Adherent Bacteria and Parasiticidal Secretion Products of Human Cervicovaginal Microbiota-Associated Lactobacillus gasseri Confer Non-Identical Cell Protection against Trichomonas vaginalis-Induced Cell Detachment

Abstract

Trichomonas vaginalis, a protozoan parasite specific to the human genital tract, is one of the most common sexually transmitted pathogens. Its pathogenicity is strongly associated with its expression of a broad array of proteases triggering cytotoxic effects in host epithelial cells. Vaginal microbiota-associated Lactobacillus, including those of L. gasseri in particular, can counteract T. vaginalis pathogenesis, but the mechanisms involved have yet to be clarified. T. vaginalis strain G3 (Tv G3) cytotoxicity was assessed by examining cell morphology, cell detachment, and fluorescent labeling of the F-actin cytoskeleton and immunolabeling of vinculin-position focal adhesions (FAs) by confocal laser scanning electron microscopy on confluent cervicovaginal epithelial HeLa cell monolayers. The inhibitory effects of bacterial cells and secreted products of L. gasseri ATCC 9857 and KS 120.1 on the Tv G3 viability and parasite deleterious effects on HeLa cells were investigated. Pre-adhering L. gasseri cells delayed but did not inhibit Tv G3-induced cell detachment, F-actin cytoskeleton disorganization and the disappearance of vinculin-positive focal FAs. L. gasseri KS 120.1 secretion products had a rapid parasiticide activity by killing time- and concentration-dependent Tv G3 parasites after direct contact. By killing Tv G3 parasites already associated with the epithelial cells, secretion products have abolished parasite-induced cell detachment. Our findings suggest that vagina microbiota-associated L. gasseri creates a physical barrier and exerts pharmacological-type mechanisms to counteract the deleterious cytotoxic effects of T. vaginalis.

Keywords: HeLa cells; Lactobacillus gasseri; Trichomonas vaginalis; adhesion; cytotoxicity; motility; parasiticidal activity; released products; vaginal microbiota.

Figure 1

The F-actin network, cell-to-cell contacts, and focal adhesion contacts are altered in a time-dependent manner by Tv G3, leading to the detachment of cervicovaginal HeLa epithelial cells. In (A), progression of Tv G3-induced cell detachment over a time course. In (B), Tv G3-induced cell detachment with and without a combination of protease inhibitors (the E64 cysteine protease inhibitor at 100 µM, the PMSF serine protease inhibitor at 2 mM, and the 1,10-PT metalloprotease inhibitor at 5 mM) (24 h post-infection (pi)). In (C), representative differential interference contrast (DIC) micrographs of cells showing a rounding of the cell surface and alterations to cell-to-cell contacts. In (D), percentages of cells displaying rounding and alterations to cell-to-cell contacts (6 h pi). In (E), representative confocal laser scanning microscopy (CLSM) images of the F-actin network. In (F), decrease in F-actin relative fluorescence intensity (RFI)/cell over time. In (G), representative CLSM images of vinculin-positive focal adhesions (FAs). In (H), decrease in vinculin RFI/cell over a time course. In (I), decrease in the number of vinculin-positive FAs/cell over time. In (J), length of vinculin-positive FAs (6 hpi). The micrographs shown are representative of two independent experiments. In (G), the white box indicates the cell viewed at high magnification in the adjacent image. The dashed white line delineates the area occupied by the cell cytoplasm. Ctl: Control. Each value shown is the mean ± SD of at least two independent experiments. In (A), * p < 0.01 for comparisons with T₀. In (B), * p < 0.01 for comparisons with Tv G3. In (D,F,H–J), * p < 0.01 in comparisons with Control.

Figure 2

The prior adhesion of L. gasseri to cells delays Tv G3-induced cell detachment. (A) Levels of parasites adhering to HeLa cells with and without prior colonization with L. gasseri ATCC 9857 or KS 120.1 cells. In (B), representative differential interference contrast micrographs of cells. In (C), progression of cell detachment over time for cells with and without prior colonization with L. gasseri ATCC 9857 or KS 120.1 cells. The micrographs shown are representative of two independent experiments. Each value shown is the mean ± SD of three separate experiments. In (A,C), * p < 0.01 for comparisons with Tv G3.

Figure 3

The prior adhesion of L. gasseri bacterial cells delays Tv G3-induced alterations to the F-actin network, cell-to-cell contacts, and focal adhesion contacts. In (A), representative differential interference contrast micrographs of T. vaginalis-exposed cells precolonized with L. gasseri ATCC 9857 or L. gasseri KS 120.1 cells. In (B), percentages of cells displaying rounding and alteration to cell-to-cell contacts (12 h post-infection). In (C), confocal laser scanning microscopy (CLSM) images of the F-actin network. In (D), decrease in F-actin relative immunofluorescence intensity (RFI) over time. In (E), representative CLSM micrographs of vinculin-positive FAs. In (F), decrease in vinculin RFI/cell over time. In (G), decrease in the number of vinculin-positive FAs/cell over time. The micrographs shown are representative of three independent experiments. Ctl: Control. Each value shown is the mean ± SD of three independent experiments. In (D,F,G), * p < 0.01 for comparisons with Tv G3.

Figure 4

Strain-specific anti-trichomonadal activity of secreted L. gasseri KS 120.1 product(s). In (A), changes in the swimming motility of untreated and treated axenic trophozoites over the period of direct contact. In (B), concentration-dependent inhibition of parasite swimming motility. CFCS: cell-free culture supernatant. Each value shown is the mean ± SD of two experiments. In (A), * p < 0.01 for comparisons with Tv G3.

Figure 5

The strain-specific anti-trichomonadal activity of secreted L. gasseri KS 120.1 products abolishes Tv G3-induced cell detachment. In (A), quantification of viable parasites adhering to HeLa cells. In (B), progression of cell detachment over the course of infection. In (C), representative differential interference contrast micrographs of cells. The micrographs shown are representative of three independent experiments. CFCS: cell-free culture supernatant. Each value shown is the mean ± SD of two experiments. In (A,B), * p < 0.01 for comparisons with Tv G3.

Figure 6

The strain-specific anti-trichomonadal activity of secreted L. gasseri KS 102.1 products abolishes Tv G3-induced alterations to the F-actin network and vinculin-positive focal adhesions. In (A), confocal laser scanning microscopy (CLSM) images of the F-actin network. In (B), change in F-actin relative fluorescence intensity (RFI) during the course of infection. In (C), representative CLSM images of vinculin-positive FAs. In (D), change in vinculin RFI/cell over the course of infection. In (E), change in the number of vinculin-positive FAs/cell over the course of infection. In (F), lengths of vinculin-positive FAs at 12 h post-infection. The micrographs shown are representative of three independent experiments. CFCS: cell-free culture supernatant. Ctl: Control. Each value shown is the mean ± SD of three independent experiments. In (B,D–F), * p < 0.01 in comparisons with Tv G3.