonizationin human hosts are the nasal mucosa and the skin, and they are avaluable model to understand S. aureus adhesion process [59,126].Colonization of anterior nares can be intermittent or persistent.Persistent subjects seem to have large numbers of carried bacteriaand a single dominant strain while intermittent carriers seem tohave a low amount of bacteria and different strains [127–129]. Ithas been suggested that difference in carrier state could be relatedto different ligands for the adhesins of S. aureus [130]. A similarpattern was observed in bovine intramammary infections [12].

Fibronectin-binding proteins (FnBPs), clumping factors (Clfs),protein A (SpA) and collagen-binding protein (Cna) are the major S.aureus MSCRAMMs. S. aureus synthesizes two fibronectin-bindingproteins: FnBPA and FnBPB. These adhesins bind fibrinoctein andelastin, and FnBPA can bind fibrinogen [52,53,59,60]. FnBPs tiefibronectin on the surface of the bacteria; this structure allows abond between S. aureus and host integrins [131]. FnBPA and FnBPBhave an important role in the colonization of the host. However,their relevance in the infections has been not completely eluci-dated [131,132]. Clumping factors A and B bind fibrinogen of thehost and promote platelets aggregation [52,53,59,60]. Neverthe-less, soluble fibrin seems to be the major protein responsible for theformation of S. aureus-platelets aggregates mediated by Clfs [133].ClfB aids colonization of anterior nares because of its ability to bindcytokeratin 10 [59,60]. Besides, ClfB seems to give a further aid tobacteria–host adhesion by binding cytokeratin 8 [134]. Collagen-binding protein mediates adhesion between S. aureus and host’scollagen; this adhesin provides help in the invasion of diverse tis-sues, and it acts as a virulence factor in different type of infections[52,53,59,60]. Several S. aureus surface proteins can bind the ECMwith a covalent link, however, the ligands of the majority of theseproteins need to be clarified [53,60]. Surface protein G (SasG) seemsto contribute in the colonization of anterior nares [60]; moreover,SasG plays a role in the formation of biofilm when zinc is avail-able for the bacteria [63,135]. Finally, protein A plays mainly a rolein immune evasion than in adhesion and it will be furthermoreconsidered in the next section.

4.4. Adhesins as effector molecules to evade from innate immune

response

Many molecules involved in adhesion are also involved in dif-ferent immune evasion tactics (Tables 2a–d). The role of adhesins

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n evading immune defences is not related to the specific processf adhesion, but their activities interfere with several mechanismsperated by innate immune defences. The role of the differentolecules was summarized in Tables 2a–d, under the specific

mmune mechanism mainly affected by each adhesin. As for otherirulence factors, adhesin expression varies depending on the strainnd on the environment, but some of them are very common andeserve a more detailed description.

Protein A (SpA) is undoubtedly one of the most studiedSCRAMMs, and this protein binds different important molecules

nvolved in the immune response. Indeed, N terminal of SpAas five subdomains biding with Fc region of IgG, thus impair-

ng opsonization and then phagocytosis. SpA also binds to vonillebrand factor and complement protein C3, increasing bacteria

dhesion to platelets. Furthermore, SpA promotes C3–C3b con-ersion [52,60,72]. More recently Spa showed to bind to TNFR1,he receptor for TNF-�, thus reducing TNF-� proinflammatoryignaling [136].

Second immunoglobulin-binding protein (Sbi) has ligands sim-lar to SpA [72,137,138]. This protein can perform its function notnly when secreted but also when it remains anchored to the bacte-ial cell wall due to its ability to bind teichoic acid [72]. Moreover,bi, together with extracellular fibrinogen-binding protein (Efb),inds plasmin in a process that leads to the degradation of C3a, C3bnd C3 [73].

Complement formation is impaired also by other adhesins suchs ClfA, Ecb and SdrE. ClfA binds to complement regulator factor 1,nd this process increases the conversion of C3b to an inactivateorm (iC3b) and impairs phagocytosis of S. aureus [139,140]. Ecbinds C3d and thus inhibits C3 (alternative pathway) and C5 con-ertases (all pathways). This inhibition determines a reduction ofhagocytosis and PMNs migration [141]. SdrE binds to complementegulator factor H, reducing C3 deposition and C5a synthesis, thisrocess impairs also phagocytosis [142].

