BACKGROUND
malaria is a major health problem in the world.
although global disease incidence is currently on decline, malaria remains responsible for at least  <dig> million infections and half a million deaths every year, in particular among immune-naïve african children under the age of five  <cit> .
human malaria is an infectious disease caused by five different species of eukaryotic parasites of the genus plasmodium and is transmitted by anopheles mosquitoes.
plasmodium falciparum is the most prevalent and virulent human malaria parasite accounting for almost all malarial deaths worldwide.
plasmodium vivax is the most prevalent malaria parasite outside africa where despite much lower mortality rates it represents a huge socioeconomic burden in many countries  <cit> .
plasmodium ovale and plasmodium malariae cause a more benign form of human malaria and are responsible for only a small percentage of global infections.
plasmodium knowlesi, although traditionally considered a non-human parasite, is responsible for a potentially life-threatening zoonotic form of human malaria acquired from southeast asian macaque monkeys  <cit> .
other plasmodium parasites are important model organisms in malaria research, including plasmodium yoelii, plasmodium chabaudi, and plasmodium berghei  as well as plasmodium gallinaceum .
a tree detailing the phylogenetic relationship of these and other plasmodium species is provided in additional file  <dig>
one of the most pressing issues in malaria research is the development of an effective antimalarial vaccine.
unfortunately, despite many decades of research, this goal has yet to be achieved.
rts,s is currently the most promising p. falciparum vaccine candidate, but latest results from clinical trials showed that rts,s provides only modest protection against both clinical and severe malaria in young infants  <cit> .
for p. vivax the situation looks even grimmer with currently no vaccine candidate in advanced clinical trials  <cit> .
in  <dig>  publication of the first two plasmodium genomes, p. falciparum and p. yoelii, promised to revolutionize vaccine development by laying out the complete map of p. falciparum genes, including a comprehensive inventory of putative antigens that could serve as vaccine targets  <cit> .
this monumental achievement was followed by the publication of the genomes of four additional plasmodium species, including p. chabaudi and p. berghei in  <dig>  <cit>  as well as p. vivax and p. knowlesi in  <dig>  <cit> .
the p. gallinaceum genome was sequenced to three-fold coverage in  <dig> and is currently unpublished.
although the availability of so many plasmodium genome sequences provides now a rich resource for  genomics studies to learn more about parasite biology and immune evasion strategies  <cit> , the promise of an effective antimalarial vaccine has yet to be fulfilled.
one major obstacle in malaria vaccine development is the notorious variability of parasite antigens.
these antigens are expressed at the surface of the parasite or of the infected erythrocyte and are typically encoded by large gene families located at subtelomeric regions of chromosomes.
we now know that each of the sequenced plasmodium genomes possesses an extensive and often species-specific array of variant surface antigens   <cit> .
the clinically most important and best studied plasmodium vsa gene family is var, which encodes for about  <dig> erythrocyte surface-expressed proteins known as p. falciparum erythrocyte membrane protein  <dig>   <cit> .
in a process called antigenic variation, var gene expression switches from one family member to another over the course of an infection, allowing the parasite to evade the host immune system and to establish a chronic infection  <cit> .
in addition, pfemp <dig> mediates adherence of infected erythrocytes to both uninfected erythrocytes and endothelial cells, which is responsible for the most severe clinical complications of p. falciparum malaria and makes pfemp <dig> the prime virulence factor in this species.
besides pfemp <dig>  plasmodium parasites express many additional vsas throughout their complex life cycle.
in p. falciparum, this includes the largest p. falciparum gene family rif/stevor   <cit> , the surfin gene family   <cit> , and pfmc-2tm   <cit> .
in p. vivax, the largest gene family by far is vir   <cit> , which is related to homologous vsa gene families named kir  in p. knowlesi <cit>  and yir , bir , and cir  in p. yoelii, p. berghei and p. chabaudi, respectively  <cit> .
together, these five gene families form the large pir superfamily, the largest known gene family in plasmodium parasites  <cit> .
to date no pir genes have been identified in p. falciparum.
p. knowlesi possesses an additional large vsa gene family named sicavar , the first plasmodium gene family demonstrated to undergo antigenic variation  <cit> .
in rodent malaria parasites the second largest vsa gene family after pir is pyst-a, which in primate parasites consists of only a single member suggesting extensive expansion of this family in the rodent malaria species  <cit> .
besides vsas, plasmodium genomes encode another large repertoire of proteins termed the ‘exportome’ that is of potential interest for vaccine development  <cit> .
proteins in this set carry an n-terminal sequence motif termed plasmodium export element  or vacuolar transport signal  that targets these proteins beyond the parasitophorous vacuole to the cytosol of the infected erythrocyte  <cit> .
exported proteins are then trafficked further to the erythrocyte surface or remain in the cytosol to help remodeling the infected host cell.
like vsas, exported proteins are typically organized into subtelomeric variant gene families, most of which are species subset-specific  <cit> .
probably the most prominent exported gene family is the large and highly divergent gene family phist, which has ~40- <dig> known members in each of the three primate parasites but only a single known member in rodent parasites  <cit> .
although their large numbers suggest that vsas and exported proteins are of major importance for the parasite, we currently know surprisingly little about their biological functions.
for example, proposed roles for pir proteins include antigenic variation, immune evasion, signaling, trafficking, protein folding, and adhesion, but direct evidence for any pir function is still lacking  <cit> .
similarly, apart from expression and localization information, the exact functions of pfmc-2tm, surfin, pyst-a, and phist proteins are currently unknown.
furthermore, the p. falciparum genome contains over a dozen exported gene families named hyp <dig> to hyp <dig> whose functions have yet to be determined.
the difficulty in elucidating the function of these gene families is due in part to the presence of many functionally redundant paralogs, which makes gene knockout studies challenging.
in such cases it would help if one can identify and work with low copy number orthologous gene families in more accessible model parasites.
besides unknown functions, the evolutionary history of many plasmodium variant gene families is also poorly understood.
for example, standard sequence similarity searches reveal no obvious homologs for the major surface antigens of p. falciparum  and p. knowlesi  outside their respective species, raising the question about their evolutionary origin.
similarly, there are currently no known pir homologs in p. falciparum, although rif/stevor has been suggested as related gene family based on shared sequence motifs and secondary structural features  <cit> .
the identification of functional homologs of vsas across plasmodium species is important because it aids comparative immunological studies, gives new insights into the evolutionary adaptation of malaria parasites to their respective hosts, and provides a means to transfer functional annotations from model to human parasites and vice versa.
in this study, we use a recently developed comparative gene family classification strategy  <cit>  to classify vsas and exported proteins across seven plasmodium genomes, including p. falciparum, p. vivax, p. knowlesi, p. yoelii, p. chabaudi, p. berghei, and p. gallinaceum.
we hypothesized that the sensitive sequence-based clustering of the entire body of currently available plasmodium proteins will yield new insights into vsa function and evolution, which in turn could open up new avenues for vaccine development.
in this strategy, protein sequences from plasmodium genomes are first clustered into a hierarchical tree using average-linkage clustering and the resulting tree is then searched for clusters corresponding to known vsa gene families.
finally, identified clusters are closely analyzed for gene content and inter-cluster relationships.
this analysis resulted in several noteworthy findings, including the identification of unusually well conserved pir orthologs that are of potential interest for vaccine development, prediction of the likely function of pyst-a proteins, discovery of a novel and putatively functional pfmc-2tm domain named mc-tyr, new conclusive evidence supporting the common evolutionary origin of pfemp <dig>  sicavar, and surfin proteins, and the identification of many new vsa gene family members, including new phist gene family members in rodent parasites.
collectively, these findings provide vital starting points for future experimental studies.
RESULTS
curation and comparative classification of variant gene families in plasmodium genomes
we curated vsa and exported gene families by reviewing the literature as well as gene annotations in plasmodb  <dig>   <cit> .
we were interested in all plasmodium gene families that met one of the following criteria:  expressed at the parasite surface or the surface of infected erythrocytes;  predicted host cell localization by virtue of the presence of a pexel/vts export motif; any other gene family that is  species -specific or  located at subtelomeric regions of chromosomes.
using these criteria, we compiled a list of  <dig> gene families , which from now on we refer to as plasmodium variant gene families.
plasmodium
variant gene families and classification performance
fifty-nine plasmodium variant gene families were curated from the literature and public databases and categorized as follows:  vsas ;  vsas linked to erythrocyte invasion ;  predicted to be exported to the host erythrocyte ;  other subtelomeric or species-specific gene families .
better studied gene families are shown at the top of each category.
classification performance of each gene family is measured in terms of sensitivity , specificity , and jaccard index .
the remaining columns show how many genes of each species have been classified to belong to this gene family .
numbers in parentheses indicate how many genes of each species served as reference gene family members.
a more comprehensive version of this table including numbers of identified genes in non-plasmodium species is available in additional file  <dig>  abbreviations: vsa… variant surface antigen; pfal… p. falciparum; pviv… p. vivax; pkno… p. knowlesi; pyoe… p. yoelii; pber… p. berghei; pcha… p. chabaudi; pgal… p. gallinaceum.
after curation, we classified plasmodium variant gene families using a comparative gene family classification strategy described previously  <cit> .
briefly, we hierarchically clustered the combined set of all protein sequences with mc-upgma  <cit>  and extracted those sub-trees that maximally overlapped with the curated gene families.
most of the  <dig> curated gene families were resolved with both high sensitivity  and specificity  .
average and median jaccard indices  of extracted clusters were  <dig>  and  <dig> , respectively.
almost half of gene families  were clustered perfectly with a jaccard index of  <dig> .
an additional  <dig> gene families  clustered with a good jaccard index ≥  <dig> .
only  <dig> gene families clustered with lower quality  due to low sensitivity , low specificity  or both .
overall, we conclude that our clustering strategy works well on plasmodium variant gene families and produces high-quality protein sequence clusters.
pir contains single ultra-conserved ortholog in each plasmodium species
the large pir gene family separates nicely into two large non-overlapping clusters of high sensitivity and specificity .
one cluster represents the vir and kir subfamilies of p. vivax and p. knowlesi, respectively , and the other cluster represents the yir, bir, and cir subfamilies from p. yoelii, p. berghei, and p. chabaudi, respectively .
the somewhat reduced specificity of the vir/kir gene cluster is due to inclusion of the pv-fam-c gene family  that likely represents a bona fide vir subfamily .
in addition, the vir/kir cluster contains  <dig> p. vivax hypothetical proteins, many of which carry a predicted vir domain and are thus likely true but currently unannotated members of the vir gene family .
