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Author response: Role of cytoneme structures and extracellular vesicles in Trichomonas vaginalis parasite-parasite communication

Year: 2023

Type: peer-review

Abstract: Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Trichomonas vaginalis, the etiologic agent of the most common non-viral sexually transmitted infection worldwide. With an estimated annual prevalence of 276 million new cases, mixed infections with different parasite strains are expected. Although it is known that parasites interact with their host to enhance their own survival and transmission, evidence of mixed infections call into question the extent to which unicellular parasites communicate with each other. Here, we demonstrated that different T. vaginalis strains can communicate through the formation of cytoneme-like membranous cell connections. We showed that cytonemes formation of an adherent parasite strain (CDC1132) is affected in the presence of a different strain (G3 or B7RC2). Our findings provide evidence that this effect is contact-independent and that extracellular vesicles (EVs) are responsible, at least in part, of the communication among strains. We found that EVs isolated from G3, B7RC2, and CDC1132 strains contain a highly distinct repertoire of proteins, some of them involved in signaling and communication, among other functions. Finally, we showed that parasite adherence to host cells is affected by communication between strains as binding of adherent T. vaginalis CDC1132 strain to prostate cells is significantly higher in the presence of G3 or B7RC2 strains. We also observed that a poorly adherent parasite strain (G3) adheres more strongly to prostate cells in the presence of an adherent strain. The study of signaling, sensing, and cell communication in parasitic organisms will enhance our understanding of the basic biological characteristics of parasites, which may have important consequences in pathogenesis. Editor's evaluation This study presents novel information and describes fundamental findings in various fields of biology. It is of interest to a wide audience, including parasitology, cell-to-cell communication, and the roles of extracellular vesicles. The manuscript presents compelling evidence that will contribute to the advancement of our understanding of how parasitic extracellular vesicles (and cytoneme structures) are formed and participate in intra-species communication. https://doi.org/10.7554/eLife.86067.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The flagellated protozoan parasite Trichomonas vaginalis is the causative agent of trichomoniasis, the most common non-viral sexually transmitted infection worldwide, with an estimated 276 million new cases annually (WHO, 2018). Although asymptomatic infection is common, multiple symptoms and pathologies can arise in both men and women, including vaginitis, urethritis, prostatitis, low birth weight infants and preterm delivery, premature rupture of membranes, and infertility (Fichorova, 2009; Swygard et al., 2004). T. vaginalis has also emerged as an important cofactor in amplifying the HIV spread as individuals infected with T. vaginalis have a significantly increased incidence of HIV transmission (McClelland et al., 2007; Van Der Pol et al., 2008). Additionally, T. vaginalis infection increases the risk of cervical and aggressive prostate cancer (Gander et al., 2009; Twu et al., 2014). Due to its great prevalence in some communities, mixed infections with several parasite strains are anticipated. In this sense, an analysis performed of 211 T. vaginalis samples isolated in five different continents identified 23 cases of mixed infections (10.9%; Conrad et al., 2012). The extent to which parasites communicate with each other in mixed infections has been severely underestimated. The fundamental ability to sense, process, and respond to extracellular signals is shared by all living forms. However, little is known about the mechanism of sensing and signaling in protozoan parasites compared to what is known in other organisms (Roditi, 2016). Although it is widely accepted that pathogens interact with their host to enhance their own survival and transmission, communication between unicellular parasites has been poorly studied (Roditi, 2016). Although it was originally believed that single-celled microorganisms do not need to cooperate with other members of their own species, in recent years, it has become clear that microbes are social organisms capable of communicating with one another and engaging