Cellular and molecular basis of TV virulence

A number of recent reviews have discussed various aspects of well-characterised and more speculative virulence factors of TV.10–13 Only a selection of TV virulence factors will be considered here and integrated with some of the most recent data on TV molecular cell biology and human innate and adaptive responses to TV. Some important gaps in our knowledge of TV pathobiology will also be highlighted. The evolutionary origin of the best-studied organelle of the parasite, the hydrogenosome, and the roles of hydrogenosomal enzymes in the parasite's physiology and as a drug target were recently reviewed10 ,14 and are not covered here.

TV is a flagellated microbial eukaryote known to exist in several cellular forms (figure 1). The two best characterised forms are the trophozoite, a free-swimming, flagellated, pear-like cell, and an amoeboid form, with a pancake shape characterised by an important increase in surface contact; this is rapidly induced upon trophozoite contact in vivo with epithelial cells from the vagina, cervix, urethra, prostate and extracellular matrix (ECM) proteins (figure 1).1 ,11 ,12 Trophozoites are typically considered as the infective form. A third cellular form called pseudocysts can be induced in vitro upon exposure of trophozoites to cold and other stressors.15 However, the significance of this form during the infection process is currently unknown.1 Data for the related bovine parasite Tritrichomonas foetus, which leads to various reproductive complications including abortion and sterility, suggest that pseudocysts are highly relevant corresponding to aggressive forms facilitating transmission to, and the initial colonisation of, a new host.16 ,17 Additionally, aggregates of parasites are also known to form (figure 1). Very little is known concerning the triggers and cellular signalling that orchestrate TV differentiations into these different forms.11 Identifying specific molecular markers for the different TV cellular forms and aggregates will be an essential prerequisite to investigate their relevance during the infection process. The draft genome sequence and its annotation represent an invaluable resource to investigate the molecular cell biology of TV by providing specific molecular leads and allowing comparative transcriptomics and proteomics investigations.10 ,18 An initial study of the phospho-proteome of TV trophozoites, amoeba and pseudocysts grown in vitro suggested differential protein phosphorylation profiles, consistent with specific signalling mechanisms occurring in the different cellular forms of the parasite that are induced upon specific environmental triggers.19

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Figure 1

Various cellular forms of Trichomonas vaginalis (TV). The TV trophozoite is characterised with four anterior and a fifth recurrent flagella. Upon contact with human tissue, TV can rapidly differentiate into an amoeboid form. In vitro trophozoites can differentiate into pseudocysts upon exposure to cold. Pseudocysts are spherical cells without visible flagella (they are internalised), they can divide and redifferentiate into trophozoites.15 It is not known if amoeboid form can differentiate into pseudocysts (?). Trophozoites are also known to form large cellular aggregates, in a process called swarming. Such groups of cells are thought to have stronger binding and cytotoxicity properties towards human tissues, and could also represent a form of defensive reaction to the host immune responses.21.

Clinical isolates of TV have been accumulated over the years from many regions of the world.20 ,21 Comparisons of their capacity to bind and kill human (and other species) cell lines in vitro have demonstrated important variations between TV isolates.13 ,21 Accordingly, these capacities are considered important virulent traits for TV.11–13 One of the currently best-characterised adhesins mediating parasite binding to host tissue are TV lipoglycans (TvLG), the most abundant surface molecules of the parasite.13 Investigating TvLG also led to the identification of galectin-1, the only identified human receptor for TV13 so far. TvLGs are also known to modulate inflammatory responses of epithelial cells and macrophages.13 A proteomics survey of TV surface proteins identified a total of 411 proteins,22 confirming a number of in silico-predicted surface proteins, and also a host of novel and important candidate virulent factors.11 ,13 ,22 By contrasting strains with low versus high level of adhesion to vaginal epithelial cells (VECs) in vitro, 11 proteins were shown to be more abundant on the cell surface of the highly adhering strains.22 These include proteins annotated as hypotheticals—which have no sequence similarity to any other proteins in protein databases. A high level of expression of two of these hypothetical proteins increased the binding to VECs of a poorly binding TV strain.22 Other novel candidate cell surface virulence factors identified in this proteomics survey included three tetraspanins, which are membrane proteins involved in signalling modulating adhesion, motility and tissue invasion in other systems; all these are key processes underlying TV pathobiology.13 The characterisation of one TV tetraspanin demonstrated that its expression and cellular localisation was modulated upon TV binding to VEC, and that it plays a role in regulating migration of the parasite through a surrogate ECM gel, strongly supporting tetraspanins as important TV virulence factors.23 These studies highlight how little we know about key aspects of TV biology, and how genomics and allied omics provide important tools to investigate host-parasite interactions.10 Related proteomics studies on TV are reviewed elsewhere.10–13