to our surprise we found three rodent pir proteins nested deep within the vir/kir cluster instead of clustering with their respective paralogs .
this unexpected result suggested that some of the otherwise highly divergent pir proteins are well conserved across species.
subsequent comprehensive analysis of pir sequence conservation revealed that each of the five plasmodium species contains a single and likely orthologous pir protein that is clearly better conserved between species than any other member of the pir family .
the five genes are: p. vivax vir14-related protein pvx_113230; p. knowlesi kir protein pkh_114850; p. yoelii hypothetical protein py06119; p. berghei bir protein pbanka_010050; and p. chabaudi cir protein pchas_ <dig>  orthology of these five pir proteins is supported by orthomcl db   <cit>  and by the fact that gene synteny is conserved among the five species .
multiple sequence alignment of the five conserved pir orthologs reveals a  <dig> aa long, gapless block recognized as the plasmodium_vir domain .
the block spans 56% of the average sequence length and has  <dig> columns  perfectly conserved .
this exceptional high degree of sequence conservation within an otherwise highly divergent gene family suggests that these genes are direct descendants of the founder member of the large pir gene family and that they serve an ancestral and probably special function in malaria parasites.
identification of the likely prototypical members of the large pir gene family allowed us to re-examine whether there exists conserved pir homologs outside p. vivax/p.
knowlesi and rodent malaria parasites, in particular within the laverania clade including p. falciparum.
ncbi psi-blast search  seeded with the five conserved pir orthologs did not reveal significant sequence similarity within p. falciparum or any other species not currently known to contain pir proteins.
similarly, multiple rounds of jackhmmer searches  <cit>  seeded with the alignment from figure  <dig> did not identify convincing homologs.
thus, despite extensive sequence conservation between the primate and rodent parasites, pir is apparently not conserved in other plasmodium or apicomplexan species, suggesting that pir surface antigens perform an important function unique to the p. vivax/p.
knowlesi and rodent clade of malaria parasites.
pyst-a proteins predicted to be involved in lipid binding and transfer
gene family expansion is an important genomic process by which parasites adapt to different lifestyles and host environments  <cit> .
our classification strategy readily identifies differentially expanded gene families in several plasmodium species , including rhoph1/clag, surfin and acs , msp- <dig>  msp- <dig>  and pv-fam-d , trythra/pvtrag , phist and hyp <dig> , rbp/235 kda and pyst-c , and pcema <dig>  pc-fam, and pst-a .
one of the most striking examples of a differentially expanded gene family in plasmodium parasites is the p. yoelii gene family pyst-a .
pyst-a has been shown to contain only a single member in primate malaria parasites but over one hundred members in p. yoelii and p. chabaudi <cit> .
in our analysis, pyst-a proteins cluster almost perfectly  within a larger cluster containing also pc-fam- <dig> and pb-fam- <dig> proteins, clearly showing that these three gene families are equivalent  in the three rodent parasite species.
total gene numbers in the combined pyst-a/pc-fam-1/pb-fam- <dig> sequence cluster are  <dig>   <dig>   <dig> genes in p. yoelii, p. chabaudi, and p. berghei, respectively.
as expected, we found only a single pyst-a gene family member in primate malaria parasites .
a single copy of pyst-a was also detected in the bird parasite p. gallinaceum, supporting the idea of extensive proliferation of the pyst-a gene family in the rodent malaria parasite lineage  <cit> .
interestingly, while examining annotated pyst-a gene models , we noticed that most pyst-a gene family members carry a predicted bet v1-like superfamily domain  <cit>  , suggesting a role of pyst-a proteins in lipid transport.
in search for further bioinformatics support for this possibility, we used i-tasser to predict the tertiary and secondary structure of p. falciparum gene family member pf14_ <dig> .
the predicted protein structure is very similar to several human proteins carrying the steroidogenic acute regulatory-related lipid transfer  domain, including pdb protein 1em <dig> .
taken together, our bioinformatics analysis suggests that the second largest rodent malaria parasite gene family pyst-a is involved in lipid binding and transport, probably playing a role in host cholesterol salvage .
pfmc-2tm proteins are conserved in rodent parasites and related to hyp <dig> and hyp <dig> through the novel mc-tyr domain
hierarchical protein sequence clustering is a powerful approach to elucidate distant evolutionary relationships between gene families  <cit> .
table  <dig> summarizes known and novel evolutionary links between plasmodium variant gene families as suggested by hierarchical clustering.
novel links of note include links between pfmc-2tm and hyp <dig>  = 100%; average e-value of blast match pairs  =  <dig> ), pfmc-2tm/hyp <dig> and hyp <dig> , hyp <dig> and hyp <dig> , hyp <dig> and hyp <dig> , hyp <dig>  pv-fam-h, and pk-fam-c , as well as pk-fam-a, pk-fam-b and pv-fam-d .
thus, several plasmodium variant gene families currently assumed to be species-specific have in fact putative members in other plasmodium species, opening up new avenues for elucidating gene function.
of note, we found that pv-fam-c is entirely nested within the larger vir/kir cluster  and thus likely represents a novel vir subfamily that we named vir <dig>  pyst-d , a family of ~ <dig> short  p. yoelii proteins, was found to be completely nested within the large pir gene family.
inspection of pyst-d and neighboring gene models revealed that this gene family is likely spurious and consists entirely of misannotated exons belonging to adjacent yir gene models.
other novel links in table  <dig> are of lower connectivity and should be regarded as tentative.
plasmodium
variant gene families predicted by hierarchical clustering
the hierarchical tree was searched for neighboring and parental clusters where both clusters  map to variant gene families as identified in table  <dig>  such cluster pairs are shown if  their blast match pair percentage  is ≥ 1% or  they share at least  <dig> blast match pairs .
avg.
e-value and avg.
pid denote the average e-value and percent identity of all blast match pairs between the two clusters.
size a and size b indicate numbers of genes in respective clusters, excluding annotated pseudogenes and gene fragments.
an asterisk next to cluster a indicates that it is a parental cluster of cluster b, i.e.
cluster b is fully contained in cluster a. our search predicts both previously established  and novel evolutionary links  between plasmodium variant gene families.
pfmc-2tm is an actively studied p. falciparum vsa gene family  <cit> , which motivated us to investigate its putative link with the two p. falciparum hypothetical gene families hyp <dig> and hyp <dig> in detail.
the  <dig> annotated pfmc-2tm proteins cluster first with two p. falciparum gametocyte-exported proteins of the hyp <dig> gene family  and then with a cluster of three p. falciparum exported proteins of unknown function, one of which is a member of the hyp <dig> gene family .
the second annotated hyp <dig> gene family member  is not found in this cluster and instead groups with hyp <dig> and pv-fam-h proteins, supporting the previously expressed idea that hyp <dig> does not constitute a real gene family  <cit> .
the combined pfmc-2tm/hyp8/hyp <dig> cluster contains a total of  <dig> proteins and is shown in figure 3a.
notably, this cluster also includes six rodent parasite genes of unknown function that share the typical two-exon gene structure with pfmc-2tm genes, including a signal peptide on the short first exon and a conserved pexel-like motif  on the 5′ end of the larger second exon.
most importantly, phobius  <cit>  predicts two adjacent c-terminal transmembrane  domains for four of the six rodent parasite proteins  as well as traces of a second tm domain in the remaining two .
no tm domains are present in the three p. falciparum proteins clustering with  hyp <dig> protein pfb0926c.
further inspection of the pfmc-2tm/hyp8/hyp <dig> gene cluster using multiple protein sequence alignments identified a hitherto unknown ~ <dig> aa long conserved domain located between the pexel motif and the two tm domains .
the domain has a predicted three-alpha helical structure and contains two almost invariant tyrosine residues  of potential functional importance.
only in three proteins, one of the two tyrosine residues is conservatively replaced with phenylalanine .
based on this observation, we name this novel domain the maurer’s clefts tyrosine  domain.
to explore the possibility that mc-tyr is conserved beyond the seed cluster shown in figure  <dig>  we carried out sensitive profile-based searches using psi-blast.
five rounds of ncbi psi-blast initiated with any one of the  <dig> pfmc-2tm mc-tyr protein sequences consistently identified three additional p. falciparum hits, albeit above psi-blast’s inclusion threshold .
these three hits include one of three annotated hyp <dig> proteins  and two other exported proteins of unknown function .
like the three proteins of the hyp <dig> cluster, these three genes share the typical two-exon gene structure with pfmc-2tm genes except for the tm domains.
this data suggests that mc-tyr is likely conserved in additional exported p. falciparum proteins and probably includes the p. falciparum hyp <dig> gene family.
we should mention that neither protein sequence clustering nor psi-blast searches identified a relationship of pfmc-2tm with hyp <dig>  pyst-b or vird proteins, which has been proposed previously based on shared gene architectural features  <cit> .
taken together, we provide first evidence that pfmc-2tm proteins carry a novel and putative functional domain named mc-tyr.
mc-tyr is conserved in other p. falciparum exported proteins and additional plasmodium species, opening up new opportunities to experimentally characterize the function of this important vsa gene family.
pfemp <dig>  sicavar, and surfin are interrelated through a modular and structurally conserved intracellular tryptophan-rich domain
as shown in table  <dig>  hierarchical clustering supports the previously proposed link between the well-studied plasmodium vsa gene families var , sicavar, and surfin/pvstp <dig> <cit> .
unexpectedly, the surfin/pvstp <dig> cluster includes two genes from p. gallinaceum  that have very similar gene structures to p. falciparum surfin genes , suggesting that surfins are conserved outside human malaria parasites.
further up the tree, surfin/pvstp <dig> proteins first group with p. knowlesi sicavar proteins  and then with another large cluster  containing the two p. falciparum gene families var  and dbl  as well as six other dbl domain-containing proteins, including two msp <dig> proteins , maebl , ama <dig> , giant protein pf <dig> , and one protein of unknown function .
notably, still further up the tree the surfin/pvstp1/sicavar/var gene cluster is joined by the large gene family pir , uniting all major plasmodium vsa gene families except rifin/stevor in a single gene cluster.
however, with only  <dig> blast high-scoring segment pairs  in total and an average e-value of  <dig> the connectivity between surfin/pvstp1/sicavar/var and pir was found to be extraordinarily low .
because rifin/stevor genes are not part of this unified vsa gene cluster we conclude that there is no sequence-based evidence supporting a link of rifin/stevor and pir.
however, as did others before us  <cit> , we noticed suspicious similarities in some protein features  that make such a link plausible .
to better understand the nature of the relationship among surfin/pvstp <dig>  sicavar, pfemp <dig>  and the dbl-containing proteins, we investigated the sequence similarity matrix of this cluster as well as pairwise blast sequence alignments.
