Research Article Comparative Analysis of Apicoplast

Research ArticleComparative Analysis of Apicoplast-Targeted Protein ExtensionLengths in Apicomplexan Parasites

Alexandr V Seliverstov Oleg A Zverkov Svetlana N IstominaSergey A Pirogov and Philip S Kitsis

Institute for Information Transmission Problems of the Russian Academy of Sciences (Kharkevich Institute)Bolshoy Karetny Pereulok 19 Moscow 127994 Russia

Correspondence should be addressed to Alexandr V Seliverstov slvstviitpru

Received 10 September 2014 Accepted 25 December 2014

Academic Editor William H Piel

Copyright copy 2015 Alexandr V Seliverstov 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

In general the mechanism of protein translocation through the apicoplast membrane requires a specific extension of a functionallyimportant region of the apicoplast-targeted proteins The corresponding signal peptides were detected in many apicomplexans butnot in the majority of apicoplast-targeted proteins in Toxoplasma gondii In T gondii signal peptides are either much diverged ortheir extension region is processed which in either casemakes the situation different fromother studied apicomplexansWeproposea statistic method to compare extensions of the functionally important regions of apicoplast-targeted proteins More specificallywe provide a comparison of extension lengths of orthologous apicoplast-targeted proteins in apicomplexan parasites We focus onresults obtained for the model species T gondii Neospora caninum and Plasmodium falciparum With our method cross speciescomparisons demonstrate that in average apicoplast-targeted protein extensions in T gondii are 15-fold longer than inN caninumand 2-fold longer than in P falciparum Extensions in P falciparum less than 87 residues in size are longer than the correspondingextensions in N caninum and reversely are shorter if they exceed 88 residues

1 Introduction

In general the mechanism of protein translocation throughthe apicoplast membrane requires a specific extension ofa functionally important region of the apicoplast-targetedproteins InT gondii signal peptides are eithermuch divergedor their extension region is processed which in either casemakes the situation different from other studied apicomplex-ans We propose a statistic method to compare extensionsof the functionally important regions of apicoplast-targetedproteins More specifically we provide a comparison ofextension lengths of orthologous apicoplast-targeted proteinsin apicomplexan parasites We ground on the notion thatthe majority of cyanobacterial proteins lack such extensions(including signal peptides) and consist of only functionalsequences

Sporozoans comprise a monophyletic lineage of api-complexan parasites Among them Toxoplasma gondii isan important medical and veterinary pathogen commonly

causing morbidity in HIV patients [1 2] The study [3 4]describes the propagation mechanism of T gondii in varioushosts worldwide including several aquatic mammal specieswhere it may provoke abortion and lethal systemic diseaseThe observation that the apicoplast of T gondii significantlyvaries in shape and protein expression patterns at differentlife stages of the parasite suggests its important role invirulence the apicoplast of T gondii is also involved in thepathogen stage conversion and the parasite proliferation [5]Due to bacterial origin of the apicoplast proteins they presenta natural target for selective treatment in the eukaryotichost The sporozoans contain a semiautonomous organellethe apicoplast acquired by secondary endosymbiosis withancient red algae plastid organelles of the red algae originatefrom cyanobacteria [6ndash8]

Elucidating the molecular mechanism that underlies therole of apicoplast in the parasite invasion conversion andproliferation is important for development of novel thera-peutics to control infection and reactivation of the parasite

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 452958 6 pageshttpdxdoiorg1011552015452958

2 BioMed Research International

Further analysis of unique features of apicoplast-targetedproteins (particularly regions involved in translocation pro-cesses) in T gondii can add to the effective design of drug-based or genetic strategies to control the pathogen develop-ment and proliferation At the reported stage of the studywe analyze only extensions in length of orthologs amongapicomplexan parasites

Note that the coccidian Cryptosporidium parvum lacksthe apicoplast [9] and the apicoplast in piroplasmids Babesiabovis andTheileria parva largely differs from that in commoncoccidians and the haemosporidian Plasmodium spp [10 11]

The majority of apicoplast proteins are encoded in thenucleus and only few in its own genome Most of theseproteins can be identified due to their cyanobacterial originTransport of nuclear-encoded proteins to the apicoplast in Tgondii is significantly less documented experimentally com-pared to Plasmodium falciparum Among the documentedcases is the nuclear-encoded lipoic acid synthetase LipA[12] other examples are described in [13ndash15] A mechanismof protein import into secondary plastids is also describedin [16] where many orthologous proteins involved in thisprocess were shown to be presented in the sporozoans Pfalciparum and T gondii cryptophyte alga Guillardia thetaand diatom Phaeodactylum tricornutum Plastids also possessthe bacterial system to translocate folded proteins [17 18]

