Trichomonas vaginalis vast BspA-like gene family: evidence for functional diversity from structural organisation and transcriptomics
© Noël et al; licensee BioMed Central Ltd. 2010
Received: 2 October 2009
Accepted: 8 February 2010
Published: 8 February 2010
Trichomonas vaginalis is the most common non-viral human sexually transmitted pathogen and importantly, contributes to facilitating the spread of HIV. Yet very little is known about its surface and secreted proteins mediating interactions with, and permitting the invasion and colonisation of, the host mucosa. Initial annotations of T. vaginalis genome identified a plethora of candidate extracellular proteins.
Data mining of the T. vaginalis genome identified 911 BspA-like entries (TvBspA) sharing TpLRR-like leucine-rich repeats, which represent the largest gene family encoding potential extracellular proteins for the pathogen. A broad range of microorganisms encoding BspA-like proteins was identified and these are mainly known to live on mucosal surfaces, among these T. vaginalis is endowed with the largest gene family. Over 190 TvBspA proteins with inferred transmembrane domains were characterised by a considerable structural diversity between their TpLRR and other types of repetitive sequences and two subfamilies possessed distinct classic sorting signal motifs for endocytosis. One TvBspA subfamily also shared a glycine-rich protein domain with proteins from Clostridium difficile pathogenic strains and C. difficile phages. Consistent with the hypothesis that TvBspA protein structural diversity implies diverse roles, we demonstrated for several TvBspA genes differential expression at the transcript level in different growth conditions. Identified variants of repetitive segments between several TvBspA paralogues and orthologues from two clinical isolates were also consistent with TpLRR and other repetitive sequences to be functionally important. For one TvBspA protein cell surface expression and antibody responses by both female and male T. vaginalis infected patients were also demonstrated.
The biased mucosal habitat for microbial species encoding BspA-like proteins, the characterisation of a vast structural diversity for the TvBspA proteins, differential expression of a subset of TvBspA genes and the cellular localisation and immunological data for one TvBspA; all point to the importance of the TvBspA proteins to various aspects of T. vaginalis pathobiology at the host-pathogen interface.
Trichomonas vaginalis is a flagellated protist responsible for the most prevalent non-viral sexually transmitted infection (STI), with an annual estimate of 174 millions new infections worldwide , corresponding to at least the combined estimates of Chlamydia trachomatis and Neisseria gonorea infections, and which has, paradoxically, attracted so far relatively little attention from health agencies worldwide [2, 3]. The parasite is capable of causing severe vaginal, ectocervical, prostatic and urethral inflammations, and is linked with sterility, pelvic inflammatory disease, adverse pregnancy outcomes, postnatal complications and cervical cancers [4–7]. Furthermore T. vaginalis also contributes, along with other STI, to the HIV pandemic by boosting the efficiency of virus transmission through several possible mechanisms including induction of inflammatory response resulting in neutrophils and macrophages recruitment into urogenital mucosa, compromising the mucosal barrier through microhaemoragia, increasing viral load in urogenital mucosa secretions and as a carrier (a Trojan horse) of infective HIV particles [6, 8, 9]. Hence, T. vaginalis is capable of invading and colonising the heavily defended host urogenital mucosa from both sexes, braking through the primary innate defences and withstanding induced innate and adaptive responses, about which little is known in relation to T. vaginalis infections . Notably, T. vaginalis infections are often considered non-self limiting in females and recent data even suggest that persistent, undetected infections can persist even after successful treatments .
The pathobiology of T. vaginalis is complex and multifaceted with adhesion to, and alterations of, the various mucosal landmarks (mucus, epithelial cell barrier, extracellular matrix [ECM], innate and adaptive immune cells, bacterial microflora) thought to be essential to initiate and maintain infections [4, 12, 13]. T. vaginalis cells are also known to form large cell aggregates (in a process called swarming or rosetting), which could represent an important process for pathogenesis , suggesting that specific cell-cell interactions also take place between cells of the parasite. When the mucosal tissue is damaged the parasite can bind to host ECM proteins  and during menstruation or parasite induced microhaemoragia, T. vaginalis also binds to various plasma proteins . Adhesion to host tissue also induces a cellular differentiation of T. vaginalis into amoeboid forms [16, 17]. Furthermore the parasite endocytoses host proteins (e.g. lactoferrin and laminin) [4, 18], as well as various human viruses [9, 19], and phagocytoses the autochthonous mucosal microflora and various host cells [20, 21], including spermatozoids ; key cellular processes for nutrient uptake thought to dependent on specific surface proteins. However, little is known about the molecular and cellular basis of these various processes, with the pathogen lipophosphoglycan (LPG), various adhesions, surface and secreted enzymes and toxins all thought to be involved, but existing data are limited when not controversial [12, 13, 15, 23–27]. A so far unique human receptor for T. vaginalis, galectin-1, was only recently identified when investigating the role of T. vaginalis LPG in binding to ectocervical epithelial cell lines .
An initial gene survey of expressed sequence tags (EST) identified T. vaginalis cDNA encoding 65 distinct proteins we named BspA-like (TvBspA) , due to their similarity with the BspA protein from Tannerella forsythensis, and led us to further characterise in silico one complete open reading frame (ORF) encoding a potential surface protein TvBspA625 . TvBspA proteins are characterised by a specific type of leucine-rich repeats (LRR), named TpLRR after a membrane protein from Treponema pallidium, shared with T. forsythensis BspA and Treponema denticola LrrA proteins . This discovery was particularly appealing for T. vaginalis because the BspA and LrrA proteins were shown to be expressed on the bacteria cell surface and to be involved in the colonisation of the oral mucosa; BspA binds to ECM protein fibronectin and to the clotting factor fibrinogen and both BspA and LrrA stimulate co-aggregation between the two bacteria, and promote their adhesion to and invasion of epithelial cells [31–34]. Furthermore, the TpLRR of the T. forsythensis BspA protein was shown to trigger an innate immune response by inducing IL-8 secretion in epithelial cells via toll-like receptor 2 (TLR2) and TLR1 signalling . The BspA protein was also shown to elicit a strong antibody responses in T. forsythensis infected patients . Hence, TvBspA proteins could play similar roles and mediate important interactions with mucosal features including its microflora and host cells and proteins. The availability of one T. vaginalis genome sequence , ~70,000 EST from several different growth conditions and 75 distinct TvBspA genes spotted on microarrays gave us the opportunity to investigate the TvBspA genome complement and perform a first exploration of their corresponding transcripts to gain further insight into their potential importance in host-pathogen interactions. Here, we present an updated survey of genomes encoding BspA-like proteins and the first detailed bioinformatic characterisations of TvBspA genomic distribution and exceptional structural diversity. We demonstrated differential expression at the transcript level of selected TvBspA genes upon T. vaginalis binding to ECM proteins or exposed to different iron concentration; and TvBspA625 cell surface expression and host antibody response during infection. Together these data strongly indicate that TvBspA proteins are likely to play several important and distinct roles in T. vaginalis pathobiology and provide the fundamental data for future TvBspA genes and proteins comparison between various T. vaginalis clinical isolates and to initiate TvBspA proteins detailed functional characterisation.
The T. vaginalis genome encodes an exceptionally large putative BspA-like protein family
Taxonomic distribution of genomes encoding BspA-like proteins
Best Bit score
Trichomonas vaginalis G3
Entamoeba dispar SAW760
Entamoeba histolytica HM-1:IMSS
Methanosarcina barkeri str Fusaro
Aquatic & rumen
Methanosarcina acetivorans C2A
Aquatic & rumen
Methanococcus vannielii SB
Methanococcus maripaludis C7
Eubacterium siraeum DSM 15702
Intestine (human, HMP)
Flavobacterium psychrophilum JIP02/86
Clostridium leptum DSM 753
Intestine (human, HMP)
Syntrophomonas wolfei e
Aquatic & rumen
Clostridium spiroforme DSM 1552
Intestine (human, HMP)
Clostridium beijerinckii NCIMB 8052
Epulopiscium sp 'N.t morphotype B'
Victivallis vadensis ATCC BAA-548
Shewanella pealeana ATCC 700345
Nidamental glands (squid)
Anaerofustis stercorihominis DSM 17244
Intestine (human, HMP)
Bacteroides fragilis NCTC 9343
Treponema denticola ATCC 35405
Alistipes putredinis DSM 17216
Intestine (human, HMP)
Synechococcus sp. WH 7805
Marine & host associated
Ruminococcus torques ATCC 27756
Intestine (human, HMP)
Bacteroides ovatus ATCC 8483
Intestine (human, HMP)
Clostridium sp. L2-50
Intestine (human, HMP)
Ruminococcus torques ATCC 27756
Intestine (human, HMP)
Clostridium butyricum 5521
Coprococcus eutactus ATCC 27759
Intestine (human, HMP)
Photobacterium sp. SKA34
Aquatic, host associated
Kordia algicida OT-1
Aquatic, algae pathogen
Bacteroides stercoris ATCC 43183
Intestine (human, HMP)
Clostridium phytofermentans ISDg
Clostridium bartlettii DSM 16795
Intestine (human, HMP)
Desulfitobacterium hafniense Y51
Features of the 18 TvBspA proteins encoded by scaffold DS113361
Only one TvBspA gene is annotated to possess two exons and 98 entries are annotated as pseudogenes due to stop codons or frame shifts interrupting the inferred ORF (additional file 1, Table S1). Eight TvBspA putative proteins do not possess a starting methionine and three of these are very similar to longer proteins with a starting methionine. There were also 14 TvBspA genes with some ambiguous sequencing data (with tandem repeats of N: A, C, G or T). In addition, a total of 17 TvBspA proteins were derived from ORF that start or end at the extremity of a scaffold suggesting they represent partial sequences, two of which have EST support (additional file 1, Table S1). Hence a total of 137 TvBspA ORF correspond to either mis-annotated, error-containing sequences, derived from genes with overlooked introns, pseudogenes, gene fragments, partially sequenced genes, or have sequencing errors/ambiguities. We focused our more detailed analyses on TvBspA protein sequences most likely derived from full-length genes based on their sequence features, genome context and evidence for transcription.
To allow comparisons of TvBspA protein sequence features and to provide estimates of their phylogenetic relationships, protein alignments and protein subfamilies were computed. These data were used to rationalise the vast structural diversity of the TvBspA proteins and contextualise their genomic organization. Two multi alignments of TvBspA proteins were generated to allow their comparisons: (i) all 911 sequences (additional file 6, Figure S1) and (ii) all 193 proteins with transmembrane domains (TMD) and inferred C-terminal cytoplasmic tails (CT, CCT) (additional file 7, Figure S2). The order of the sequences in the alignment is a reflection of their relatedness with the most similar sequences typically being aligned next to each other and the more divergent sequences overall tend to be aligned last during the alignment estimation and these are located towards the bottom of the alignment  (additional file 1, Table S1). The position of the TvBspA sequences in the two alignments were contrasted with TvBspA protein subfamilies derived from an alignment-free clustering algorithm designed to deal, to some extent, with hard to cluster (and align) repeat containing proteins . A total of 397 subfamilies were identified for the 911 TvBspA (additional file 1, Table S1). In numerous cases there was a good agreement between the subfamilies membership and their juxtaposed position in the alignments (additional file 1, Table S1).