as revealed by the sequence similarity heat map , sicavar and pfemp <dig> proteins share no direct pairwise sequence similarity with each other but exhibit weak similarity with surfin/pvstp <dig> proteins .
notably, one p. knowlesi sicavar protein  and two of the dbl-containing proteins  stand out by having high sequence similarity not only with paralogs of their respective gene families but also with surfin/pvstp <dig> proteins .
by examining the underlying blast alignments we found that blast consistently identified hsps within the c-terminal intracellular regions of proteins, where, in surfin/pvstp <dig> proteins, the hsps overlapped with the previously described tryptophan-rich domain   <cit> .
in pfemp <dig> proteins, the hsps overlapped with the pfemp <dig> intracellular region also known as the acidic terminal sequence  or varc  <cit> .
subsequent multiple sequence alignments of surfin, pfemp <dig> and sicavar intracellular regions revealed a more complete picture of the relationship of these proteins than described previously  <cit> .
we find that surfin wrd has a modular architecture consisting of two distinct and structurally conserved subdomains, which we named wrd-a and wrd-b .
wrd-a and wrd-b occur in variable numbers and configurations in different gene families .
the typical wrd consist of one wrd-a  and two wrd-b subdomains , which is found in p. falciparum  and p. gallinaceum  surfin proteins, the two full-length p. vivax pvstp <dig> proteins , and the two p. falciparum dbl-containing proteins, including pf <dig>  and pfa0665w .
where wrds occur in tandem, the first of the two wrds is typically better conserved, as illustrated by the partially empty dashed rectangles representing cases where complete wrds but not individual subdomains reached statistical significance in a hmmer <dig> search .
in other gene families, wrd is only partially conserved.
the typical sicavar protein contains a single wrd-a but no wrd-b subdomain, while pfemp <dig> proteins carry one copy of both wrd-a and wrd-b separated by a ~ <dig> aa long variable region.
during our studies an experimentally determined three-dimensional structure of the pfemp <dig> intracellular ats domain became available, which shows that ats consists of a conserved core composed of four alpha helices  <cit>  .
these four helices map nicely to the two conserved homology blocks corresponding to wrd-a and wrd-b , supporting the biological significance of these alignments.
notably, we identified at least one p. knowlesi sicavar protein  with a completely conserved wrd , which explains the high blast sequence similarity of this protein with p. falciparum surfins and provides, for the first time, compelling alignment-based evidence that the two antigenically variant gene families var and sicavar are evolutionarily linked via shared intracellular wrd domains.
a tblastn search against the p. knowlesi genome using wrd-b of pkh_ <dig> as query identified one additional sicavar antigen  carrying a wrd-b subdomain, but this gene appears to be severely truncated  and lacks wrd-a, suggesting that pkh_ <dig> is possibly a pseudogene.
new members of variant gene families predicted by sequence clustering
sequence clustering complements consensus-based strategies  for the identification of gene family members.
we therefore examined gene clusters of sufficient quality  for the presence of putative new gene family members.
table  <dig> lists newly predicted members of several p. falciparum gene families, including phist , hyp4/hyp <dig> , tsp_ <dig> , msp- <dig> , msp- <dig> , etramp , hyp <dig> , hyp <dig> , and hyp <dig> .
in none of these cases could we identify obvious cases of misclassifications, suggesting that most of these genes are indeed potential novel members of these gene families.
p. falciparum
gene family members of variant gene families
clusters corresponding to variant gene families were searched for p. falciparum proteins that are currently not annotated as members of these gene families.
only clusters with a jaccard index of at least  <dig>  were considered in this search .
annotated pseudogenes and gene fragment not shown.
mp # and mp% indicate number and percentage of blast match pairs between the putative new member and all other proteins in the cluster representing that gene family.
avg.
e-value and avg.
pid denote the average e-value and the average pid of these blast match pairs, respectively.
aunusual phist gene family member carrying an n-terminal acyl-coa-binding protein domain .
to verify the credibility of these predictions, we examined the clustering result of the large and highly divergent phist gene family in more detail .
phist  is a large subtelomeric gene family of exported proteins of unknown function with ~ <dig> members in p. falciparum, ~ <dig> members in p. vivax, and ~ <dig> members in p. knowlesi.
so far only one gene family member has been identified in each of the three rodent parasite genomes, suggesting extensive radiation of the phist gene family in primate parasites  <cit> .
in p. falciparum, phist has been divided into three subfamilies .
another chromosome-internally expanded p. vivax gene family of  <dig> genes named rad  has later been shown to be closely related to phist <cit> .
in our results, the phist-a, phist-b, phist-c, and rad gene families are resolved with high accuracy  and as expected cluster together into the larger phist gene family  .
phist-c first clusters with rad followed by phist-b and phist-a, which differs from an earlier report suggesting that phist-a and phist-b form a clade with phist-c being the out-group  <cit> .
in total, the phist cluster contains  <dig> full-length proteins that are currently not annotated as phist gene family members, including the four genes from p. falciparum mentioned earlier , and pf10_0015), two from p. vivax ,  <dig> from p. knowlesi , and one from each p. berghei and p. chabaudi .
the two novel rodent parasite phist family members cluster with the phist-c subfamily and could be interesting targets for experimental studies of phist function in the rodent model system.
multiple protein sequence alignment of these putative new phist gene family members  shows that key residues of the phist presan domain  are well conserved, suggesting that these  <dig> genes are likely true members of the phist gene family.
notably, the phist cluster also contains two proteins from p. gallinaceum, supporting previous findings that phist is conserved in the bird parasite  <cit> .
we conclude that our clustering procedure resolves even challenging gene families correctly and is a successful strategy to identify putative new gene family members in p. falciparum and other plasmodium species.
discussion
in this study, we classified and investigated vsa and exported gene families across seven plasmodium genomes.
this led to several interesting findings, some of which may have application value.
conserved pir proteins are targets for functional studies and vaccine development
the identification of a single ultra-conserved pir ortholog in each of the pir-containing plasmodium species  was highly unexpected given that pir is the largest and one of the most diverse gene families in plasmodium parasites.
pir gene family sizes range from  <dig> in p. knowlesi to  <dig> in p. yoelii, with amino acid similarities ranging from 30-50% between p. chabaudi, p. yoelii and p. berghei and from 20-30% between p. chabaudi and p. vivax <cit> .
the same conserved pir orthologs were found recently in an independent work studying genetic diversity of the global p. vivax population  <cit> .
it was shown that p. vivax gene pvx_ <dig>  is completely invariant across four strains sampled from different sites around the world and also exhibits conserved synteny in rodent malaria parasites.
the fact that these highly conserved pir orthologs could be identified in our study clearly highlights the value of comparative genomics in general and of comparative gene family classification in particular.
the discovery of ultra-conserved pir orthologs has two important implications.
firstly, these genes can now serve as starting points to elucidate pir function, which is still unknown.
for example, gene knockouts of the conserved pir orthologs in the more accessible rodent model system may result in important and measurable phenotypes.
alternatively, tagging the conserved pir orthologs with different fluorescence markers coupled with the manipulation of their expression levels should also help clarifying pir localization and function.
pvx_ <dig> has a distinct expression profile in erythrocytes relative to most other vir genes  <cit>  and therefore may serve a different, ancestral function.
if true, insights from the proposed experiments may not be directly applicable to other members of the gene family, but should nevertheless be informative.
secondly, the finding of a conserved pir ortholog in p. vivax has potential ramifications for p. vivax vaccine development.
if, like other vir proteins  <cit> , pvx_ <dig> is confirmed as surface-exposed antigen, antibodies raised either against pvx_ <dig> or its hypothetical host receptor could elicit exquisite cross-strain immunity.
that parasite genes with limited genetic diversity can be effective vaccine targets was recently demonstrated in p. falciparum, where antibodies against an essential and highly conserved ligand for erythrocyte invasion  elicited exquisite neutralizing cross-strain immunity  <cit> .
this seemingly paradoxical situation, that a highly conserved parasite protein can be successfully targeted by the host immune system, makes sense if one imagines pfrh <dig> as functionally important and therefore conserved component of a host-parasite interaction complex.
within such a complex, pfrh <dig> would not be under direct immune attack because it is efficiently shielded by immunodominant paralogs that act as red herrings or “smoke-screen”  <cit> .
a similar model has been proposed for rif/stevor and surfin genes in the context of the merozoite invasion process  <cit> .
if this model applies to pir proteins, then pvx_ <dig> could be a promising target for the development of a p. vivax vaccine.
lipid-binding pyst-a proteins potentially involved in cholesterol salvage
another family of surface antigens that is currently without known function is the large rodent parasite gene family pyst-a .
studies of pyst-a expression have shown that the pyst-a family member and glutamate-rich protein pc <dig>  <dig>  pc <dig>  and pch105/resa  <cit> ) is the immunodominant protein within red blood cell  membranes and localizes to the cytoplasmic face of the membrane  <cit> .
more recently, rbc membrane localization of a pyst-a homolog  has also been demonstrated in p. berghei <cit> .
pyst-a genes are concurrently expressed in large numbers without evidence of differential expression in response to different host environments  <cit> , suggesting that altering expression of pyst-a gene family members is not an immune evasion strategy of the parasite.
using bioinformatics sequence and structural analysis we predict the existence of a star-related lipid-transfer  domain in pyst-a proteins .
start is the archetypical domain found in the steroidogenic acute regulatory  protein, which is part of a multi-protein complex termed the “transduceosome” whose function in mammals is to transfer cholesterol across the two mitochondrial membranes and to initiate the first enzymatic step in steroid biosynthesis  <cit> .
cholesterol is also a major lipid fraction in the membrane bilipid layer and is required for membrane genesis.
plasmodium parasites cannot synthesize cholesterol de novo <cit> .
it has been shown that host cell membranes of p. falciparum-infected erythrocytes show a 50% decrease in the cholesterol/phospholipid ratio compared to uninfected cells  <cit> .
in addition, malaria infections result in hypocholesterolaemic conditions  <cit> .
together these observations suggest that the parasite scavenges required cholesterol from both host erythrocyte membranes and plasma.
how this is accomplished by the parasite is currently not understood.
based on the presence of a cholesterol-binding start domain in rbc membrane-localized pyst-a proteins we hypothesize that pyst-a is part of a molecular machinery that diverts cholesterol away from erythrocyte membranes to fuel parasite growth within erythrocytes.
notably, p. chabaudi pcema <dig> proteins could constitute a second component of this proposed cholesterol import machinery.
we found five pc-fam- <dig> proteins  that share sequence similarity with both pyst-a  and pcema <dig> proteins .
