NlpC/P60 peptidoglycan hydrolases of Trichomonas vaginalis have complementary activities that empower the protozoan to control host-protective lactobacilli.

Trichomonas vaginalis is a human protozoan parasite that causes trichomoniasis, a prevalent sexually transmitted infection. Trichomoniasis is accompanied by a shift to a dysbiotic vaginal microbiome that is depleted of lactobacilli. Studies on co-cultures have shown that vaginal bacteria in eubiosis (e.g. Lactobacillus gasseri) have antagonistic effects on T. vaginalis pathogenesis, suggesting that the parasite might benefit from shaping the microbiome to dysbiosis (e.g. Gardnerella vaginalis among other anaerobes). We have recently shown that T. vaginalis has acquired NlpC/P60 genes from bacteria, expanding them to a repertoire of nine TvNlpC genes in two distinct clans, and that TvNlpCs of clan A are active against bacterial peptidoglycan. Here, we expand this characterization to TvNlpCs of clan B. In this study, we show that the clan organisation of NlpC/P60 genes is a feature of other species of Trichomonas, and that Histomonas meleagridis has sequences related to one clan. We characterized the 3D structure of TvNlpC_B3 alone and with the inhibitor E64 bound, probing the active site of these enzymes for the first time. Lastly, we demonstrated that TvNlpC_B3 and TvNlpC_B5 have complementary activities with the previously described TvNlpCs of clan A and that exogenous expression of these enzymes empower this mucosal parasite to take over populations of vaginal lactobacilli in mixed cultures. TvNlpC_B3 helps control populations of L. gasseri, but not of G. vaginalis, which action is partially inhibited by E64. This study is one of the first to show how enzymes produced by a mucosal protozoan parasite may contribute to a shift on the status of a microbiome, helping explain the link between trichomoniasis and vaginal dysbiosis. Further understanding of this process might have significant implications for treatments in the future.

Lactobacillus gasseri and Gardnerella vaginalis produce extracellular vesicles that contribute to the function of the vaginal microbiome and modulate host-Trichomonas vaginalis interactions.

Trichomonas vaginalis is an extracellular protozoan parasite of the human urogenital tract, responsible for a prevalent sexually transmitted infection. Trichomoniasis is accompanied by a dysbiotic microbiome that is characterised by the depletion of host-protective commensals such as Lactobacillus gasseri, and the flourishing of a bacterial consortium that is comparable to the one seen for bacterial vaginosis, including the founder species Gardnerella vaginalis. These two vaginal bacteria are known to have opposite effects on T. vaginalis pathogenicity. Studies on extracellular vesicles (EVs) have been focused on the direction of a microbial producer (commensal or pathogen) to a host recipient, and largely in the context of the gut microbiome. Here, taking advantage of the simplicity of the human cervicovaginal microbiome, we determined the molecular cargo of EVs produced by L. gasseri and G. vaginalis and examined how these vesicles modulate the interaction of T. vaginalis and host cells. We show that these EVs carry a specific cargo of proteins, which functions can be attributed to the opposite roles that these bacteria play in the vaginal biome. Furthermore, these bacterial EVs are delivered to host and protozoan cells, modulating host-pathogen interactions in a way that mimics the opposite effects that these bacteria have on T. vaginalis pathogenicity. This is the first study to describe side-by-side the protein composition of EVs produced by two bacteria belonging to the opposite spectrum of a microbiome and to demonstrate that these vesicles modulate the pathogenicity of a protozoan parasite. Such as in trichomoniasis, infections and dysbiosis co-occur frequently resulting in significant co-morbidities. Therefore, studies like this provide the knowledge for the development of antimicrobial therapies that aim to clear the infection while restoring a healthy microbiome.

Vaginal dysbiotic bacteria act as pathobionts of the protozoal pathogen Trichomonas vaginalis.