During the infection process, TV actively phagocytoses human cells, bacteria and fungi to obtain nutrients.11 ,12 Similarly, receptor-mediated endocytosis by TV is also considered important in order to internalise nutrients and iron, and to neutralise host defence proteins.11 ,12 However, there are currently no functionally characterised TV genes encoding surface proteins mediating the specific binding process underlying phagocytosis or endocytosis.11 ,12 Bioinformatic analyses of candidate transmembrane proteins have identified a number of cytoplasmic tails possessing classic signals for endocytosis, some of which were supported by proteomics data (table 1).11 ,13 In silico identification of candidate genes regulating membrane trafficking have also identified a surprisingly large repertoire of proteins, with some gene families unexpectedly larger than those encoding human homologues (eg, ∼300 TvRab vs ∼70 HsRab GTPases), further suggesting that phagocytosis and endocytosis are important processes for the parasite and represent promising targets for interfering with parasite virulence.11 ,12 ,18

TV interactions with human-associated bacteria and viruses

The phagocytosis of human microbiota by TV, including bacteria and fungal cells, has the potential to induce dysbiosis (imbalanced microbial community7) in the human microbiota, for example, as observed during bacterial vaginosis (BV). Consistent with this possibility, a correlation between the presence of TV and a vaginal microbiota characterised with low abundance of protective lactobacilli and higher proportions of Mycoplasma, Prevotella and other bacteria typically observed in BV, has been observed.9 Future work will be required to establish temporality and causality between BV and TV, and their contribution (possibly synergistic) to increase host susceptibility to HIV.9 In addition, antibiotics can significantly disturb the host-microbiota-viruses balances, and are also increasingly recognised to contribute to various disease conditions.7 Hence, the potential negative impact of the treatment of TV with metronidazole/tinidazole should also be considered in this global framework of human–microbes interactions.

TV has been shown to form a symbiosis with Mycoplasma hominis in in vitro cultures, and Mycoplasma cells carried by TV could infect human cells, suggesting that TV could be a Trojan horse for the bacterium.24 Intriguingly, coinfections with TV-Mycoplasma were shown to consume larger amounts of free arginine than TV alone, which could contribute to reducing nitric oxide production by macrophages through depletion of free arginine in the vagina, thus potentially interfering with an important host defence mechanism.25

Another likely consequence of TV actively feeding on host microbiota, is the acquisition of a number of bacterial genes through lateral gene transfer (LGT). Of particular interest is an almost complete set of enzymes of bacterial origin capable of degrading glycans, and mostly acquired from members of the bacteroidetes,26 one of the most abundant bacterial lineage of mucosal microbiota and known to encode a rich set of glycan-targeting enzymes27 (table 2). These TV enzymes of bacterial origins are thought to play important roles in degrading mucins and other human glycans, including those from the glycocalyx of epithelial cells, which represent the initial barrier the parasite needs to deal with to be able to colonise the host mucosal surfaces.28 Glycan degradation is also likely to provide an important source of energy for the parasite.

Tissue damage and inflammatory responses due to TV infections are thought to facilitate HIV entry and, in TV-HIV dually infected patients, to stimulate HIV production.1 Additionally, TV can also internalise human viruses through endocytosis.11 Viruses carried by TV could contribute to their transmission to a new host, as viruses internalised by TV were shown to be infectious for human cells for 2–6 days. The viruses are either released upon parasite death or secreted from TV-loaded viruses through the recycling route of the endocytic pathway.11 However, there is no evidence that any human virus can replicate in TV. Hence, as suggested for Mycoplasma, TV could be a Trojan horse for HIV and possibly other viruses.

These different considerations clearly illustrate the importance of studying a pathogen in the broader context of human–microbes interactions (figure 2).7

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Figure 2

Interactions between Trichomonas vaginalis (TV), viruses, microbiota and the human host and their impact on human health. The diagram illustrates a TV-infected pregnant woman and her partner in the global context of human–microbe interactions.7 TV infections can affect the health status of both the adults and the embryo (eg, preterm birth and HIV transmission in utero).1 ,3 Rather then single/linear human–pathogen interactions, it is the complex network of interactions between viruses-bacteria-eukaryotes and the human host that should be considered when investigating disease conditions.7

TV viruses

Adding to its capacity to physically interact with human viruses and facilitate HIV transmission through damaging mucosal surfaces, TV can be infected with four dsRNA viruses (TVV) members of the Totiviridae, thought to be transmitted mainly vertically.29 Various data suggest, collectively, that TVV modulate the parasite virulence by influencing TV gene expression; however, additional studies are required to firmly establish this fascinating possibility.29 Two recent developments are of particular interest in relation to a potential role of TVV in TV pathobiology. A study investigating TV genetic diversity and population structure identified two major genotypes for the parasite (TV1 and TV2) with distinct properties.20 TV1 isolates are more sensitive to metronidazole, and are also significantly more likely to be infected with TVV than TV2, in a ratio ∼3:1.20 In another study investigating the potential role of TVV in modulating the human innate immune system, it was established that TVV are sensed by the human toll-like receptor 3 (TLR3), and that this induces an innate response, including proinflammatory cascades, previously implicated in preterm birth and HIV-1 susceptibility.30 These new aspects of TV pathobiology should be taken into account in future research on TV pathobiology, and influence, for example, how clinical samples are processed for research and, possibly, diagnostics. For instance, to allow quantification of TVV, processing of both total DNA (to investigate TV, bacteria, etc…) and RNA will be required.