A variety of protein localization prediction methods areused to identify apicoplast-targeted proteins Some of themutilize the notion that translocation across the four mem-branes surrounding the apicoplast is mediated by an N-terminal bipartite targeting sequence a special N-terminalsignal and a transit peptide [13] The algorithm ApicoAPdescribed in [19] predicts apicoplast-targeted proteins con-taining the signal peptide because it trains on a learning sam-ple of signal peptide-containing proteins Other apicoplast-targeted proteins are predicted neither with this algorithmnor with ApicoAMP [20] The comprehensive ToxoDBdatabase constructed using the SignalP algorithm containsproteins with the information on presenceabsence of thesignal peptide According to this database some nuclear-encoded proteins in T gondii that are experimentally shownto reach the apicoplast should do not contain signal peptidesalbeit bearing housekeeping functions in the apicoplast Themethods in application to Plasmodium spp are describedfor example in [19 21ndash23] The PlasmoAP algorithm [24]is designed specifically for Plasmodium spp and is of littleapplicability to coccidians Hence these two widely useddatabases may be considered of limited use to identifyapicoplast-targeted proteins not containing the standard sig-nal peptide in coccidians We therefore applied a crude tech-nique to compare apicomplexan proteins with their orthologsin a cyanobacterium Namely orthology between nuclear-encoded sporozoan proteins and cyanobacterial proteins isused as a basis to suggest the apicoplast-targeted natureof the proteins As our study relies on statistic estimatesits predictions are hopefully not affected by the chosenparameters of global protein alignment

In this work the lengths of sporozoan proteins are com-pared with each other and with the length of their orthologsin the cyanobacterium Synechocystis sp PCC 6803 We

consider lengths of the sporozoan proteins that extend out-side the conserved alignment regionwhich usually covers theentire cyanobacterial sequence We focus on results obtainedfor the model species T gondii Neospora caninum (the twococcidian sporozoanswith completed genomeprojects as perthe end of 2013) and the malaria agent P falciparum from theHaemosporidia

Based on the total comparison we conclude that T gondiiin most cases contains longer proteins compared to bothN caninum and P falciparum We also surmised that atleast some of them undergo processing in the cytoplasmto facilitate transporting into the apicoplast The extendedportions of proteins may also be involved in gene expressionregulation at the level of protein-protein interaction

As an argument the regulation of plastid-encoded genesycf24 and rps4 affects the general functionality of the api-coplast in T gondii [25] The expression regulation of ycf24(the SufB factor mediating the Fe-S cluster assembly in manynuclear proteins) was suggested to take place in the apicom-plexans Eimeria tenella T gondii RH and Plasmodium sppas well as in Gracilaria tenuistipitata Porphyra purpurea andPorphyra yezoensis [25] The same type of regulation wassuggested for rps4 (ribosomal protein S4) in T gondii RH[25]

2 Materials and Methods

Protein data for T gondii and N caninum was extractedfrom the ToxoDBdatabase (version 82) data forPlasmodiumspp from the PlasmoDB database (version 93) and datafor Synechocystis sp PCC 6803 from GenBank NCBI [26]ToxoDB and PlasmoDB are specialized regularly updatedand nonoverlapping databases Conserved domains weredetected according to the Pfam database [27] The locationof regions enriched with a certain amino acid was establishedusing the PROSITE database [28]

We compared the proteomes of three apicomplexanparasites (T gondii ME49 N caninum Liverpool and Pfalciparum 3D7) and the cyanobacterium Synechocystis spPCC 6803 For each pair of proteomes pairs of orthologousproteins were computed on the basis of an alignment qualityscore using theNeedleman-Wunschmethod andBLOSUM62matrix [29 30]

Our method to study the lengths of the apicomplexanprotein extensions is as follows For each cyanobacterialprotein with length 119904 and its two orthologs (from a fixedpair of apicomplexans) with lengths 119886 and 1198861015840 the point withcoordinates (119886 minus 119904 1198861015840 minus 119904) is computed In some cases oneor both of the coordinates are negative which indicates asporadic case of a shorter length of the sporozoan proteinversus the cyanobacteria

