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. 2021 Dec 24;79(1):11.
doi: 10.1007/s00018-021-04083-3.

VPS32, a member of the ESCRT complex, modulates adherence to host cells in the parasite Trichomonas vaginalis by affecting biogenesis and cargo sorting of released extracellular vesicles

Nehuén Salas  1 , Veronica M Coceres  1 , Tuanne Dos Santos Melo  2 , Antonio Pereira-Neves  2 , Vanina G Maguire  1 , Tania M Rodriguez  1 , Bruna Sabatke  3 , Marcel I Ramirez  3 , Jihui Sha  4 , James A Wohlschlegel  4 , Natalia de Miguel  5
Affiliations

VPS32, a member of the ESCRT complex, modulates adherence to host cells in the parasite Trichomonas vaginalis by affecting biogenesis and cargo sorting of released extracellular vesicles

Nehuén Salas et al. Cell Mol Life Sci. .

Abstract

Trichomonas vaginalis is a common sexually transmitted extracellular parasite that adheres to epithelial cells in the human urogenital tract. Extracellular vesicles (EVs) have been described as important players in the pathogenesis of this parasite as they deliver proteins and RNA into host cells and modulate parasite adherence. EVs are heterogeneous membrane vesicles released from virtually all cell types that collectively represent a new dimension of intercellular communication. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery contributes to several key mechanisms in which it reshapes membranes. Based on this, some components of the ESCRT have been implicated in EVs biogenesis in other cells. Here, we demonstrated that VPS32, a member of ESCRTIII complex, contribute to the biogenesis and cargo sorting of extracellular vesicles in the parasite T. vaginalis. Moreover, we observe that parasites overexpressing VPS32 have a striking increase in adherence to host cells compared to control parasites; demonstrating a key role for this protein in mediating host: parasite interactions. These results provide valuable information on the molecular mechanisms involved in extracellular vesicles biogenesis, cargo-sorting, and parasite pathogenesis.

Keywords: ESCRTIII complex; Extracellular vesicles; Trichomonas vaginalis; VPS32.

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Conflict of interest statement

The authors declare that no competing interests exist.

