Insight into trichomonas vaginalis genome evolution through metabolic pathways comparison

Trichomonas vaginalis causes the trichomoniasis, in women and urethritis and prostate cancer in men. Its genome draft published by TIGR in 2007 presents many unusual genomic and biochemical features like, exceptionally large genome size, the presence of hydrogenosome, gene duplication, lateral gene transfer mechanism and the presence of miRNA. To understand some of genomic features we have performed a comparative analysis of metabolic pathways of the T. vaginalis with other 22 significant common organisms. Enzymes from the biochemical pathways of T. vaginalis and other selected organisms were retrieved from the KEGG metabolic pathway database. The metabolic pathways of T. vaginalis common in other selected organisms were identified. Total 101 enzymes present in different metabolic pathways of T. vaginalis were found to be orthologous by using BLASTP program against the selected organisms. Except two enzymes all identified orthologous enzymes were also identified as paralogous enzymes. Seventy-five of identified enzymes were also identified as essential for the survival of T. vaginalis, while 26 as non-essential. The identified essential enzymes also represent as good candidate for novel drug targets. Interestingly, some of the identified orthologous and paralogous enzymes were found playing significant role in the key metabolic activities while others were found playing active role in the process of pathogenesis. The N-acetylneuraminate lyase was analyzed as the candidate of lateral genes transfer. These findings clearly suggest the active participation of lateral gene transfer and gene duplication during evolution of T. vaginalis from the enteric to the pathogenic urogenital environment.


Background:
Trichomonas vaginalis was first described by Donne in 1836 [1].T. vaginalis cause of trichomoniasis with an estimate, number one nonviral and second most sexually transmitted disease (STD) [2].According to WHO trichomoniasis results women with 250 million infections in the world each year.T. vaginalis transmitted mostly by sexual contact it results in bad pregnancy outcome and also increases the chances of HIV infection [3,4].Infection is treated and cured with metronidazole or tinidazole, and is prescribed to any sexual partner(s) as well because they may be asymptomatic carriers [5].The draft genome sequence of the Trichomonas vaginalis, a sexually transmitted human pathogen has been published in 2007 [6].Its genome presents many unusual genomic and biochemical feature like, an abnormally large genome size of 160 Mb.T. vaginalis has been evolved to the pathogenic urogenital environment from the enteric environment.Therefore it is important to get insight into the mechanism of genome evolution that enabled T. vaginalis in acquiring the adaptations essential for the pathogenic behavior.Essential enzymes are those which are indispensable for the survival of an organism, and therefore are considered a foundation of life.Therefore the identified orthologous and paralogous enzymes were also analyzed whether they belongs to essential gene category or not.The N-acetylneuraminate lyase was investigated as the candidate of lateral genes transfer.Here, we present a comparative metabolic pathway analysis 1) To identify metabolic pathways in T. vaginalis which are common in selected organisms; 2) To identify orthologous and paralogous enzymes among common identified metabolic pathways; 3) To identify the essential enzymes.

Methodology:
The enzymes and metabolic pathways of T. vaginalis and other 22 common selected organisms were retrieved from KEGG pathway database