in cooperative behavior (Oberholzer et al., 2010; Roditi, 2016). In this sense, social interactions in a population offer advantages over a unicellular lifestyle, including increased protection from host defenses, access to nutrients, exchange of genetic information, and enhanced ability to colonize, differentiate, and migrate as a group (Oberholzer et al., 2010; Roditi, 2016). Cells communicate over short or long distances in different ways. Extracellular vesicles (EVs), soluble secreted factors, membrane protrusions, and direct contact between cells are all different forms of cell communication (Buszczak et al., 2016; Matthews, 2021; Regev-Rudzki et al., 2013). Plasma membrane serves as the primary interface between a cell and its environment, playing an essential role in mediating direct contact, sensing environmental factors, and releasing signaling molecules. Cellular protrusions have emerged as a way for cells to communicate with one another. Among different types of cellular protrusions, filopodia are thin cellular extensions that have been observed in many cell types and have been assigned different roles like cell migration, cell adhesion, force generation, wound healing, environmental sensing, antigen presentation, and neuronal pathfinding (Roy and Kornberg, 2015). Although their physical properties vary (2–400 µm in length and 0.1–0.3 µm diameter), all are actin-based, they extend and retract at velocities that have been measured as much as 25 µm/min, and their tips can contact other cells (Roy and Kornberg, 2015). Their different shapes and roles are reflected in the many names that have been coined: thin filopodia (Miller et al., 1995), thick filopodia (McClay, 1999), invadopodia (Chen, 1989), telopodes (Popescu and Faussone-Pellegrini, 2010), tunneling nanotubes (Rustom et al., 2004), and cytonemes (Ramírez-Weber and Kornberg, 1999). Specifically, cytonemes are considered as thin specialized filopodia that have been shown to traffic signaling proteins such as morphogens, growth factors, and cell determination factors (Roy and Kornberg, 2015). Although cytonemes have similar diameters to conventional filopodia (typically smaller than 200 nm), they have the potential to extend up to ~300 nm from the originating cell body and have been observed in both vertebrate and invertebrate systems (Kornberg and Roy, 2014). Alternatively, EVs are also considered key mediators in intercellular communication in many types of cells. They are a group of heterogeneous particles formed by a lipid bilayer containing proteins and nucleic acids (Abels and Breakefield, 2016; Yáñez-Mó et al., 2015). As proposed by the International Society for EVs, the term ‘‘EVs’’ is referred to all sub-populations of EVs, so it is recommended to use it collectively and universally (Théry et al., 2018). Among the various subtypes of EVs, the particles can be defined according to the mode of biogenesis, size, and function into three major categories: (1) exosomes formed due to plasma membrane invagination into multivesicular bodies with size ranging from 40 to 100 nm; (2) microvesicles (MVs), also called shedding vesicles, microparticles or ectosomes, originated from the budding and extrusion of the plasma membrane, with sizes between 50 and 1000 nm and an asymmetric structure; and (3) apoptotic bodies, with greater sizes (up to 1000 nm) originated from cells in the process of programmed cell death (Kalra et al., 2012). Different to cell membrane protrusions, EVs modulate short- and long-range events, allowing cells to communicate even at long distances. These particles regulate physiological processes such as blood coagulation, cell differentiation, and inflammation, as well as pathological processes caused cancer, neurological, cardiovascular, and infectious diseases (Kao and Papoutsakis, 2019; Raposo and Stoorvogel, 2013; Yáñez-Mó et al., 2015). EVs are relevant for the communication between pathogens and host cells (Drurey and Maizels, 2021; Khosravi et al., 2020; Nievas et al., 2020, p. 2021; Sabatke et al., 2021; Torrecilhas et al., 2020). Specifically, in T. vaginalis, the analysis of EVs, both exosomes and MVs, has become a very exciting field in the study of parasite: host interaction, as it has been shown that the formation of EVs increase in the presence of host cells and modulate parasite adherence (Nievas et al., 2020; Nievas et al., 2018a; Olmos-Ortiz et al., 2017; Rai and Johnson, 2019; Twu et al., 2013). Although the role of EVs in T. vaginalis: host interaction has been deeply analyzed (Nievas et al., 2020; Rada et