Figures

Fig. 1
Fig. 1
Localization of VPS32 protein. a–c Electron micrographs of ultrathin cryosections of TvVPS32-HA transfected parasites immunogold-labelled with anti-HA antibodies demonstrate that VPS32 is localized in ILVs inside MVBs (a), microvesicles (MVs) protruding from the cell surface (b) and EVs being endocytosed or exocytosed in or out of the cell (c). d EVs isolation protocol. e VPS32 localization in isolated EVs analyzed by immunoelectron-microscopy using an anti-HA antibody. Arrows indicate colloidal gold particles
Fig. 2
Fig. 2
Large-scale isolation and characterization of EVs secreted by TvVPS32 and TvEpNEO parasites. a EVs isolated from TvVPS32 (VPS32-EVs) and TvEpNEO (EpNEO-EVs) cell culture supernatants were negatively stained and analyzed using transmission electron microscopy. Scale bar, 500 nm. b The total amounts of proteins in the EVs pellet isolated from large-scale cultures of TvVPS32 and TvEpNEO control parasites were quantified by Bradford Reagent (Sigma-Aldrich) and are presented as the values per 107 secreting parasites. Data are the mean ± SD. from three independent experiments (p < 0.01). c Characterization of EVs proteins by SDS-PAGE. Isolated EVs secreted by equal numbers of TvVPS32 or TvEpNEO parasites were analyzed by SDS-PAGE. Same volume of samples was loaded on the gel. d Characterization of EVs proteins by immunoblotting. Whole cell lysate (Total extract) and isolated EVs secreted by equal numbers of TvVPS32 or TvEpNEO were analyzed by immunoblotting to evaluate the upresence of three proteins previously detected in the exosome and MVs proteomes (TvTSP1, TvMIF and TvTCTP proteins). Same number of initial cells and same volume of sample was used for comparison. Hmp33 (a hydrogenosomal protein) was used as loading control. One representative experiment out of three is shown. e Nanoparticle tracking analysis of isolated VPS32-EVs and EpNEO-EVs samples. The concentration of particles detected in each sample is shown. Three independent set of samples were analyzed
Fig. 3
Fig. 3
a Differential EVs isolation protocol. b Concentration and size of EVs isolated at 10.000 × g from TvVPS32 and TvEpNEO control parasites analyzed by NTA. c Size of isolated EVs analyzed using negative staining EM. The diameter of 500 EVs was measured
Fig. 4
Fig. 4
Attachment of TvEpNEO and TvVPS32 parasites to NhPRE1 cells. a TvEpNEO and TvVPS32 parasites were labeled with Cell Tracker Blue CMAC (Invitrogen). Labeled parasites were then incubated for 30 min with NhPRE1 prostate cell monolayers grown on coverslips in 24-well plates at 37 °C and 5% CO2. Coverslips were washed to remove non-adherent parasites, mounted, and visualized by fluorescence microscopy. b Adherence of TvEpNEO (red bar) and TvVPS32 (green bar) parasites to NhPRE1 cells. Data are from three experiments performed in triplicate and show the fold increase in the number of parasites attached per coverslip standardized using TvEpNEO parasites with SD (p < 0.05). c Schematic representation of the experimental design. EVs from TvEpNEO (red) and TvVPS32 (green) were isolated. NhPRE1 cells were pre-incubated with increasing amount of EVs. Then, parasite attachment was analyzed by fluorescent microscopy. d Preincubation of NhPRE1 cells with VPS32-EVs or EpNEO-EVs increases adherence of a poorly adherent T. vaginalis strain (G3). Note that a stronger effect in increasing parasite adherence is observed when the cells are pre-incubated with EVs isolated from parasites transfected with VPS32. Statistical analyses were performed by comparing the adherence values of each treatment against the control treatment (*p < 0.05, **p < 0.01, ***p < 0.001). Besides, statistical analyses within each treatment were performed comparing between EVs concentration
Fig. 5
Fig. 5
Analysis of EVs uptake by prostate cells. a T. vaginalis VPS32-EVs or EpNEO-EVs were labeled with PKH67 dye and incubated with NhPRE1cells for 3 h. EV binding was visualized using fluorescence microscopy by incubating NhPRE1 cells with PKH67-labeled EVs or PKH67-labeled PBS as control (green). The nucleus was stained with DAPI (blue). The images are representative of 20 images viewed under similar conditions. b Quantification of EVs binding. Data shown represent the mean ± SD from 3 independent experiments, each performed in duplicate. The maximal fluorescence after incubating with labeled EVs was arbitrarily set at 100%. c Effect on uptake by temperature that block endocytosis of fluorescently labeled VPS32-EVs or EpNEO-EVs detected using flow cytometry. T. vaginalis VPS32-EVs or EpNEO-EVs were labeled with PKH67 dye, incubated with NhPRE1 cells at 37 °C or 4 °C for 45 min and uptake was quantified by flow cytometry. d Quantification of VPS32-EVs or EpNEO-EVs uptake at 37 °C or 4 °C. The maximal fluorescence after incubating with labeled VPS32-EVs was arbitrarily set at 100%. e T. vaginalis VPS32-EVs or EpNEO-EVs were labeled with PKH67 dye, incubated with NhPRE1cells for 45 min at 37 °C and the binding quantified using flow cytometry. Three independent experiments were performed. A representative experiment is shown. f Quantification of PKH67-labeled EVs uptake to NhPRE1 cells using flow cytometry. The maximal fluorescence after incubating the NhPRE1 cells with labeled VPS32-EVs was set at 100%. Data are presented are the mean ± SD from three independent experiments
Fig. 6
Fig. 6
EVs proteins identified by LC–MS/MS analysis. a A volcano plot of − log10(p-value) versus − log2(fold change) depicting the 483 proteins identified in the EVs isolated from TvVPS32 and TvEpNEO was constructed. Data points to the right, colored green, denote proteins which exhibited fold changes of VPS32-EVs/EpNEO-EVs greater than 2 (29 proteins). Data points to the left, colored red, denote proteins which exhibited fold changes of EpNEO/VPS32 greater than 2 (7 proteins). b Proteins differentially detected in the EVs isolated from TvVPS32 were sorted into functional groups using genome annotation. Most representative groups are shown
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References

    1. WHO (2018) Report on global sexually transmitted infection surveillance. pp 63
    1. Fichorova RN. Impact of T. vaginalis infection on innate immune responses and reproductive outcome. J Reprod Immunol. 2009;83:185–189. doi: 10.1016/j.jri.2009.08.007. - DOI - PMC - PubMed
    1. Van Der Pol B, Kwok C, Pierre-Louis B, et al. Trichomonas vaginalis infection and human immunodeficiency virus acquisition in African women. J Infect Dis. 2008;197:548–554. doi: 10.1086/526496. - DOI - PubMed
    1. McClelland RS, Sangaré L, Hassan WM, et al. Infection with Trichomonas vaginalis increases the risk of HIV-1 acquisition. J Infect Dis. 2007;195:698–702. doi: 10.1086/511278. - DOI - PubMed
    1. Twu O, Dessi D, Vu A, et al. Trichomonas vaginalis homolog of macrophage migration inhibitory factor induces prostate cell growth, invasiveness, and inflammatory responses. Proc Natl Acad Sci USA. 2014;111:8179–8184. doi: 10.1073/pnas.1321884111. - DOI - PMC - PubMed

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