we could not find obvious problems with the gene models of these five proteins, suggesting that they are genuine pyst-a/pcema <dig> protein hybrids.
co-occurrence of pyst-a and pcema <dig> domains within the same polypeptide suggests physical interaction between pyst-a and pcema <dig> proteins.
this possibility is supported by the fact that pcema <dig> proteins also localize to the cytoplasmic face of the rbc membrane  <cit> .
it remains to be shown why rodent malaria parasites maintain so many pyst-a proteins compared to primate malaria parasites.
pyst-a proteins are probably immunogenic in rodent but not primate parasites, putting pyst-a proteins under strong selective pressure to diversify.
alternatively, massive pyst-a gene amplification could increase the efficiency of cholesterol salvage, probably satisfying an elevated need for cholesterol in rodent parasites or compensating for lower cholesterol levels in rodent blood.
pfmc-2tm – conserved beyond p. falciparum
another interesting erythrocyte surface-expressed gene family of currently unknown function is the p. falciparum gene family pfmc-2tm.
the  <dig> members of the pfmc-2tm gene family encode putative functional and highly similar  basic membrane proteins that, based on structural features, are grouped within the larger 2tm superfamily.
the 2tm superfamily consists of 200– <dig> aa long proteins that on their first exon carry a signal peptide followed by a n-terminal pexel motif and two c-terminal tm domains on their second exon.
besides pfmc-2tm, the 2tm superfamily includes rif/stevor as well as genes in p. knowlesi, p. vivax, and the rodent parasite genomes that share similar architectural features  <cit> .
it is important to note that, other than pir, the large 2tm superfamily is currently united solely based on gene structural features and not sequence similarity; in fact, to date no pfmc-2tm sequence homologs have been identified in p. falciparum or any other species.
pfmc-2tm proteins have been localized to the maurer’s clefts  <cit> , the parasitophorous vacuole, parasitophorous vacuole membrane  <cit> , and the erythrocyte surface  <cit> .
at this latter location, 2tm proteins including pfmc-2tm are thought to interact with the host immune system through a surface-exposed and hypervariable loop region that is located between the two tm domains  <cit> .
pfmc-2tm proteins are most highly expressed in the mid-trophozoite stage and have been shown to undergo expression switching, suggesting that the pfmc-2tm gene family plays a role in antigenic variation  <cit> .
we provide first evidence that the pfmc-2tm gene family has additional members both within and outside the p. falciparum species .
experimental clues for the existence of potential pfmc-2tm homologs in the rodent plasmodium genome already existed, but the identity of these genes remained unknown  <cit> .
furthermore, we showed that pfmc-2tm proteins contain a conserved and putative functional domain located between pexel and the two tm domains, which we named mc-tyr.
these two findings have important implications for future studies of pfmc-2tm function.
experimental double-knockout studies can now be attempted in rodent parasites on the two pfmc-2tm gene family members to see if they produce an observable phenotype.
given the reduced number of pfmc-2tm gene family members in rodent malaria parasites compared to p. falciparum and the more tractable rodent model system, such studies should now be feasible.
two additional observations suggest that hyp <dig>  hyp <dig>  and the six rodent malaria proteins are genuine members of the extended pfmc-2tm gene family.
first, all but three of the putative new pfmc-2tm members  have a similar basic isoelectric point  as known pfmc-2tm proteins.
second, all newly identified members that have two tm domains carry a single proline residue within the second tm domain, another known characteristic of pfmc-2tm proteins  <cit> .
proline residues internal to helices are often found in transporters, channels, and receptors, and tend to be conserved between homologous proteins.
in contrast, we did not observe conservation of the two cysteine residues preceding the tm domains  <cit> .
also the fact that no tm domains are present in three of the proteins carrying mc-tyr is intriguing and suggests that the presence of two tm domains is not essential for a possible mc-tyr function.
it is also noteworthy that most of the hyp <dig> and hyp <dig> gene family members are more broadly expressed throughout the intra-erythrocytic life cycle than pfmc-2tm proteins, showing expression not only in the mid-trophozoite stage but also in early ring and schizont stages  as well as in gametocytes  .
this suggests that the function of mc-tyr containing proteins is not restricted to trophozoites.
our clustering results do not provide evidence for a link between pfmc-2tm and the pyst-b gene family as has been proposed previously based on gene structural features  <cit> .
wrd as missing link between major plasmodium vsa gene families
besides the large 2tm superfamily, malaria parasites express additional vsa gene families on cell surfaces.
among them are the var gene family in p. falciparum and sicavar in p. knowlesi, both of which encode proteins expressed at the erythrocyte surface and are known to undergo antigenic variation.
surfin, a family of  <dig> p. falciparum proteins, has been shown to be expressed on the surfaces of both infected erythrocytes and merozoites  <cit> .
by examining intracellular regions of co-clustered surfins, pfemp <dig> and sicavar proteins, we now better understand the sequence conservation pattern present among these major plasmodium vsas .
we found that surfin proteins contain twice as many wrds as reported previously  <cit>  and that each wrd consists of three conserved blocks.
the first block, named wrd-a , is found once in each of the wrds, whereas block two and three represent two copies of a second subdomain that we named wrd-b .
winter et al.
previously described the conservation pattern among wrd-containing proteins in terms of shared conserved elements denoted s <dig>  s <dig>  s2*, s2**, and pexel-like motif  <cit> .
compared to this earlier study, we find that wrd-a corresponds to segment s <dig> plus five additional c-terminal amino acids in wrd-a, which map to the second alpha helix of the protein structure .
wrd-b corresponds roughly to the c-terminal half of segment s <dig>  and segment s2*  is in fact the first of the two wrd-b copies found within this protein.
the short s2** subsegment, indicating another duplication at the end of each wrd in surfin  <dig> , is in fact the most conserved part of the second copy of the wrd-b subdomain in this protein.
the pexel-like motif localized within the s <dig> segment was also found to be well conserved in our study .
interestingly, this pexel-like motif currently has no correspondence in the reported tertiary structure , which might be explained by the fact that the ats-core deletion construct that was used to determine the ats structure lacked this pexel-like motif  <cit> .
it thus remains to be shown if the pexel-like motif is in fact part of ats-core or serves another conserved function in wrds.
importantly, our data provides the first conclusive alignment-based evidence that the two major antigenically variant gene families in p. falciparum  and in p. knowlesi  share common evolutionary origin, substantiating the usefulness of the p. knowlesi-rhesus monkey model for the in vivo study of p. falciparum antigenic variation.
in  <dig>  winter et al.
provided the first bioinformatics evidence that pfemp <dig> and sicavar proteins are probably evolutionarily linked through their intracellular domains, which share partial sequence similarity with the intracellular tryptophan-rich domain  of surfin proteins  <cit> .
short conserved sequence motifs have also been identified in var and sicavar introns  <cit>  as well as in extracellular regions of these proteins  <cit> .
however, at present the picture remains highly fragmented and reported sequence similarity of intracellular regions between pfemp <dig> and sicavar is essentially limited to a highly conserved six amino acid long pexel-like motif, questioning the robustness of these observations.
our study provides new evidence for common ancestry of pfemp <dig> and sicavar, which rests on three critical observations.
firstly, not only wrd-b  but also wrd-a  is entirely conserved in pfemp <dig> proteins.
secondly, at least one sicavar protein  has a completely conserved wrd, consisting of one wrd-a and two wrd-b subdomains.
thirdly, complete wrds are conserved in at least two other p. falciparum proteins , which are clearly related to pfemp <dig> proteins through n-terminal dbl domains.
why were these sequence similarities not detected previously?
unlike surfins, pfemp <dig> proteins have wrd-a and wrd-b separated by a larger  variable insertion, which in the protein structure does not adopt a stable fold  <cit> .
similarly, compared to surfins, pfemp <dig> proteins carry additional insertions both within wrd-a  and wrd-b  .
in wrd-a, this insertion splits the highly conserved pexel-like motif from the rest of the wrd-a subdomain .
the presence of these insertions in pfemp <dig> proteins combined with the fact that wrd-b is absent in all but one sicavar protein likely thwarted earlier attempts to find significant sequence conservation between pfemp <dig> and sicavar proteins  <cit> .
it is not too surprising that larger blocks of conserved sequence between pfemp <dig> and sicavar are confined to intracellular regions as extracellular regions are likely under strong diversifying selection pressure from the host immune system.
consistent with earlier reports, we did not find evidence that wrd or any of its subdomains is conserved within the large pir gene superfamily of plasmodium surface antigens  <cit> .
what is the function of wrd?
despite this now much improved picture of the conservation structure of pfemp <dig>  sicavar, and surfin intracellular domains, many fundamental questions remain.
why do surfins have multiple copies of complete wrds while most other gene families carry only a single wrd?
what is the functional significance of having two wrd-b subdomains  vs. having only one wrd-b subdomain ?
why is wrd-b found in only a single sicavar protein  while all other members of the sicavar gene family carry only the wrd-a subdomain?
unfortunately, the molecular function of wrd is currently not well understood, so these questions are difficult to answer.
studies have shown that ats of pfemp <dig> interacts either directly or indirectly via the knob-associated histidine-rich protein  with erythrocyte membrane skeletal proteins, including actin and spectrin  <cit> .
ats is thus believed to anchor pfemp <dig> at the membrane of infected rbcs, which strengthens and stabilizes the roots of pfemp1-endothelial receptor interaction.
similarly, pf <dig>  which contains a single complete wrd near its c-terminal end, was found to contribute to the overall deformability of p. falciparum-infected erythrocytes through anchoring and scaffolding  <cit> , probably through wrd-mediated binding of pf <dig> to actin  <cit> .
it seems therefore plausible that the main function of wrd is to provide a scaffold for anchoring surface antigens like pfemp <dig> to the rbc cytoskeleton.
the function of having many wrds resulting from gene family expansion and/or intra-protein domain amplification is then probably to increase the strength of this scaffold.
this would explain why particularly p. falciparum, which compared to other malaria parasites has strong cytoadherence properties, expresses so many wrds in form of the surfin gene family.
surfins have not yet been implicated in anchoring surface antigens to the erythrocyte cytoskeleton, but we think that this is a plausible hypothesis that should be tested experimentally.
it is also worth mentioning that the abbabbabb-like repeat structure of the surfin wrds is reminiscent of similar repeats present in other cytoskeleton-binding proteins, including the vertebrate titin and the invertebrate twitchin muscle proteins  <cit> .
regardless of the exact function of wrd, its modular nature suggests that different proteins carrying distinct wrd subdomains act in concert to perform this function.
p. knowlesi protein pkh_ <dig>  which appears to be the only functional protein in the p. knowlesi genome carrying both wrd-a and wrd-b, thus probably interacts physically with other, wrd-a-containing sicavar proteins to anchor sicavar to the cytoskeleton.
with the refined domain structure presented here it should now be possible to test this prediction experimentally.
ultimately, such experiments will hopefully expand our knowledge of how p. falciparum accomplishes cytoadherence at the molecular level, which is responsible for much of the virulence of this parasite.