Due to the highly repetitive nature of the T. vaginalis genome the current genome sequence data are fragmented over 17,000 scaffolds , consequently it was only possible to generate a partial picture of the genome organisation for the TvBspA gene family. The 911 TvBspA candidate genes were scattered over 440 scaffolds (size range 1 kbp to 585 kbp, mean 101.4 kbp), with 245 scaffolds encoding one TvBspA and 195 scaffolds encoding two or more TvBspA (up to 18 TvBspA, Table 2) making up the majority and remaining 666 entries. Many TvBspA genes are organised in clusters either in tandem repeats, or in close proximity to each other, with the largest cluster made of 17 TvBspA genes (and five unrelated interspersed genes) over a genomic segment of 46.5 kbp (Table 2; additional file 8, Figure S3). Some of the clustered genes encoded proteins highly similar to each other and are also recovered in the same protein subfamily and/or were aligned beside each other in the global (911 TvBspA) alignment (additional file 6, Figure S1; additional file 1, Table S1). Such patterns suggest local gene duplications generating tandem repeats . We also identified in several cases closely related paralogues (additional file 1, Table S1), which were encoded by genes present on different scaffolds, reminiscent of ectopic duplications events (Table 2 lists one example).
The extensive size of the TvBspA gene family is currently unparalleled. The combined PHI- and PSI-Blast searches that recovered 908 T. vaginalis proteins recovered one to 298 BspA-like sequences in 154 additional RefSeq annotated genomes (Table 1; additional file 2, Table S2, additional file 3, Table S3 and additional file 9, Table S6). The next largest BspA-like gene families were found in Entamoeba dispar (298 EdBspA entries) and Entamoeba histolytica (124 EhBspA entries), the only other eukaryotic genomes, with T. vaginalis, currently known to encode proteins with TpLRR [26, 40] (Tables 1, additional file 3, Table S3). In contrast, prokaryotes encoded fewer BspA-like genes (one to 19 entries) but their taxonomic diversity was much broader including five archaeal species and 147 bacterial species/strains, with the majority of bacterial taxa being members of Firmicutes (70%) or the Bacteroidetes (12%) (additional file 9, Table S6).
Structural diversity of TvBspA proteins
TpLRR containing proteins are thought to be extracellular either as surface exposed or secreted proteins [26, 41]. Notably, T. vaginalis encodes none of the enzymes required for glycosylphosphatidylinositol (GPI)-anchors synthesis and mediating their anchoring to proteins , hence we focused our more detailed sequence analyses on entries with potential signal peptides (SP), TMDs or conserved features located towards the C-terminal end in the absence of evidence for TMD (Figure 1; additional file 1, Table S1). The 193 entries with TMD-CCT (additional file 7, Figure S2) have an inferred membrane topology implying that the entire TpLRR would be exposed to the extracellular milieu if these were to be expressed on the cell surface. Of these 193 TvBspA-TMD-CCT entries 35 also had an inferred SP, defining type I membrane proteins and the 158 entries with no inferred SP defined potential membrane proteins of type III . Among the 719 TvBspA proteins that were considered not to possess TMD, 92 had a detectable SP (Figure 1) and several of these also formed co-aligned subfamilies characterised by conserved C-termini often ending with hydrophobic residues and possessing conserved cysteines and other residues within motifs located ~20-30 residues from the C-terminus (additional file 6, Figure S1).
To initiate the rationalisation of the potential functional significance of the considerable TvBspA protein family, we further investigated their structural diversity and gene expression at the transcript level in different in vitro culture conditions. Selected TvBspA proteins with notable sequence features or included in large subfamilies were also investigated in more details. Our bioinformatic analyses also included the identification of repetitive sequences (in addition to the TpLRR) that are often linked with surface proteins important for host-pathogen interactions in many pathogenic bacteria and microbial eukaryotes and are directly implicated in virulence and pathogenicity, including adhesion to host tissues and immune evasion [43, 44].
The first investigated TvBspA protein sequence, TvBspA625  (Genbank accession AAM51159, corresponding to TVAG_073760 and XP_001321233, TrichDB and RefSeq accession numbers respectively, see additional file 1, Table S1 for all 911 accessions numbers) was recovered among the nine sequences of subfamily #13. TvBspA805 (TVAG_154640) (the number after TvBspA indicates the inferred number of amino acids) was the most similar to TvBspA625 (72% identity) when compared to other members of subfamily #13 (range of pairwise identity: 37% to 54%). The two sequences share a TpLRR-TMD linker segment made of proline and asparagines-rich repeats (P/NRR) (Figure 2) and they are encoded on different scaffolds and separated by at least 67 kbp (additional file 1, Table S1) if not located on different chromosomes. Variations in the number of TpLRR and the P/NRR between TvBspA625 and TvBspA805 indicate differential contractions and expansions of these repetitive segments between the two paralogues (Figure 2). The variations in repetitive sequences of proteins from microbial pathogens are an important source of genetic variations between species/isolate/strains and are thought to correspond to dynamic adaptive responses in host-pathogen interactions (e.g. [43, 44]). Hence we PCR cloned a 3'end segment of the TvBspA625 gene from the clinical isolate SS-22  that encompasses the P/NRR, TMD and CT to compare it with the corresponding G3 sequence (Figure 2C). Although the TvBspA625 protein sequences of isolate G3 and C-1:NIH were identical (Figure 2C) the amplicon of isolate SS-22 was slightly smaller compared to the control amplicon obtained from isolate G3 (data not shown). Sequencing revealed that this difference was due to a reduced number of ENP [NS]QPG repeats (12× P/NRR in protein from isolate G3 and C-1:NIH) with five fewer repeats in isolate SS-22 (7× P/NRR) (Figure 2C). For reason we currently don't understand, several attempts to PCR clone the entire TvBspA625 ORF from strain SS-22 failed using both genomic DNA and cDNA as template. As the entire TvBspA625 ORF could be amplified and sequenced from isolate G3 genomic DNA in control PCR this suggests differences in the 5'end of the TvBspA625 gene between the two clinical isolates and together with the differences in P/NRR indicate that this gene readily accumulate changes between clinical isolates. All members of subfamily #13 did also co-align in the global TvBspA alignment with one intercalated aligned sequence not included in subfamily #13 (additional file 1, Table S1). Four members of subfamily #13 possess a TMD-CCT. In addition to TvBspA625 and TvBspA805, TvBspA786 (TVAG_234090) was also characterised by a PRR in the TpLRR-TMD linker (16 prolines over 49 residues including two NPTPETP repeats) (additional file 6, Figure S1; additional file 10, Table S7).
Two highly similar paralogues TvBspA515 and TvBspA575 (92% identity - TVAG_244780 and TVAG_244800, respectively, members of subfamily #384) were characterised by TpLRR-TMD linker sequences with serine-rich repeats (SRR). The length variation between the two proteins was essentially restricted to the SRR (additional file 6, Figure S1; additional file 11, Figure S4) reminiscent of the variation identified between TvBspA625 P/NRR from two clinical isolates (Figure 2C).
Taxonomic distribution of proteins sharing a glycine-rich domain found in 12 TvBspA.
Top hit annotation
Trichomonas vaginalis G3
Surface antigen BspA-like
Flavobacteria bacterium MS024-3C
Clostridium phage phiCD27
Clostridium phage phi CD119
Clostridium difficile ATCC 43255
Clostridium difficile QCD-63q42
Clostridium difficile QCD-37×79
Clostridium difficile 630
Bacillus cereus Rock4-18
FG-GAP repeat protein
Evidence for TvBspA genes transcription
Microarray data for 13 TvBspA with significant modulation in their mRNA concentration upon exposure to different iron concentration
Subfamily membershipa, b
High iron culture condition
#43, 10 memberse
1.5 × 10-4
2.16 ± 0.002
BspA-like, SP, TMD
#374, singleton, divergent TpLRR
8.4 × 10-3
2.02 ± 0.04
BspA-like, SP, PG, GRD
#168, 12 members
5.5 × 10-3
#44, 15 members*
4.8 × 10-3
#44, 15 members*
8.5 × 10-3
Low iron culture condition
#184, 3 members
2.9 × 10-3
#69, 3 members *
6.8 × 10-3
#384, 6 members
3.7 × 10-3
#30, 15 members
2.4 × 10-5
#99, 2 members
2.5 × 10-3
-2.06 ± 0.02
#215, 3 members
3.7 × 10-3
#341, singleton, divergent TpLRR
7.8 × 10-3
3.9 × 10-4
Hydrogenosomal malic enzyme subunit B
1.5 × 10-4
2.51 ± 0.01
Cytosolic malate dehydrogenase
2.8 × 10-4
-1.62 ± 0.03
Consistent with these differences in pattern of expression for the tested conditions, marked differences in the upstream sequences to the TvBspA start codon could be observed, suggesting different promoters for each gene potentially mediating differential transcriptional regulation (additional file 1, Table S1). The majority of TvBspA genes possessed known T. vaginalis core promoter elements including an initiator (Inr) (735 entries) or TATA-boxes only (17 entries) with 159 genes possessing neither (additional file 1, Table S1). Interestingly there was no significant difference in the proportion of TvBspA genes with EST or without EST among entries positive (41%) or negative (47%) for Inr/TATA-box suggesting that other core promoter elements for transcription exist in TvBspA genes. It will be interesting to investigate promoter sequence features for TvBspA and other protein coding genes when global tanscriptomics data will be generated for T. vaginalis.
Cellular localization of TvBspA625 and its expression during infection
In order to investigate the expression and cellular localization of one TvBspA-like candidate surface protein four synthetic peptides were produced derived from the TvBspA625 sequence (Figure 2C) and used to raise mouse antisera. Three peptides are likely to generate antisera specific for TvBspA625 (CT-1, EXT-2 and CT-2) whereas the fourth could possibly lead to antibodies cross-reacting with the paralogues TvBsp805 (EXT-1) (Figure 2 and see Methods). Western blot analyses on T. vaginalis total protein extracts with the mouse antisera raised against one of the cytoplasmic located peptides (CT-1) recognized a consistent major protein with an apparent molecular mass of ~52 kDa for both isolate G3 and SS-22, while all presera did not detect any material (additional file 17, Figure S6). However, the other antisera did not identify the same material and were often characterised by more complex banding patterns (G3: CT-2, EXT-1, EXT-2) or had a distinct major band ~40 kDa (SS-22: EXT-1). Since the pre-sera did not detect any proteins and the apparent molecular masses of the proteins detected by the different antisera did not match the theoretical one for TvBspA625 (nor TvBspA805) from the G3 isolate (67 kDa) the lower apparent molecular mass could be explained by aberrant gel migration (sometime observed in proteins with repeats) or represent proteolytic fragments. As we currently don't know the corresponding genome sequence for isolate SS-22 it is difficult to interpret the observed differences between the two isolates.
Patients antisera response to peptides derived from TvBspA625.