Trichomoniasis, a prevalent sexually transmitted infection caused by the protozoan parasite Trichomonas vaginalis, is often accompanied by a vaginal dysbiotic microbiota of pathogenic potential. Our objective was to investigate whether these dysbiotic bacteria act as pathobionts of T. vaginalis infection by altering pathogenic capabilities of the parasite, particularly in regard to adhesion to vaginal substrates and viability of human ectocervical cells. Assays interrogated the performance of T. vaginalis adhesion to biofilm produced by vaginal dysbiotic bacteria and whether these bacteria were capable of altering the ability of the parasite to bind to mucins and cells. The binding activities of T. vaginalis were quantified by flow cytometry. Host cell viability and apoptosis, as affected by T. vaginalis with or without the bacteria, were also measured experimentally. An in vitro biofilm was shown to provide adhesion for T. vaginalis. The binding of parasites to mucins and cells was modulated by the vaginal dysbiotic bacteria. Parasite cytoadhesion was significantly increased by these bacteria. In addition, these bacteria enhanced the pathogenic effects of the parasite to host cells. Together, this study showed that dysbiotic bacteria accompanying T. vaginalis infection in the vagina function as pathobionts as they are capable of enhancing the pathogenic capabilities of this parasite. This study highlights the importance of understanding the contribution of the vaginal microbiome to trichomoniasis.

Cooperative Interactions between Trichomonas vaginalis and Associated Bacteria Enhance Paracellular Permeability of the Cervicovaginal Epithelium by Dysregulating Tight Junctions.

The human protozoan Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection, which is accompanied by a species-diversified vaginal microbiota named community state type IV (CST-IV). Coincidently, CST-IV includes species associated with bacterial vaginosis (e.g. Gardnerella vaginalis, Atopobium vaginae, and Prevotella bivia). Both diseases are linked to the transmission of human immunodeficiency virus (HIV) and preterm birth, which complications are likely to result from the disruption of the cervicovaginal epithelial barrier. Here, we show that paracellular permeability of fluorescein isothiocyanate (FITC)-dextran through a monolayer of human ectocervical cells (hECs) is increased as a consequence of the activity of T. vaginalis and the aforementioned species of CST-IV bacteria cooperatively. T. vaginalis enhances paracellular permeability of hECs two times more than the individual bacterial species, by up to ∼10% versus ∼5%, respectively. However, any two or all three bacterial species are capable of synergizing this effect. T. vaginalis and the bacteria together increase the paracellular permeability of hECs by ∼50%, which is 5 to 10 times more than the results seen with the protozoan or bacteria alone. This effect is accompanied by enhancement of phosphatase activity, while phosphatase inhibition results in preservation of the integrity of the ectocervical cell monolayer. In addition, these microorganisms induce changes in the expression of tight junction proteins, particularly occludin, and of proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α). Together, our findings establish that cooperative interactions between CST-IV bacteria and T. vaginalis enhance the paracellular permeability of the cervicovaginal epithelium by disturbing the integrity of the tight junction complex. Our study results highlight the importance of understanding the contribution of the vaginal microbiota to trichomoniasis.

A Cell Surface Aggregation-Promoting Factor from Lactobacillus gasseri Contributes to Inhibition of Trichomonas vaginalis Adhesion to Human Vaginal Ectocervical Cells.

Trichomoniasis, a prevalent sexually transmitted infection, is commonly symptomatic in women. The causative agent is Trichomonas vaginalis, an extracellular protozoan parasite. The host-protective mechanisms and molecules of vaginal lactobacilli that counteract this pathogen are largely unknown. This study examines the inhibition promoted by Lactobacillus gasseri against the adhesion of T. vaginalis to host cells, a critical virulence aspect of this pathogen. We observed that the vaginal strain L. gasseri ATCC 9857 is highly inhibitory by various contact-dependent mechanisms and that surface proteins are largely responsible for this inhibitory phenotype. We found that the aggregation-promoting factor APF-2 from these bacteria significantly contributes to inhibition of the adhesion of T. vaginalis to human vaginal ectocervical cells. Understanding the molecules and mechanisms used by lactobacilli to protect the host against T. vaginalis might help in the development of novel and specific therapeutic strategies that take advantage of the natural microbiota.