Extracellular adherence protein (Eap/MaP) is an adhesin withifferent roles such as the formation of bonds between S. aureusells; bonds between bacteria and host’s cells; biofilm formation;ngiogenesis and wound healing inhibition [52,64–67]. Eap showsrystals structure similar to superantigens; however, it do noteems to possess superantigen activities [143]. Furthermore, Eapcts as a MHC-II analog and has several immunomodulatory prop-rties like pro-inflammatory cytokines stimulation (Il-6 and TNF�)nd inhibition of neutrophil migration and adhesion [66,68,69,143].

Adhesins have been targeted as a major antigen for vaccineevelopment both in human and veterinary medicine [43,46,144],ut still the efficacy of S. aureus vaccines based on adhesion isontroversial. A lot of studies were focused on the adhesion mech-nisms and relatively less on investigating the interferences ofdhesins on the innate immune response. This latter aspect can-ot be neglected, and a more holistic approach investigating at theame time the complex adhesion process and the interference withnnate immunity is needed to increase the chances to develop anfficacious vaccine.

.5. Host defense peptides and polypeptides

Once the PAMPs had been recognized and while the bacteriattempt to adhere to host cells, these latter ones react activatingnnate immune defenses and releasing two classes of molecules:ntimicrobial peptides (i.e. cathelicidins) and polypeptides withntimicrobial activity (i.e. lysozyme). The type and amount of theseolecules are dependent from the host, the infected tissues and

. aureus virulence characteristics. Nevertheless, the efficacy ofhis response is pivotal to control the infection and, eventually, toestore the host health both in human and animal S. aureus infec-ions [36,39,145–147].

Letters 150 (2013) 12– 22 17

Host defense peptides (HDPs) are a group of molecules also clas-sically defined as antimicrobial peptides. This latter definition ismisleading, being more related to their history of discovery than tothe potent influence these molecules have on cell immune behav-ior [148]. These molecules are used by multicellular organisms todefend themselves against infective microorganism, and they arewidely present in nature, from plants to humans [81,149–151]. Alarge number of such peptides have been identified, and there isconsistent evidence of their pivotal role in innate immune system[152,153]. The expression of these antimicrobial peptides can beconstitutive or can be induced by infectious stimuli such as bacteriaand bacterial molecules (i.e. LPS, lipoteichoic acid) or inflamma-tory stimuli, such as proinflammatory cytokines [154]. Indeed,HDPs are important effector and modulator molecules of the innateimmune system, being able to enhance phagocytosis, stimulatethe prostaglandin release, neutralize the septic effects of LPS,promote recruitment and accumulation of various immune cellsat inflammatory sites, promote angiogenesis, and induce woundrepair [152–155]. The activity against bacteria is mainly related totheir electrostatic binding to negatively charged bacterial surfacemolecules: LPS in Gram-negative bacteria, lipoteichoic acid andteichoic acid in Gram-positive bacteria. After attracting the pep-tides to the bacterial cell surface, the membrane is made permeable.This involves pore or gap formation by aggregation of the peptidesin the membrane, resulting in bacterial cell death [152–154].

HDPs have a relative low number of aminoacids (12–100), arepositively charged and amphiphilic. HDPs were discovered in theearly 20th century; however, a characterization of these moleculeswas started only recently [150,151]. These peptides can be classi-fied according to their chemicals characteristics: molecules with�-helix structures; molecules with �-sheet structures; moleculeswith extended structures and molecules with loop structures [156].

Among HDPs two large families of antimicrobial peptides havebeen defined: defensins and cathelicidins. Defensins are widelydistributed in mammalian epithelia and phagocytes, while otherpeptides, including cathelicidins, histatins, dermicidin, and anionicpeptides have a more restricted tissue and animal species distribu-tion [146,147,155,157].