T. vaginalis positive patients
T. vaginalis negative patients
Positive ≥ 1 peptide
Negative to all peptides
HIV positive & positive ≥ 1 peptide
HIV positive & negative all peptides
HIV negative & positive ≥ 1 peptide
HIV negative & negative all peptides
Initial analyses of the draft genome sequence of T. vaginalis (isolate G3) identified a plethora of candidate surface/secreted proteins among which the largest family was made of the TvBspA proteins with over 650 entries that share a type of LRR [26, 36]. Here we describe additional TvBspA candidate proteins further extending the size of this considerable protein family and the first detailed analyses of the sequence structural diversity of all 911 TvBspA candidate proteins. We also investigated selected TvBspA transcripts from parasites grown in different conditions and provide for one TvBspA protein the first cellular localisation data as well as identify sequence variations between clinical isolates and patients antibody responses during T. vaginalis infection.
TvBspA gene family tremendous diversity and origins
More comprehensive protein searches of the annotated T. vaginalis draft genome, made more sensitive and specific by combining pattern and profile based searches for TpLRR containing proteins, extended the TvBspA gene family by over 250 to a total of 911 entries. Searching the RefSeq protein database for BspA-like proteins with the same search strategy identified a taxonomically divers set of organisms, including two additional eukaryotes (E. dispar and E. histolytica) and a great majority (82%) of species derived from two major bacterial lineages, Firmicutes (70%) and Bacteroidetes (12%). Interestingly, Firmicutes and Bacteroidetes are the most prevalent taxa identified by gene surveys in the gut of vertebrates, including humans . Indeed, the great majority of taxa encoding BspA-like proteins, including the three eukaryotes, are known to share the capacity to thrive on mucosal surfaces either as pathogens, commensals or mutualists (79% of taxa in Table 1). These data clearly reinforced earlier observations, made on a much more restricted set of sequenced genomes , that BspA-like proteins are preferentially encoded by mucosal microbes.
This pattern strengthened the hypotheses that lateral gene transfers (LGT) of BspA-like genes between microorganisms thriving on mucosal surfaces took place  and that BspA-like proteins are involved in important aspects of microbes-mucosa interactions. Mucosal surfaces; which include the colon, the niche with the highest known density of microbes ; harbour a large diversity of cellular microbes (mainly Bacteria but also Archaea and various microbial eukaryotes) between which LGT could take place, a mechanism thought to contribute to adaptations to a mucosal life style [26, 52, 53]. As the taxonomic diversity of genomes encoding BspA-like proteins is higher among prokaryotes we suggest that the only three eukaryotes (T. vaginalis and two Entamoeba species) currently known to encode BspA-like proteins acquired the corresponding genes from prokaryotic donors, likely bacterial species. The three TvBspA proteins with the highest level of sequence identity in BlastP alignments with prokaryotic proteins are with the Firmicute Eubacterium siraeum (TVAG_495790, 57% identity and TVAG_057870, 56% identity) and the Bacteroidetes Tannerella forsythia (TVAG_225790, 52% identity) (additional file 4, Table S4). Interestingly a bias was observed among candidate LGT genes identified in T. vaginalis with Firmicutes and Bacteroridetes being the most common identifiable candidate donors as supported by detailed phylogenetics (with proteins that lead to reliable alignments, without repeats) [36, 54]. The extensive eukaryotic BspA-like gene families (911 in T. vaginalis, 298 in E. dipsar and 124 in E. histolytica), compared with the much restricted gene families found in prokaryotes (1-19 entries per genome) could be explained by one or a few LGT acquisitions from prokaryotic donors followed by large numbers of gene duplication events within the eukaryotic genomes, so called "conservative" gene duplications that are thought (along with LGT) to contribute to an organism adaptations to its environment . Alternatively, the larger gene families observed in eukaryotes could be explained by several LGTs followed by less dramatic sets of gene duplication events. We favour the former hypothesis as few eukaryotic BspA-like entries show higher scores with their prokaryotic counterparts (only 15 TvBspA had prokaryotic proteins as top hits from nine different Bacteria and one Archaea) with the great majority (93%) of individual TvBspA protein recovering as top BlastP hits other TvBspA (additional file 4, Table S4). Trichomonas sequences are also rather distinct from the Entamoeba sequences both in terms of their TpLRR and overall structural organisation, consistent with independent gene acquisition and amelioration (functional integration) programs - only 19 TvBspA proteins have Entamoeba entries (one EhBspA and 18 EdBspA) as top Blast hit (additional file 4, Table S4).
Contrasting TvBspA gene positions on scaffolds with a TvBspA global alignment and subfamily composition (as surrogate to phylogeny) of the corresponding proteins indicated that both tandem and ectopic gene duplications events took place, as discussed for the large disease resistance gene family encoding proteins with LRR in plants . The largest gene cluster made of 17 TvBspA genes was generated by a combination of a few ectopic and several tandem duplication events. More detailed information on the genome distribution of the TvBspA genes, for instance their potential locations in subtelomeric regions as known for important surface variant proteins in other pathogenic microbial eukaryotes , will await more extensive clustering of the >17,000 scaffolds and their mapping onto chromosomes [36, 57].
TvBspA protein structural diversity
Following gene duplications, TvBspA paralogues differentiated dramatically. This diversity was identified in both the sequence and number of the TpLRR with extensive overall length variation between TvBspA proteins from less then 100 to over 1800 residues. The compression and/or extensions of the TpLRR segments contributed to most of the observed length diversity but variation of other type of sequences are also involved including non-LRR repeats, TMD and CT, indicating an evolutionary highly dynamic gene family and suggesting that these proteins play several distinct functions. As TvBspA proteins are characterised by TpLRR typically present on extracellular proteins in other taxa , and additional repeats are also present in several cases, they are potentially involved in various aspects of host-pathogen interactions as shown for many repeat containing proteins including LRR [43, 44, 53, 58, 59]. A total of 193 TvBspA were inferred to possess TMD and CCT, supporting the hypothesis that the N-terminal ends of the proteins, including the TpLRR, face the extracellular milieu if expressed on the cell surface. Potential SPs were also detected for TvBspA with and without TMD. For proteins without TMD and with SP this suggests that these could be secreted or bound to the cell surface with unknown anchors - as there are no GPI-anchors in T. vaginalis. Notably the current complete absence of experimental data for T. vaginalis SP probably contributes to underestimating the number of SP positive TvBspA entries in silico as SignalP3.0 and PHOBIUS were trained with a restricted diversity of eukaryotes [60, 61]. Entries genuinely without SP and TMD could be secreted or anchored to the cell surface through unknown mechanisms or could represent non-functional proteins, perhaps corresponding to pseudogenes, although EST suggest that all types of TvBspA proteins are transcribed and could be functional. Several TvBspA without TMD (with and without detected SP) were also characterised by conserved C-terminal ends, including motifs with conserved cysteines and other residues and in some cases were ending with hydrophobic residues. Such conserved C-terminal motifs could be implicated in anchoring TvBspA proteins via unknown lipids (as GPI-anchor do not exist in T. vaginalis). Such motifs included the sequences [TS]C K in members of subfamilies #20, 22, 24, 33, 34 and 35, SC HIA for some members of subfamilies #44 and 45 or TC QC R in subfamily #26 (additional file 1, Table S1; additional file 6, Figure S1). These TvBspA C-termini could be modified by hypothetical and 'atypical' lipid anchors for a eukaryotic surface exposed protein, as shown, or hypothesized, for some surface proteins from E. histolytica including EhBspA proteins . In the case of several EhBspA, the cysteine of a C-terminal CAAX motif (cysteine followed by two aliphatic residues and any terminal residue ) could be implicated and one CAAX containing EhBspA protein was indeed demonstrated to be expressed on the cell surface . None of the TvBspA possessed a C-terminal CAAX box.
The important sequence variation observed between the TvBspA paralogues contrast dramatically with the high level of sequence conservation observed among copies of the highly repetitive genes made of virus-like, transposable elements (TE) and unclassified gene families, with a 2.4% average pairwise difference for an average gene copy number of 660, identified in T. vaginalis G3 genome (listed in Table two in Carlton et al. ). This suggests that the TvBspA genes are under some level of positive selective pressure whereas the virus-like, TE and unclassified highly repetitive genes families are evolving neutrally, as would be expected for proteins involved in host-pathogen interactions (potentially TvBspA) and selfish genes, respectively. It will be of great interest to contrast the level of selection pressure (neutral, positive or negative selection using ω = dN/dS - nonsynonymous-synonymous substitution rate ratio tests) on TvBspA genes and contrast these with other genes across several T. vaginalis isolate and one or more closely related species - required to generate DNA alignments of ORF to calculate ω values, the more sequences the better for tests measuring selection - e.g. .
One TvBspA subfamily was characterised by a shared N-terminal GRD followed by the TpLRR. BlastP searches with the GRD from TvBspA-GRD identified hypothetical proteins encoded by a very restricted set of genomes including T. vaginalis, Flavobacteria bacterium, four strains of Clostridium difficile, two C. difficile phages and Bacillus cereus. These data defined a new protein domain of unknown function that is shared between proteins with distinct C-termini currently encoded by few and distantly related taxa, T. vaginalis (a eukaryote), F. bacterium (Bacteroidetes-Chlorobi), C. difficile and B. cereus (both Firmicute), and two C. difficile phages, with all cellular organisms sharing the capacity to be potentially pathogenic to human mucosa. Clostridium difficile is the most common source of nosocomial diarrhea  and F. bacterium and B. cereus are commonly found in soil and water systems with B. cereus being a common opportunistic pathogen also causing pathologies in the digestive tract . Members of the Flavobacteria are often pathogens (e.g. Flavobacteria psychrophilum, a virulent fish pathogen listed in Table 1) or opportunistic pathogens, including in humans . This highly restricted and biased taxonomic distribution among specialised mucosal pathogens and potential opportunist mucosal pathogens is intriguing. In addition many prophages encode toxins and other virulent factors in pathogenic bacteria  and the phage GRD containing protein (ORF 30 in phage PhiCD119 ) is located beside of the holin protein, which is part of the cell lysis casset, and that corresponds to one of the preferential locations of toxins encoded by lambda-like prophages as know for the shiga toxin in subsets of E. coli strains . Indeed, the two GRD containg proteins in the complete genome of C. difficile 630 are encoded by genes (CD0967 and CD2397) with the same location (as in phage PhiCD119) within the two highly conserved prophages identified during annotation. Holin is also homologous to the TcdE gene of the C. difficile pathogenicity locus harbouring the five genes tcdABCDE that is known to regulate the expression of the C. difficile toxins tcdA and tcdB . Taken together, these different considerations suggest that proteins containing the newly identified GRD could be involved in some aspects of T. vaginalis-host interactions possibly by contributing to damaging bacteria of the vaginal microflora or human cells. It will be of interest to directly test this hypothesis experimentally with both T. vaginalis and C. difficile GRD-containing proteins.