al., 2022; Rai and Johnson, 2019; Salas et al., 2021; Twu et al., 2013), the understanding of the role of EVs in communication between different parasite strains and the implications in the infection process is still scarce. Here, we demonstrated that different T. vaginalis strains are capable of communicating with one another through the formation of cytoneme-like membranous cell connections. We observed that cytoneme formation of an adherent parasite strain (CDC1132) is affected in the presence of a different strain (G3 or B7RC2). Furthermore, we have shown that this effect on cytoneme formation is contact-independent and that EVs are responsible, at least in part, of the communication between strains. To explain the differential response in cytoneme formation due to the presence of EVs isolated from different strains, we analyzed the EVs’ protein content using mass spectrometry and demonstrated that highly specific protein cargo was detected in EVs isolated from different strains. Finally, we showed that parasite adherence to host cells is affected by this communication as binding of adherent T. vaginalis CDC1132 strain to prostate cells is significantly higher in the presence of G3 or B7RC2 strains. Importantly, we observed that in the presence of an adherent strain, a poorly adherent parasite strain (G3) adheres more strongly to prostate cells, suggesting that interaction between isolates with distinct phenotypic characteristics may have significant clinical repercussions. The study of signaling, sensing, and cell communication in parasitic organisms will surely enhance our understanding of the basic biological characteristics of parasites and reveal new potential clinical outcomes. Results T. vaginalis adherent strains form abundant membrane protrusions Visualization of T. vaginalis by fluorescent and live-cell microscopy revealed the presence of filamentous structures extending from the surface of some cells (Video 1). Due to their morphological appearance (Figure 1A), these structures resembled previously described filopodia and cytonemes (Kornberg and Roy, 2014). To determine if these structures were related to pathogenesis, we evaluated the presence of filopodia and cytonemes in strains with different adherence capacities to host cells: two poorly adherent strains (G3 and NYU209) and two highly adherent strains (B7RC2 and CDC1132) using the membrane binding lectin wheat germ agglutinin (WGA). When the number of membrane protrusions was quantified, we observed that the highly adherent strains B7RC2 and CDC1132 have greater number of filopodia and cytonemes compared to poorly adherent strains G3 and NYU209 (Figure 1A). In concordance, SEM revealed that multiple filopodia and cytonemes originated from the surface of highly adherent CDC1132 parasites and almost no such protrusions were observed in the poorly adherent G3 strain (Figure 1B). These tubular extensions appear to be close ended and can be seen protruding from the region where the flagella emerge as well as from the cell body, with a length up to 4.6 µm (Figure 1B; Figure 1—figure supplement 1). The cytonemes from the flagellar base appear unbranched, displaying a homogeneous morphology and size ranging from 70 nm to 100 nm in thickness (Figure 1B; Figure 1—figure supplement 1). Their ultrastructural features are similar to those flagellar projections previously described in the related veterinary trichomonad Tritrichomonas foetus (Benchimol et al., 2021). However, the tubular extensions from the cell body are heterogeneous, with diameters that vary from 70 nm to 540 nm and some of them furcated (Figure 1B; Figure 1—figure supplement 1; Figure 2—figure supplement 1). Altogether, these findings demonstrate that T. vaginalis contains different types of membrane protrusions arising from their surface and suggest that its abundance depends, at least in part, on the phenotype of the strain. Figure 1 with 1 supplement see all Download asset Open asset Adherent T. vaginalis strains form abundant membrane protrusions. (A) Quantification of the percentage of parasites containing filopodia (top) or cytonemes (bottom) in their cell surface. The presence of filopodia/cytonemes in two poorly adherent (G3 and NYU209) and two highly adherent strains (B7RC2 and CDC1132) was analyzed. Three independent experiments by duplicate were performed, and 100 parasites were randomly counted per sample. Data are expressed as percentage of parasites with filopodia and cytonemes ± standard deviation. ANOVA followed by Tukey’s post hoc test (α=0.95) was used to determine significant differences. (B) SEM reveals a myriad of projections originating from the surface of parasites from CDC1132 strain. Cytonemes protruding from cell body and flagellar base region are indicated by white and green arrowheads, respectively. Pseudopodia (*) and filopodia (arrow) are also seen. The surface protuberances appear to be close ended. Almost no protrusions were observed arising from the surface of poorly adherent parasites (G3). F, flagella; Ax, axostyle. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Live-cell microscopy revealed the presence of filamentous structures extending from the surface of T. vaginalis. Cytonemes are associated with parasite clumps formation As previously shown (Coceres et al., 2015; Lustig et al., 2013), formation of clumps in cell culture generally correlates with the ability of the strain to adhere and be cytotoxic to host cells. Specifically, highly adherent strains tend to aggregate when cultured in the absence of host cells in contrast to poorly adherent strains that generally do not form clumps in vitro (Coceres et al., 2015; Nievas et al., 2018b). Based on this observation and our SEM results demonstrating that cytonemes are usually detected connecting different parasites inside the clumps (Figure 2A), we evaluated whether the formation of clumps (Figure 2B) is accompanied by an increase in the number of parasites containing cytonemes (Figure 2C). As can be observed in Figure 2, the number of parasites containing cytonemes is higher at 106 parasites/ml (Figure 2C); a cell culture condition where usually clumps are formed (Figure 2B). These results suggest that cytonemes might be involved in parasite: parasite communication. Consistent with this, cytonemes are frequently observed connecting two parasites (Figure 2D). The closed tip of cytonemes was frequently seen in contact with any region of the surface of an adjacent parasite (Figure 2—figure supplement 1), including other cytonemes (Figure 2A and D). Figure 2 with 1 supplement see all Download asset Open asset Cytonemes are associated to clumps formation. (A) Cytonemes emerging from flagella base (green arrowheads) and cell body (white arrowheads) are frequently observed connecting adjacent cells inside clumps of parasites by SEM. F, flagella; AF, anterior flagella; RF, recurrent flagellum. (B) Quantification of clumps per field at different parasite densities (parasites/ml). Twenty fields were counted by duplicate in three independent experiments. A clump was defined as an aggregate of ∼5 or more parasites. Data are expressed as number of clumps for field ± standard deviation. ANOVA followed by Tukey’s post hoc test (α=0.95) was used to determine significant differences. (C) Quantification of the number of CDC1132 parasites containing cytonemes at different parasite densities (parasites/ml). Three independent experiments by duplicate were performed, and 100 parasites were randomly counted per sample. Data are expressed as −fold change compared to the number of parasites containing cytonemes at density 1×105 parasites/ml ± the standard deviation of the mean. Student T-tests (α=0.95) were used to determine significant difference between treatments. (D) Cytonemes (orange) are observed connecting two parasites (blue) by SEM (arrowheads). F, flagella; AF, anterior flagella; RF, recurrent flagellum; Ax, axostyle. Interaction between different strains induce formation of membrane protrusions Due to the high prevalence of T. vaginalis, mixed infections with different parasite strains have been observed (Conrad et al., 2012). However, the extent to which parasites communicate and interact with each other during infection has never been analyzed. To evaluate if different parasite strains are able to interact, CDC1132 parasites stained with CFSE(Carboxyfluorescein succinimidyl ester) (green) were co-incubated for 1 hr with a highly adherent strain (B7RC2) or a poorly adherent strain (G3) stained with CellTracker CMTPX Dye (red). As control, CDC1132 stained with CFSE was also co-incubated with CDC1132 stained with CellTracker CMTPX Dye (red). As observed in Figure 3A, CDC1132 parasites are able to interact and form clumps with parasites from B7RC2 or G3 strains (Figure 3A). As G3 is a poorly adherent strain and usually do not form clumps (Coceres et al., 2015), the formation of smaller clumps when incubated with CDC1132 is expected (Figure 3A). On the basis of these findings and the observation that cytonemes are usually found connecting parasites (Figure 2D), we hypothesized that communication among different