CONCLUSIONS
sensitive clustering of protein sequences followed by manual examination of clustering results revealed many new insights into variant surface antigens of malaria parasites, including atypical and new gene family members, novel domains, new protein function predictions, and a better understanding of the evolutionary relationship and origin of these important proteins.
the findings presented in this work can now jump-start follow-up experimental research around the world, most importantly to elucidate the function of pvx_ <dig> and to explore its potential value as p. vivax vaccine target.

 malaria is a major health problem in the world. although global disease incidence is currently on decline, malaria remains responsible for at least million infections and half a million deaths every year, in particular among immune-naïve african children under the age of five. human malaria is an infectious disease caused by five different species of eukaryotic parasites of the genus plasmodium and is transmitted by anopheles mosquitoes. plasmodium falciparum is the most prevalent and virulent human malaria parasite accounting for almost all malarial deaths worldwide. plasmodium vivax is the most prevalent malaria parasite outside africa where despite much lower mortality rates it represents a huge socioeconomic burden in many countries. plasmodium ovale and plasmodium malariae cause a more benign form of human malaria and are responsible for only a small percentage of global infections. plasmodium knowlesi, although traditionally considered a non-human parasite, is responsible for a potentially life-threatening zoonotic form of human malaria acquired from southeast asian macaque monkeys. other plasmodium parasites are important model organisms in malaria research, including plasmodium yoelii, plasmodium chabaudi, and plasmodium berghei as well as plasmodium gallinaceum. a tree detailing the phylogenetic relationship of these and other plasmodium species is provided in additional file one of the most pressing issues in malaria research is the development of an effective antimalarial vaccine. unfortunately, despite many decades of research, this goal has yet to be achieved. rts,s is currently the most promising p. falciparum vaccine candidate, but latest results from clinical trials showed that rts,s provides only modest protection against both clinical and severe malaria in young infants. for p. vivax the situation looks even grimmer with currently no vaccine candidate in advanced clinical trials. in publication of the first two plasmodium genomes, p. falciparum and p. yoelii, promised to revolutionize vaccine development by laying out the complete map of p. falciparum genes, including a comprehensive inventory of putative antigens that could serve as vaccine targets. this monumental achievement was followed by the publication of the genomes of four additional plasmodium species, including p. chabaudi and p. berghei in as well as p. vivax and p. knowlesi in. the p. gallinaceum genome was sequenced to three-fold coverage in and is currently unpublished. although the availability of so many plasmodium genome sequences provides now a rich resource for genomics studies to learn more about parasite biology and immune evasion strategies, the promise of an effective antimalarial vaccine has yet to be fulfilled. one major obstacle in malaria vaccine development is the notorious variability of parasite antigens. these antigens are expressed at the surface of the parasite or of the infected erythrocyte and are typically encoded by large gene families located at subtelomeric regions of chromosomes. we now know that each of the sequenced plasmodium genomes possesses an extensive and often species-specific array of variant surface antigens. the clinically most important and best studied plasmodium vsa gene family is var, which encodes for about erythrocyte surface-expressed proteins known as p. falciparum erythrocyte membrane protein. in a process called antigenic variation, var gene expression switches from one family member to another over the course of an infection, allowing the parasite to evade the host immune system and to establish a chronic infection. in addition, pfemp mediates adherence of infected erythrocytes to both uninfected erythrocytes and endothelial cells, which is responsible for the most severe clinical complications of p. falciparum malaria and makes pfemp the prime virulence factor in this species. besides pfemp plasmodium parasites express many additional vsas throughout their complex life cycle. in p. falciparum, this includes the largest p. falciparum gene family rif/stevor, the surfin gene family, and pfmc-2tm. in p. vivax, the largest gene family by far is vir, which is related to homologous vsa gene families named kir in p. knowlesi and yir, bir, and cir in p. yoelii, p. berghei and p. chabaudi, respectively. together, these five gene families form the large pir superfamily, the largest known gene family in plasmodium parasites. to date no pir genes have been identified in p. falciparum. p. knowlesi possesses an additional large vsa gene family named sicavar, the first plasmodium gene family demonstrated to undergo antigenic variation. in rodent malaria parasites the second largest vsa gene family after pir is pyst-a, which in primate parasites consists of only a single member suggesting extensive expansion of this family in the rodent malaria species. besides vsas, plasmodium genomes encode another large repertoire of proteins termed the ‘exportome’ that is of potential interest for vaccine development. proteins in this set carry an n-terminal sequence motif termed plasmodium export element or vacuolar transport signal that targets these proteins beyond the parasitophorous vacuole to the cytosol of the infected erythrocyte. exported proteins are then trafficked further to the erythrocyte surface or remain in the cytosol to help remodeling the infected host cell. like vsas, exported proteins are typically organized into subtelomeric variant gene families, most of which are species subset-specific. probably the most prominent exported gene family is the large and highly divergent gene family phist, which has ~40- known members in each of the three primate parasites but only a single known member in rodent parasites. although their large numbers suggest that vsas and exported proteins are of major importance for the parasite, we currently know surprisingly little about their biological functions. for example, proposed roles for pir proteins include antigenic variation, immune evasion, signaling, trafficking, protein folding, and adhesion, but direct evidence for any pir function is still lacking. similarly, apart from expression and localization information, the exact functions of pfmc-2tm, surfin, pyst-a, and phist proteins are currently unknown. furthermore, the p. falciparum genome contains over a dozen exported gene families named hyp to hyp whose functions have yet to be determined. the difficulty in elucidating the function of these gene families is due in part to the presence of many functionally redundant paralogs, which makes gene knockout studies challenging. in such cases it would help if one can identify and work with low copy number orthologous gene families in more accessible model parasites. besides unknown functions, the evolutionary history of many plasmodium variant gene families is also poorly understood. for example, standard sequence similarity searches reveal no obvious homologs for the major surface antigens of p. falciparum and p. knowlesi outside their respective species, raising the question about their evolutionary origin. similarly, there are currently no known pir homologs in p. falciparum, although rif/stevor has been suggested as related gene family based on shared sequence motifs and secondary structural features. the identification of functional homologs of vsas across plasmodium species is important because it aids comparative immunological studies, gives new insights into the evolutionary adaptation of malaria parasites to their respective hosts, and provides a means to transfer functional annotations from model to human parasites and vice versa. in this study, we use a recently developed comparative gene family classification strategy to classify vsas and exported proteins across seven plasmodium genomes, including p. falciparum, p. vivax, p. knowlesi, p. yoelii, p. chabaudi, p. berghei, and p. gallinaceum. we hypothesized that the sensitive sequence-based clustering of the entire body of currently available plasmodium proteins will yield new insights into vsa function and evolution, which in turn could open up new avenues for vaccine development. in this strategy, protein sequences from plasmodium genomes are first clustered into a hierarchical tree using average-linkage clustering and the resulting tree is then searched for clusters corresponding to known vsa gene families. finally, identified clusters are closely analyzed for gene content and inter-cluster relationships. this analysis resulted in several noteworthy findings, including the identification of unusually well conserved pir orthologs that are of potential interest for vaccine development, prediction of the likely function of pyst-a proteins, discovery of a novel and putatively functional pfmc-2tm domain named mc-tyr, new conclusive evidence supporting the common evolutionary origin of pfemp sicavar, and surfin proteins, and the identification of many new vsa gene family members, including new phist gene family members in rodent parasites. collectively, these findings provide vital starting points for future experimental studies.  curation and comparative classification of variant gene families in plasmodium genomes we curated vsa and exported gene families by reviewing the literature as well as gene annotations in plasmodb. we were interested in all plasmodium gene families that met one of the following criteria: expressed at the parasite surface or the surface of infected erythrocytes; predicted host cell localization by virtue of the presence of a pexel/vts export motif; any other gene family that is species -specific or located at subtelomeric regions of chromosomes. using these criteria, we compiled a list of gene families, which from now on we refer to as plasmodium variant gene families. plasmodium variant gene families and classification performance fifty-nine plasmodium variant gene families were curated from the literature and public databases and categorized as follows: vsas; vsas linked to erythrocyte invasion; predicted to be exported to the host erythrocyte; other subtelomeric or species-specific gene families. better studied gene families are shown at the top of each category. classification performance of each gene family is measured in terms of sensitivity, specificity, and jaccard index. the remaining columns show how many genes of each species have been classified to belong to this gene family. numbers in parentheses indicate how many genes of each species served as reference gene family members. a more comprehensive version of this table including numbers of identified genes in non-plasmodium species is available in additional file abbreviations: vsa… variant surface antigen; pfal… p. falciparum; pviv… p. vivax; pkno… p. knowlesi; pyoe… p. yoelii; pber… p. berghei; pcha… p. chabaudi; pgal… p. gallinaceum. after curation, we classified plasmodium variant gene families using a comparative gene family classification strategy described previously. briefly, we hierarchically clustered the combined set of all protein sequences with mc-upgma and extracted those sub-trees that maximally overlapped with the curated gene families. most of the curated gene families were resolved with both high sensitivity and specificity. average and median jaccard indices of extracted clusters were and, respectively. almost half of gene families were clustered perfectly with a jaccard index of. an additional gene families clustered with a good jaccard index ≥ . only gene families clustered with lower quality due to low sensitivity, low specificity or both. overall, we conclude that our clustering strategy works well on plasmodium variant gene families and produces high-quality protein sequence clusters. pir contains single ultra-conserved ortholog in each plasmodium species the large pir gene family separates nicely into two large non-overlapping clusters of high sensitivity and specificity. one cluster represents the vir and kir subfamilies of p. vivax and p. knowlesi, respectively, and the other cluster represents the yir, bir, and cir subfamilies from p. yoelii, p. berghei, and p. chabaudi, respectively. the somewhat reduced specificity of the vir/kir gene cluster is due to inclusion of the pv-fam-c gene family that likely represents a bona fide vir subfamily. in addition, the vir/kir cluster contains p. vivax hypothetical proteins, many of which carry a predicted vir domain and are thus likely true but currently unannotated members of the vir gene family. to our surprise we found three rodent pir proteins nested deep within the vir/kir cluster instead of clustering with their respective paralogs. this unexpected result