Among relatively recently duplicated TvBspA genes (paralogues encoding proteins with high level of protein sequence identity, >70%, 198 pairs of paralogues - additional file 4, Table S4) we identified cases where paralogues accumulated differences in both the number of TpLRR and/or other repetitive sequences as often observed for proteins known to be involved in host-pathogen interactions [43, 44, 53]. Such variations in repetitive segments were also identified between TvBspA625 from two clinical isolates, with isolate SS-22 possessing a shorter P/NRR in the TpLRR-TMD linker sequence when compared to TvBspA625 from isolate G3. The incapacity to PCR clone the entire TvBspA625 ORF from isolate SS-22 also suggested variations at the 5'end of the gene corresponding to the TpLRR domain. Western blot analyses did detect the same major band with the anti-CT-1 antisera but identified some differences with the other antisera (CT-2, EXT-1/2) and sequencing of the full TvBspA625 holomologue of isolate SS-22 will be required to try to rationalise these results. Taken together these data demonstrated that TvBspA paralogues within a given genome and orthologues between clinical isolates readily accumulate changes in repetitive sequences as would be expected for proteins involved in host-pathogen interactions and possibly under selection pressures such as in the case of host immune responses directed against them [43, 44, 53, 56]. In addition variations in repetitive sequence of surface proteins can also lead to important quantitative alterations of their functions, such as variation of adhesion properties to substrates as in cell-cell adhesions during parasite swarming (possibly induced by TvBspA-TvBspA interactions) with dramatic cases described in yeast involving surface proteins with different type of tandem repeats [70, 71]. Variants in TpLRR could also lead to different functions altogether (such as binding to different substrates), and possibly contribute to rapid adaptations to environmental changes (as for example between the male and female urogential tracts or between the various mucosal landmarks) as known, or suggested, for different parasitic and other microbial eukaryotes [43, 44, 71, 72]. It will be particularly interesting to compare in the future the extent of global variations of TvBspA TpLRR and non-LRR repeats between various clinical isolates to gain a better insight into their potential roles in evading host immune response and possible link between T. vaginalis genetic diversity and virulence . As such, TvBspA-like genes could represent valuable markers for epidemiological studies to type clinical isolates .
Another interesting aspect of the diversity among TvBspA proteins with TMD was identified in two subfamilies with conserved CT. Each subfamily was characterised with a classic sorting signals in their CT with one possessing a di-leucine-like signal and the other an NPXY-like signal, both known to mediate rapid endocytosis in other eukaryotes . In both cases the sequence and their position in the cytoplasmic tails suggested that these are functional . The NPXY containing sequences in particular were characterised by flanking sequences FDNP [LIF]F (similar to FDNPVY of the human LDL receptors where the important tyrosine can be replace by a phenylalanine) known to form potent signals for endocytosis further supporting their functionality . Hence these TvBspA proteins could represent receptors mediating endocytosis of various host (or others) proteins with their TpLRR likely involved in binding to ligands. These TvBspA proteins represent to our knowledge the first candidate receptor potentially mediating endocytosis of various host or other proteins.
Some TvBspA genes are potential pseudogenes with 98 entries currently being annotated as such as they possess ORF disrupting sequence features. In addition, other TvBspA sequences have sequencing ambiguities or are obviously truncated due to sequencing problems or missing data. However, very little is currently known about the structure-function relationship for TvBspA proteins and some of these annotated pseudogenes could actually correspond to functional genes such as TVAG_191490, which correspond to a C-terminal truncated form compared to a longer TvBspA-GRD. Hence some 'pseudogenes' could actually encode functional proteins (a shorter one in the case of TVAG_191490 when compared to close paralogues) or correspond to a reservoir used to generate TvBspA diversity through recombination or other processes facilitating the creation of new functional genes distinct from existing ones .
Evidence for expression
For a total of over 270 TvBspA entries (~30% of all 911 TvBspA genes) we obtained evidence for transcription through EST, RT-PCR and microarray analyses. Differential expression was suggested from EST surveys and demonstrated for a subset of genes by microarray and RT-PCR analyses for two conditions, change in iron concentration and binding of parasite to ECM proteins, consistent with different functions for the analysed TvBspA. Due to the large gene family size a global approach based on microarrays designed to cover all 911 TvBspA genes combined with testing key stages of T. vaginalis infections of the urogenital tract of both sexes will be required in the future to gain further insight into the functional relevance of the vast TvBspA gene family. The microarray data presented here indicate that this approach will be an important one to contrast transcripts abundance.
The use of antisera directed against peptide derived from the TvBspA625 sequence demonstrated by IFA the expression of the proteins and its cell surface location, consistent with the bioinformatics analyses, indicating that the TpLRR and the P/NRR of TvBspA625 are exposed to extracellular milieu where they could mediate binding to host or other proteins as demonstrated for the bacterial proteins BspA and LrrA [29, 31, 33].
The same peptides used to raise the mouse anti-TvBspA625 antisera were also used in ELISA assays to test the presence of antibodies recognizing TvBspA625 from clinical patients. Our data strongly suggest that this protein is indeed expressed and triggers antibody responses in both sexes during the majority of tested T. vaginalis infections. Variations of the length of the P/NRR of TvBspA625 also suggested that the protein is under selective pressure possibly due to the immune response it stimulates. Contrasting the TvBspA625 sequences and their expression (along with all other TvBspA) between additional T. vaginalis clinical isolates from patients with and without an antibody response to this protein would be particularly interesting to further test this possibility. The patients without detected antibodies directed against TvBspA625 (~12% of T. vaginalis investigated patients) could correspond to T. vaginalis isolates that don't express that protein at all or with differences in the epitopes tested here. From these different considerations, we predict that differential expression of TvBspA gene sets combined with differences in the TpLRR and other repeats between TvBspA proteins will be identified between different clinical isolates.
Finally TvBspA could play important roles in regulating the innate immune responses in the urogential tracts, as demonstrated in vitro for T. vaginalis total cell surface proteins , since the BspA protein from T. forsythia was shown to regulate in a CD4 and TLR2 dependent manner cytokine induction . The TpLRR of some TvBspA could be directly involved in binding the LRR of TLRs and induce specific innate response signalling.
Considering the phenotypes of organisms encoding BspA-like proteins, the majority being pathogens, commensal or mutualists thriving on vertebrate mucosal surfaces; the established function of BspA-like proteins in the pathogenicity of two mouth mucosal bacteria (T. forsythia and T. denticola); the extraordinarily large TvBspA protein family size with its vast structural diversity, the differential expression patterns demonstrated for some TvBspA genes and the cell surface expression and induction of an antibody response during infections for one TvBspA protein; together strongly suggest that the TvBspA proteins play various and important roles in T. vaginalis' pathobiology by contributing to the invasion and long term infections of the human urogenital tract. TvBspA-like proteins represent strong candidate surface proteins mediating interaction with various mucosal landmarks including the mucus; VEC, urethra epithelial cell and other host cells; ECM proteins and vaginal microflora or cell-cell adhesion during parasite swarming. TvBspA could also mediate endocytosis of various host proteins and viruses, as well as underpin phagocytosis of bacteria and various host cells. Finally TvBspA proteins could orchestrate the modulation of the innate immune system through TLRs signalling during infection and mediate immune evasion through differential expression.
Genome data mining and other bioinformatic analyses
A Wu-BlastP (expectation value ≤ 0.001) search at TrichDB  with the TpLRR of TvBspA625 as query (TVAG_073760, XP_001321233, positions 1-420, ) identified 885 TvBspA candidate proteins. A PHI-Blast  (NCBI Blast server, with same query as for the BlastP, expect threshold ≤ 0.001, with the TpLRR pattern [LIV]xx [LIV]x [LIV]xxx [LIV]xx [LIV]xxxAFxx [CNST]xx)  followed by two iterations of PSI-Blast searches  recovered 908 T. vaginalis annotated proteins in RefSeq (20 August 2008) , which included all but 13 entries of the Wu-BlastP list and 26 additional sequences, defining a total of 911 distinct TvBspA candidate proteins. We also performed BlastP searched with all putative TvBspA proteins and investigated their respective Blast hit lists using SPyPhy to annotate them and investigate their level of similarity with the proteins they hit .
Profile based searches  were also used to investigate the presence of TpLRR. All entries that hit the LRR of BspA-like proteins from other taxa in the BlastP searches or were positive for the TpLRR Profile search (TpLRR profile accession: PS50505 http://www.isrec.isb-sib.ch/cgi-bin/get_pstprf?PS50505) or the TpLRR pattern were all considered as BspA-like candidate proteins and were hence named TvBspA.
The first 150 bp of the 5' upstream regions for all TvBspA-like genes were extracted from TrichDB to allow their comparisons and identify potential Inr sequences (consensus: [ATC]CA+1 [ATGC] [AT]) or TATA-box (consensus: TATA [AT]A [AT]) typically located in T. vaginalis genes within 30 bp or 50 bp, respectively, upstream of the translation initiation codon [36, 82].
In order to identify candidate surface and secreted proteins the potential presence of SP (SignalP3.0 ) and TMD (TMHMM2.0 ) was investigate using the annotation and retrieval tool at TrichDB. These data were complemented with TMD identified with SPLIT4  and PHOBIUS [61, 86], with the latter also investigating the presence of SP. Proteins positive for SP with either SignalP3.0 or PHOBIUS where considered to possess a SP. The position of TMD and protein topology in the membrane was established using the consensus between the three methods TMHMM2.0, SPLIT4 and PHOBIUS and entries were annotated as TMD-CCT when at least two methods overlapped with the TMD position and agreed with the protein topology, as such consensus approaches can provide better predictions . In order to investigate the potential presence of CAAX motif at the C-terminus in TvBspA-like proteins without TMD domains we used PrePS .
SAPS [88, 89], RepSeq  and REPTILE  analyses were performed to investigate the potential presence of repeats and their features (in addition to the TpLRR) and low complexity segments among TvBspA-like proteins.
Protein alignments were performed with ClustalW2.0.9  for all or selected subsets of TvBspA sequences and took advantage of the iteration process (iteration on the final alignment for all 911 TvBspA or for each step for reduced set of sequences) that allow improvement of the alignment quality. Clustal was also used to generate table of protein pairwise sequence identity (%). SEAVIEW  was used to view, edit and manipulate the alignments for figure preparation.
To identify potential protein domains and functional sites the TvBspA proteins were analyses with InterProScan .
The T. vaginalis clinical isolate G3, for which a draft genome was published , and the clinical isolate SS-22  were used for most of the molecular cell biology experimental work. In addition other isolates described below were used for EST libraries and microarray analyses. Cells were grown axenically at 37°C in Modified Diamond Medium  without agar, supplemented with 10%(v/v) heat-inactivated horse serum (Gibco, Invitrogen) and 50,000 units/l penicillin and 50 mg/l streptomycin (Penicillin-Streptomycin Solution, Sigma).
Total RNAs were isolated from different culture conditions indicated below. cDNAs primed with oligo-dT were synthesized by using a ZAP-cDNA synthesis kit and directionally cloned into the EcoRI and XhoI sites of Uni-ZAP XR (Stratagene) vector. The quality of the unidirectional cDNA library was assessed by colony PCR of 96 randomly picked clones to determine the average insert size and percentage of clones without inserts. Then 384 randomly picked clones were sequenced to determine the percentage of vector contamination, valid average length and redundancy of the cDNA library. Plasmids were in-vivo excised from the cDNA library by using helper phage and transformed into E. coli DH10B (Invitrogen) as described by the manufacturer (Stratagene). Around 10,000 ESTs were randomly picked from each cDNA library which represents different stages of cell cycle, pathogenesis and specific nutrient requirements . The five culture conditions used are described below:
Condition : TvEST Library (10,749 ESTs), T. vaginalis isolate ATCC30236 (JH 31A#4).