T. vaginalis strains might involve the formation of filopodia and/or cytonemes. To evaluate this, CDC1132 strain was stained with CellTracker CMTPX Dye (red) and co-incubated with varying amount of unstained G3, B7RC2, and CDC1132 strains (ratios of 1:1; 1:2; 1:9). Then, co-incubated parasites were stained with WGA, and the formation of filopodia and cytonemes in CDC1132 strain was evaluated by fluorescent microscopy (CDC1132 was visualized as the cells stained with red and green). Although we could not observe a clear increase in filopodia formation in CDC1132 strain upon exposure to G3 or B7RC2, the formation of cytonemes in CDC1132 is clearly affected by the presence of a different strain compared to the exposure to CDC1132 itself (Figure 3B). These results suggest that cytonemes are specifically involved in parasite: parasite communication. Figure 3 Download asset Open asset Cytoneme formation is induced by interaction between different strains. (A) CDC1132 parasites stained with CFSE (green) were co-incubated with CDC1132, G3, or B7RC2 stained with Cell Tracker CMTPX Dye (red) for 1 hr. The interaction between different strains was evaluated by analyzing the capacity to form clumps. As G3 is a poorly adherent strain, the observation of smaller clumps when incubated with CDC1132 is expected. (B) Percentage of CDC1132 parasites containing filopodia or cytonemes during co-incubation with different strains. CDC1132 parasites were stained with Cell Tracker Red (red) and co-incubated for 1 hr with different ratio (1:1, 1:2, and 1:9) of unstained CDC1132, G3, and B7RC2. Then, all the parasites were stained with wheat germ agglutinin (WGA; green), and the number of CDC1132 parasites (identified as the parasites stained red and green) containing filopodia (middle panel) and cytoneme (lower panel) were analyzed. As control, the number of cytonemes and filopodia of CDC1132 parasites (without co-incubation) was quantified. Data are expressed as percentage of CDC1132 parasites with filopodia and cytonemes ± standard deviation of three independent experiments. Student T-tests (α=0.95) were used to determine significant difference between treatments. Strain specific parasite-secreted EVs promote cytoneme formation To evaluate if the observed increase in cytoneme formation in CDC1132 strain due to the presence of a different strain is the result of physical cell contact, we exposed the CDC1132 strain to G3, B7RC2, and CDC1132 (control) strains using a 1 µm porous membrane that prevent direct contact between parasites. This system allows for secreted factors and small EVs to pass between the two cell populations but keeps the parasites (10–15 µm in diameter) loaded in the inserts from contacting the parasites inoculated in the bottom. To measure paracrine signal activation, the number of CDC1132 parasites in the bottom of the well containing cytonemes and filopodia was measured by WGA staining after 1 hr incubation (Figure 4A). As can be observed in Figure 4A, the number of receiving parasites containing cytonemes and filopodia significantly increases when G3 or B7RC2 strains are loaded in the inserts, compared to the control where CDC1132 strain was loaded in the chamber. These results demonstrated that the effect in cytoneme formation is, at least partially, contact independent. Additionally, these results also suggest that cytoneme formation is affected by paracrine communication among T. vaginalis strains. Figure 4 with 1 supplement see all Download asset Open asset Cytoneme formation is induced by paracrine communication. (A) Using a cell culture insert assay, CDC1132 parasites (bottom) were with CDC1132, G3, and B7RC2 strains for 1 hr. Then, the number of CDC1132 parasites (bottom) containing filopodia and cytonemes was by wheat germ agglutinin Data are expressed as percentage of parasites with filopodia and cytonemes ± standard deviation of three independent experiments. Student T-tests (α=0.95) were used to determine significant difference between treatments. (B) Using a cell culture insert assay, CDC1132 parasites (bottom) were with CDC1132, G3, G3 with an and G3 with for 1 hr. Then, the number of CDC1132 parasites (bottom) containing filopodia and cytonemes was by WGA Data are expressed as percentage of parasites with filopodia and cytonemes ± standard deviation of three independent experiments. Student T-tests (α=0.95) were used to determine significant difference between treatments. (C) Extracellular vesicles isolated from G3, B7RC2, and CDC1132 parasites were incubated with CDC1132 parasites for 1 hr. As