Among the different HDPs, cathelicidins represent a family ofheterogeneous antimicrobial peptides. Cathelicidins can be foundin several animals [158–161]; furthermore, in bovines, swineand equines, different cathelicidins in the same species havebeen identified [162–164]. However, in humans, only one gene(CAMP) codifying for cathelicidin was identified, and this geneis similar to mouse CRAMP gene [165,166]. CAMP gene codifiesfor hCAP18, the precursor of active form of human cathelicidin,namely LL-37 [167,168], which is probably the most investigatedhuman cathelicidin. LL-37 seems to adopt a �-helical structurein fluids such as plasma, interstitial liquid and intracellular fluid[169]. Human cathelicidin alters the membrane of sensible bacte-ria through creation of channels and pores [172]. FurthermoreLL-37 has immunomodulatory and chemotactic effects on neu-trophils, T lymphocytes and monocytes [158], and it is involved incutaneous wound healing [148]. Numerous cells like neutrophils,lymphocytes, monocytes, mast cells and natural killer can produceLL-37 [170–172]. Epithelial cells of skin [173], and of respiratory[174,175], gastro-enteric [176] and urogenital tract [177] are alsocapable of secrete this cathelicidin.

4.6. Host defence polypeptides – lysozyme

Host defense polypeptides, usually defined as natural antimi-

crobial enzymes, were discovered since many years [148], but theirrole as immunomodulators has been discovered only more recently[81,148]. Among these molecules, one of the most investigatedand important one is lysozyme (1,4-8-N-acetylmuraminidases)

1 nology

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8 A. Zecconi, F. Scali / Immu

hich we will use as a focused example of this category of sub-tances. Lysozyme has a wide distribution in nature being presentoth in plants and animals, and exhibits antimicrobial activitiesgainst different microorganisms. The bacterial killing mecha-ism of lysozyme consists in the lytic and non-lytic mechanisms.ysozyme provokes direct cell lysis by hydrolyzing the peptido-lycan layers of bacteria and induction of autolysins [178]. Theon-lytic mechanism is principally based on the properties of

ysozyme to cause membrane perturbation through the binding of specific domain of lysozyme within the bacterial surface [179].

The antibacterial activity of lysozyme depends on the acces-ibility of substrate. Gram-positive bacteria are more susceptibleecause peptidoglycan represents about 90% of the cell wall, whileram-negative bacteria, having <10% peptidoglycan, are more

esistant to the enzyme.The real role of lysozyme in vertebrates is still under dis-

ussion. Generally, the antibacterial activity is considered itsajor role. More recently it has been suggested that lysozyme is

n immunomodulator. Indeed, breakdown products of lysozymection on peptidoglycan demonstrated immunological properties39]. Moreover, recent studies suggest that LL-37 and lysozymexhibit antimicrobial activities synergistically or additively againstnvading microorganisms, in particular S. aureus [180], supportinghe importance of these soluble components of innate immuneefenses.

.7. Host defense peptides and polypeptides resistance

Antimicrobial peptides are conserved in their structure, functionnd mechanisms of action. At high concentration could be bacteri-idal, while at lower concentration, a role in immunomodulation inore plausible [154]. Due to their characteristics, it was assumed

hat resistance to antimicrobial peptides was less probable thanor conventional antimicrobials, but S. aureus infections, once more,howed that this was not the case. Some resistance mechanisms rel-tively common among S. aureus strains were already known, evenf their pathways were elucidated only recently. Moreover, new

echanisms related to the newly discovered HDPs have been iden-ified and more are expected to be discovered in the near future.

Staphylococci can modulate their sensitivity to defensins, and to broad range of antimicrobial peptides, by altering the com-osition and net charge of lipoteichoic acid, wall teichoic acidnd phospholipids [49,181]. A three-component regulatory systemalled the antimicrobial peptide sensor (Aps) has been identifiedn S. epidermidis [182]. The Aps system showed to be conserved intaphylococci, and a similar system is present also among S. aureusirulence factors repertoire [183]. The Aps system includes a histi-ine protein kinase sensory component (ApsS) a response regulatorApsR), and a third protein component (ApsX), which functionsre not yet fully understood [182,183]. Several cationic peptidesan activate Aps system which upregulates expression of the dtlperon and the mrpF gene. Dlt operon expression cause a d-alanineubstitutions of ribitol teichoic acid and lipoteichoic acid [184].prf operon expression adds a l-lysine residue to phosphatidyl-

lycerol exposed on the outer face of the cytoplasmic membrane185].