suggested that some of the otherwise highly divergent pir proteins are well conserved across species. subsequent comprehensive analysis of pir sequence conservation revealed that each of the five plasmodium species contains a single and likely orthologous pir protein that is clearly better conserved between species than any other member of the pir family. the five genes are: p. vivax vir14-related protein pvx_113230; p. knowlesi kir protein pkh_114850; p. yoelii hypothetical protein py06119; p. berghei bir protein pbanka_010050; and p. chabaudi cir protein pchas_ orthology of these five pir proteins is supported by orthomcl db and by the fact that gene synteny is conserved among the five species. multiple sequence alignment of the five conserved pir orthologs reveals a aa long, gapless block recognized as the plasmodium_vir domain. the block spans 56% of the average sequence length and has columns perfectly conserved. this exceptional high degree of sequence conservation within an otherwise highly divergent gene family suggests that these genes are direct descendants of the founder member of the large pir gene family and that they serve an ancestral and probably special function in malaria parasites. identification of the likely prototypical members of the large pir gene family allowed us to re-examine whether there exists conserved pir homologs outside p. vivax/p. knowlesi and rodent malaria parasites, in particular within the laverania clade including p. falciparum. ncbi psi-blast search seeded with the five conserved pir orthologs did not reveal significant sequence similarity within p. falciparum or any other species not currently known to contain pir proteins. similarly, multiple rounds of jackhmmer searches seeded with the alignment from figure  did not identify convincing homologs. thus, despite extensive sequence conservation between the primate and rodent parasites, pir is apparently not conserved in other plasmodium or apicomplexan species, suggesting that pir surface antigens perform an important function unique to the p. vivax/p. knowlesi and rodent clade of malaria parasites. pyst-a proteins predicted to be involved in lipid binding and transfer gene family expansion is an important genomic process by which parasites adapt to different lifestyles and host environments. our classification strategy readily identifies differentially expanded gene families in several plasmodium species, including rhoph1/clag, surfin and acs, msp- msp- and pv-fam-d, trythra/pvtrag, phist and hyp, rbp/235 kda and pyst-c, and pcema pc-fam, and pst-a. one of the most striking examples of a differentially expanded gene family in plasmodium parasites is the p. yoelii gene family pyst-a. pyst-a has been shown to contain only a single member in primate malaria parasites but over one hundred members in p. yoelii and p. chabaudi. in our analysis, pyst-a proteins cluster almost perfectly within a larger cluster containing also pc-fam- and pb-fam- proteins, clearly showing that these three gene families are equivalent in the three rodent parasite species. total gene numbers in the combined pyst-a/pc-fam-1/pb-fam- sequence cluster are genes in p. yoelii, p. chabaudi, and p. berghei, respectively. as expected, we found only a single pyst-a gene family member in primate malaria parasites. a single copy of pyst-a was also detected in the bird parasite p. gallinaceum, supporting the idea of extensive proliferation of the pyst-a gene family in the rodent malaria parasite lineage. interestingly, while examining annotated pyst-a gene models, we noticed that most pyst-a gene family members carry a predicted bet v1-like superfamily domain, suggesting a role of pyst-a proteins in lipid transport. in search for further bioinformatics support for this possibility, we used i-tasser to predict the tertiary and secondary structure of p. falciparum gene family member pf14_. the predicted protein structure is very similar to several human proteins carrying the steroidogenic acute regulatory-related lipid transfer domain, including pdb protein 1em. taken together, our bioinformatics analysis suggests that the second largest rodent malaria parasite gene family pyst-a is involved in lipid binding and transport, probably playing a role in host cholesterol salvage. pfmc-2tm proteins are conserved in rodent parasites and related to hyp and hyp through the novel mc-tyr domain hierarchical protein sequence clustering is a powerful approach to elucidate distant evolutionary relationships between gene families. table  summarizes known and novel evolutionary links between plasmodium variant gene families as suggested by hierarchical clustering. novel links of note include links between pfmc-2tm and hyp  = 100%; average e-value of blast match pairs  =  ), pfmc-2tm/hyp and hyp, hyp and hyp, hyp and hyp, hyp pv-fam-h, and pk-fam-c, as well as pk-fam-a, pk-fam-b and pv-fam-d. thus, several plasmodium variant gene families currently assumed to be species-specific have in fact putative members in other plasmodium species, opening up new avenues for elucidating gene function. of note, we found that pv-fam-c is entirely nested within the larger vir/kir cluster and thus likely represents a novel vir subfamily that we named vir pyst-d, a family of ~ short p. yoelii proteins, was found to be completely nested within the large pir gene family. inspection of pyst-d and neighboring gene models revealed that this gene family is likely spurious and consists entirely of misannotated exons belonging to adjacent yir gene models. other novel links in table  are of lower connectivity and should be regarded as tentative. plasmodium variant gene families predicted by hierarchical clustering the hierarchical tree was searched for neighboring and parental clusters where both clusters map to variant gene families as identified in table  such cluster pairs are shown if their blast match pair percentage is ≥ 1% or they share at least blast match pairs. avg. e-value and avg. pid denote the average e-value and percent identity of all blast match pairs between the two clusters. size a and size b indicate numbers of genes in respective clusters, excluding annotated pseudogenes and gene fragments. an asterisk next to cluster a indicates that it is a parental cluster of cluster b, i.e. cluster b is fully contained in cluster a. our search predicts both previously established and novel evolutionary links between plasmodium variant gene families. pfmc-2tm is an actively studied p. falciparum vsa gene family, which motivated us to investigate its putative link with the two p. falciparum hypothetical gene families hyp and hyp in detail. the annotated pfmc-2tm proteins cluster first with two p. falciparum gametocyte-exported proteins of the hyp gene family and then with a cluster of three p. falciparum exported proteins of unknown function, one of which is a member of the hyp gene family. the second annotated hyp gene family member is not found in this cluster and instead groups with hyp and pv-fam-h proteins, supporting the previously expressed idea that hyp does not constitute a real gene family. the combined pfmc-2tm/hyp8/hyp cluster contains a total of proteins and is shown in figure 3a. notably, this cluster also includes six rodent parasite genes of unknown function that share the typical two-exon gene structure with pfmc-2tm genes, including a signal peptide on the short first exon and a conserved pexel-like motif on the 5′ end of the larger second exon. most importantly, phobius predicts two adjacent c-terminal transmembrane domains for four of the six rodent parasite proteins as well as traces of a second tm domain in the remaining two. no tm domains are present in the three p. falciparum proteins clustering with hyp protein pfb0926c. further inspection of the pfmc-2tm/hyp8/hyp gene cluster using multiple protein sequence alignments identified a hitherto unknown ~ aa long conserved domain located between the pexel motif and the two tm domains. the domain has a predicted three-alpha helical structure and contains two almost invariant tyrosine residues of potential functional importance. only in three proteins, one of the two tyrosine residues is conservatively replaced with phenylalanine. based on this observation, we name this novel domain the maurer’s clefts tyrosine domain. to explore the possibility that mc-tyr is conserved beyond the seed cluster shown in figure  we carried out sensitive profile-based searches using psi-blast. five rounds of ncbi psi-blast initiated with any one of the pfmc-2tm mc-tyr protein sequences consistently identified three additional p. falciparum hits, albeit above psi-blast’s inclusion threshold. these three hits include one of three annotated hyp proteins and two other exported proteins of unknown function. like the three proteins of the hyp cluster, these three genes share the typical two-exon gene structure with pfmc-2tm genes except for the tm domains. this data suggests that mc-tyr is likely conserved in additional exported p. falciparum proteins and probably includes the p. falciparum hyp gene family. we should mention that neither protein sequence clustering nor psi-blast searches identified a relationship of pfmc-2tm with hyp pyst-b or vird proteins, which has been proposed previously based on shared gene architectural features. taken together, we provide first evidence that pfmc-2tm proteins carry a novel and putative functional domain named mc-tyr. mc-tyr is conserved in other p. falciparum exported proteins and additional plasmodium species, opening up new opportunities to experimentally characterize the function of this important vsa gene family. pfemp sicavar, and surfin are interrelated through a modular and structurally conserved intracellular tryptophan-rich domain as shown in table  hierarchical clustering supports the previously proposed link between the well-studied plasmodium vsa gene families var, sicavar, and surfin/pvstp. unexpectedly, the surfin/pvstp cluster includes two genes from p. gallinaceum that have very similar gene structures to p. falciparum surfin genes, suggesting that surfins are conserved outside human malaria parasites. further up the tree, surfin/pvstp proteins first group with p. knowlesi sicavar proteins and then with another large cluster containing the two p. falciparum gene families var and dbl as well as six other dbl domain-containing proteins, including two msp proteins, maebl, ama, giant protein pf, and one protein of unknown function. notably, still further up the tree the surfin/pvstp1/sicavar/var gene cluster is joined by the large gene family pir, uniting all major plasmodium vsa gene families except rifin/stevor in a single gene cluster. however, with only blast high-scoring segment pairs in total and an average e-value of the connectivity between surfin/pvstp1/sicavar/var and pir was found to be extraordinarily low. because rifin/stevor genes are not part of this unified vsa gene cluster we conclude that there is no sequence-based evidence supporting a link of rifin/stevor and pir. however, as did others before us, we noticed suspicious similarities in some protein features that make such a link plausible. to better understand the nature of the relationship among surfin/pvstp sicavar, pfemp and the dbl-containing proteins, we investigated the sequence similarity matrix of this cluster as well as pairwise blast sequence alignments. as revealed by the sequence similarity heat map, sicavar and pfemp proteins share no direct pairwise sequence similarity with each other but exhibit weak similarity with surfin/pvstp proteins. notably, one p. knowlesi sicavar protein and two of the dbl-containing proteins stand out by having high sequence similarity not only with paralogs of their respective gene families but also with surfin/pvstp proteins. by examining the underlying blast alignments we found that blast consistently identified hsps within the c-terminal intracellular regions of proteins, where, in surfin/pvstp proteins, the hsps overlapped with the previously described tryptophan-rich domain. in pfemp proteins, the hsps overlapped with the pfemp intracellular region also known as the acidic terminal sequence or varc. subsequent multiple sequence alignments of surfin, pfemp and sicavar intracellular regions revealed a more complete picture of the relationship of these proteins than described previously. we find that surfin wrd has a modular architecture consisting of two distinct and structurally conserved subdomains, which we named wrd-a and wrd-b. wrd-a and wrd-b occur in variable numbers and configurations in different gene families. the typical wrd consist of one wrd-a and two wrd-b subdomains, which is found in p. falciparum and p. gallinaceum surfin proteins, the two full-length p. vivax pvstp proteins, and the two p. falciparum dbl-containing proteins, including pf and pfa0665w. where wrds occur in tandem, the first of the two wrds is typically better conserved, as illustrated by the partially empty dashed rectangles representing cases where complete wrds but not individual subdomains reached statistical significance in a hmmer search. in other gene families, wrd is only partially conserved. the typical sicavar protein