Role of a novel uropod-like cell membrane protrusion in the pathogenesis of the parasite Trichomonas vaginalis Free

The parasite Trichomonas vaginalis is the primary cause of trichomoniasis, the most prevalent non-viral sexually transmitted disease (STD) in the world (WHO, 2021, https://www.who.int/publications/i/item/9789240027077). The symptoms are generally mild, manifesting as a general irritation and/or swelling of the urogenital tract and surrounding tissues (Fichorova, 2009; Petrin et al., 1998). However, infections can lead to severe complications such as cervical erosion and premature birth during pregnancy (Fichorova, 2009; Petrin et al., 1998). Furthermore, it can cause infertility in both men and women (Fichorova, 2009). T. vaginalis has also come to light as a crucial component in accelerating the spread of the human immunodeficiency virus (HIV), as those who are infected with it have a markedly higher risk of virus transmission (McClelland et al., 2007; Van Der Pol et al., 2008). In addition, the infection also increases the possibility of aggressive prostate and cervical cancer (Gander et al., 2009; Stark et al., 2009; Sutcliffe et al., 2009; Twu et al., 2014). These severe complications and high incidence of infection support the need to better understand the mechanism of pathogenesis, a crucial knowledge for adopting more dynamic strategies to identify novel drug and vaccine targets. Despite the prevalence of trichomoniasis, the fundamental biochemical processes driving its pathogenesis remain poorly elucidated.

As an extracellular organism, T. vaginalis must adhere to the epithelial lining of the urogenital tract of the host to survive (Ryan et al., 2011). This process, termed cytoadherence, is thus crucial for its lifestyle. Often, cytoadherence is followed by killing of the host cell. During host cell attachment, the trophozoite undergoes a transition from a pear-shaped morphology to an ameboid state (Arroyo and Alderete, 1989; Arroyo et al., 1993; González-Robles et al., 1995; Kusdian and Gould, 2014). This transformation, characterized by the enlargement of its surface area and alterations in its cytoskeleton, is crucial for facilitating parasite migration (Kusdian et al., 2013). Cell migration is a key function in different biological processes, such as development, invasion and the immune response of eukaryotic organisms (Vicente-Manzanares and Horwitz, 2011). Cells can migrate in various forms, either individually or as groups. Regardless of the chosen mode, the migrating cell is required to undergo polarization. This characteristic is based on the dynamic assembly and disassembly of actin filaments, together with specific activities in the anterior and posterior regions of the cell (Schaks et al., 2019). The cellular polarization gives rise to the development of protrusive structures during the migration and invasion process, including lamellipodia (Ma and Baumgartner, 2013), filopodia (Om Alblazi and Siar, 2015), cytonemes (Roy et al., 2014), pseudopods (van Haastert, 2020) and uropods (Hind et al., 2016; Sánchez-Madrid and Serrador, 2009). Lamellipodia, characterized as broad and flat membranous structures formed by the F-actin network, are typically situated at the leading edge (Machesky, 2008; Sadhu et al., 2023). Filopodia are actin rich finger-like protrusions of the plasma membrane that usually arise from lamellipodia and have a diameter of 0.1–0.3 µm (Mellor, 2010; Bischoff and Bogdan, 2021). They serve as tentacles for cells to explore their environment and adhere to contact surfaces. Cytonemes, considered specialized filopodia, can reach longer sizes, and have the function of transporting signaling proteins (Roy et al., 2014). Pseudopodia are tubular cellular extensions, perceived as sensory organelles, produced by chemotactic cells in random directions to sense chemoattractants (Weber, 2006). Finally, the uropod is defined as the rearward part of the cell that includes a distinctive trailing protrusion or knob-like structure (Hind et al., 2016). Although most eukaryotic cells exhibit these different forms of membrane protrusion, efficient migration demands a carefully balanced coordination between protrusive and retractile activities (Webb et al., 2002). Although research carried out on the migration process focuses on understanding the events that occur mainly at the anterior pole of the cell, the rear part is equally important because without the uncoupling of adhesion and recycling factors from the receptors, the cell would not move (Bisaria et al., 2020; Schaks et al., 2019; Webb et al., 2002). In protozoan parasites, prior studies have shown that the posterior region of the cell plays a crucial role in replenishing membrane components in Entamoeba histolytica (Arhets et al., 1995). Furthermore, the posterior part of the cell in flagellated parasites has also been associated with the removal of ligands from the surface (Krüger and Engstler, 2015), a mechanism that appears to be strategic to evade host immune responses. In T. vaginalis, it has been observed that adherent parasite strains use the posterior part to explore the surfaces when exposed to human vaginal endothelial cells (hVECs), which would indicate an important role of this structure in the initial adherence of the parasite (Hsu et al., 2023).