Also the regulatory system graRS plays a crucial role in reg-lation of dlt operon. Indeed, S. aureus graRS-mutants are moreusceptible to the bactericidal action of LL-37 [186]. The expres-ion of these genes leads S. aureus to reduce his superficial negativeharge in teichoic acids, resulting in a less effective attraction of

L-37 and other cationic HDPs. Modification of teichoic acids andhospholipids with d-alanine and l-lysine, respectively, has addi-ional consequences for the pathogenicity of S. aureus. Indeed, the

odifications also interfere with the susceptibility to glycopeptide

Letters 150 (2013) 12– 22

antibiotics such as vancomycin [187,188] and may contribute to thedevelopment of intermediate vancomycin-resistant strains [48].

Similarly, the resistance to lysozyme is related to composi-tional changes of constitutive proteins. Resistance to lysozymeis due to the changes in N-acetylmuramic acid of staphylococ-cal peptidoglycan which is O-acetylated at position C6-OH byan O-acetyltansferase that is an integral membrane protein. Theresponsible gene, oatA, was recently identified, and the oatA dele-tion mutant had an increased sensitivity to lysozyme [189].

Moreover, many S. aureus strains secrete staphylokinase (Sak), aprotein that, in addition to its plasminogen-activating role, showedto be able to inactivate several defensins [190]. The binding ofdefensins by Sak inhibits their bactericidal effects and, in vivo,staphylococcal strains producing Sak were protected against thebactericidal effect of a-defensins. Notably, the site within Sak thatbinds �-defensins is different from its plasminogen-binding site.Sak seems to be also able to form a complex with LL-37 that increaseits fibrinolytic action and improve the invasivity of S. aureus inhuman airways [191]. Therefore, due to its different activities inter-fering with innate immune defenses, Sak is considered to be animportant virulence factor of S. aureus [192].

As described previously, cathelicidin LL-37 is one of the fewhuman bactericidal peptides with potent anti-staphylococcal activ-ity. Until some years ago, LL-37 seemed to show a good activityagainst S. aureus [193]. However, in recent years, different strainsof S. aureus showed to be resistant to human cathelicidin [194].Indeed, aureolysin, a metalloproteinase in S. aureus, was found toinactivate LL-37 in a time- and concentration-dependent manner[194]. Aureolysin A cleaves LL-37 at positions 19–20, 23–24 and31–32, thereby directly degrading human cathelicidin and impair-ing its activity [194]. On the contrary, the V8 proteinase of S. aureusalso cleaves LL-37 but the resulting LL-17–37 fragment retainedthe antibacterial activity against S. aureus [194]. Some of theseresistance mechanisms showed to be related to MRSA resistance.Indeed, when MSSA with MRSA strains were compared, the MRSAstrains showed lower susceptibility to HDPs, indicating that someMRSA strains are resistant not only to various antibiotics, includingmethicillin, but also to some of HDPs [165,174]. More specifically,recent studies showed a correlation between methicillin and LL-37resistance [195,196].

Staphylococcal resistance against HDPs seems to be a multifac-torial process and, while some bacterial factors limiting the activityof HDPs have been identified, many others are still unknown. Thereare increasing research activities in the area of HDPs discovery,as a way to overcome the widespread problem of antimicrobialresistance. However, the S. aureus models show as the evolutionof pathogens could already have affected the sensitivity to thesenatural molecules. Therefore, before applying them as therapeu-tics, we should be very careful in avoiding the mistakes we made inchemotherapeutical approach in human and veterinary medicine.

5. Conclusions

Even if innate immunity is the oldest and most widespreaddefense system in mammals, our knowledge on it is far to be com-pleted. Moreover, the studies on the relationship between thissystem and invading pathogens require a holistic approach whichshould include both the host defense mechanisms and the pathogencapability to avoid them. Undoubtedly, it is hard to believe thatwe will be able to elucidate all the potential relationships, becausethese latter ones will co-evolute naturally, as long as immune sys-

tem will be efficient. However, the still increasing importance ofS. aureus in human and veterinary medicine, the problems relatedto antimicrobial resistance and the need to have efficacious vac-cines or molecules against S. aureus, strongly support the ongoing

nology

raph

R

A. Zecconi, F. Scali / Immu

esearch on the innate immune defenses and their role against S.ureus. This will allow to have new and, hopefully, more powerfulreventive and therapeutical tools to reduce S. aureus impact onealth both in humans and animals.

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