contains a single wrd-a but no wrd-b subdomain, while pfemp proteins carry one copy of both wrd-a and wrd-b separated by a ~ aa long variable region. during our studies an experimentally determined three-dimensional structure of the pfemp intracellular ats domain became available, which shows that ats consists of a conserved core composed of four alpha helices. these four helices map nicely to the two conserved homology blocks corresponding to wrd-a and wrd-b, supporting the biological significance of these alignments. notably, we identified at least one p. knowlesi sicavar protein with a completely conserved wrd, which explains the high blast sequence similarity of this protein with p. falciparum surfins and provides, for the first time, compelling alignment-based evidence that the two antigenically variant gene families var and sicavar are evolutionarily linked via shared intracellular wrd domains. a tblastn search against the p. knowlesi genome using wrd-b of pkh_ as query identified one additional sicavar antigen carrying a wrd-b subdomain, but this gene appears to be severely truncated and lacks wrd-a, suggesting that pkh_ is possibly a pseudogene. new members of variant gene families predicted by sequence clustering sequence clustering complements consensus-based strategies for the identification of gene family members. we therefore examined gene clusters of sufficient quality for the presence of putative new gene family members. table  lists newly predicted members of several p. falciparum gene families, including phist, hyp4/hyp, tsp_, msp-, msp-, etramp, hyp, hyp, and hyp. in none of these cases could we identify obvious cases of misclassifications, suggesting that most of these genes are indeed potential novel members of these gene families. p. falciparum gene family members of variant gene families clusters corresponding to variant gene families were searched for p. falciparum proteins that are currently not annotated as members of these gene families. only clusters with a jaccard index of at least were considered in this search. annotated pseudogenes and gene fragment not shown. mp # and mp% indicate number and percentage of blast match pairs between the putative new member and all other proteins in the cluster representing that gene family. avg. e-value and avg. pid denote the average e-value and the average pid of these blast match pairs, respectively. aunusual phist gene family member carrying an n-terminal acyl-coa-binding protein domain. to verify the credibility of these predictions, we examined the clustering result of the large and highly divergent phist gene family in more detail. phist is a large subtelomeric gene family of exported proteins of unknown function with ~ members in p. falciparum, ~ members in p. vivax, and ~ members in p. knowlesi. so far only one gene family member has been identified in each of the three rodent parasite genomes, suggesting extensive radiation of the phist gene family in primate parasites. in p. falciparum, phist has been divided into three subfamilies. another chromosome-internally expanded p. vivax gene family of genes named rad has later been shown to be closely related to phist. in our results, the phist-a, phist-b, phist-c, and rad gene families are resolved with high accuracy and as expected cluster together into the larger phist gene family. phist-c first clusters with rad followed by phist-b and phist-a, which differs from an earlier report suggesting that phist-a and phist-b form a clade with phist-c being the out-group. in total, the phist cluster contains full-length proteins that are currently not annotated as phist gene family members, including the four genes from p. falciparum mentioned earlier, and pf10_0015), two from p. vivax, from p. knowlesi, and one from each p. berghei and p. chabaudi. the two novel rodent parasite phist family members cluster with the phist-c subfamily and could be interesting targets for experimental studies of phist function in the rodent model system. multiple protein sequence alignment of these putative new phist gene family members shows that key residues of the phist presan domain are well conserved, suggesting that these genes are likely true members of the phist gene family. notably, the phist cluster also contains two proteins from p. gallinaceum, supporting previous findings that phist is conserved in the bird parasite. we conclude that our clustering procedure resolves even challenging gene families correctly and is a successful strategy to identify putative new gene family members in p. falciparum and other plasmodium species. discussion in this study, we classified and investigated vsa and exported gene families across seven plasmodium genomes. this led to several interesting findings, some of which may have application value. conserved pir proteins are targets for functional studies and vaccine development the identification of a single ultra-conserved pir ortholog in each of the pir-containing plasmodium species was highly unexpected given that pir is the largest and one of the most diverse gene families in plasmodium parasites. pir gene family sizes range from in p. knowlesi to in p. yoelii, with amino acid similarities ranging from 30-50% between p. chabaudi, p. yoelii and p. berghei and from 20-30% between p. chabaudi and p. vivax. the same conserved pir orthologs were found recently in an independent work studying genetic diversity of the global p. vivax population. it was shown that p. vivax gene pvx_ is completely invariant across four strains sampled from different sites around the world and also exhibits conserved synteny in rodent malaria parasites. the fact that these highly conserved pir orthologs could be identified in our study clearly highlights the value of comparative genomics in general and of comparative gene family classification in particular. the discovery of ultra-conserved pir orthologs has two important implications. firstly, these genes can now serve as starting points to elucidate pir function, which is still unknown. for example, gene knockouts of the conserved pir orthologs in the more accessible rodent model system may result in important and measurable phenotypes. alternatively, tagging the conserved pir orthologs with different fluorescence markers coupled with the manipulation of their expression levels should also help clarifying pir localization and function. pvx_ has a distinct expression profile in erythrocytes relative to most other vir genes and therefore may serve a different, ancestral function. if true, insights from the proposed experiments may not be directly applicable to other members of the gene family, but should nevertheless be informative. secondly, the finding of a conserved pir ortholog in p. vivax has potential ramifications for p. vivax vaccine development. if, like other vir proteins, pvx_ is confirmed as surface-exposed antigen, antibodies raised either against pvx_ or its hypothetical host receptor could elicit exquisite cross-strain immunity. that parasite genes with limited genetic diversity can be effective vaccine targets was recently demonstrated in p. falciparum, where antibodies against an essential and highly conserved ligand for erythrocyte invasion elicited exquisite neutralizing cross-strain immunity. this seemingly paradoxical situation, that a highly conserved parasite protein can be successfully targeted by the host immune system, makes sense if one imagines pfrh as functionally important and therefore conserved component of a host-parasite interaction complex. within such a complex, pfrh would not be under direct immune attack because it is efficiently shielded by immunodominant paralogs that act as red herrings or “smoke-screen”. a similar model has been proposed for rif/stevor and surfin genes in the context of the merozoite invasion process. if this model applies to pir proteins, then pvx_ could be a promising target for the development of a p. vivax vaccine. lipid-binding pyst-a proteins potentially involved in cholesterol salvage another family of surface antigens that is currently without known function is the large rodent parasite gene family pyst-a. studies of pyst-a expression have shown that the pyst-a family member and glutamate-rich protein pc pc and pch105/resa ) is the immunodominant protein within red blood cell membranes and localizes to the cytoplasmic face of the membrane. more recently, rbc membrane localization of a pyst-a homolog has also been demonstrated in p. berghei. pyst-a genes are concurrently expressed in large numbers without evidence of differential expression in response to different host environments, suggesting that altering expression of pyst-a gene family members is not an immune evasion strategy of the parasite. using bioinformatics sequence and structural analysis we predict the existence of a star-related lipid-transfer domain in pyst-a proteins. start is the archetypical domain found in the steroidogenic acute regulatory protein, which is part of a multi-protein complex termed the “transduceosome” whose function in mammals is to transfer cholesterol across the two mitochondrial membranes and to initiate the first enzymatic step in steroid biosynthesis. cholesterol is also a major lipid fraction in the membrane bilipid layer and is required for membrane genesis. plasmodium parasites cannot synthesize cholesterol de novo. it has been shown that host cell membranes of p. falciparum-infected erythrocytes show a 50% decrease in the cholesterol/phospholipid ratio compared to uninfected cells. in addition, malaria infections result in hypocholesterolaemic conditions. together these observations suggest that the parasite scavenges required cholesterol from both host erythrocyte membranes and plasma. how this is accomplished by the parasite is currently not understood. based on the presence of a cholesterol-binding start domain in rbc membrane-localized pyst-a proteins we hypothesize that pyst-a is part of a molecular machinery that diverts cholesterol away from erythrocyte membranes to fuel parasite growth within erythrocytes. notably, p. chabaudi pcema proteins could constitute a second component of this proposed cholesterol import machinery. we found five pc-fam- proteins that share sequence similarity with both pyst-a and pcema proteins. we could not find obvious problems with the gene models of these five proteins, suggesting that they are genuine pyst-a/pcema protein hybrids. co-occurrence of pyst-a and pcema domains within the same polypeptide suggests physical interaction between pyst-a and pcema proteins. this possibility is supported by the fact that pcema proteins also localize to the cytoplasmic face of the rbc membrane. it remains to be shown why rodent malaria parasites maintain so many pyst-a proteins compared to primate malaria parasites. pyst-a proteins are probably immunogenic in rodent but not primate parasites, putting pyst-a proteins under strong selective pressure to diversify. alternatively, massive pyst-a gene amplification could increase the efficiency of cholesterol salvage, probably satisfying an elevated need for cholesterol in rodent parasites or compensating for lower cholesterol levels in rodent blood. pfmc-2tm – conserved beyond p. falciparum another interesting erythrocyte surface-expressed gene family of currently unknown function is the p. falciparum gene family pfmc-2tm. the members of the pfmc-2tm gene family encode putative functional and highly similar basic membrane proteins that, based on structural features, are grouped within the larger 2tm superfamily. the 2tm superfamily consists of 200– aa long proteins that on their first exon carry a signal peptide followed by a n-terminal pexel motif and two c-terminal tm domains on their second exon. besides pfmc-2tm, the 2tm superfamily includes rif/stevor as well as genes in p. knowlesi, p. vivax, and the rodent parasite genomes that share similar architectural features. it is important to note that, other than pir, the large 2tm superfamily is currently united solely based on gene structural features and not sequence similarity; in fact, to date no pfmc-2tm sequence homologs have been identified in p. falciparum or any other species. pfmc-2tm proteins have been localized to the maurer’s clefts, the parasitophorous vacuole, parasitophorous vacuole membrane, and the erythrocyte surface. at this latter location, 2tm proteins including pfmc-2tm are thought to interact with the host immune system through a surface-exposed and hypervariable loop region that is located between the two tm domains. pfmc-2tm proteins are most highly expressed in the mid-trophozoite stage and have been shown to undergo expression switching, suggesting that the pfmc-2tm gene family plays a role in antigenic variation. we provide first evidence that the pfmc-2tm gene family has additional members both within and outside the p. falciparum species. experimental clues for the existence of potential pfmc-2tm homologs in the rodent plasmodium genome already existed, but the identity of these genes remained unknown. furthermore, we showed that pfmc-2tm proteins contain a conserved and putative functional domain located between pexel and the two tm domains, which we