Box H/ACA snoRNAs are preferred substrates for the trimethylguanosine synthase in the divergent unicellular eukaryote Trichomonas vaginalis.

The 2,2,7-trimethylguanosine caps of eukaryal snRNAs and snoRNA are formed by the enzyme Tgs1, which catalyzes sequential guanine-N2 methylations of m(7)G caps. Atypically, in the divergent unicellular eukaryote Trichomonas vaginalis, spliceosomal snRNAs lack a guanosine cap and the recombinant T. vaginalis trimethylguanosine synthase (TvTgs) produces only m(2,7)G in vitro. Here, we show by direct metabolic labeling that endogenous T. vaginalis RNAs contain m(7)G, m(2,7)G, and m(2,2,7)G caps. Immunodepletion of TvTgs from cell extracts and TvTgs add-back experiments demonstrate that TvTgs produces m(2,7)G and m(2,2,7)G caps. Expression of TvTgs in yeast tgs1Δ cells leads to the formation of m(2,7)G and m(2,2,7)G caps and complementation of the lethality of a tgs1Δ mud2Δ strain. Whereas TvTgs is present in the nucleus and cytosol of T. vaginalis cells, TMG-containing RNAs are localized primarily in the nucleolus. Molecular cloning of anti-TMG affinity-purified T. vaginalis RNAs identified 16 box H/ACA snoRNAs, which are implicated in guiding RNA pseudouridylation. The ensemble of new T. vaginalis H/ACA snoRNAs allowed us to predict and partially validate an extensive map of pseudouridines in T. vaginalis rRNA.

Unconventional features in the transcription and processing of spliceosomal small nuclear RNAs in the protozoan parasite Trichomonas vaginalis.

Trichomonas vaginalis is a medically important protozoan parasite, and a deep-branching, evolutionarily divergent unicellular eukaryote that has conserved several key features of eukaryotic gene expression. Trichomonas vaginalis possesses a metazoan/plant-like capping apparatus, mRNAs with a cap 1 structure and spliceosomes containing the five small nuclear RNAs (snRNAs). However, in contrast to metazoan and plant snRNAs, the structurally conserved T. vaginalis snRNAs were initially identified as lacking the canonical guanosine cap nucleotide. To explain this unusual condition, we sought to investigate transcriptional and processing features of the spliceosomal snRNAs in this protist. Here, we show that T. vaginalis spliceosomal snRNA genes mostly lack typical eukaryotic promoters. In contrast to other eukaryotes, the putative TATA box in the T. vaginalis U6 snRNA gene was found to be dispensable for transcription or RNA polymerase selectivity. Moreover, U6 transcription in T. vaginalis was virtually insensitive to tagetitoxin compared with other cellular transcripts produced by the same RNA polymerase III. Most important and unexpected, snRNA transcription in T. vaginalis appears to bypass capping as we show that these transcripts retain their original 5'-triphosphate groups. In conclusion, transcription and processing of spliceosomal snRNAs in T. vaginalis deviate considerably from the conventional rules of other eukaryotes.

The adherence of Trichomonas vaginalis to host ectocervical cells is influenced by lactobacilli.