The adherence of T. vaginalis to host cells has been established to rely, at least partially, on different surface localized proteins, such as BAP1 and BAP2 (De Miguel et al., 2010; Pachano et al., 2017), a cadherin-like protein (Chen et al., 2019), bacteroides surface protein A (BspA) (Handrich et al., 2019; Noël et al., 2010), polymorphic membrane protein (Pmp) (Handrich et al., 2019) and tetraspanin (TSP) proteins (Coceres et al., 2015; de Miguel et al., 2012; Nievas et al., 2018a,b), among others. Specifically, different members of the T. vaginalis TSP family have been characterized, and their functions appear to be related to pathogenesis, such as being involved in cell aggregation (Nievas et al., 2018a), extracellular vesicle formation (Twu et al., 2013), host–parasite interaction and migration (Coceres et al., 2015; de Miguel et al., 2012). The extensive spectrum of biological functions in which TSPs participate indicates their functional importance. In mammalian cells, a common feature of many TSPs is their ability to regulate cell adhesion, migration and proliferation (Hemler, 2003; Jiang et al., 2015). Specifically related to migration and motility, TSPs have been shown to directly interact with several integrins modulating membrane compartmentalization, intracellular trafficking, recycling and downstream signaling in response to migratory signals (Jiang et al., 2015). As an example, the tetraspanin protein CD82 inhibits cancer cell motility by decreasing integrin-mediated signaling (Liu and Zhang, 2006). Increased expression of CD82 causes more elongated cell extensions in migrating cells and inhibits the formation of lamellipodia at the cell edge (Liu and Zhang, 2006). Similarly, it has been demonstrated that CD81 is essential to drive the development of filopodia and stress fibers in neuroblastoma cells (Martins et al., 2019), and decreased expression of CD37 is associated with a reduced ability to generate short actin protrusions in dendritic cells (De Winde et al., 2018). In immune cells, CD81 induces phosphorylation of ezrin-radixin-moesin proteins (Sala-Valdés et al., 2006) and leads to natural killer (NK) cell polarization, thereby facilitating NK cell migration towards chemoattractants (Krämer et al., 2009). These findings indicate that TSPs play a role in modulating the formation of cell membrane protrusions in different cell types, suggesting a potential regulatory influence on the dynamics of cell migration.

Here, we demonstrate that adherent T. vaginalis strains form a membranous protrusion at the posterior end of the cells, resembling the previously described uropod, which appears to play a role as an anchoring structure during the attachment process. Interestingly, overexpression of the surface-localized tetraspanin protein T. vaginalis (Tv)TSP5, leads to an increase in the formation of this uropod-like structure as well as the formation of other membranous structures, such as pseudopods, filopodia and cytonemes. Moreover, parasites overexpressing TvTSP5 exhibit an enhanced aggregation capacity between parasites and an elevated adherence to host cells. In alignment with the notion of the uropod-like protrusion serving as an anchoring structure, TvTSP5-overexpressing parasites also display reduced migration on agar plates. These findings significantly contribute to advancing our comprehension of the mechanisms employed by the parasite in the infection process and its intricate interactions with host cells.