named mc-tyr. these two findings have important implications for future studies of pfmc-2tm function. experimental double-knockout studies can now be attempted in rodent parasites on the two pfmc-2tm gene family members to see if they produce an observable phenotype. given the reduced number of pfmc-2tm gene family members in rodent malaria parasites compared to p. falciparum and the more tractable rodent model system, such studies should now be feasible. two additional observations suggest that hyp hyp and the six rodent malaria proteins are genuine members of the extended pfmc-2tm gene family. first, all but three of the putative new pfmc-2tm members have a similar basic isoelectric point as known pfmc-2tm proteins. second, all newly identified members that have two tm domains carry a single proline residue within the second tm domain, another known characteristic of pfmc-2tm proteins. proline residues internal to helices are often found in transporters, channels, and receptors, and tend to be conserved between homologous proteins. in contrast, we did not observe conservation of the two cysteine residues preceding the tm domains. also the fact that no tm domains are present in three of the proteins carrying mc-tyr is intriguing and suggests that the presence of two tm domains is not essential for a possible mc-tyr function. it is also noteworthy that most of the hyp and hyp gene family members are more broadly expressed throughout the intra-erythrocytic life cycle than pfmc-2tm proteins, showing expression not only in the mid-trophozoite stage but also in early ring and schizont stages as well as in gametocytes. this suggests that the function of mc-tyr containing proteins is not restricted to trophozoites. our clustering results do not provide evidence for a link between pfmc-2tm and the pyst-b gene family as has been proposed previously based on gene structural features. wrd as missing link between major plasmodium vsa gene families besides the large 2tm superfamily, malaria parasites express additional vsa gene families on cell surfaces. among them are the var gene family in p. falciparum and sicavar in p. knowlesi, both of which encode proteins expressed at the erythrocyte surface and are known to undergo antigenic variation. surfin, a family of p. falciparum proteins, has been shown to be expressed on the surfaces of both infected erythrocytes and merozoites. by examining intracellular regions of co-clustered surfins, pfemp and sicavar proteins, we now better understand the sequence conservation pattern present among these major plasmodium vsas. we found that surfin proteins contain twice as many wrds as reported previously and that each wrd consists of three conserved blocks. the first block, named wrd-a, is found once in each of the wrds, whereas block two and three represent two copies of a second subdomain that we named wrd-b. winter et al. previously described the conservation pattern among wrd-containing proteins in terms of shared conserved elements denoted s s s2*, s2**, and pexel-like motif. compared to this earlier study, we find that wrd-a corresponds to segment s plus five additional c-terminal amino acids in wrd-a, which map to the second alpha helix of the protein structure. wrd-b corresponds roughly to the c-terminal half of segment s and segment s2* is in fact the first of the two wrd-b copies found within this protein. the short s2** subsegment, indicating another duplication at the end of each wrd in surfin, is in fact the most conserved part of the second copy of the wrd-b subdomain in this protein. the pexel-like motif localized within the s segment was also found to be well conserved in our study. interestingly, this pexel-like motif currently has no correspondence in the reported tertiary structure, which might be explained by the fact that the ats-core deletion construct that was used to determine the ats structure lacked this pexel-like motif. it thus remains to be shown if the pexel-like motif is in fact part of ats-core or serves another conserved function in wrds. importantly, our data provides the first conclusive alignment-based evidence that the two major antigenically variant gene families in p. falciparum and in p. knowlesi share common evolutionary origin, substantiating the usefulness of the p. knowlesi-rhesus monkey model for the in vivo study of p. falciparum antigenic variation. in winter et al. provided the first bioinformatics evidence that pfemp and sicavar proteins are probably evolutionarily linked through their intracellular domains, which share partial sequence similarity with the intracellular tryptophan-rich domain of surfin proteins. short conserved sequence motifs have also been identified in var and sicavar introns as well as in extracellular regions of these proteins. however, at present the picture remains highly fragmented and reported sequence similarity of intracellular regions between pfemp and sicavar is essentially limited to a highly conserved six amino acid long pexel-like motif, questioning the robustness of these observations. our study provides new evidence for common ancestry of pfemp and sicavar, which rests on three critical observations. firstly, not only wrd-b but also wrd-a is entirely conserved in pfemp proteins. secondly, at least one sicavar protein has a completely conserved wrd, consisting of one wrd-a and two wrd-b subdomains. thirdly, complete wrds are conserved in at least two other p. falciparum proteins, which are clearly related to pfemp proteins through n-terminal dbl domains. why were these sequence similarities not detected previously? unlike surfins, pfemp proteins have wrd-a and wrd-b separated by a larger variable insertion, which in the protein structure does not adopt a stable fold. similarly, compared to surfins, pfemp proteins carry additional insertions both within wrd-a and wrd-b. in wrd-a, this insertion splits the highly conserved pexel-like motif from the rest of the wrd-a subdomain. the presence of these insertions in pfemp proteins combined with the fact that wrd-b is absent in all but one sicavar protein likely thwarted earlier attempts to find significant sequence conservation between pfemp and sicavar proteins. it is not too surprising that larger blocks of conserved sequence between pfemp and sicavar are confined to intracellular regions as extracellular regions are likely under strong diversifying selection pressure from the host immune system. consistent with earlier reports, we did not find evidence that wrd or any of its subdomains is conserved within the large pir gene superfamily of plasmodium surface antigens. what is the function of wrd? despite this now much improved picture of the conservation structure of pfemp sicavar, and surfin intracellular domains, many fundamental questions remain. why do surfins have multiple copies of complete wrds while most other gene families carry only a single wrd? what is the functional significance of having two wrd-b subdomains vs. having only one wrd-b subdomain? why is wrd-b found in only a single sicavar protein while all other members of the sicavar gene family carry only the wrd-a subdomain? unfortunately, the molecular function of wrd is currently not well understood, so these questions are difficult to answer. studies have shown that ats of pfemp interacts either directly or indirectly via the knob-associated histidine-rich protein with erythrocyte membrane skeletal proteins, including actin and spectrin. ats is thus believed to anchor pfemp at the membrane of infected rbcs, which strengthens and stabilizes the roots of pfemp1-endothelial receptor interaction. similarly, pf which contains a single complete wrd near its c-terminal end, was found to contribute to the overall deformability of p. falciparum-infected erythrocytes through anchoring and scaffolding, probably through wrd-mediated binding of pf to actin. it seems therefore plausible that the main function of wrd is to provide a scaffold for anchoring surface antigens like pfemp to the rbc cytoskeleton. the function of having many wrds resulting from gene family expansion and/or intra-protein domain amplification is then probably to increase the strength of this scaffold. this would explain why particularly p. falciparum, which compared to other malaria parasites has strong cytoadherence properties, expresses so many wrds in form of the surfin gene family. surfins have not yet been implicated in anchoring surface antigens to the erythrocyte cytoskeleton, but we think that this is a plausible hypothesis that should be tested experimentally. it is also worth mentioning that the abbabbabb-like repeat structure of the surfin wrds is reminiscent of similar repeats present in other cytoskeleton-binding proteins, including the vertebrate titin and the invertebrate twitchin muscle proteins. regardless of the exact function of wrd, its modular nature suggests that different proteins carrying distinct wrd subdomains act in concert to perform this function. p. knowlesi protein pkh_ which appears to be the only functional protein in the p. knowlesi genome carrying both wrd-a and wrd-b, thus probably interacts physically with other, wrd-a-containing sicavar proteins to anchor sicavar to the cytoskeleton. with the refined domain structure presented here it should now be possible to test this prediction experimentally. ultimately, such experiments will hopefully expand our knowledge of how p. falciparum accomplishes cytoadherence at the molecular level, which is responsible for much of the virulence of this parasite.  sensitive clustering of protein sequences followed by manual examination of clustering results revealed many new insights into variant surface antigens of malaria parasites, including atypical and new gene family members, novel domains, new protein function predictions, and a better understanding of the evolutionary relationship and origin of these important proteins. the findings presented in this work can now jump-start follow-up experimental research around the world, most importantly to elucidate the function of pvx_ and to explore its potential value as p. vivax vaccine target. 
plasmodium parasites, the causative agents of malaria, express many variant antigens on cell surfaces. to gain further insights into vsa function and evolution, we comparatively classified and examined known vsa gene families across seven plasmodium species. variant surface antigens are typically organized into large subtelomeric gene families that play critical roles in virulence and immune evasion. furthermore, we predict a lipid-binding domain in erythrocyte surface-expressed pyst-a proteins, suggesting a role of this second largest rodent parasite gene family in host cholesterol salvage. additionally, it was found that pfmc-2tm proteins carry a novel and putative functional domain named mc-tyr, which is conserved in other p. falciparum gene families and rodent parasites. many important aspects of vsa function and evolution remain obscure, impeding our understanding of virulence mechanisms and vaccine development. we identified a set of ultra-conserved orthologs within the largest plasmodium gene family pir, which should be considered as high-priority targets for experimental functional characterization and vaccine development. our comparative analysis of plasmodium vsa gene families revealed important functional and evolutionary insights, which can now serve as starting points for further experimental studies. malaria parasitesplasmodiumcomparative genomicsvariant surface antigensgene family classificationvaccine target
 plasmodium parasites, the causative agents of malaria, express many variant antigens on cell surfaces. variant surface antigens are typically organized into large subtelomeric gene families that play critical roles in virulence and immune evasion. many important aspects of vsa function and evolution remain obscure, impeding our understanding of virulence mechanisms and vaccine development. to gain further insights into vsa function and evolution, we comparatively classified and examined known vsa gene families across seven plasmodium species.  we identified a set of ultra-conserved orthologs within the largest plasmodium gene family pir, which should be considered as high-priority targets for experimental functional characterization and vaccine development. furthermore, we predict a lipid-binding domain in erythrocyte surface-expressed pyst-a proteins, suggesting a role of this second largest rodent parasite gene family in host cholesterol salvage. additionally, it was found that pfmc-2tm proteins carry a novel and putative functional domain named mc-tyr, which is conserved in other p. falciparum gene families and rodent parasites. finally, we present new conclusive evidence that the major plasmodium vsas pfemp sicavar, and surfin are evolutionarily linked through a modular and structurally conserved intracellular domain.  our comparative analysis of plasmodium vsa gene families revealed important functional and evolutionary insights, which can now serve as starting points for further experimental studies. malaria parasitesplasmodiumcomparative genomicsvariant surface antigensgene family classificationvaccine target 