OBJECTIVES: Trichomoniasis is a common sexually transmitted disease, and adhesion of the pathogen Trichomonas vaginalis to the host vaginal cells is the first step in establishing infection. For this to happen, the pathogen has to overcome a natural protective barrier composed mostly of lactobacilli. The objective of this study was to understand the role of lactobacilli in the adhesion of T vaginalis to host cells. METHODS: Adhesion assays were carried out by incubating vaginal epithelial cells (VECs) with T vaginalis and lactobacilli together and compared with non-lactobacilli recipient controls. By varying incubation parameters and testing several microbial isolates, the number of pathogens that adhered to the VECs was determined by flow cytometry. RESULTS: Overall, but with few exceptions, lactobacilli caused inhibition of T vaginalis adhesion to a variable degree. Lactobacillus gasseri ATCC 9857 and CBI3 (ambiguous Lactobacillus plantarum or Lactobacillus pentosus) caused the highest level of parasite adhesion inhibition and enhancement, respectively. These isolates of Lactobacillus can profoundly alter the adhesive properties of low-adherent and high-adherent strains of T vaginalis in a dose-dependent manner. Additionally, the effects of lactobacilli on T vaginalis adhesion are strictly contact-dependent, and surface lipoglycans of T vaginalis are most likely not involved in this modulation of adhesion mediated by the bacteria. CONCLUSIONS: Lactobacilli can modulate adhesion of T vaginalis by significantly modifying the natural adhesive properties of various T vaginalis strains. This study highlights the importance of considering the role of the vaginal microbiota in the pathogenesis of trichomoniasis.

A metazoan/plant-like capping enzyme and cap modified nucleotides in the unicellular eukaryote Trichomonas vaginalis.

The cap structure of eukaryotic messenger RNAs is initially elaborated through three enzymatic reactions: hydrolysis of the 5'-triphosphate, transfer of guanosine through a 5'-5' triphosphate linkage and N7-methylation of the guanine cap. Three distinctive enzymes catalyze each reaction in various microbial eukaryotes, whereas the first two enzymes are fused into a single polypeptide in metazoans and plants. In addition to the guanosine cap, adjacent nucleotides are 2'-O-ribose methylated in metazoa and plants, but not in yeast. Analyses of various cap structures have suggested a linear phylogenetic trend of complexity. These findings have led to a model in which plants and metazoa evolved a two-component capping apparatus and modification of adjacent nucleotides while many microbial eukaryotes maintained the three-component system and did not develop modification of adjacent nucleotides. Here, we have characterized a bifunctional capping enzyme in the divergent microbial eukaryote Trichomonas vaginalis using biochemical and phylogenetic analyses. This unicellular parasite was found to harbor a metazoan/plant-like capping apparatus that is represented by a two-domain polypeptide containing a C-terminus guanylyltransferase and a cysteinyl phosphatase triphosphatase, distinct from its counterpart in other microbial eukaryotes. In addition, T. vaginalis mRNAs contain a cap 1 structure represented by m(7)GpppAmpUp or m(7)GpppCmpUp; a feature typical of metazoan and plant mRNAs but absent in yeast mRNAs. Phylogenetic and biochemical analyses of the origin of the T. vaginalis capping enzyme suggests a complex evolutionary model where differential gene loss and/or acquisition occurred in the development of the RNA capping apparatus and cap modified nucleotides during eukaryote diversification.

A Microbial Piñata: Bacterial Endosymbionts of Trichomonas vaginalis Come in Different Flavors.

The protozoan parasite Trichomonas vaginalis causes trichomoniasis, a prevalent human urogenital infection with significant morbidity that is commonly associated with vaginal dysbiosis. Exacerbation of T. vaginalis pathogenicity has been related to endosymbionts, including mycoplasma, and thought for a while to be solely attributable to Mycoplasma hominis. In a recent publication, Margarita and colleagues (https://journals.asm.org/doi/10.1128/mbio.00918-22) showed that endosymbiosis extends to a second species of mycoplasma known as "Candidatus Mycoplasma girerdii." Those authors confirmed the strong association of T. vaginalis with both species of mycoplasma by reassessing clinical samples. Additionally, they showed that in vitro symbiosis of protozoa and bacteria resulted in the modulation of gene expression of T. vaginalis and enhancement of parasite cytoadhesion and hemolytic activity in culture assays. In this commentary, we portray T. vaginalis as a synergistically interacting multimicrobe organism-a "microbial piñata"-whose endosymbionts contribute significantly to the pathophysiology of this medically important protozoan parasite.