Discussion:
The comparative analysis of T. vaginalis metabolic pathways was carried out with the selected 22 common organisms.Most of the selected organisms were having maximum conserved metabolic pathways (Figure 1).We have found that although the selected organisms belong to different kingdoms like Eukaryota, Animalia, Fungi and Bacteria most of the metabolic pathways were common.Some metabolic pathways like Caffeine metabolism, N-Glycan biosynthesis and Glycosaminoglycan degradation were conserved only in few organisms like Aspergillus niger, Bacillus subtilis, Homo sapiens, Candida albicans, Entamoeba histolytica, Plasmodium vivax, Giardia lamblia etc, Table 1 (see supplementary material).The orthologous and paralogous T.vaginalis enzymes which were and found in the selected organism were also identified Table 2 (see supplementary material).In most of the organisms almost all metabolic pathway are conserved, except some organism, like Entamoeba histolytica, Plasmodium vivax, Salmonella enteric, Haemophilus influenza and Helicobacter pylori where there was difference in the metabolic pathways (Figure 1).Red pattern represents total metabolic pathways present in selected organism while blue pattern represent common metabolic pathway.
T. vaginalis has evolved from enteric environment to the urinary tract environment where it causes the infection.The evolution of a parasitic lifestyle of T. vaginalis necessitates adaptations to specialized features essential for pathogenesis [9].Examples of common adaptive features include host interaction systems, metabolic pathways that allow the acquisition of nutrients from the host and mechanisms to evade host defenses.Such traits could originate by a process of gradual change, but there are mechanisms that would allow potential parasites to adapt very quickly.One of these mechanisms is lateral gene transfer.Lateral transfer is the process by which genetic information is passed from one genome to an unrelated genome, where it is stably integrated and maintained.There is growing evidence from whole-genome analyses, which concludes this process as an important mechanism in genome evolution [10].In the present study we have found total 101 orthologous enzymes Table 2 (see supplementary material), present in different organisms.Presence of large number of orthologous proteins across wide range of different organisms indicates the active participation of lateral gene transfer events in the T. vaginalis from other genomes.This may have helped T. vaginalis in acquiring features required for pathogenicity as lateral transfer could allow a previously harmless organism to rapidly colonize a new environment by acquiring highly specific biochemical functionality by gradual adaptation.
Herein, the important identified orthologous enzymes which are possible of lateral gene transfer and also playing key roles like host interaction systems, metabolic pathways that allow the acquisition of nutrients from the host and mechanisms to evade host defenses, are discussed.TVAG_044970 (Nacetylneuraminate lyase) identified as orthologous enzymes.Nacetylneuraminate lyase is an enzyme involved in the metabolism of sialic acids.N-acetylneuraminate lyase is an enzyme involved in the metabolism of sialic acids.Nacetylneuraminate lyase is the final enzyme in the sialic acid degradative pathway therefore, plays significant role in the sialic acid metabolism.Moreover, this event may represent an important factor in the evolution of parasitism [11].The physicochemical properties variations.The percentage of different secondary structures (alpha helix, extended strand & random coil) was also found to be similar (Figure 2).Similarly no significant variation was also found in the number of motif, their length and their composition  Similarly other important enzymes like TVAG_261970 Carbamate kinase and TVAG_387920 cysteine synthase were also identified as orthologous.Carbamate kinase catalyzes the reversible reaction of carbamoyl phosphate, ADP to ATP and ammonium phosphate which is then hydrolyzed to ammonia and carbonate.This enzyme has involved in the different metabolic pathways like purine, arginine, proline, and nitrogen metabolisms of the T.vaginalis.These metabolic pathways in trichomonads cause the rapid depletion of arginine in vaginal and seminal fluid with an accompanied production of putrescine.This carbamate kinase pathway has been reported in many prokaryotes and two primitive eukaryotes namely Giardia lamblia and T. vaginalis [14].TVAG_387920 (cysteine synthase) is also identified as orthologous.T. vaginalis is an anaerobic protozoan parasite of humans that relies heavily on cysteine, a major redox buffer, as it lacks glutathione.This has been reported that for synthesis of cysteine from sulfide, T. vaginalis relies upon cysteine synthase.This parasite enzyme could be an exploitable drug target [15], TVAG_474980 (thioredoxin reductase) also identified as orthologous protein.
The thioredoxin system is one of the important defense mechanisms in trichomonads as if offers major antioxidant activity in response to environmental changes.Increase in the levels of thioredoxin and thioredoxin peroxidase has been reported.Sequence data indicate that the thioredoxin reductase of trichomonads differs fundamentally in structure from that of its human host and thus may represent as a useful drug target [16].
In the present study 75 orthologous enzymes were identified which were found as essential enzymes while 26 enzymes as non-essential Table 2 (see supplementary material).Essential genes constitute a minimal genome, forming a set of functional modules, which play key roles in activities essential for the survival of an organism.Therefore essential gene products also comprise excellent targets for antibacterial drugs.It has been reported that the most recent common ancestor of T. vaginalis underwent a population bottleneck during its transition from an enteric environment (the habitat of most trichomonads) to the urogenital tract.During this time, the decreased effectiveness of selection resulted in repeat accumulation and differential gene family expansion.One of the possible mechanisms behind genome expansion is gene duplication.In case of gene duplication, a non functional copy of a gene gets incorporated in the host genome.Many protein families underwent massive duplication [17].Pseudogenes are DNA sequences that were derived from a functional copy of a gene but which have acquired mutations that are deleterious to function.This duplicated copy of original functional gene gets incorporated into a new chromosomal location leading to expansion of the existing gene family and hence can lead to genome expansion [9].The pseudogenes get expressed in the form of paralogous proteins.In present study we have found that except TVAG_243770 (hypoxanthine phosphoribosyltransferase) and TVAG_248810 (Glutathione peroxidase) all identified orthologous enzymes were also identified as paralogous proteins.In case of T. vaginalis it is an interesting finding because the genome draft of T. vaginalis reveals exceptionally large genome size of 160 Mb nearly ten times larger than predicted earlier.The genome expansion has been thought to be cause of exceptionally large genome [17].
It will be interesting to predict and validate other examples of lateral gene transfer and investigate whether these examples are involved in pathogenesis or not.Due to a lack of supporting data in the literature, about other identified orthologous and paralogous enzymes in T. vaginalis, these predicted enzymes reported in this paper are only a "first order guess" for probable candidate of lateral gene transfer mechanism.These enzymes are of particular interest for further characterization to verify the roles and essentiality for T. vaginalis survival.

