Trichomonas vaginalis contact-dependent cytolysis of epithelial cells

Abstract

Trichomonas vaginalis is an extracellular protozoan parasite that binds to the epithelium of the human urogenital tract during infection. In this study, we examined the propensities of 26 T. vaginalis strains to bind to and lyse prostate (BPH-1) and ectocervical (Ect1) epithelium and to lyse red blood cells (RBCs). We found that only three of the strains had a statistically significant preference for either BPH-1 (MSA1103) or Ect1 (LA1 and MSA1123). Overall, we observed that levels of adherence are highly variable among strains, with a 12-fold range of adherence on Ect1 cells and a 45-fold range on BPH-1 cells. Cytolysis levels displayed even greater variability, from no detectable cytolysis to 80% or 90% cytolysis of Ect1 and BPH-1, respectively. Levels of adherence and cytolysis correlate for weakly adherent/cytolytic strains, and a threshold of attachment was found to be necessary to trigger cytolysis; however, this threshold can be reached without inducing cytolysis. Furthermore, cytolysis was completely blocked when we prevented attachment of the parasites to host cells while allowing soluble factors complete access. We demonstrate that hemolysis was a rare trait, with only 4 of the 26 strains capable of lysing >20% RBCs with a 1:30 parasite/RBC ratio. Hemolysis also did not correlate with adherence to or cytolysis of either male (BPH-1)- or female (Ect1)-derived epithelial cell lines. Our results reveal that despite a broad range of pathogenic properties among different T. vaginalis strains, all strains show strict contact-dependent cytolysis.

Fig 1

Visualization of T. vaginalis adherence to and cytolysis of human ectocervical (Ect1) or benign hyperplasia prostate (BHP-1) cell monolayers. (A) Representative adherence fields of T. vaginalis strain T1 (panels A and C) or MSA1132 (panels B and D) parasites labeled with Cell Tracker Blue to monolayers of Ect1 (panels A and B) and BPH-1 (panels C and D) cells. Images were captured with a 10× objective. (B) Cell Tracker Blue-labeled MSA1132 (panels A, B, E, and F) or T1 (panels C, D, G, and H) parasites were exposed to Cell Tracker Red-labeled Ect1 monolayers (panels A to H) either directly (panels A to D) or with a 0.4-μm porous membrane barrier between them (panels E to H) for 4 h. (C) Cell Tracker Blue-labeled MSA1132 (panels A, B, E, and F) or T1 (panels C, D, G, and H) parasites were exposed to Cell Tracker Red-labeled BPH-1 (panels A to H) monolayers either directly (panels A to D) or with a 0.4-μm porous membrane barrier between them (panels E to H) for 4 h. All fluorescent images are of the red (epithelial cells) and blue (parasites) images merged; all the phase panels are the same field as the fluorescent image to the left. All images were taken with a 40× objective.

Fig 2

Adherence to and cytolysis of human Ect-1 or BPH-1 cell monolayers by T. vaginalis. (A) Adherence of different strains of T. vaginalis to either Ect1 (white bars) or BPH-1 (gray bars) cells. Data are from three experiments performed in triplicate and show the average percentage of parasites attached per coverslip with standard deviation. Data are arranged in order of ability to attach to Ect1, and this order is maintained throughout the report. (B) Cytolysis of Ect1 (white bars) and BPH-1 (gray bars) cells by different T. vaginalis strains. Parasites were exposed to monolayers of the epithelial cells, and cytolysis was measured by determining LDH release. Data are from three experiments performed in triplicate, showing the average percentage of lysis with standard deviation. Lysis with Triton X-100 (+C) was used as a positive control for complete monolayer lysis, and the data set was fit from 0 to 100%.

Fig 3

Correlation of attachment and cytolysis. The T. vaginalis strains were ordered according to increasing attachment and independently for increasing cytolysis, and ranked values were plotted for Ect-1 (A) and BHP-1 (B). The top scatter plot shows all strains assayed. Strains with low and high adherence were independently examined in the middle and bottom scatter plots, respectively. Lines are the best-fit trendlines. The Pearson coefficient (r) and the significance value (p) are on the upper left of each plot.

Fig 4

Contact-dependent cytolysis of Ect1 and BPH-1 cells by T. vaginalis, showing cytolysis of Ect1 and BPH-1 cells by T. vaginalis strains when contact was prevented by a 0.4-μm membrane. Parasites were exposed to monolayers of Ect1 (white bars) and BPH-1 (gray bars) cells with contact prevented by a 0.4-μm membrane. Cytolysis was measured by determining LDH release, and LDH background levels in the absence of parasites is shown (Bkgd). Data are from three experiments performed in triplicate, showing the average percentage of lysis with standard deviation. Complete lysis of the monolayer was achieved with Triton X-100 (+C), and the data set was fit from 0 to 100%. The control for direct monolayer lysis by T. vaginalis is shown as MSA1132(*), in which MSA1132 strain was placed under the membrane.

Fig 5

Hemolysis by T. vaginalis strains. Hemolysis was carried out for 1 h with a parasite/RBC ratio of 1:30 with constant rotation. Hemolysis was measured by hemoglobin release by absorbance at 404 nm. Data are from three experiments performed at least in triplicate, showing the average percentage of lysis with standard deviation. Complete RBC lysis was with Triton X-100, and the data set was fit from 0 to 100%.

Fig 6

Correlation of hemolysis and cytolysis. The T. vaginalis strains were independently ordered according to increasing cytolysis and hemolysis (see Table S2 in the supplemental material). Ranked values were plotted for Ect-1 and hemolysis (A), BPH-1 and hemolysis (B), and Ect1 and BPH-1 cytolysis (C). The inset in panel C shows the correlation of Ect1 and BPH-1 attachment data. Lines are the best-fit trendlines. The Pearson coefficient (r) and the significance value (p) are in the upper left of each plot.