In Figures 1ndash3 the cases ofN caninum-T gondii (N-T) Pfalciparum-T gondii (P-T) and P falciparum-N caninum (P-N) are analyzed using three sets of points Each coordinateis the difference in lengths between the sporozoan andcyanobacterial orthologs T gondii versus Synechocystis (T-S) N caninum versus Synechocystis (N-S) and P falciparumversus Synechocystis (P-S) The sets of points are then sta-tistically analyzed The following statistic was used to test

BioMed Research International 3

900700500300100 1100 1300 1500minus500 minus300 minus100

y

x

Neos

pora

cani

num

Live

rpoo

l

Toxoplasma gondii ME49100

300

500

700

900

1100

1300

1500

minus500

minus300

minus100

Figure 1 Plot of protein length extensions in T gondiiME49 versusN caninum relative to their orthologs in Synechocystis sp PCC6803 Differences in lengths between sporozoan and cyanobacterialorthologous proteins are plotted as follows T-S on the 119909-axis N-Son the 119910-axis

the hypotheses that ldquoa constant is better compatible with theset of points (119909

119894 119910119894) | 1 le 119894 le 119899 than the nontrivial

affine function 119910 = 119896119909 + 119887 119896 = 0rdquo and ldquothe linearfunction 119910 = 119896119909 is better compatible with this set of pointsthan the affine function 119910 = 119896119909 + 119887 119887 = 0rdquo 119865 =radic((sum119894(119910119894minus 1199100(119909119894))2sum119894(119910119894minus 119910(119909

119894))2) minus 1) sdot (119899 minus 2) where 119910

0

in the numerator is a constant (mean 119910 over all 119910) or linearregression 119910 = 119896119909 and 119910 in the denominator is affineregression 119910 = 119896119909 + 119887 This statistic can be explained moreclearly it determines whether there is a correlation betweenthe difference 119910

119894minus 119910(119909

119894) and 119909

119894 This statistic is standard

and substantiated in [31 32] The value of 119865 was comparedagainst a threshold defined as the Student random variable atsignificance level 120572 119905(119899 minus 2 120572) Under the number of degreesof freedom 119899 minus 2 gt 30 the Student and standard Gaussiandistributions approximate each other and the threshold119905(266 005) equals 196 An analogous statistic was used totest the hypothesis ldquoaffine function versus general polynomialof second degreerdquo The confidence interval radius and theradius of the intercept (further referred to as radius) for theaffine regression slope as well as the slope coefficient radiusfor linear regression were calculated in a standard fashion[32]The Student test statistic 119878was used as well [32] Demingregression and screening singular points were tested as well

Regions of proteins with a predominance of one aminoacid were determined by using the PROSITE programThe distribution of amino acid pairs separated by a fixeddistance 119896 in a given set of amino acid sequences wasestablished using the simple computer program availablefrom httplab6iitpruutilsaapf Namely frequencies ofall amino acid pairs occurring in the given sequences at


y

xToxoplasma gondii ME49100

300

500

700

900

1100

1300

1500

minus500

minus300

minus100

Plas

mod

ium

falci

paru

m3

D7

Figure 2 Plot of protein length extensions in T gondiiME49 versusP falciparum 3D7 relative to their orthologs in Synechocystis sp PCC6803 Differences in lengths between sporozoan and cyanobacterialorthologous proteins are plotted as follows T-S on the 119909-axis P-Son the 119910-axis

y

xNeospora caninum Liverpool

Plas

mod

ium

falci

paru

m3

D7

900700500300100 1100 1300 1500minus500 minus300 minus100minus100

100

300

500

700

900

1100

1300

1500

minus500

minus300

Figure 3 Plot of protein length extensions inN caninum Liverpoolversus P falciparum 3D7 relative to their orthologs in Synechocystissp PCC 6803 Differences in lengths between sporozoan andcyanobacterial orthologous proteins are plotted as follows N-S onthe 119909-axis P-S on the 119910-axis

the distance of 119896 residues (specified in the interval from0 to 255) are computed and averaged over all sequencesThe output is a frequency matrix of amino acid pairs Thismatrix can be used to characterize nonstandard types of theputative signal peptide This way it also appeared impossibleto determine specificity of the N-terminus of apicoplast-targeted proteins in T gondii refer to Figure 4