Medium: YIS, pH 6.0 supplemented with 100 μM ferric ammonium citrate. Growth condition: unsynchronized culture harvested at mid-log phase, trophozoites forms.
Condition : TvG Library (10,470 ESTs), T. vaginalis isolate: ATCC30001 (C-1:NIH). Medium: YIS, pH 6.0 supplemented with 100 μM ferric ammonium citrate. Growth condition: mid-log phase culture cold stressed for 4 hours at 4°C, then return to 37°C for 6 hours, synchronised G2 phase trophozoites forms.
Condition : TvCS Library (9,865 ESTs), T. vaginalis isolate ATCC30001 (C-1:NIH).
Medium: YIS, pH 6.0 supplemented with 100 μM ferric ammonium citrate. Growth condition: Mid-log phase culture cold stressed for 4 hours at 4°C, pseudocyst forms.
Condition : TvFN Library (9,955 ESTs), T. vaginalis isolate isolate TO16. Medium: DMEM:TYM. Growth condition: parasites were grown in fibronectin coated T-75 flask for 3 hours at 37°C, amoeboid forms.
Condition : TvLI Library (10,505 ESTs), T. vaginalis isolate T1. Medium: TYM, pH 6.2. Growth condition: parasites were grown in culture medium supplemented with 80 μM Dipyridyl (DIP) for 10 passages at 37°C, trophozoite forms.
We also took advantage of the ~24,000 EST stored in GenBank dbEST that include those generated by TIGR  and our 4003 in house EST generated from T. vaginalis (G3) trophozoites (mid log-phase). The latter were deposited in dbEST with the accession numbers (GT107175-GT111177) http://www.ncbi.nlm.nih.gov/nucest?term=Harriman_N%20Hirt_RP%20trichomonas%20vaginalis.
Microarray and quantitate RT-PCR
Corning® UltraGAPST coated slides were spotted with cDNA derived from the ~70,000 EST characterised from the various growth condition described above. A total of 7,680 cDNA (4,938 distinct entries) were successfully amplified by PCR from plasmids and spotted on the array once, twice or in triplicate. A total of 8 arrays were used to contrast expression levels - dye-swap experimental design for four independent experiments. T. vaginalis cells were grown in two conditions contrasting high iron (TYM medium supplemented with 150 μM iron nitrilotriacetate) and low iron (TYM medium with 80 μM DIP) and grown for 10 passages in the respective conditions. Total RNA was extracted using QuickPrep Total RNA Extraction Kit (Amersham Biosciences) and cleaned up using Rneasy CleanUp Kit (Quiagen). Total RNA concentration and purity was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). The cDNA probes were synthesized from 2 μg of total RNA using primers contained in 3DNA Array 900 Expression Array Detection Kit (Genisphere). The hybridization was carried out following 3DNA Array 900 Expression Array Detection Kit (Genisphere) protocol. The signal was transformed with natural log (ln) and normalized by LOWESS normalization method in the TIGR microarray data analysis system (MIDAS) version 2.19 . Entries with p values <1e-3 were considered to have significant differences in transcript concentration. Mean and standard deviation for the 13 TvBspA genes with significant difference in level of expression and appropriate control genes are listed in Table 4. qRT-PCR of selected genes was used to validate the microarray data and included three TvBspA genes and 14 control unrelated genes (unpublished data) two of which are listed in Table 4. All primers used for semi-quantitative RT-PCR and qRT-PCR are listed in additional file 19, Table S12. All tested genes by qRT-PCR were in qualitative agreement with the microarray data. The Microarray data were deposited into ArrayExpress with the accession number E-MTAB-126.
Extracellular matrix binding assay and semi-quantitative RT-PCR
Culture cell flasks (25 cm2, Greiner Bio-One) were coated with a PBS solution containing 500 μg/ml Collagen I (rat tail, Marathon Lab), 40 μg/ml laminin (Engelbreth-Holm-Swarm murine sarcoma, basement membrane, Sigma), 50 μg/ml fibronectin (human plasma, Sigma) and 50 μg/ml phenol red (Sigma) as pH indicator. The solidification of the coating solution in a gel was obtained by incubation at 37°C for 10 min and coated flasks were stored at room temperature. The resulting gel matrix was an ultra-thin pink coloured layer of proteins homogenously covering the entire bottom surface of the flask. Trichomonas vaginalis cells used for binding assays were harvested from mid-log phase growth (~24 hrs) by centrifugation at 750 × g min-1 and washed twice in minimal binding buffer (MBB) . Parasites were counted using a Neubauer Haemocytometer and coated flasks were seeded with 5 × 106 cells in 5 ml of MBB and incubation carried out at 37°C for 60 min. Amoeboid shaped cells could be observed upon binding to the substrate after ~10 min. After 60 min, flasks were washed twice with warm MBB in order to remove unbound cells and the majority of cells were tightly bound to the substrate and showed a large proportion of amoeba forms or had pseudopodial-like cellular extensions. The population of cells were diverse in their morphology and movements with some cells actively roaming the substrate (including some with long pseudopodia up ~1/3 of the length of the cell), whereas others were static.
Expression of selected TvBspA-like protein coding genes was analysed by semi-quantitative RT-PCR contrasting ECM bound cells and trophozoites not exposed to the substrate. Total RNA extractions were performed in parallel on both ECM bound cells and trophozoites not exposed to the substrate, using SV Total RNA Isolation System (Promega) according to manufacturer specifications (that includes a DNAase treatment step to insure the absence of any genomic DNA) and total RNA were quantified by absorbance at 260 nm. The polyA+ mRNA were then purified using Dynabeads® mRNA Purification Kit (Dynal Biotech) and The RT-PCR THERMOSCRIPT Kit (Invitrogen) was used for the RT-PCR reactions with specific primer pairs previously tested on genomic DNA - all producing the expected size amplicons without detectable background. The same amounts of cDNA from either ECM bound cells or trophozoites were used for each the PCR reactions. The amount of cDNA used for bound cells and trophozoites was normalized based on the total RNA concentrations since the smaller amount of cDNA obtained from the ECM proteins bound cells did not allow its quantification. Controls RT-PCRs consisted of actin (loading control) and alpha-actinin (up-regulated gene upon amoeba transformation) amplifications using specific primers previously described . These procedures were carried out in five independent binding assays and independent PCR reactions (at least three per binding assays) and gave similar expression patterns for the tested genes. All primers are listed in additional file 19, Table S12. PCR reactions were run on 1% agarose gel for analysis.
Peptide synthesis and mouse anti-peptide antisera
Four peptides were designed from the TvBspA625 sequence  and their sequence features to optimized peptide synthesis, solubility and antigenicity and to differentiate TvBspA625 from other Trichomonas proteins. Two peptides sequences were derived from the inferred extracellular domain and two peptides from the cytoplasmic tail (Figure 2). BlastP with all four peptides as query established that three peptides are likely to generate TvBspA625 specific antisera. One TvBspA625 peptide (EXT-1) could possibly generate antisera cross-reacting with TvBspA805 or be recognised by patients antibodies directed against TvBspA805 as a stretch of 10 identical contiguous residues was shared between TvBspA625 and TvBspA805. The four peptides were synthesised by adding a N-terminal cysteine, to allow cross-linking with the maleimide-activated carrier proteins. Peptides were coupled to both keyhole limpet hemocyanin (KLH) and bovine serum albumine (BSA) by using the Imject Maleimide Activated Immunogen kit (Pierce, Rockford, IL, USA), following the manufacturer instruction. Immunization of eight BALB/c mice (two mice/peptide) five weeks old was performed by both subcutaneous and intraperitoneal inoculation of 20 μg of KHL conjugated peptide proteins in complete Freund's adjuvant per inoculum. Mice were inoculated three times with an interval of 10 days, and sacrificed six days after the final intravenous boost with 10 μg of BSA-coupled peptides. Sera were collected and specific reactivity against each peptide was tested by ELISA using plates coated with KLH and BSA coupled peptides. The antisera titre ranged from 1:100,000 to 1:500,000. Presera and sera were collected and used for indirect immunofluorescence and Western blot analyses using indicated dilutions.
Western blot analyses
Total cell extracts from T. vaginalis cell cutlures were submitted to SDS-PAGE and immunoblotting as previously described . Briefly, 3 × 105 washed cells from exponentially growing cultures were resuspended in 100 μl of Laemmli lysis buffer and boiled for 3 min. 10 μl of each sample were then loaded in each well of a 7.5% SDS-PAGE gel, electrophoresed, blotted onto nitrocellulose, blocked, and separately incubated with the mouse anti-peptide sera at 1: 2000 dilution. After washing, membranes were incubated with a goat anti-mouse immunoglobulin sera, conjugated with alkaline phosphatase (Sigma, S. Louis, USA). Bound antibodies were detected by soaking the nitrocellulose membrane in AP buffer (0.1 M tris pH 9.5, 0.1 M NaCl, 0.005 M MgCl2) to which 0.33 mg/ml nitroblue tetrazolium (NBT), and 0.165 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP) were added.
Indirect immunofluorescence analyses (IFA)
Trophozoites of T. vaginalis grown in vitro were collected during the exponential growth phase at 350 × g for 5 min and washed twice with Ringer (NaCl 0.12 M, KCl 3.5 mM, CaCl2 2 mM, NaHCO3 2.5 mM, pH 7.2) and fixed with either 3% formaldehyde (Sigma) 10 min at room temperature or 70% ethanol at -20°C, 20 min. Fixed cells were pelleted by centrifugation at 250 × g for 10 min at 4°C, washed twice with PBS pH 7.2, and allowed to adhere to poly-L-lysine coated slides for 1 h. Formaldehyde fixed cells, once bound, were further incubated with 50 mM NH4Cl to quench protein side groups exposed by this fixation procedure and then washed twice in PBS. Slides were incubated with blocking buffer (3% bovine serum albumin in PBS) for 30 min before incubation with primary mouse anti-peptide antisera over night at 4°C. As control we used VG2, a mouse anti-T. vaginalis-α-tubulin monoclonal antibody  and a rabbit anti-T. vaginalis-hydrogenosomal malic enzyme antisera . After three washes of 10 min each with PBS, the slides were incubated with both Alexa-Fluor-488 conjugated goat anti-mouse IgGs and Alexa-Fluor-594 conjugated goat anti-rabbit IgGs (both from Invitrogen, Molecular Probes) diluted 1:200 for 1 h at 37°C. After rinsing three times with PBS, the coverslips were mounted with VECTASHIELD® Mounting Medium with DAPI (Vector Laboratories, UK) prior microscopic observation with a confocal laser scanning microscopy Leica TCS SP2UV. Images were captured and processed using Leica CS Lite program version 2.61.