As an extracellular parasite, the process of T. vaginalis adherence to the host cell is a key step in the establishment of infection (Ryan et al., 2011). Based on the importance of cell membrane protrusions in the process of migration and adherence in other cells, we evaluated the cell morphological appearance of T. vaginalis strains with different host cell adherence capacities. By using wheat germ agglutinin (WGA) membrane-binding lectin staining, we evaluated the formation of cell membrane protrusions in three previously characterized T. vaginalis strains, which vary in their in vitro pathogenesis-related phenotypes: a poorly adherent strain (G3), a moderately adherent strain (B7RC2) and a highly adherent strain (CDC1132) (Lustig et al., 2013) (Fig. 1A). We observed that the formation of a posterior cell membrane protrusion, resembling the uropod-like structure (Hind et al., 2016), correlates with the level of adherence to host cell of the strain. Parasites derived from the highly adherent strain CDC1132 exhibited a higher abundance of uropod-like protrusions in comparison to parasites from strains B7RC2 (medium adherence) and G3 (poor adherence) (Fig. 1A; Fig. S1). This is observed both in the presence (Fig. 1A) and absence (Fig. S1) of fibronectin (FN) or poly-L-lysine (Fig. S1). When highly adherent CDC1132 parasites are observed by live-cell imaging (Fig. 1B; Movie 1), these uropod-like membrane extensions seem to have a role in tethering the parasites to the attaching surface. In agreement, scanning electron microscopy (SEM) analysis of T. vaginalis in suspension confirmed that numerous highly adherent CDC1132 parasites contained this posterior membrane cell protrusion and almost no such protrusions were observed in the poorly adherent G3 strain (Fig. 1C). In G3, only small bulbous structures, ranging from 0.6 µm to 1.5 µm in thickness, are found in the posterior region of some parasites (Fig. 1C). However, in CDC1132 parasites, the posterior projections were much more prominent and extensive, with a length that could reach more than 20 µm (Fig. 1C). Those uropod-like projections protruded next to region where the axostyle emerges and exhibited a heterogeneous morphology with size ranging from 0.1 µm to more than 4 µm in thickness (Fig. 1C). The main ultrastructural features observed were: (1) single or branched slender projections displaying a twisted surface; (2) slender projections with different sizes and tubular extensions emanating from the main projections, such that they could be braided together or exhibit an anastomosed appearance; and (3) single wide projections with a smooth and/or flattened surface (Fig. 1C). The uropod-like projections were frequently seen in contact with the surface of flagella, cell body or other surface protrusions of adjacent parasites (Fig. 1C). These ultrastructural data were confirmed and extended using negative staining for transmission electron microscopy (TEM) (Fig. 1D). In twisted slender and anastomosed projections, tubular extensions, and bulbous- and vesicle-like structures are seen emerging from their surfaces, whereas the surface of a wide projection exhibits a smooth surface (Fig. 1D). In addition, by this technique, we observed the longest uropod-like projection found by us, with more than 65 µm in length (Fig. 1D). The uropod-like projections are markedly distinguishable from the axostyle posterior tail-like tip. The axostyle tip had a homogeneous, thin, unbranched, tubular structure, that is less prominent than uropod-like projections, and had visible microtubules when analyzed by negative staining (Fig. 1C; Fig. S2). As observed by live-cell imaging (Movie 1), the uropod-like projection can quickly vary its morphology and size. In this sense, it is very likely that the morphological differences observed using electron microscopy could be attributed to different points or phases of this highly dynamic process. The posterior protrusions observed in parasites in suspension, as well as those prepared for negative staining or adhered to FN-coated coverslips in WGA assays, originated from the region where the axostyle emerges and exhibited an elongated, heterogeneous morphology. Taking these results into account, we hypothesized that this uropod-like protrusion could play an important role in the adhesion of the parasite to different surfaces, including other parasites and host cells.

As mammalian TSPs have been previously demonstrated to have a role in the biogenesis of posterior cell protrusions (De Winde et al., 2018; Martins et al., 2019), we evaluated whether different cell surface-localized T. vaginalis TSPs (TvTSPs) (Coceres et al., 2015; de Miguel et al., 2012) could have a role in modulating the formation of this posterior structure using a gain-of-function analysis. To this end, five different previously characterized surface-localized TvTSPs (Coceres et al., 2015; de Miguel et al., 2012; Nievas et al., 2018a) were cloned and transfected under the control of the α-SCS promoter as a C-terminally hemagglutinin (HA)-tagged fusion protein in the CDC1132 strain. Then, the number of parasites containing a uropod-like protrusion was evaluated using WGA staining by fluorescence microscopy. Our results showed that only TvTSP5, and not TvTSP1, TvTSP2, TvTSP6 or TvTSP8, increased the formation of this structure as ∼2.5-fold more CDC1132 parasites with uropod-like protrusions were observed when TvTSP5 was overexpressed compared to parasites transfected with the empty vector control (EpNEO) (Fig. 2A). We then determined the localization of the TvTSP5-tagged protein in transfected cells using immunofluorescence assays with an anti-HA antibody and demonstrated that TvTSP5 was localized on the cell surface, including the uropod-like protrusion (Fig. 2B). To further evaluate whether TvTSP5 had a role in the formation of the uropod-like protrusion, we transfected TvTSP5 in the medium-adherent strain B7RC2 and evaluated whether the overexpression was able to modulate the protrusion formation. The results indicate that TvTSP5 overexpression resulted in ∼2 times more parasites with uropod-like posterior protrusions than seen in parasites transfected with the empty vector (EpNEO) (Fig. 2C). Similar to what was found in CDC1132 strains, TvTSP5 localized at the cell surface as well as to the uropod of the parasite when transfected into a B7RC2 strain (Fig. 2D). To validate these results, CDC1132 parasites that overexpressed TvTSP5 as well as EpNEO control were visualized by SEM. The results showed that TvTSP5-overexpressing parasites form ∼4-times more posterior protrusions than parasites expressing control EpNEO (Fig. 2E). Additionally, many uropod-like projections were seen in contact with the surface of flagella, axostyle, cell body or other surface protrusions of adjacent parasites (Fig. 2E; Fig. S3). These results suggest that TvTSP5 has a role in the formation of the uropod structure in adherent strains of T. vaginalis.