646 aa

338 aa

712 aa

574 aa

543 aa

SER SER

SER

SER

SERSE

R

SER

Tg IspA 6ndash143267ndash309 412ndash477

Tg IspF 145ndash172

Tg LytB 31ndash385

Tg SufC 56ndash143

Tg LipA 56ndash143

Figure 4 Some apicoplast-targeted proteins in T gondii containingserine-rich regionsThe region coordinates indicated on the left andthe protein lengths on the rightThe serine-rich regions are arrangedirregularly which is confirmed on a larger number of proteins fordifferent pairs triples and so forth of amino acids

3 Results and Discussion

Orthologs of Synechocystis sp PCC 6803 were identified for515 of 8319 (sim6) nuclear proteins in T gondii 560 of 7122(sim8) nuclear proteins inN caninum and 390 of 5538 (sim7)nuclear proteins in P falciparum Only 877 of 3179 (sim28)proteins in Synechocystis sp were found to be orthologousagainst at least one of the three apicomplexan species (seeSupplementary Material available online at httpdxdoiorg1011552015452958)

The identified orthologs are putative apicoplast-targetedproteins Among them are proteins with either experimen-tally shown or anticipated apicoplast affinity such as thebacterial type RNA polymerase sigma subunit (RpoD) DNAligase aminoacyl-tRNA synthetases cell-cycle-associatedprotein kinase PRP4 enzymes IspA IspB IspE IspF IspG(GpcE) and IspH (LytB) of the mevalonate-independentpathway of isoprenoid biosynthesis sulphur mobilizationprotein SufC from a Fe-S cluster assembly pathway andLipA and LipB enzymes of lipoic acid synthesis (refer tothe Introduction [5 12 14 15]) In pairwise alignments thesporozoan and cyanobacterial proteins usually align well attheir C-termini and the cyanobacterial sequence is fullycovered by the alignment In many cases the N-termini ofsporozoan proteins extend outside the alignment (data notshown)

In most cases sporozoan proteins are longer comparedto their bacterial orthologs Figures 1ndash3 We demonstratestatistically that the majority of proteins in T gondii areconsiderably longer compared to their orthologs in N can-inum Liverpool and P falciparum 3D7 which was evidencedpreviously only for selected proteins [12 33]

The hypotheses ldquoa constant is better than the nontrivialaffine functionrdquo and ldquoaffine function versus general polyno-mial of second degreerdquo were rejected for every three setsof points shown in Figures 1ndash3 The hypothesis ldquothe linearfunction is better than the affine functionrdquo was compatiblewith the first two sets (119865 = 015 and 119865 = 157 refer tothe designation in Materials and Methods section) and wasrejected for the third set (119865 = 340) Thus the third setwas tested against the hypothesis ldquothe mean over all x-coordinates coincides with the mean over all y-coordinatesrdquo

this hypothesis was accepted with the Student test statistic 119878at the same significance level 120572 (with S = 1547) [32]

Hence the following regressions were justified For set1 119910 = 06711119909 with radius 00468 for set 2 119910 = 0528119909with radius 00590 for set 3 119910 = 05685119909 + 37756 (linearregression rejected with 119865 = 340) with radii 00926 and217521 respectively

The Deming regression gives approximately the sameestimates screening singular points does not significantlyaffect the results (data not shown)

So the following conclusions can be drawn for theapicoplast protein orthologs that have orthologs in thecyanobacterium

(1) Protein extensions in T gondii are on average 15-foldlonger compared to the corresponding extensions inN caninum with almost 10 confidence (Figure 1)

(2) Protein extensions in T gondii are on average 2-foldlonger compared to the corresponding extensions inP falciparum with high confidence (Figure 2)

(3) Set 3 (Figure 3) is compatible with the hypothesis thatthe average of protein extension lengths in N can-inum equals that in P falciparum Extension lengthsin P falciparum being less than 87 residues are longerthan the corresponding extensions inN caninum andreversely are shorter if they exceed 88 residues Inunits the dependency between extension lengths inP falciparum versus N caninum is an affine function119910 = 0 5685119909 + 37 756 where 119910 runs over extensionlengths in P falciparum and 119909 in N caninum Theaffinity but not the linearity of the regression testifieson behalf of the difference of T gondii from herimmediate species P falciparum andN caninum onceagain