control, CDC1132 parasites were incubated with the of the or of EVs from different strains. Then, the parasites were stained with WGA, and the number of parasites containing cytonemes and filopodia was quantified. Data are expressed as a −fold increase compared to control (without ± standard deviation of three independent experiments. Student T-tests (α=0.95) were used to determine significant difference between and As the cell culture inserts system allows for secreted factors as well as EVs to pass the we evaluated whether EVs have a role in cytoneme and filopodia formation of parasites. To this we a using parasites that the a of the which has been demonstrated to regulate EVs and protein cargo in the parasite T. vaginalis et al., 2021). As shown in Figure the number of CDC1132 parasites in the bottom of the well containing cytonemes and filopodia is significantly higher when parasites (G3 are loaded into the inserts compared to the incubation with G3 parasites with control or G3 parasites (Figure As more EVs than parasites, these results may that EVs have a role in the formation of membrane protrusions in parasites. In to these we isolated EVs from G3, B7RC2, and CDC1132 strains using a previously et al., and EVs samples were incubated with CDC1132 parasites (Figure The of of EVs from G3 or B7RC2, but not EVs from CDC1132, increased the formation of cytonemes and filopodia of CDC1132 parasites (Figure As a control, CDC1132 parasites were incubated with as well as EVs by et al., 2020; Figure as previously described where the effect of EVs in T. vaginalis was observed at et al., 2013), we not observe any increase in the formation of cytonemes and filopodia when increased the of EVs to (Figure supplement 1). These results that EVs are key factors for communication between different strains and suggest that specific are loaded in EVs isolated from different strains. specific protein cargo was detected in EVs isolated from different strains To explain the differential response in cytoneme and filopodia formation induced by the presence of EVs isolated from different strains, we analyzed the protein content of the EVs using mass Three samples of EVs isolated from G3, B7RC2, and CDC1132 parasite strains were analyzed (Figure analysis that the of EVs samples isolated from the different strains were clearly distinct (Figure we identified and proteins in the EVs samples isolated from G3, CDC1132, and B7RC2 strains, (Figure and 1). Although EVs have been isolated from the number of parasites, the number of proteins identified in the from G3 and CDC1132 was higher than the number of proteins in B7RC2 samples (Figure of proteins detected in the EVs from G3, B7RC2, and CDC1132 with previously T. vaginalis et al., extracellular et al., 2020; Rada et al., 2022; Twu et al., 2013), EVs et al., (Nievas et al., and/or surface et al., showed significant as of the proteins identified were previously detected in other 1). When the protein content was the results indicated that a of proteins is shared among EVs isolated from different strains (Figure as the that from a analysis of proteins using as a demonstrated that the proteins of EVs samples from the different strains were different from each other (Figure As involved in several biological processes were highly using the analysis 1 and Figure the of proteins detected in the EVs of each strain from one the analysis that the biological processes detected in the different EVs are highly (Figure Specifically, proteins associated to cellular and response to signaling, process, and have been detected in similar among the proteins detected in samples isolated from each of the analyzed strains (Figure EVs from parasites from G3, CDC1132, and B7RC2 strains contain several proteins that roles in the formation of filopodia and/or cytonemes in other cells. These proteins proteins and members in G3, CDC1132, and B7RC2, proteins and 3 members in G3, CDC1132, and B7RC2, 2, and 1 in G3, CDC1132, and B7RC2, and and 4 members in G3, CDC1132, and B7RC2, as in the EVs isolated from all three strains also several proteins that could be involved in biogenesis, including members of the and 2 members in G3, CDC1132, and B7RC2, and members in G3, CDC1132, and B7RC2, and protein and 2 members in G3, CDC1132, and B7RC2, 1). These data suggest that EVs may be loaded with specific of proteins that may be specific as of a for of to cells. Figure Download asset Open asset Extracellular vesicles (EVs) isolated from different strains contain different protein EVs’

Source: eLife

DOI: 10.7554/elife.86067.sa2