Highly adherent T. vaginalis strains form clumps in cell culture in contrast to poorly adherent strains (Coceres et al., 2015). Our previous data suggest that cell membrane protrusions, particularly cytonemes, are involved in the formation of clumps in adherent T. vaginalis strains (Salas et al., 2023). Based on this observation, we evaluated whether overexpression of TvTSP5 promoted parasite aggregation in the CDC1132 strain. To this end, we assessed the aggregation capacity of parasites that overexpressed TvTSP5, parasites transfected with an empty vector (EpNEO) and a mixture (50:50) of these transfected parasites (Fig. 3A). Supporting a role in clump formation, we found that the number of clumps was higher in parasites overexpressing TvTSP5 compared to that in parasites transfected with an empty plasmid (EpNEO) (Fig. 3A). Then, we assessed the composition of clumps formed when TvTSP5 parasites were co-incubated with EpNEO parasites. To this end, TvTSP5 and EpNEO parasites were stained with CellTracker Red (red) and CFSE (green), respectively, and co-cultured in a 1:1 ratio. Then, we evaluated whether the resulting clumps consisted of TvTSP5 parasites, EpNEO parasites or a combination of both (Fig. 3B). If TvTSP5 parasites had a higher clumping ability, we would expect to see a greater number of TvTSP5 parasites in the clumps when these were co-incubated with EpNEO. As controls, we also assessed the composition of clumps in two scenarios: (1) when EpNEO parasites (stained green) were co-incubated with EpNEO parasites (stained red) in a 1:1 ratio, and (2) when TvTSP5 parasites (stained green) were co-incubated with TvTSP5 parasites (stained red) in a 1:1 ratio. Our results showed that when EpNEO parasites stained green were co-cultured with EpNEO parasites stained red, most clumps were mixed, consisting of both green and red parasites (Fig. 3B, left bars). Similarly, co-culturing TvTSP5 parasites stained green with TvTSP5 parasites stained red resulted in mostly mixed clumps (Fig. 3B, middle bars). However, when EpNEO parasites stained green were co-incubated with TvTSP5 parasites stained red in a 1:1 ratio, the clumps were mostly formed by TvTSP5 parasites (60% of predominantly red clumps) (Fig. 3B, right bars). These results indicate that TvTSP5 parasites have a higher tendency to form clumps than EpNEO parasites, indicating a role in modulating clump formation.

As an extracellular parasite, attachment to urogenital epithelial cells is a crucial step in the pathogenesis of T. vaginalis. However, the characterization of the parasite-specific proteins and structures involved in host attachment is not well defined. To evaluate the role of the uropod-like protrusion during the process of parasite–host interaction, highly adherent CDC1132 parasites were co-incubated with benign prostate-1 (BPH-1) cells and visualized by video microscopy. In concordance with our above observation suggesting a role as a tether structure (Fig. 1B), observation by time-lapse video microscopy suggested that the uropod-like protrusion formed at the posterior pole served as an anchor structure that might be playing a crucial role in the attachment of the parasite to the host cell (Fig. 4A; Movie 2). In agreement, SEM analysis confirmed that many parasites adhered to BPH-1 cells through the posterior uropod-like projections, whereas the anterior pole and flagella were free (Fig. 4B). The uropod-like projections exhibit strong adhesion to the host cell, as evidenced by the fact that repeated washes and the critical point drying procedure for SEM failed to detach the parasites. In this latter procedure, there is a powerful flush of gases in the interior of the equipment. Thin tubular extensions branching from the uropod-like projections are also seen in close contact with the BPH1 cells (Fig. 4B). Based on these observations and our results demonstrating that TvTSP5 regulated the formation of the uropod-like protrusion, we performed adherence assays to evaluate whether the sole gain of expression of TvTSP5 influences adherence capacity. Overexpression of TvTSP5 in transfected parasites was capable of increasing attachment to BPH1 cells ∼2-fold in CDC1132 (Fig. 5A) and ∼3-fold in B7RC2 (Fig. 5B) parasites compared with that seen for control EpNEO parasites. In concordance, qualitative analysis of TvTSP5-overexpressing CDC1132 parasites by SEM showed that more parasites adhered to host cells compared to EpNEO control (Fig. 5C). Many of the TvTSP5-transfected CDC1132 parasites established a tight adhesion with posterior uropod-like projections (Fig. 5C). These results indicate that TvTSP5 has a role in modulating adherence to host cells, probably through modulation of cell membrane protrusions.