Among other specific features of apicoplast-targeted pro-teins is the abundance of serine-rich regions revealed inanalyses with PROSITE (Figure 4) Each of the 3551 proteinsin T gondii ME49 possesses at least one 27 amino acid-long region with at least 9 serine residues and 39 proteinspossess at least one region with 27 or more continuous serineresidues Contrary to our expectations larger-scale searchingfor serine-rich motifs in T gondii showed their presence invarious protein families thus suggesting a selectively neutralnature of their origin In other words serine-rich regions arenot specific to N-termini of apicoplast-targeted proteins Thesame is also observed for other amino acids This approachdoes not allowdetecting a novel type of theN-terminal signal

Earlier preliminary results are reported in [33]

4 Conclusions

For apicomplexan parasites we suggest a statistically basedmethod to compare the extension lengths of orthologousproteins that have orthologs in the cyanobacterium

With this method we demonstrate that the majority ofcyanobacterium orthologs in Toxoplasma gondii are signifi-cantly longer compared to those in both Neospora caninumand Plasmodium falciparum These proteins commonly lack


signal sequences typical for Plasmodium spp [34] Thecorresponding extensions might be essential for regulationof the apicoplast proteins and their translocation into theapicoplast This notion conforms well with the observationthat the apicoplast membrane in T gondii is known to beless permissible at least against drugs compared to thatin P falciparum (personal communication with GamaleyaResearch Institute of Epidemiology and Microbiology) Dif-ferences in protein extension lengths between T gondii andother apicomplexan species may suggest different membranetransport mechanisms in these sporozoan groups Mecha-nism of regulation and translocation in T gondii may bebased on protein processing in the cytoplasm to mature theirextended N-termini

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are deeply respectful to the editor for valuablecomments that improved the paperThey also thank L Rusinfor valuable discussions and help with preparing the paperResearch was funded by the Russian Scientific Fund (Project14-50-00150)

References

[1] T N Ermak A B Peregudova V I Shakhgilrsquodian and D BGoncharov ldquoCerebral toxoplasmosis in the pattern of secondaryCNS involvements in HIV-infected patients in the Russian fed-eration clinical and diagnostic featuresrdquoMeditsinskaia Parazi-tologiia i Parazitarnye Bolezni no 1 pp 3ndash7 2013

[2] H N Luma B C N Tchaleu Y NMapoure et al ldquoToxoplasmaencephalitis in HIVAIDS patients admitted to the Doualageneral hospital between 2004 and 2009 a cross sectionalstudyrdquo BMC Research Notes vol 6 article 146 2013

[3] N Andenmatten S Egarter A J Jackson N Jullien J PHerman andMMeissner ldquoConditional genome engineering inToxoplasma gondii uncovers alternative invasion mechanismsrdquoNature Methods vol 10 no 2 pp 125ndash127 2013

[4] G Di Guardo and S Mazzariol ldquoToxoplasma gondii clues fromstranded dolphinsrdquo Veterinary Pathology vol 50 no 5 p 7372013

[5] R J M Wilson K Rangachari J W Saldanha et al ldquoParasiteplastids maintenance and functionsrdquo Philosophical Transac-tions of the Royal Society B Biological Sciences vol 358 no 1429pp 155ndash164 2003

[6] S Kohler C F Delwiche P W Denny et al ldquoA plastid ofprobable green algal origin in Apicomplexan parasitesrdquo Sciencevol 275 no 5305 pp 1485ndash1489 1997

[7] J Janouskovec AHorakMObornık J Lukes and P J KeelingldquoA common red algal origin of the apicomplexan dinoflagellateand heterokont plastidsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 107 no 24 pp10949ndash10954 2010

[8] S Sato ldquoThe apicomplexan plastid and its evolutionrdquo Cellularand Molecular Life Sciences vol 68 no 8 pp 1285ndash1296 2011

[9] G Zhu M J Marchewka and J S Keithly ldquoCryptosporidiumparvum appears to lack a plastid genomerdquo Microbiology vol146 no 2 pp 315ndash321 2000

[10] K A Brayton A O T Lau D R Herndon et al ldquoGenomesequence of Babesia bovis and comparative analysis of apicom-plexan hemoprotozoardquo PLoS Pathogens vol 3 no 10 pp 1401ndash1413 2007

[11] M J Gardner R Bishop T Shah et al ldquoGenome sequence ofTheileria parva a bovine pathogen that transforms lympho-cytesrdquo Science vol 309 no 5731 pp 134ndash137 2005

[12] NThomsen-Zieger J Schachtner and F Seeber ldquoApicomplexanparasites contain a single lipoic acid synthase located in theplastidrdquo FEBS Letters vol 547 no 1ndash3 pp 80ndash86 2003