Conclusion:
The comparison of metabolic pathways of T. vaginalis with 22 common organism revealed 101 enzymes present in T. vaginalis as orthologous while 99 enzymes as paralogous.It is significant finding as 75 of these enzymes were also identified as essential for the survival of T. vaginalis.Identification of orthologous and paralogous enzymes clearly indicates the involvement of lateral gene transfer (indicated by case study of N-acetylneuraminate lyase) and gene duplication events during the genome evolution of T. vaginalis.We can conclude that the expansion of genetic material may be due to the adaptations of the T. vaginalis during its transition to a urogenital environment from enteric environment (the habitat of most trichomonads).Aspergillus niger Eukaryota 3.

Homo sapiens Animalia
Caffeine metabolism

[ 7 ]
. Metabolic pathways which were common with T. vaginalis were identified.Each T. vaginalis enzyme in common metabolic pathways are used for the database search against all 22 selected organisms for the identification of orthologous enzymes.The database search was performed with BLAST program [8].Using sequence identity more than 40 %, query coverage more than 80 % and having significantly low e value.Paralogous enzymes were identified from selected orthologous enzymes by performing the database search of selected orthologous enzymes against the T. vaginalis proteome.The identified orthologous & paralogous enzymes were again screened against the Database of Essential Genes.BLASTP search for N-acetylneuraminate lyase of T. vaginalis (Q27818) was performed to select the related sequences in different organisms.Four other N-acetylneuraminate lyase sequences in Haemophilus parainfluenzae (E1W1D4), Pasteurella dagmatis (C9PLY8), Haemophilus influenza (A4N656) and Aggregatibacter actinomycetemcomitans (G4B7P9) were selected.The physicochemical properties were calculated by ProtParam, motif prediction by motif search (www.genome.jp/tools/motif/),secondary structures prediction by GOR IV method, predicting Pfam families and conserved domain by (NCBI CD search tool).

Figure 1 :
Figure 1: Comparison of metabolic pathway comparison with selected organism

Figure 2 :
Figure 2: Secondary Structure of N-acetylneuraminate lyase in selected organisms.TVAG_318670 (AP33), TVAG_318670 (AP33), TVAG_144730 (AP51) and TVAG_183500 (AP51) were identified as orthologous proteins, all are adhesin proteins helping the T. vaginalis in adhering the site of infection and colonization of vaginal mucosa.T. vaginalis is a mucosal parasite of the urogenital vaginal tract thus it needs pathogenesis of adherence to the cervicovaginal epithelium and get colonization therefore these adhesin proteins are playing key role in host-pathogen interaction.These proteins mediate the interaction of the parasite to the receptor molecules [12, 13].Similarly other important enzymes like TVAG_261970 Carbamate kinase and TVAG_387920 cysteine synthase were also identified as orthologous.Carbamate kinase catalyzes the reversible reaction of carbamoyl phosphate, ADP to ATP and ammonium phosphate which is then hydrolyzed to ammonia and carbonate.This enzyme has involved in the different metabolic pathways like purine, arginine, proline, and nitrogen metabolisms of the T.vaginalis.These metabolic pathways in trichomonads cause the rapid depletion of arginine in vaginal and seminal fluid with an accompanied production of putrescine.This carbamate kinase pathway has been reported in many prokaryotes and two primitive eukaryotes namely Giardia lamblia and T. vaginalis[14].TVAG_387920 (cysteine synthase) is

Table 5 (see supplementary material). The PfamTable 6 (see supplementary material
[11]d conserved domain Table7(see supplementary material) predicted for Nacetylneuraminate lyase in different organisms was also found to be same.From the sequence (physicochemical properties), pattern (motif & conserved domain), Pfam and structural (secondary structures) analysis, it is clear that the Nacetylneuraminate lyase of T. vaginalis was very similar with Nacetylneuraminate lyase in other selected organisms (bacteria).It thus supports the role of lateral gene transfer mechanism in the transfer of N-acetylneuraminate lyase from bacterial genome and to the T. vaginalis genome[11].Similar studies can also be carried out to investigate other identified enzymes as the possible candidate of lateral gene transfer.

Table 1 :
Metabolic pathway common in few organism

Table 2 :
T. vaginalis orthologous genes identified by metabolic pathway comparison

Table 3 :
Physicochemical properties of N-acetylneuraminate lyase in selected organisms

Table 4 :
Amino acid composition N-acetylneuraminate lyase in selected organisms.

Table 5 :
Motifs predicted for N-acetylneuraminate lyase in selected organisms.

Table 6 :
Pfam predicted for N-acetylneuraminate lyase in selected organisms

Table 7 :
Conserved domain predicted for N-acetylneuraminate lyase in selected organisms