As we propose that the uropod-like protrusion might have a role in tethering the parasites to the attaching surface, we hypothesize that altering the formation of this structure could impact the migration of the parasites. To evaluate this, we assessed the migration capacity of TvTSP5- and EpNEO-transfected parasites on a solid agar surface. To do this, B7RC2 and CDC1132 parasites transfected with EpNEO and TvTSP5 were seeded on solid agar and their migration capacity was analyzed by measuring the size of the halo diameter from the inoculation point to the periphery of the plate after 3 days of incubation (Fig. 6A,B). The observed results indicate that the parasites that overexpressed TvTSP5 had significantly less migration capacity than those that were transfected with EpNEO in both analyzed strains (Fig. 6C,D). To exclude the possibility that the observed difference was an effect of differences in cell growth, we analyzed the kinetic of growth of parasites transfected with TvTSP5 and EpNEO (Fig. S4). No differences in growth were observed in parasites transfected with TvTSP5 and EpNEO in any of the strains (Fig. S4). This result on the observed differences in migration on solid agar further support a role for TvTSP5 in a tethering structure. Our results underscore the essential contribution of TvTSP5 in the formation of the novel anchor structure and also highlight the role of this protein in modulating parasite pathogenesis.

The data presented here provide new evidence of the dynamic behavior of the T. vaginalis parasite during the process of infection (Arroyo et al., 1993; González–Robles et al., 1995; Hsu et al., 2023). We identified a novel membrane cell protrusion, distinctively formed at the posterior pole of the cell that morphologically resembles the previously described uropod of migrating cells (Andreata et al., 2023; Hind et al., 2016; Sánchez-Madrid and Serrador, 2009). Uropods, the rearward part of the cell that includes a distinctive knob-like structure, have been previously described in Dictyostelium discoideum, Entamoeba histolytica and leukocytes (Gudima et al., 1988; Markiewicz et al., 2011; Sánchez-Madrid and Del Pozo, 1999; Sánchez-Madrid and Serrador, 2009). In leukocytes, the uropod has emerged as a crucial structure for proper motility that can also be the driving center for cell polarization and migration (Hind et al., 2016). In addition to its role in motility, the neutrophil uropod has also been implicated in cellular communication, particularly by modulating adhesion (Cera et al., 2009). In this sense, our results suggest that this uropod-like protrusion appears to function as a tethering structure that might be important to module parasite migration and attachment. We also demonstrated that the formation of this protrusion at the posterior pole depends, at least in part, on the abundance of tetraspanin protein TvTSP5. Several adhesion receptors and TSPs are concentrated at the uropod in other cells, raising the possibility that tetraspanin-enriched microdomains could be responsible for organization of adhesion receptors in this structure (Barreiro et al., 2008). Similarly, the participation of TSPs in the formation of different cell membrane protrusions, which are crucial for processes such as cell polarization, adhesion and migration, has been previously described (De Winde et al., 2018; Liu and Zhang, 2006; Martins et al., 2019). TSPs can affect cellular behaviors by interacting with cell adhesion molecules and growth factor receptors (Liu and Zhang, 2006). Acting as mediators, TSPs receive signals from the cellular environment and transmit them into the interior of the cell to modulate various cellular functions (Hemler, 2003). Along with their associated molecules, TSPs exert their influence on motility-related cellular events as well as cell migration (Kovalenko et al., 2005; Maecker et al., 1997). Studies have shown that TSPs interact directly with several integrins, modulating their membrane compartmentalization, intracellular trafficking and recycling, and subsequent signaling in response to migratory signals (Jiang et al., 2015). In concordance with these antecedents, we observed that TvTSP5 had a role in modulating parasite migration. Specifically in T. vaginalis, our previous report demonstrated that another TSP protein, TvTSP6, primarily localized in the flagella, has a role in modulating parasite migration (de Miguel et al., 2012). In this sense, supporting a transmembrane linker model, our previous results proposed that the C-terminal tail of TvTSP6 establishes connections with intracellular pathways, modulating both parasite migration and sensory reception throughout the infection process (de Miguel et al., 2012). In unicellular organisms, cell migration and motility play a vital role in actively seeking signals and localizing optimal adhesion sites in response to extracellular signals (Colin et al., 2021; Ginger et al., 2008). In concordance, studies have demonstrated that the movement capacity of Entamoeba histolytica trophozoites is reduced in the presence of fibronectin and this phenomenon is attributed to the increased formation of membrane protrusions (Sierra-López et al., 2018; Talamás-Lara et al., 2020). In contrast, non-invasive Entamoeba dispar trophozoites do not exhibit this inhibition to the same extent (Talamás-Lara et al., 2020). Additionally, the enteric parasite E. histolytica has the ability to generate membranous blister-like protrusions (Krishnan and Ghosh, 2020), a characteristic considered a fundamental mode of motility in certain types of eukaryotic cells (Liu et al., 2015; Zatulovskiy et al., 2014). Interestingly, the formation of these protuberances in E. histolytica was specifically placed in the uroid, a structure located at the posterior part of the parasite that has a role in the elimination of waste products and replacement of membrane components (Bailey et al., 1992; Espinosa-Cantellano et al., 1998). It has been suggested that E. histolytica forms uropods to escape the immune response of the host (Markiewicz et al., 2011). In a proteome study of uropods isolated from E. histolytica, Rho and Ras proteins were identified, which have been described as key mediators in membrane protrusion formation (Koizumi et al., 2012; Kozma et al., 1995; Pellegrin and Mellor, 2005), along with the protein calreticulin, which is known to be involved in controlling cell adhesion and in the initiation, stabilization and turnover of focal contacts in fibroblasts (Opas et al., 1996; Villagomez et al., 2009). Here, we propose that the increased formation of uropod-like protrusions at the posterior pole of TvTSP5-transfected parasites might serve as a tethering structure, consequently leading to reduced migration in semi-solid agar. Migration of unicellular organisms has been demonstrated to have an influence on cooperative behaviors such as the formation of biofilms, social motility (SoMo) and quorum sensing (Colin et al., 2021; Ginger et al., 2008; Shrout et al., 2011). Building upon these findings, it is indeed promising to explore whether T. vaginalis has the capability to demonstrate social motility on solid surfaces.

