Fig 1.
Murine susceptibility to vaginal T. vaginalis and T. foetus infection.
Young (3–4 weeks) female BALB/cJ mice were intravaginally inoculated with the indicated strains of T. vaginalis (A, B) or T. foetus (C, D). In the experiments in panel B, mice were pretreated by vaginal instillation (topical) of iron or intraperitoneal (i.p.) injection of iron, estrogen, or estrogen and dexamethasone (dex) before infection, at dosages detailed in the Methods. Vaginal lavages were taken on day 3 or 4 of infection (A-C) or on the indicated days (D), and motile trophozoites were counted in a hemocytometer. Data points represent counts from individual mice, dashed lines indicate the detection threshold of the assays, and red diamonds show geometric means. For select samples without countable trophozoites, lavages were cultured in trichomonad growth medium with antibiotics (blue symbols). Positive cultures are indicated by solid blue dots, while negative cultures (no growth) are depicted by open blue dots.
Fig 2.
In vitro growth characteristics of T. foetus and T. vaginalis strains.
Trophozoites of the indicated strains were cultured in vitro in trypticase-yeast extract-maltose (TYM) medium. (A) Cell numbers were counted before and after a 24-hour time incubation period and doubling times were calculated. Data are mean ± SE; *p<0.05 (unpaired t-test for all T. vaginalis strains vs. all T. foetus strains). (B) Media were adjusted to the indicated pH and trophozoites were grown in each medium for 24 hours and counted. Data are mean ± SE.
Fig 3.
Comparison of T. foetus and T. vaginalis susceptibility to antimicrobial drugs of different classes.
Activity (pIC50) of the indicated compounds from each of the indicated three drug classes against multiple strains of T. foetus and T. vaginalis was determined by 24 h growth and survival assay (the detailed data are provided in S1 Table). Each point represents the mean pIC50 of four (T. foetus) or three (T. vaginalis) different strains for the drugs listed below each graph. ΔpIC50 was calculated for each drug class by subtracting the mean T. foetus pIC50s from the mean T. vaginalis pIC50s. Red lines represent geometric means. Significance was calculated with Tukey’s multiple comparisons test (*p <0.01).
Fig 4.
Correlation of drug susceptibility between different T. foetus and T. vaginalis strains.
The graphs show correlations of the pIC50 values for individual drugs between the indicated pairs of trichomonad strains (the detailed data on drug activities used for these analyses are provided in S1 Table). Each point represents one drug and is color-coded by drug class. Plots are shown with a linear regression line (black) and line of identity (dotted red).
Table 1.
Comparisons of drug responses in different T. foetus and T. vaginalis strains.
Table 2.
MLC assays of T. foetus and T. vaginalis.
Fig 5.
In vivo efficacy of selected antimicrobial compounds against T. foetus.
(A) Timeline of infection and treatment. Mice were treated five times over the course of 2.5 days, starting pno day after infection. (B/C) Mice were infected with the indicated T. foetus strains and treated intravaginally with 0.05 mg/dose (5 μL of a 10 mg/mL suspension) of either the gold(I) compound CPD4 (B) or bortezomib (C), or were left untreated as controls. After 4 days, trophozoite numbers were determined. Each point represents an individual mouse. Dashed black lines represent the detection threshold. Red lines represent geometric means. Significance was calculated by Mann-Whitney test (*p <0.01).
Fig 6.
Phylogenetic analysis of trichomonad 20S proteasome subunits.
Phylogenetic analysis of 20S proteasome α- and β-subunits from T. foetus (TF, bolded), T. vaginalis (TV), Plasmodium falciparum (PF), Mus musculus (MM), and Homo sapiens (HS). Branches are labeled with UniProt accession numbers when available, or otherwise GenBank accession numbers. Subunit groups are highlighted with the same color. Bootstrap values were calculated with 100 replications. Nodes supported with values ≥95% are marked with color-coded circles. Distance scale is shown on the bottom left.
Fig 7.
Functional analysis of trichomonad proteasomes.
(A) The depicted trichomonad strains were incubated for 1 hour with the indicated final concentrations of bortezomib or left without inhibitor (0 μM). Total cell lysates were prepared and analyzed by western blotting with an anti-ubiquitin antibody (left panels). As expected, multiple proteins of different sizes are labeled with ubiquitin, leading to the apparent “smear” on the blots. Total protein was visualized by Coomassie Blue staining (right panels). (B) Total cell lysates of T. foetus and T. vaginalis were incubated for 1 hour with the indicated concentrations of bortezomib and subsequently labeled for 2 hour with the fluorescent activity-based proteasome probe, MV151. Lysates were fractionated by SDS-PAGE and labeled proteins were visualized by fluorescent imaging. The band labeled with an asterisk corresponds to a non-specific target of MV151 that could not be competed for with any available proteasome inhibitor and is thus unlikely to be a proteasome subunit. Raw images of the blots and gels are shown in S1 Raw images.