Gas chromatography-mass spectrometry (GC/MS) reveals urine metabolites associated to light and heavy infections by Schistosoma mansoni in mice.

High-throughput profiling of metabolites has been used to identify metabolic changes in murine models as a response to the infection by the parasitic trematode Schistosoma. These investigations have contributed to our understanding on the pathogenesis of this tropical neglected disease, with a potential of helping diagnosis. Here, our study aimed to investigate the application of gas chromatography-mass spectrometry (GC/MS) on the profiling of urine metabolites from mice carrying infections by Schistosoma mansoni. Two larval infection doses created distinctive infection intensities in mice, whereby the heavily infected animals were found to release 25 times more eggs in faeces than lightly infected animals. Over 200 urine metabolites were identified from these animals by GC/MS, following two complementary derivatisation methods. A list of 14 individual metabolites with altered relative abundances between groups were identified. Most of the altered metabolites showed a trend of increased abundances in response to infection intensity, indicating host-specific metabolic alterations as a result of the disease. Hippurate, a metabolite which concentration is intimately modulated by the gut microbiota, was found to be highly correlated to infection intensity. Our study showed that urine metabolic profiling by GC/MS can distinguish non-infected animals from those carrying light and heavy infections by S. mansoni, revealing metabolites associated to the infection and providing insights on the pathogenesis of schistosomiasis.

Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5'-cap structure.

Few genes in the divergent eukaryote Trichomonas vaginalis have introns, despite the unusually large gene repertoire of this human-infective parasite. These introns are characterized by extended conserved regulatory motifs at the 5' and 3' boundaries, a feature shared with another divergent eukaryote, Giardia lamblia, but not with metazoan introns. This unusual characteristic of T. vaginalis introns led us to examine spliceosomal small nuclear RNAs (snRNAs) predicted to mediate splicing reactions via interaction with intron motifs. Here we identify T. vaginalis U1, U2, U4, U5, and U6 snRNAs, present predictions of their secondary structures, and provide evidence for interaction between the U2/U6 snRNA complex and a T. vaginalis intron. Structural models predict that T. vaginalis snRNAs contain conserved sequences and motifs similar to those found in other examined eukaryotes. These data indicate that mechanisms of intron recognition as well as coordination of the two catalytic steps of splicing have been conserved throughout eukaryotic evolution. Unexpectedly, we found that T. vaginalis spliceosomal snRNAs lack the 5' trimethylguanosine cap typical of snRNAs and appear to possess unmodified 5' ends. Despite the lack of a cap structure, U1, U2, U4, and U5 genes are transcribed by RNA polymerase II, whereas the U6 gene is transcribed by RNA polymerase III.

The divergent eukaryote Trichomonas vaginalis has an m7G cap methyltransferase capable of a single N2 methylation.

Eukaryotic RNAs typically contain 5' cap structures that have been primarily studied in yeast and metazoa. The only known RNA cap structure in unicellular protists is the unusual Cap4 on Trypanosoma brucei mRNAs. We have found that T. vaginalis mRNAs are protected by a 5' cap structure, however, contrary to that typical for eukaryotes, T. vaginalis spliceosomal snRNAs lack a cap and may contain 5' monophophates. The distinctive 2,2,7-trimethylguanosine (TMG) cap structure usually found on snRNAs and snoRNAs is produced by hypermethylation of an m(7)G cap catalyzed by the enzyme trimethylguanosine synthase (Tgs). Here, we biochemically characterize the single T. vaginalis Tgs (TvTgs) encoded in its genome and demonstrate that TvTgs exhibits substrate specificity and amino acid requirements typical of an RNA cap-specific, m(7)G-dependent N2 methyltransferase. However, recombinant TvTgs is capable of catalysing only a single round of N2 methylation forming a 2,7-dimethylguanosine cap (DMG) as observed previously for Giardia lamblia. In contrast, recombinant Entamoeba histolytica and Trypanosoma brucei Tgs are capable of catalysing the formation of a TMG cap. These data suggest the presence of RNAs with a distinctive 5' DMG cap in Trichomonas and Giardia lineages that are absent in other protist lineages.