We propose that the influence of TvTSP5 on the formation of cell membrane protrusions could consequently impact parasite migration as well as attachment to host cells, and the aggregation of parasites. Clumping is a distinct adhesive phenotype that is exhibited by some but not all parasite strains and which correlates with the capacity of the strain to adhere to host cells. Highly adherent T. vaginalis strains form clumps in cell culture in contrast to poorly adherent strains (Coceres et al., 2015; Lustig et al., 2013). Based on earlier findings, it appears that the formation of clumps in T. vaginalis is mediated, at least partially, by TvTSP8 (Coceres et al., 2015) and cadherin-like (CLP) proteins (Chen et al., 2019). Our previous results suggest that increased clumping capacity of TvTSP8-transfected parasites can be attributed, at least in part, to the increase of TSP8 on the cell surface of these parasites, with this effect being mediated by the C-terminal tail of the protein. This observation aligns with studies involving the mammalian TSP family protein CD9, where it has been demonstrated that it promotes homotypic cell–cell aggregation and microvilli formation, whereas a mutant lacking the C-terminal tail did not exhibit the same effect (Wang et al., 2011). Similarly, CLP overexpression also induced parasite aggregation, with this effect being further amplified in the presence of Ca²⁺ (Chen et al., 2019). Our recent data also indicate that cytonemes are involved in the formation of clumps in adherent T. vaginalis strains (Salas et al., 2023). Building upon these previous observations, it is plausible that the involvement of TvTSP5 in clump formation is mediated by the increased formation of cell protrusions when the protein is overexpressed.