ELISA assays to measure human patients immune response against TvBspA625 peptides
Sera from a total of 591 humans with high risk of sexually transmitted diseases were selected and kept frozen at -20°C until enzyme-linked immunosorbent assay (ELISA) experiments; 356 sera were from female patients, while 235 from males. Consent was obtained from the patients and the material was databased and processed anonymously. ELISA were performed following a published method . Cells from T. vaginalis isolate SS-22 were used as the source of total antigen since it is characterised by a low degree of phenotypic variation and is not infected by Mycoplasma hominis. Parasites (viability 99%) were resuspended at the density of 1 × 106/ml in phosphate-buffered saline (PBS), and 50 μl of T. vaginalis suspension were seeded in each well of PVC microtiter-well plates (Becton Dickinson, Lincoln Park, NJ) and allowed to dry. 50 μl of ice-cold 95% ethanol were added to each well and allowed to dry, then washed in distilled water, and stored at 4°C until use. Prior to use, wells were pretreated for 2 hours with a PBS-0.05% Tween 20 (PBS-T) solution containing 5% nonfat dry milk. 100 μl of sera diluted 1:200 in the same solution were then added and incubated for two hours at room temperature. After extensive rinsing with PBS-T, 100 μl of goat anti-human IgG or IgM antibodies conjugated with alkaline phosphatase (Sigma, St. Louis) were added. After two hours, the color reaction was induced with specific substrates and absorbance measured. Cutoff was established as twice the mean value obtained with sera from 10 healthy male volunteers distinct from the 591 tested humans. Sera were classified as negative if the ELISA readings were lower than the cutoff value, or positive if at least twice above the cutoff value.
In order to evaluate the immunogenicity of TvBspA625 protein during T. vaginalis infections, the presence of specific antibodies against the four immunogenic peptides were tested by ELISA in 161 human sera positive for T. vaginalis total proteins, as defined above. In addition, 61 sera negative for T. vaginalis total proteins were used for comparisons. ELISA plates were coated over night separately with 1 μg of BSA or KLH-linked peptide in 50 μl carbonate buffer, pH 8,6. Plates were then washed with PBS 0.05% Tween 20 (PBS-T) and saturated with BPS-T containing 5% nonfat dry milk. All sera were diluted 1:200 and separately tested for reactivity against each peptide and immune complexes detected as described above. Statistical tests on the ELISA data (2-way contingency table, Pearson chi-square test) were performed at .
This work was funded by a Wellcome Trust University Award to RPH (grant #060068) and a European Union Marie Curie Individual Fellowship to CN (contract #HPMF-CT-2002-02071). Funds provided by the Italian ministry of University PRIN 2007 (to PLF) and Czech Ministry of Education (MSM0021620858, LC07032) (to JT) also contributed to this work. The following individual for help with some bioinformatics analyses: Dan Swan, Sebastian Maurer-Stroh (PrePs and biG-PI), Dan Depledge (RepSeq) and Corin Yeats (SPLIT4). Thanks to the reviewers for constructive comments and Colin Harwood for discussion on lambda prophages encoding toxins.
- WHO: Global Prevalence and Incidence of Selected Curable Sexually Transmitted Infections: Overview and Estimates. 2001, Geneva, Switzerland: World Health Organization, [http://www.who.int/hiv/pub/sti/pub7/en/index.html]Google Scholar
- McClelland RS: Trichomonas vaginalis infection: can we afford to do nothing?. J Infect Dis. 2008, 197: 487-489. 10.1086/526498.PubMed CentralPubMedView ArticleGoogle Scholar
- Pol Van der B: Trichomonas vaginalis infection: the most prevalent nonviral sexually transmitted infection receives the least public health attention. Clin Infect Dis. 2007, 44: 23-25. 10.1086/509934.PubMedView ArticleGoogle Scholar
- Petrin D, Delgaty K, Bhatt R, Garber G: Clinical and microbiological aspects of Trichomonas vaginalis . Clin Microbiol Rev. 1998, 11: 300-317.PubMed CentralPubMedGoogle Scholar
- Schwebke JR, Burgess D: Trichomoniasis. Clin Microbiol Rev. 2004, 17: 794-803. 10.1128/CMR.17.4.794-803.2004. table of contents.PubMed CentralPubMedView ArticleGoogle Scholar
- Johnston VJ, Mabey DC: Global epidemiology and control of Trichomonas vaginalis . Curr Opin Infect Dis. 2008, 21: 56-64. 10.1097/QCO.0b013e3282f3d999.PubMedView ArticleGoogle Scholar
- Nanda N, Michel RG, Kurdgelashvili G, Wendel KA: Trichomoniasis and its treatment. Expert Rev Anti Infect Ther. 2006, 4: 125-135. 10.1586/1478718.104.22.168.PubMedView ArticleGoogle Scholar
- Galvin SR, Cohen MS: The role of sexually transmitted diseases in HIV transmission. Nat Rev Microbiol. 2004, 2: 33-42. 10.1038/nrmicro794.PubMedView ArticleGoogle Scholar
- Rendon-Maldonado J, Espinosa-Cantellano M, Soler C, Torres JV, Martinez-Palomo A: Trichomonas vaginalis : in vitro attachment and internalization of HIV-1 and HIV-1-infected lymphocytes. J Eukaryot Microbiol. 2003, 50: 43-48. 10.1111/j.1550-7408.2003.tb00104.x.PubMedView ArticleGoogle Scholar
- Russell MW, Sparling PF, Morrison RP, Cauci S, Fidel PLJ, Martin D, Hook EWI, Mestecky J: Mucosal immunology of sexually transmitted diseases. Mucosal immunity. Edited by: Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR, Mayer L. 2005, Burlington, MA, USA: Elsevier, Academic Press, 1693-1720. full_text. [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B84D8-4NH7HG8-41&_rdoc=9&_hierId=700000012&_refWorkId=802&_explode=700000007,700000012&_fmt=high&_orig=na&_docanchor=&_idxType=TC&view=c&_ct=14&_acct=C000014659&_version=1&_urlVersion=0&_userid=7229486&md5=b73cf192b284644dac51a75c183ee7a3]3View ArticleGoogle Scholar
- Peterman TA, Tian LH, Metcalf CA, Malotte CK, Paul SM, Douglas JM: Persistent, undetected Trichomonas vaginalis infections?. Clin Infect Dis. 2009, 48: 259-260. 10.1086/595706.PubMedView ArticleGoogle Scholar
- Lehker MW, Alderete JF: Biology of trichomonosis. Curr Opin Infect Dis. 2000, 13: 37-45.PubMedView ArticleGoogle Scholar
- Fiori PL, Rappelli P, Addis MF: The flagellated parasite Trichomonas vaginalis : new insights into cytopathogenicity mechanisms. Microbes Infect. 1999, 1: 149-156. 10.1016/S1286-4579(99)80006-9.PubMedView ArticleGoogle Scholar
- Honigberg BM: Host cell-Trichomonad interactions and virulence assays in in vitro systems. Trichomonads parasitic in humans. Edited by: Honigberg BM. 1990, New York: Springer-Verlag, 155-212.View ArticleGoogle Scholar
- Alderete JF, Benchimol M, Lehker MW, Crouch ML: The complex fibronectin--Trichomonas vaginalis interactions and Trichomonosis. Parasitol Int. 2002, 51: 285-292. 10.1016/S1383-5769(02)00015-6.PubMedView ArticleGoogle Scholar
- Arroyo R, Gonzalez-Robles A, Martinez-Palomo A, Alderete JF: Signalling of Trichomonas vaginalis for amoeboid transformation and adhesion synthesis follows cytoadherence. Mol Microbiol. 1993, 7: 299-309. 10.1111/j.1365-2958.1993.tb01121.x.PubMedView ArticleGoogle Scholar
- Lal K, Noel CJ, Field MC, Goulding D, Hirt RP: Dramatic reorganisation of Trichomonas endomembranes during amoebal transformation: a possible role for G-proteins. Mol Biochem Parasitol. 2006, 148: 99-102. 10.1016/j.molbiopara.2006.02.022.PubMedView ArticleGoogle Scholar
- Sutak R, Lesuisse E, Tachezy J, Richardson DR: Crusade for iron: iron uptake in unicellular eukaryotes and its significance for virulence. Trends Microbiol. 2008, 16: 261-268. 10.1016/j.tim.2008.03.005.PubMedView ArticleGoogle Scholar
- Pindak FF, Mora de Pindak M, Hyde BM, Gardner WA: Acquisition and retention of viruses by Trichomonas vaginalis . Genitourin Med. 1989, 65: 366-371.PubMed CentralPubMedGoogle Scholar
- Rendon-Maldonado JG, Espinosa-Cantellano M, Gonzalez-Robles A, Martinez-Palomo A: Trichomonas vaginalis : in vitro phagocytosis of lactobacilli, vaginal epithelial cells, leukocytes, and erythrocytes. Exp Parasitol. 1998, 89: 241-250. 10.1006/expr.1998.4297.PubMedView ArticleGoogle Scholar
- Pereira-Neves A, Benchimol M: Phagocytosis by Trichomonas vaginalis : new insights. Biol Cell. 2007, 99: 87-101. 10.1042/BC20060084.PubMedView ArticleGoogle Scholar
- Benchimol M, de Andrade Rosa I, da Silva Fontes R, Burla Dias AJ: Trichomonas adhere and phagocytose sperm cells: adhesion seems to be a prominent stage during interaction. Parasitol Res. 2008, 102: 597-604. 10.1007/s00436-007-0793-3.PubMedView ArticleGoogle Scholar
- Lehker MW, Sweeney D: Trichomonad invasion of the mucous layer requires adhesins, mucinases, and motility. Sex Transm Infect. 1999, 75: 231-238. 10.1136/sti.75.4.231.PubMed CentralPubMedView ArticleGoogle Scholar
- Okumura CY, Baum LG, Johnson PJ: Galectin-1 on cervical epithelial cells is a receptor for the sexually transmitted human parasite Trichomonas vaginalis . Cell Microbiol. 2008, 10: 2078-2090. 10.1111/j.1462-5822.2008.01190.x.PubMed CentralPubMedView ArticleGoogle Scholar
- Klemba M, Goldberg DE: Biological roles of proteases in parasitic protozoa. Annu Rev Biochem. 2002, 71: 275-305. 10.1146/annurev.biochem.71.090501.145453.PubMedView ArticleGoogle Scholar
- Hirt RP, Noel CJ, Sicheritz-Ponten T, Tachezy J, Fiori PL: Trichomonas vaginalis surface proteins: a view from the genome. Trends Parasitol. 2007, 23: 540-547. 10.1016/j.pt.2007.08.020.PubMedView ArticleGoogle Scholar