[13] C Y He B Striepen C H Pletcher J M Murray and D SRoos ldquoTargeting and processing of nuclear-encoded apicoplastproteins in plastid segregation mutants of Toxoplasma gondiirdquoJournal of Biological Chemistry vol 276 no 30 pp 28436ndash28442 2001

[14] M J Crawford N Thomsen-Zieger M Ray J Schachtner DS Roos and F Seeber ldquoToxoplasma gondii scavenges host-derived lipoic acid despite its de novo synthesis in the api-coplastrdquo EMBO Journal vol 25 no 13 pp 3214ndash3222 2006

[15] J Mazumdar E H Wilson K Masek C A Hunter and BStriepen ldquoApicoplast fatty acid synthesis is essential for organ-elle biogenesis and parasite survival in Toxoplasma gondiirdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 103 no 35 pp 13192ndash13197 2006

[16] S Agrawal and B Striepen ldquoMore membranes more proteinscomplex protein import mechanisms into secondary plastidsrdquoProtist vol 161 no 5 pp 672ndash687 2010

[17] T Bruser and C Sanders ldquoAn alternative model of the twinarginine translocation systemrdquo Microbiological Research vol158 no 1 pp 7ndash17 2003

[18] D Mehner H Osadnik H Lunsdorf and T Bruser ldquoThe Tatsystem for membrane translocation of folded proteins recruitsthe membrane-stabilizing Psp machinery in Escherichia colirdquoJournal of Biological Chemistry vol 287 no 33 pp 27834ndash27842 2012

[19] G Cilingir S L Broschat and A O T Lau ldquoApicoAP thefirst computational model for identifying apicoplast-targetedproteins in multiple species of apicomplexardquo PLoS ONE vol 7no 5 Article ID e36598 2012

[20] G Cilingir AO T Lau and S L Broschat ldquoApicoAMP the firstcomputational model for identifying apicoplast-targeted trans-membrane proteins in Apicomplexardquo Journal of MicrobiologicalMethods vol 95 no 3 pp 313ndash319 2013

[21] K E Jackson J S Pham M Kwek et al ldquoDual targeting ofaminoacyl-tRNA synthetases to the apicoplast and cytosol inPlasmodium falciparumrdquo International Journal for Parasitologyvol 42 no 2 pp 177ndash186 2012

[22] B Kumar S Chaubey P Shah et al ldquoInteraction between sul-phur mobilisation proteins SufB and SufC evidence for aniron-sulphur cluster biogenesis pathway in the apicoplast ofPlasmodium falciparumrdquo International Journal for Parasitologyvol 41 no 9 pp 991ndash999 2011

[23] E V S R Ram A Kumar S Biswas S Chaubey M I Siddiqiand S Habib ldquoNuclear gyrB encodes a functional subunit ofthe Plasmodium falciparum gyrase that is involved in apicoplastDNA replicationrdquoMolecular and Biochemical Parasitology vol154 no 1 pp 30ndash39 2007


[24] B J Foth S A Ralph C J Tonkin et al ldquoDissecting apicoplasttargeting in the malaria parasite Plasmodium falciparumrdquo Sci-ence vol 299 no 5607 pp 705ndash708 2003

[25] TA Sadovskaya andAV Seliverstov ldquoAnalysis of the 51015840-Leaderregions of several plastid genes in protozoa of the phylumapicomplexa and red algaerdquo Molecular Biology vol 43 no 4pp 552ndash556 2009

[26] T Kaneko and S Tabata ldquoComplete genome structure of theunicellular cyanobacterium Synechocystis sp PCC6803rdquo Plantand Cell Physiology vol 38 no 11 pp 1171ndash1176 1997

[27] M Punta P C Coggill R Y Eberhardt et al ldquoThe Pfam proteinfamilies databaserdquo Nucleic Acids Research vol 40 no 1 ppD290ndashD301 2012

[28] C J A Sigrist E De Castro L Cerutti et al ldquoNew and continu-ing developments at PROSITErdquo Nucleic Acids Research vol 41no 1 pp D344ndashD347 2013

[29] S B Needleman and C D Wunsch ldquoA general method appli-cable to the search for similarities in the amino acid sequenceof two proteinsrdquo Journal of Molecular Biology vol 48 no 3 pp443ndash453 1970