Determinants of translation efficiency in the evolutionarily-divergent protist Trichomonas vaginalis.

BACKGROUND: Trichomonas vaginalis, the causative agent of a prevalent urogenital infection in humans, is an evolutionarily divergent protozoan. Protein-coding genes in T. vaginalis are largely controlled by two core promoter elements, producing mRNAs with short 5' UTRs. The specific mechanisms adopted by T. vaginalis to fine-tune the translation efficiency (TE) of mRNAs remain largely unknown. RESULTS: Using both computational and experimental approaches, this study investigated two key factors influencing TE in T. vaginalis: codon usage and mRNA secondary structure. Statistical dependence between TE and codon adaptation index (CAI) highlighted the impact of codon usage on mRNA translation in T. vaginalis. A genome-wide interrogation revealed that low structural complexity at the 5' end of mRNA followed closely by a highly structured downstream region correlates with TE variation in this organism. To validate these findings, a synthetic library of 15 synonymous iLOV genes was created, representing five mRNA folding profiles and three codon usage profiles. Fluorescence signals produced by the expression of these synonymous iLOV genes in T. vaginalis were consistent with and validated our in silico predictions. CONCLUSIONS: This study demonstrates the role of codon usage bias and mRNA secondary structure in TE of T. vaginalis mRNAs, contributing to a better understanding of the factors that influence, and possibly regulate, gene expression in this human pathogen.

Extracellular vesicles produced by the protozoan parasite Trichomonas vaginalis contain a preferential cargo of tRNA-derived small RNAs.

Trichomonas vaginalis is a protozoan parasite that causes trichomoniasis, the most prevalent non-viral sexually transmitted infection worldwide. Trichomonas vaginalis releases extracellular vesicles that play a role in parasite:parasite and parasite:host interactions. The aim of this study was to characterise the RNA cargo of these vesicles. Trichomonas vaginalis extracellular vesicles were found to encapsulate a cargo of RNAs of small size. RNA-seq analysis showed that tRNA-derived small RNAs, mostly 5' tRNA halves, are the main type of small RNA in these vesicles. The tRNA-derived small RNAs in T. vaginalis extracellular vesicles were shown to be derived from the specific processing of tRNAs within cells. The specificity of this RNA cargo in T. vaginalis extracellular vesicles suggests a preference for packaging. The RNA cargo of T. vaginalis was shown to be rapidly internalised by human cells via lipid raft-dependent endocytosis. The potential role of these tsRNAs - an emerging class of small RNAs with regulatory functions - on altering host cellular responses requires further examination, suggesting a new mode of parasite:host communication.

Spliceosomal introns in Trichomonas vaginalis revisited.

BACKGROUND: The human protozoan parasite Trichomonas vaginalis is an organism of interest for understanding eukaryotic evolution. Despite having an unusually large genome and a rich gene repertoire among protists, spliceosomal introns in T. vaginalis appear rare: only 62 putative introns have been annotated in this genome, and little or no experimental evidence exists to back up these predictions. RESULTS: This study revisited the 62 annotated introns of T. vaginalis derived from the genome sequencing plus previous publications. After experimental validation and a new genome-wide search, we confirmed the presence of introns in 32 genes and 18 others were concluded to be intronless. Sequence analyses classified the validated introns into two types, based on distinctive features such as length and conservation of splice site motifs. CONCLUSIONS: Our study provides an updated list of intron-containing genes in the genome of T. vaginalis. Our findings suggests the existence of two intron 'families' spread among T. vaginalis protein-coding genes. Additional studies are needed to understand the functional separation of these two classes of introns and to assess the existence of further introns in the T. vaginalis genome.

CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis.

The sexually-transmitted parasite Trichomonas vaginalis infects ~1/4 billion people worldwide. Despite its prevalence and myriad adverse outcomes of infection, the mechanisms underlying T. vaginalis pathogenesis are poorly understood. Genetic manipulation of this single-celled eukaryote has been hindered by challenges presented by its complex, repetitive genome and inefficient methods for introducing DNA (i.e. transfection) into the parasite. Here, we have developed methods to increase transfection efficiency using nucleofection, with the goal of efficiently introducing multiple DNA elements into a single T. vaginalis cell. We then created DNA constructs required to express several components essential to drive CRISPR/Cas9-mediated DNA modification: guide RNA (gRNA), the Cas9 endonuclease, short oligonucleotides and large, linearized DNA templates. Using these technical advances, we have established CRISPR/Cas9-mediated repair of mutations in genes contained on circular DNA plasmids harbored by the parasite. We also engineered CRISPR/Cas9 directed homologous recombination to delete (i.e. knock out) two non-essential genes within the T. vaginalis genome. This first report of the use of the CRISPR/Cas9 system in T. vaginalis greatly expands the ability to manipulate the genome of this pathogen and sets the stage for testing of the role of specific genes in many biological processes.

The Protozoan Trichomonas vaginalis Targets Bacteria with Laterally Acquired NlpC/P60 Peptidoglycan Hydrolases.

The human eukaryotic pathogen Trichomonas vaginalis causes trichomoniasis, a prevalent sexually transmitted infection. This extracellular protozoan is intimately associated with the human vaginal mucosa and microbiota, but key aspects of the complex interactions between the parasite and the vaginal bacteria remain elusive. We report that T. vaginalis has acquired, by lateral gene transfer from bacteria, genes encoding peptidoglycan hydrolases of the NlpC/P60 family. Two of the T. vaginalis enzymes were active against bacterial peptidoglycan, retaining the active-site fold and specificity as dl-endopeptidases. The endogenous NlpC/P60 genes are transcriptionally upregulated in T. vaginalis in the presence of bacteria. The overexpression of an exogenous copy enables the parasite to outcompete bacteria from mixed cultures, consistent with the biochemical activity of the enzyme. Our study results highlight the relevance of the interactions of this eukaryotic pathogen with bacteria, a poorly understood aspect of the biology of this important human parasite.IMPORTANCETrichomonas vaginalis is a parasitic protozoan of the human urogenital tract that causes trichomoniasis, a very common sexually transmitted disease. Despite residing extracellularly and in close association with the vaginal bacteria (i.e., the microbiota), very little is known about the nature of the parasite-bacterium interactions. Our study showed that this parasite had acquired genes from bacteria which retained their original function. They produce active enzymes capable of degrading peptidoglycan, a unique polymer of the bacterial cell envelope, helping the parasite to outcompete bacteria in mixed cultures. This study was the first to show that a laterally acquired group of genes enables a eukaryotic mucosal pathogen to control bacterial population. We highlight the importance of understanding the interactions between pathogens and microbiota, as the outcomes of these interactions are increasingly understood to have important implications on health and disease.

The fluorescent protein iLOV outperforms eGFP as a reporter gene in the microaerophilic protozoan Trichomonas vaginalis.

Trichomonas vaginalis is a flagellated protozoan causing a notorious urogenital infection in humans. Due to its anaerobic metabolism, an alternative fluorescent protein that can be readily expressed in oxygen-deprived conditions is ideal. This study assessed the performance of iLOV, which does not require oxygen to function, as compared to the conventional enhanced green fluorescent protein (eGFP) in T. vaginalis. The results indicated that iLOV outperforms eGFP in both transient and stable expression, being detectable earlier and producing higher fluorescent intensity than eGFP in T. vaginalis. This finding facilitates forthcoming genetic studies that will advance the knowledge on this human parasitic infection.