- Addis MF, Rappelli P, Fiori PL: Host and tissue specificity of Trichomonas vaginalis is not mediated by its known adhesion proteins. Infect Immun. 2000, 68: 4358-4360. 10.1128/IAI.68.7.4358-4360.2000.PubMed CentralPubMedView ArticleGoogle Scholar
- Hirt RP, Harriman N, Kajava AV, Embley TM: A novel potential surface protein in Trichomonas vaginalis contains a leucine-rich repeat shared by micro-organisms from all three domains of life. Mol Biochem Parasitol. 2002, 125: 195-199. 10.1016/S0166-6851(02)00211-6.PubMedView ArticleGoogle Scholar
- Sharma A, Sojar HT, Glurich I, Honma K, Kuramitsu HK, Genco RJ: Cloning, expression, and sequencing of a cell surface antigen containing a leucine-rich repeat motif from Bacteroides forsythus ATCC 43037. Infect Immun. 1998, 66: 5703-5710.PubMed CentralPubMedGoogle Scholar
- Kajava AV, Kobe B: Assessment of the ability to model proteins with leucine-rich repeats in light of the latest structural information. Protein Sci. 2002, 11: 1082-1090. 10.1110/ps.4010102.PubMed CentralPubMedView ArticleGoogle Scholar
- Ikegami A, Honma K, Sharma A, Kuramitsu HK: Multiple functions of the leucine-rich repeat protein LrrA of Treponema denticola . Infect Immun. 2004, 72: 4619-4627. 10.1128/IAI.72.8.4619-4627.2004.PubMed CentralPubMedView ArticleGoogle Scholar
- Sharma A, Inagaki S, Honma K, Sfintescu C, Baker PJ, Evans RT: Tannerella forsythia-induced alveolar bone loss in mice involves leucine-rich-repeat BspA protein. J Dent Res. 2005, 84: 462-467. 10.1177/154405910508400512.PubMedView ArticleGoogle Scholar
- Sharma A, Inagaki S, Sigurdson W, Kuramitsu HK: Synergy between Tannerella forsythia and Fusobacterium nucleatum in biofilm formation. Oral Microbiol Immunol. 2005, 20: 39-42. 10.1111/j.1399-302X.2004.00175.x.PubMedView ArticleGoogle Scholar
- Inagaki S, Kuramitsu HK, Sharma A: Contact-dependent regulation of a Tannerella forsythia virulence factor, BspA, in biofilms. FEMS Microbiol Lett. 2005, 249: 291-296. 10.1016/j.femsle.2005.06.032.PubMedView ArticleGoogle Scholar
- Onishi S, Honma K, Liang S, Stathopoulou P, Kinane D, Hajishengallis G, Sharma A: Toll-like receptor 2-mediated interleukin-8 expression in gingival epithelial cells by the Tannerella forsythia leucine-rich repeat protein BspA. Infect Immun. 2008, 76: 198-205. 10.1128/IAI.01139-07.PubMed CentralPubMedView ArticleGoogle Scholar
- Carlton JM, Hirt RP, Silva JC, Delcher AL, Schatz M, Zhao Q, Wortman JR, Bidwell SL, Alsmark UC, Besteiro S, Sicheritz-Ponten T, Noel CJ, Dacks JB, Foster PG, Simillion C, Peer Van de Y, Miranda-Saavedra D, Barton GJ, Westrop GD, Muller S, Dessi D, Fiori PL, Ren Q, Paulsen I, Zhang H, Bastida-Corcuera FD, Simoes-Barbosa A, Brown MT, Hayes RD, Mukherjee M, Okumura CY, Schneider R, Smith AJ, Vanacova S, Villalvazo M, Haas BJ, Pertea M, Feldblyum TV, Utterback TR, Shu CL, Osoegawa K, de Jong PJ, Hrdy I, Horvathova L, Zubacova Z, Dolezal P, Malik SB, Logsdon JM, Henze K, Gupta A, Wang CC, Dunne RL, Upcroft JA, Upcroft P, White O, Salzberg SL, Tang P, Chiu CH, Lee YS, Embley TM, Coombs GH, Mottram JC, Tachezy J, Fraser-Liggett CM, Johnson PJ: Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis . Science. 2007, 315: 207-212. 10.1126/science.1132894.PubMed CentralPubMedView ArticleGoogle Scholar
- Thompson JD, Gibson TJ, Higgins DG: Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinformatics. 2002, Chapter 2 (Unit 2): 3-PubMedGoogle Scholar
- Kelil A, Wang S, Brzezinski R: CLUSS2: an alignment-independent algorithm for clustering protein of multiple biological functions. International Journal of Computational Biology. 2008, 1: 122-140. [http://prospectus.usherbrooke.ca/CLUSS/Index.html]Google Scholar
- Leister D: Tandem and segmental gene duplication and recombination in the evolution of plant disease resistance gene. Trends Genet. 2004, 20: 116-122. 10.1016/j.tig.2004.01.007.PubMedView ArticleGoogle Scholar
- Davis PH, Zhang Z, Chen M, Zhang X, Chakraborty S, Stanley SL: Identification of a family of BspA like surface proteins of Entamoeba histolytica with novel leucine rich repeats. Mol Biochem Parasitol. 2005, 145: 111-116. 10.1016/j.molbiopara.2005.08.017.PubMed CentralPubMedView ArticleGoogle Scholar
- Kobe B, Kajava AV: The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol. 2001, 11: 725-732. 10.1016/S0959-440X(01)00266-4.PubMedView ArticleGoogle Scholar
- Goder V, Spiess M: Topogenesis of membrane proteins: determinants and dynamics. FEBS Lett. 2001, 504: 87-93. 10.1016/S0014-5793(01)02712-0.PubMedView ArticleGoogle Scholar
- Depledge DP, Lower RP, Smith DF: RepSeq--a database of amino acid repeats present in lower eukaryotic pathogens. BMC Bioinf. 2007, 8: 122-10.1186/1471-2105-8-122.View ArticleGoogle Scholar
- Fankhauser N, Nguyen-Ha TM, Adler J, Maser P: Surface antigens and potential virulence factors from parasites detected by comparative genomics of perfect amino acid repeats. Proteome Sci. 2007, 5: 20-10.1186/1477-5956-5-20.PubMed CentralPubMedView ArticleGoogle Scholar
- Fiori PL, Rappelli P, Addis MF, Mannu F, Cappuccinelli P: Contact-dependent disruption of the host cell membrane skeleton induced by Trichomonas vaginalis . Infect Immun. 1997, 65: 5142-5148.PubMed CentralPubMedGoogle Scholar
- Bonifacino JS, Traub LM: Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu Rev Biochem. 2003, 72: 395-447. 10.1146/annurev.biochem.72.121801.161800.PubMedView ArticleGoogle Scholar
- De Jesus JB, Cuervo P, Junqueira M, Britto C, Silva-Filho FC, Soares MJ, Cupolillo E, Fernandes O, Domont GB: A further proteomic study on the effect of iron in the human pathogen Trichomonas vaginalis . Proteomics. 2007, 7: 1961-1972. 10.1002/pmic.200600797.PubMedView ArticleGoogle Scholar
- Torres-Romero JC, Arroyo R: Responsiveness of Trichomonas vaginalis to iron concentrations: Evidence for a post-transcriptional iron regulation by an IRE/IRP-like system. Infect Genet Evol. 2009,Google Scholar
- Drmota T, Proost P, Van Ranst M, Weyda F, Kulda J, Tachezy J: Iron-ascorbate cleavable malic enzyme from hydrogenosomes of Trichomonas vaginalis : purification and characterization. Mol Biochem Parasitol. 1996, 83: 221-234. 10.1016/S0166-6851(96)02777-6.PubMedView ArticleGoogle Scholar
- Delgado-Viscogliosi P, Brugerolle G, Viscogliosi E: Tubulin post-translational modifications in the primitive protist Trichomonas vaginalis . Cell Motil Cytoskeleton. 1996, 33: 288-297. 10.1002/(SICI)1097-0169(1996)33:4<288::AID-CM5>3.0.CO;2-5.PubMedView ArticleGoogle Scholar
- Ley RE, Lozupone CA, Hamady M, Knight R, Gordon JI: Worlds within worlds: evolution of the vertebrate gut microbiota. Nat Rev Microbiol. 2008, 6: 776-788. 10.1038/nrmicro1978.PubMed CentralPubMedView ArticleGoogle Scholar
- Ley RE, Peterson DA, Gordon JI: Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell. 2006, 124: 837-848. 10.1016/j.cell.2006.02.017.PubMedView ArticleGoogle Scholar
- Pallen MJ, Wren BW: Bacterial pathogenomics. Nature. 2007, 449: 835-842. 10.1038/nature06248.PubMedView ArticleGoogle Scholar
- Alsmark UC, Sicheritz-Ponten T, Foster PG, Hirt RP, Embley TM: Horizontal Gene Transfer in Eukaryotic Parasites: A Case Study of Entamoeba histolytica and Trichomonas vaginalis. Methods Mol Biol. 2009, 532: 489-500. full_text.PubMedView ArticleGoogle Scholar
- Vogel C, Chothia C: Protein family expansions and biological complexity. PLoS Comput Biol. 2006, 2: e48-10.1371/journal.pcbi.0020048.PubMed CentralPubMedView ArticleGoogle Scholar
- Deitsch KW, Lukehart SA, Stringer JR: Common strategies for antigenic variation by bacterial, fungal and protozoan pathogens. Nat Rev Microbiol. 2009, 7: 493-503. 10.1038/nrmicro2145.PubMed CentralPubMedView ArticleGoogle Scholar
- Zubacova Z, Cimburek Z, Tachezy J: Comparative analysis of trichomonad genome sizes and karyotypes. Mol Biochem Parasitol. 2008, 161: 49-54. 10.1016/j.molbiopara.2008.06.004.PubMedView ArticleGoogle Scholar
- Kedzierski L, Montgomery J, Curtis J, Handman E: Leucine-rich repeats in host-pathogen interactions. Arch Immunol Ther Exp (Warsz). 2004, 52: 104-112. [http://www.iitd.pan.wroc.pl/journals/AITEFullText/5239.pdf]Google Scholar
- Butler G, Rasmussen MD, Lin MF, Santos MA, Sakthikumar S, Munro CA, Rheinbay E, Grabherr M, Forche A, Reedy JL, Agrafioti I, Arnaud MB, Bates S, Brown AJ, Brunke S, Costanzo MC, Fitzpatrick DA, de Groot PW, Harris D, Hoyer LL, Hube B, Klis FM, Kodira C, Lennard N, Logue ME, Martin R, Neiman AM, Nikolaou E, Quail MA, Quinn J, Santos MC, Schmitzberger FF, Sherlock G, Shah P, Silverstein KA, Skrzypek MS, Soll D, Staggs R, Stansfield I, Stumpf MP, Sudbery PE, Srikantha T, Zeng Q, Berman J, Berriman M, Heitman J, Gow NA, Lorenz MC, Birren BW, Kellis M, Cuomo CA: Evolution of pathogenicity and sexual reproduction in eight Candida genomes. Nature. 2009, 459: 657-662. 10.1038/nature08064.PubMed CentralPubMedView ArticleGoogle Scholar
- Nielsen H, Engelbrecht J, Brunak S, von Heijne G: A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Int J Neural Syst. 1997, 8: 581-599. 10.1142/S0129065797000537.PubMedView ArticleGoogle Scholar
- Kall L, Krogh A, Sonnhammer EL: A combined transmembrane topology and signal peptide prediction method. J Mol Biol. 2004, 338: 1027-1036. 10.1016/j.jmb.2004.03.016.PubMedView ArticleGoogle Scholar