[30] O A Zverkov A V Seliverstov and V A Lyubetsky ldquoPlastid-encoded protein families specific for narrow taxonomic groupsof algae and protozoardquoMolecular Biology vol 46 no 5 pp 717ndash726 2012

[31] J-R BarraNotions Fondamentales de StatistiqueMathematiqueMaıtrise de Mathematiques et Applications FondamentalesDunod Paris France 1971

[32] G A F Seber Linear Regression Analysis John Wiley amp SonsNew York NY USA 1977

[33] N V Kobets D B Goncharov A V Seliverstov O A Zverkovand V A Lyubetsky ldquoComparative analysis of apicoplast -targeted proteins in Toxoplasma gondii and other Apicomplexaspeciesrdquo in Proceedings of the International Moscow Conferenceon Computational Molecular Biology (MCCMB rsquo13) MoscowRussia July 2013

[34] L Lim and G I McFadden ldquoThe evolution metabolism andfunctions of the apicoplastrdquo Philosophical Transactions of theRoyal Society B Biological Sciences vol 365 no 1541 pp 749ndash763 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

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Molecular Biology International

GenomicsInternational Journal of


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BioinformaticsAdvances in

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Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



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Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Further analysis of unique features of apicoplast-targetedproteins (particularly regions involved in translocation pro-cesses) in T gondii can add to the effective design of drug-based or genetic strategies to control the pathogen develop-ment and proliferation At the reported stage of the studywe analyze only extensions in length of orthologs amongapicomplexan parasites

Note that the coccidian Cryptosporidium parvum lacksthe apicoplast [9] and the apicoplast in piroplasmids Babesiabovis andTheileria parva largely differs from that in commoncoccidians and the haemosporidian Plasmodium spp [10 11]

The majority of apicoplast proteins are encoded in thenucleus and only few in its own genome Most of theseproteins can be identified due to their cyanobacterial originTransport of nuclear-encoded proteins to the apicoplast in Tgondii is significantly less documented experimentally com-pared to Plasmodium falciparum Among the documentedcases is the nuclear-encoded lipoic acid synthetase LipA[12] other examples are described in [13ndash15] A mechanismof protein import into secondary plastids is also describedin [16] where many orthologous proteins involved in thisprocess were shown to be presented in the sporozoans Pfalciparum and T gondii cryptophyte alga Guillardia thetaand diatom Phaeodactylum tricornutum Plastids also possessthe bacterial system to translocate folded proteins [17 18]

A variety of protein localization prediction methods areused to identify apicoplast-targeted proteins Some of themutilize the notion that translocation across the four mem-branes surrounding the apicoplast is mediated by an N-terminal bipartite targeting sequence a special N-terminalsignal and a transit peptide [13] The algorithm ApicoAPdescribed in [19] predicts apicoplast-targeted proteins con-taining the signal peptide because it trains on a learning sam-ple of signal peptide-containing proteins Other apicoplast-targeted proteins are predicted neither with this algorithmnor with ApicoAMP [20] The comprehensive ToxoDBdatabase constructed using the SignalP algorithm containsproteins with the information on presenceabsence of thesignal peptide According to this database some nuclear-encoded proteins in T gondii that are experimentally shownto reach the apicoplast should do not contain signal peptidesalbeit bearing housekeeping functions in the apicoplast Themethods in application to Plasmodium spp are describedfor example in [19 21ndash23] The PlasmoAP algorithm [24]is designed specifically for Plasmodium spp and is of littleapplicability to coccidians Hence these two widely useddatabases may be considered of limited use to identifyapicoplast-targeted proteins not containing the standard sig-nal peptide in coccidians We therefore applied a crude tech-nique to compare apicomplexan proteins with their orthologsin a cyanobacterium Namely orthology between nuclear-encoded sporozoan proteins and cyanobacterial proteins isused as a basis to suggest the apicoplast-targeted natureof the proteins As our study relies on statistic estimatesits predictions are hopefully not affected by the chosenparameters of global protein alignment

In this work the lengths of sporozoan proteins are com-pared with each other and with the length of their orthologsin the cyanobacterium Synechocystis sp PCC 6803 We

consider lengths of the sporozoan proteins that extend out-side the conserved alignment regionwhich usually covers theentire cyanobacterial sequence We focus on results obtainedfor the model species T gondii Neospora caninum (the twococcidian sporozoanswith completed genomeprojects as perthe end of 2013) and the malaria agent P falciparum from theHaemosporidia