Different T. vaginalis strains exhibit significant variability in their ability to bind to and/or kill host cells (Heath, 1981; Krieger et al., 1985; Lustig et al., 2013). Although attachment to host cells is influenced by multiple factors, some of which have been identified (Riestra et al., 2022), recent attention has been directed to exploring the role of cell membrane protrusions in regulating parasite attachment (Salas et al., 2023). Specifically, T. vaginalis has been shown to form various cellular protuberances, such as pseudopodia, lamellipodia, filopodia and cytonemes, which are important for adherence to different surfaces (Lorenzo-Benito et al., 2022; Ortiz et al., 2023; Salas et al., 2023). In particular, we recently showed that cytonemes have a role in the process of T. vaginalis attachment to prostate cells (Salas et al., 2023). Consistent with these findings, we have demonstrated that the formation of uropod-like membrane protrusions correlates with the ability of the parasite to bind to host cells, as the highly adherent strain CDC1132 forms more protrusions than the poorly adherent strain G3. Notably, we observed that TvTSP5 plays a role in the biogenesis of the uropod-like protrusion, and parasites overexpressing TvTSP5 exhibited an increased capacity for attachment to prostate cells. Research by other groups has revealed that cytoskeletal proteins, mainly actin, play a crucial role in the formation of cell membrane protrusions, like pseudopodia, and cytoplasmic extensions, such as filopodia, exclusively at the cell edge of T. vaginalis (Lorenzo-Benito et al., 2022; Wang et al., 2023). Similarly, it has been shown that the related veterinary trichomonad Tritrichomonas foetus adheres to bovine oviduct epithelial cells through interactions from the posterior projections (Midlej et al., 2009), which are morphologically similar to the T. vaginalis uropod-like structure described here. In concordance with our findings, recent reports have demonstrated that cancer cells in spheroids form long protrusions to extend into the surrounding matrix. These protrusions, once attached, can use integrin adhesion and myosin II-based contractility to pull cells through the basement membrane for initial invasion (Nazari et al., 2023).

Our work has uncovered new roles for a previously undescribed uropod-like protrusion, further elucidating mechanistic interactions contributing to pathogenesis. Defining the cellular structures and proteins required for adhesive phenotypes of T. vaginalis might therefore help us to understand how the parasite colonizes the urogenital tract and how to prevent or treat infections.

Trichomonas vaginalis strains G3 (ATCC PRA-98; Beckenham, UK), B7RC2 (ATCC 50167; Greenville, NC, USA) and CDC1132 (MSA1132; Mt. Dora, Fla, USA 2008) (Mercer et al., 2016) were cultured in Diamond's trypticase-yeast extract-maltose medium supplemented with 10% horse serum and 10 U ml⁻¹ penicillin and 10 μg ml⁻¹ streptomycin (Clark). Parasites were grown at 37°C and passaged daily. The human BPH-1 cells, kindly provided by Dr Simon Hayward (NorthShore University, USA) (Jiang et al., 2010), were grown in RPMI 1640 medium containing 10% fetal bovine serum (FBS; Internegocios, Argentina) with 10 U/ml penicillin and cultured at 37°C under 5% CO₂.

The TvTSP5 constructs were generated using primers with NdeI and KpnI restriction sites engineered into the 5′- and 3′-primers as previously described (Coceres et al., 2015). Parasites were transfected in parallel with an empty plasmid (EpNEO) to be used as control. Electroporation of T. vaginalis strains B7RC2 and CDC1132 was carried out as described previously (Delgadillo et al., 1997) with 50 µg of circular plasmid DNA. Transfectants were selected with 100 µg/ml G418 (Sigma).

Cells membrane protrusion was quantified on coverslips coated with using poly-L-lysine and fibronectin and coverslips without coating. Parasites (10⁶ cells/ml) were incubated at 37°C for 1 h on glass coverslips as previously described (De Miguel et al., 2010). When using fibronectin, the coverslips were covered with 100 µl of a FN solution (10 μg/ml; Sigma) in PBS, incubated at room temperature for 1 h, and washed with sterile PBS twice. When poly-L-lysine was used, the coverslips were covered with 100 μl of a 0.01% (v/v) solution (Sigma), incubated at room temperature for 1 h, and washed with sterile PBS twice. Parasites attached to coverslips were fixed in 4% paraformaldehyde at room temperature for 20 min and labeled with wheat germ agglutinin (WGA) lectin from Triticum vulgaris conjugated to FITC (Sigma). To this end, parasites were incubated with a 1:100 dilution of WGA in PBS at 37°C for 1 h, washed three times with PBS solution and mounted using Fluoromont Aqueous Mounting Medium (Sigma). Fluorescence parasites were visualized using a Zeiss Axio Observer 7 (Zeiss) microscope.

Parasites in suspension and after interaction with prostate cells were washed with PBS solution and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. The cells were then post-fixed for 15 min in 1% OsO₄, dehydrated in ethanol and critical point-dried with liquid CO₂. The dried cells were coated with gold–palladium to a thickness of 15 nm and then observed with a Jeol JSM-5600 scanning electron microscope, 6 operating at 15 kV. The percentage of parasites that contained posterior projections was determined from counts of at least 500 parasites randomly selected per sample.