- Laughlin RC, Temesvari LA: Cellular and molecular mechanisms that underlie Entamoeba histolytica pathogenesis: prospects for intervention. Expert Rev Mol Med. 2005, 7: 1-19. 10.1017/S1462399405009622.PubMedView ArticleGoogle Scholar
- Maurer-Stroh S, Eisenhaber F: Refinement and prediction of protein prenylation motifs. Genome Biol. 2005, 6: R55-10.1186/gb-2005-6-6-r55.PubMed CentralPubMedView ArticleGoogle Scholar
- Yang Z, Wong WS, Nielsen R: Bayes empirical bayes inference of amino acid sites under positive selection. Mol Biol Evol. 2005, 22: 1107-1118. 10.1093/molbev/msi097.PubMedView ArticleGoogle Scholar
- Sebaihia M, Wren BW, Mullany P, Fairweather NF, Minton N, Stabler R, Thomson NR, Roberts AP, Cerdeno-Tarraga AM, Wang H, Holden MT, Wright A, Churcher C, Quail MA, Baker S, Bason N, Brooks K, Chillingworth T, Cronin A, Davis P, Dowd L, Fraser A, Feltwell T, Hance Z, Holroyd S, Jagels K, Moule S, Mungall K, Price C, Rabbinowitsch E, Sharp S, Simmonds M, Stevens K, Unwin L, Whithead S, Dupuy B, Dougan G, Barrell B, Parkhill J: The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat Genet. 2006, 38: 779-786. 10.1038/ng1830.PubMedView ArticleGoogle Scholar
- Stenfors Arnesen LP, Fagerlund A, Granum PE: From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev. 2008, 32: 579-606. 10.1111/j.1574-6976.2008.00112.x.PubMedView ArticleGoogle Scholar
- Duchaud E, Boussaha M, Loux V, Bernardet JF, Michel C, Kerouault B, Mondot S, Nicolas P, Bossy R, Caron C, Bessieres P, Gibrat JF, Claverol S, Dumetz F, Le Henaff M, Benmansour A: Complete genome sequence of the fish pathogen Flavobacterium psychrophilum. Nat Biotechnol. 2007, 25: 763-769. 10.1038/nbt1313.PubMedView ArticleGoogle Scholar
- Govind R, Fralick JA, Rolfe RD: Genomic organization and molecular characterization of Clostridium difficile bacteriophage PhiCD119. J Bacteriol. 2006, 188: 2568-2577. 10.1128/JB.188.7.2568-2577.2006.PubMed CentralPubMedView ArticleGoogle Scholar
- Boyd EF, Brussow H: Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved. Trends Microbiol. 2002, 10: 521-529. 10.1016/S0966-842X(02)02459-9.PubMedView ArticleGoogle Scholar
- Verstrepen KJ, Jansen A, Lewitter F, Fink GR: Intragenic tandem repeats generate functional variability. Nat Genet. 2005, 37: 986-990. 10.1038/ng1618.PubMed CentralPubMedView ArticleGoogle Scholar
- Verstrepen KJ, Klis FM: Flocculation, adhesion and biofilm formation in yeasts. Mol Microbiol. 2006, 60: 5-15. 10.1111/j.1365-2958.2006.05072.x.PubMedView ArticleGoogle Scholar
- Verstrepen KJ, Reynolds TB, Fink GR: Origins of variation in the fungal cell surface. Nat Rev Microbiol. 2004, 2: 533-540. 10.1038/nrmicro927.PubMedView ArticleGoogle Scholar
- Zheng D, Gerstein MB: The ambiguous boundary between genes and pseudogenes: the dead rise up, or do they?. Trends Genet. 2007, 23: 219-224. 10.1016/j.tig.2007.03.003.PubMedView ArticleGoogle Scholar
- Scott K, Manunta M, Germain C, Smith P, Jones M, Mitchell P, Dessi D, Branigan Bamford K, Lechler RI, Fiori PL, Foster GR, Lombardi G: Qualitatively distinct patterns of cytokines are released by human dendritic cells in response to different pathogens. Immunology. 2005, 116: 245-254. 10.1111/j.1365-2567.2005.02218.x.PubMed CentralPubMedView ArticleGoogle Scholar
- Hajishengallis G, Martin M, Sojar HT, Sharma A, Schifferle RE, DeNardin E, Russell MW, Genco RJ: Dependence of bacterial protein adhesins on toll-like receptors for proinflammatory cytokine induction. Clin Diagn Lab Immunol. 2002, 9: 403-411.PubMed CentralPubMedGoogle Scholar
- Aurrecoechea C, Brestelli J, Brunk BP, Carlton JM, Dommer J, Fischer S, Gajria B, Gao X, Gingle A, Grant G, Harb OS, Heiges M, Innamorato F, Iodice J, Kissinger JC, Kraemer E, Li W, Miller JA, Morrison HG, Nayak V, Pennington C, Pinney DF, Roos DS, Ross C, Stoeckert CJ, Sullivan S, Treatman C, Wang H: GiardiaDB and TrichDB: integrated genomic resources for the eukaryotic protist pathogens Giardia lamblia and Trichomonas vaginalis. Nucleic Acids Res. 2009, 37: D526-530. 10.1093/nar/gkn631.PubMed CentralPubMedView ArticleGoogle Scholar
- Zhang Z, Schaffer AA, Miller W, Madden TL, Lipman DJ, Koonin EV, Altschul SF: Protein sequence similarity searches using patterns as seeds. Nucleic Acids Res. 1998, 26: 3986-3990. 10.1093/nar/26.17.3986.PubMed CentralPubMedView ArticleGoogle Scholar
- Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997, 25: 3389-3402. 10.1093/nar/25.17.3389.PubMed CentralPubMedView ArticleGoogle Scholar
- Pruitt KD, Tatusova T, Maglott DR: NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 2007, 35: D61-65. 10.1093/nar/gkl842.PubMed CentralPubMedView ArticleGoogle Scholar
- SPyPhy. [http://www.cbs.dtu.dk/researchgroups/metagenomics/metagenomics.php]
- Gattiker A, Gasteiger E, Bairoch A: ScanProsite: a reference implementation of a PROSITE scanning tool. Appl Bioinformatics. 2002, 1: 107-108.PubMedGoogle Scholar
- Liston DR, Johnson PJ: Gene Transcription in Trichomonas vaginalis . Parasitol Today. 1998, 14: 261-265. 10.1016/S0169-4758(98)01264-2.PubMedView ArticleGoogle Scholar
- Bendtsen JD, Nielsen H, von Heijne G, Brunak S: Improved prediction of signal peptides: SignalP 3.0. J Mol Biol. 2004, 340: 783-795. 10.1016/j.jmb.2004.05.028.PubMedView ArticleGoogle Scholar
- Krogh A, Larsson B, von Heijne G, Sonnhammer EL: Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001, 305: 567-580. 10.1006/jmbi.2000.4315.PubMedView ArticleGoogle Scholar
- Juretic D, Zoranic L, Zucic D: Basic charge clusters and predictions of membrane protein topology. J Chem Inf Comput Sci. 2002, 42: 620-632.PubMedView ArticleGoogle Scholar
- Kall L, Krogh A, Sonnhammer EL: Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server. Nucleic Acids Res. 2007, 35: W429-432. 10.1093/nar/gkm256.PubMed CentralPubMedView ArticleGoogle Scholar
- Punta M, Forrest LR, Bigelow H, Kernytsky A, Liu J, Rost B: Membrane protein prediction methods. Methods. 2007, 41: 460-474. 10.1016/j.ymeth.2006.07.026.PubMed CentralPubMedView ArticleGoogle Scholar
- SAPS. [http://www.isrec.isb-sib.ch/software/SAPS_form.html]
- Brendel V, Bucher P, Nourbakhsh IR, Blaisdell BE, Karlin S: Methods and algorithms for statistical analysis of protein sequences. Proc Natl Acad Sci USA. 1992, 89: 2002-2006. 10.1073/pnas.89.6.2002.PubMed CentralPubMedView ArticleGoogle Scholar
- Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG: Clustal W and Clustal X version 2.0. Bioinformatics. 2007, 23: 2947-2948. 10.1093/bioinformatics/btm404.PubMedView ArticleGoogle Scholar
- Galtier N, Gouy M, Gautier C: SeaView and Phylo_win, two graphic tools for sequence alignment and molecular phylogeny. Comput Applic Biosci. 1996, 12: 543-548. [http://bioinformatics.oxfordjournals.org/cgi/content/short/12/6/543]Google Scholar
- Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, Bork P, Das U, Daugherty L, Duquenne L, Finn RD, Gough J, Haft D, Hulo N, Kahn D, Kelly E, Laugraud A, Letunic I, Lonsdale D, Lopez R, Madera M, Maslen J, McAnulla C, McDowall J, Mistry J, Mitchell A, Mulder N, Natale D, Orengo C, Quinn AF, Selengut JD, Sigrist CJ, Thimma M, Thomas PD, Valentin F, Wilson D, Wu CH, Yeats C: InterPro: the integrative protein signature database. Nucleic Acids Res. 2009, 37: D211-215. 10.1093/nar/gkn785.PubMed CentralPubMedView ArticleGoogle Scholar
- Diamond LS: The establishment of various trichomonads of animals and man in axenic cultures. J Parasitol. 1957, 43: 488-490. 10.2307/3274682.PubMedView ArticleGoogle Scholar
- TvXpress. [http://TvXpress.cgu.edu.tw]
- Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, Popov D, Ryltsov A, Kostukovich E, Borisovsky I, Liu Z, Vinsavich A, Trush V, Quackenbush J: TM4: a free, open-source system for microarray data management and analysis. Biotechniques. 2003, 34: 374-378.PubMedGoogle Scholar
- Crouch ML, Alderete JF: Trichomonas vaginalis interactions with fibronectin and laminin. Microbiology. 1999, 145 (Pt 10): 2835-2843.PubMedView ArticleGoogle Scholar
- Addis MF, Rappelli P, Delogu G, Carta F, Cappuccinelli P, Fiori PL: Cloning and molecular characterization of a cDNA clone coding for Trichomonas vaginalis alpha-actinin and intracellular localization of the protein. Infect Immun. 1998, 66: 4924-4931.PubMed CentralPubMedGoogle Scholar
- Rappelli P, Carta F, Delogu G, Addis MF, Dessi D, Cappuccinelli P, Fiori PL: Mycoplasma hominis and Trichomonas vaginalis symbiosis: multiplicity of infection and transmissibility of M. hominis to human cells. Arch Microbiol. 2001, 175: 70-74. 10.1007/s002030000240.PubMedView ArticleGoogle Scholar
- Addis MF, Rappelli P, Pinto De Andrade AM, Rita FM, Colombo MM, Cappuccinelli P, Fiori PL: Identification of Trichomonas vaginalis alpha-actinin as the most common immunogen recognized by sera of women exposed to the parasite. J Infect Dis. 1999, 180: 1727-1730. 10.1086/315095.PubMedView ArticleGoogle Scholar
- STATTEST. [http://statpages.org/ctab2x2.html]
- Turnbaugh PJ, Ley RE, Hamady M, Fraser-Liggett CM, Knight R, Gordon JI: The human microbiome project. Nature. 2007, 449: 804-810. 10.1038/nature06244.PubMed CentralPubMedView ArticleGoogle Scholar
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