Based on the total comparison we conclude that T gondiiin most cases contains longer proteins compared to bothN caninum and P falciparum We also surmised that atleast some of them undergo processing in the cytoplasmto facilitate transporting into the apicoplast The extendedportions of proteins may also be involved in gene expressionregulation at the level of protein-protein interaction

As an argument the regulation of plastid-encoded genesycf24 and rps4 affects the general functionality of the api-coplast in T gondii [25] The expression regulation of ycf24(the SufB factor mediating the Fe-S cluster assembly in manynuclear proteins) was suggested to take place in the apicom-plexans Eimeria tenella T gondii RH and Plasmodium sppas well as in Gracilaria tenuistipitata Porphyra purpurea andPorphyra yezoensis [25] The same type of regulation wassuggested for rps4 (ribosomal protein S4) in T gondii RH[25]

2 Materials and Methods

Protein data for T gondii and N caninum was extractedfrom the ToxoDBdatabase (version 82) data forPlasmodiumspp from the PlasmoDB database (version 93) and datafor Synechocystis sp PCC 6803 from GenBank NCBI [26]ToxoDB and PlasmoDB are specialized regularly updatedand nonoverlapping databases Conserved domains weredetected according to the Pfam database [27] The locationof regions enriched with a certain amino acid was establishedusing the PROSITE database [28]

We compared the proteomes of three apicomplexanparasites (T gondii ME49 N caninum Liverpool and Pfalciparum 3D7) and the cyanobacterium Synechocystis spPCC 6803 For each pair of proteomes pairs of orthologousproteins were computed on the basis of an alignment qualityscore using theNeedleman-Wunschmethod andBLOSUM62matrix [29 30]

Our method to study the lengths of the apicomplexanprotein extensions is as follows For each cyanobacterialprotein with length 119904 and its two orthologs (from a fixedpair of apicomplexans) with lengths 119886 and 1198861015840 the point withcoordinates (119886 minus 119904 1198861015840 minus 119904) is computed In some cases oneor both of the coordinates are negative which indicates asporadic case of a shorter length of the sporozoan proteinversus the cyanobacteria

In Figures 1ndash3 the cases ofN caninum-T gondii (N-T) Pfalciparum-T gondii (P-T) and P falciparum-N caninum (P-N) are analyzed using three sets of points Each coordinateis the difference in lengths between the sporozoan andcyanobacterial orthologs T gondii versus Synechocystis (T-S) N caninum versus Synechocystis (N-S) and P falciparumversus Synechocystis (P-S) The sets of points are then sta-tistically analyzed The following statistic was used to test



y

x

Neos

pora

cani

num

Live

rpoo

l


300

500

700

900

1100

1300

1500

minus500

minus300

minus100






0


119894minus 119910(119909

119894) and 119909





y


300

500

700

900

1100

1300

1500

minus500

minus300

minus100

Plas

mod

ium

falci

paru

m3

D7


y


Plas

mod

ium

falci

paru

m3

D7


100

300

500

700

900

1100

1300

1500

minus500

minus300




646 aa

338 aa

712 aa

574 aa

543 aa

SER SER

SER

SER

SERSE

R

SER


Tg IspF 145ndash172

Tg LytB 31ndash385

Tg SufC 56ndash143

Tg LipA 56ndash143
















4 Conclusions







Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



y

x

Neos

pora

cani

num

Live

rpoo

l


300

500

700

900

1100

1300

1500

minus500

minus300

minus100






0


119894minus 119910(119909

119894) and 119909





y


300

500

700

900

1100

1300

1500

minus500

minus300

minus100

Plas

mod

ium

falci

paru

m3

D7


y


Plas

mod

ium

falci

paru

m3

D7


100

300

500

700

900

1100

1300

1500

minus500

minus300




646 aa

338 aa

712 aa

574 aa

543 aa

SER SER

SER

SER

SERSE

R

SER


Tg IspF 145ndash172

Tg LytB 31ndash385

Tg SufC 56ndash143

Tg LipA 56ndash143
















4 Conclusions







Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


646 aa

338 aa

712 aa

574 aa

543 aa

SER SER

SER

SER

SERSE

R

SER


Tg IspF 145ndash172

Tg LytB 31ndash385

Tg SufC 56ndash143

Tg LipA 56ndash143
















4 Conclusions







Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgments


References











































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Comparative Analysis of Apicoplast