Parasites were washed with PBS solution and settled onto glow-discharged carbon film nickel grids for 10 min at 37°C. Next, cells were fixed in 2.5% glutaraldehyde in PEME (100 mM PIPES pH 6.9, 1 mM MgSO₄, 2 mM EGTA, 0.1 mM EDTA) for 10 min at room temperature. The grids were washed with deionized water by flotation, and negatively stained with 1% aurothioglucose (UPS Reference Standard) in deionized water for 5 s. The grids were then air-dried and observed with a FEI Tecnai Spirit transmission electron microscope. The images were randomly acquired with a CCD camera system (MegaView G2, Olympus, Germany).

Parasite clumps was analyzed in microaerophilic conditions when parasites reached a concentration of 10⁶ parasites ml⁻¹ using an inverted microscope. A clump was defined as the size corresponding to an aggrupation of at least eight parasites. For quantification of clumps, parasites were observed in 20 fields at a total magnification of 200× using a Nikon TSM (Nikon) microscope. Three independent experiments were performed.

EpNEO and TvTSP5 transfected parasites (CDC1132 strain) were labeled using CellTracker Red dye CMTPX (Thermo Fisher Scientific) and carboxyfluorescein succinimidyl ester (CFSE). The labeled parasites were then co-incubated for 1 h at 37°C with the parasites in a 1:1 ratio. The number of parasites with the capacity to form aggregates were visualized. For quantification of clumps, parasites were observed in 20 fields at a total magnification of 200× using a Zeiss Axio Observer 7 microscope (Zeiss). A red clump was defined as a clump that was formed by at least 75% red parasites. A green clump was defined as a clump that was formed by at least 75% green parasites. Three independent experiments were performed.

Cultivation on semi-solid agarose plates was composed of adapted Diamond medium (Hollander, 1976). A 50% (w/v) agar solution was made in distilled water, autoclaved and cooled. A work medium solution was made with 10% FBS and 1% antibiotic. The resulting 0.58% agarose solution was cooled to 37°C for 1 h. A 20 ml aliquot was poured into Petri dishes, which were then dried without lids for 45 min in a laminar flow hood at room temperature. Then, 2×10⁶ parasites (resuspended in 5 µl) were inoculated onto the plate. The plates were then incubated in an anaerobic chamber for 3 to 4 days.

A modified version of an in vitro assay was carried out to quantify the binding of T. vaginalis to host cell monolayers (Bastida-Corcuera et al., 2005). Briefly, prostate BPH-1 cells were seeded on 12 mm coverslips in 24-well plates with RPMI culture medium (Invitrogen) and grown to confluence at 37°C and 5% CO₂. Coverslips were washed with fresh RPMI culture medium prior to the addition of parasites. Parasites transfected with EpNEO or TvTSP5 (CDC1132 and B7RC2 strains) were labeled with 10 mM Cell Tracker Blue 7-amino-4-chloromethylcoumarin (CMAC; Invitrogen) added to confluent BPH-1 cells (10⁵ parasites/well) and incubated at 37°C and 5% CO₂ for 30 min in duplicate. Coverslips were subsequently washed in PBS solution, fixed with 4% paraformaldehyde and mounted on slides with Fluoromont Aqueous Mounting Medium (Sigma). Quantification of the number of fluorescence parasites attached to host cells were measured using a Zeiss Axio Observer 7 microscope. The scoring and counting of all images were performed by a researcher who was unaware of the experimental conditions. Thirty fields per coverslip were analyzed at a total magnification of 400×. All adhesion assay data were normalized to values for EpNEO (in each strain) and shown as changes in adhesion. The experiments were performed four independent times.

The kinetic of growth curves were assessed using EpNEO- and TvTSP5-transfected CDC1132 and B7RC2 strain parasites. For these experiments, 10⁵ trophozoites were inoculated in 10 ml of Diamond's medium and incubated at 37°C for 72 h. Cell counts were recorded starting at 18 h post inoculation using a hemocytometer. Parasites counts were collected at indicated times on the x-axis. The results represent the average of three independent experiments and error bars represent s.d.

Specific statistical considerations and the tests used are described separately for each subsection. GraphPad Prism for Windows version 8.0.2 were used for graphics. Data are given as mean±s.d. Significance was established at P<0.05. For statistical analyses, InfoStat software version 2020e was used.

During the preparation of this work the authors used ChatGPT to improve the readability and language of the manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

We thank Maria Florencia Irigoyen for her technical assistance and our colleagues in the lab for helpful discussions. We also thank Dr Karina Saraiva and Dr Cássia Docena from the Technological Platform Core of the Aggeu Magalhães Institute for their technical support.