Putrescine effect on the TvCP39 activity from <i>T. vaginalis</i>.

<p>A) Putrescine effect on the proteolytic activity of <i>T. vaginalis</i>. Zimograms using total proteinases from parasites grown in normal media (N)(lane 1), DAB-treated parasites (D)(lane 2), DAB-treated parasites transferred into exogenous putrescine (DP)(lane 3), DAB-treated trichomonads transferred into a normal medium (DN)(lane 4) and parasites grown in normal medium transferred into an exogenous putrescine media (NP)(lane 5). B) Polyamine effect on the proteinases activity bound to HeLa cells. Ligand-proteinases assays using untreated parasites grown in normal medium (N)(lane 1); DAB-treated parasites (D)(lane 2); DAB-treated parasites transferred into exogenous putrescine media (DP)(lane 3), DAB-treated parasites transferred into normal media (DN)(lane 4) and parasites grown in normal media and transferred into an exogenous putrescine media (NP)(lane 5). Arrowhead shows the TvCP30 proteolytic activity. C) Densitometry analyses of TvCP39 proteolytic activity bands from panel B. Bars indicate the average of the intensity of TvCP39 activity bands from three independent ligand-proteinases assays and error bars represent the standard deviations.</p

TvCP39 re-localization after DAB treatment and putrescine restoration.

<p>A) Cytoplasmic (Cyt) and nuclear (Nuc) protein extract from DAB-treated parasites transferred into exogenous putrescine media (DP) (lanes 1 and 2) and from untreated parasites grown in normal media (N)(lanes 3 and 4) were blotted into a nitrocellulose membrane and incubated with anti-TvCP39, anti-TveIF-5A (control of cytoplasmic protein), anti-nucleoporin (control of nuclear protein) and anti-PCNA (control of nuclear protein) antibodies. Arrowheads show TvCP39 (39 kDa), the TveIF-5A (20 kDa), the nucleoporin (53 kDa), and the PCNA (28 kDa) protein bands. B) Zymograms from Cytoplasmic (Cyt) and nuclear (Nuc) protein extract from DAB-treated parasites transferred into exogenous putrescine media (DP) (lanes 1 and 2) and from untreated parasites grown in normal media (N)(lanes 3 and 4). Arrowhead indicates the TvCP39 proteolytic activity.</p

Putrescine effect on TvCP39 transcript and protein.

<p>A) Semi-quantitative RT-PCR analysis performed with total RNA from untreated parasites grown in normal medium (N)(lane 1); DAB-treated parasites (D)(lane 2,); DAB-treated trichomonads transferred into 40 mM exogenous putrescine medium (DP)(lane 3); DAB-treated trichomonads transferred to normal medium (DN)(lane 4), and trichomonads grown in normal medium and transferred into 40 mM exogenous putrescine medium (NP)(lane 5) to amplify 238 bp from the <i>tvcp39</i>. A 112 pb amplicon from <i>β-tubulin</i> was amplify as a loading control. B) qRT-PCR of samples described in A. The Ct levels of <i>tvcp39</i> mRNA in trichomonads after DAB treatment (bar D) decreased at 20% but the <i>tvcp39</i> mRNA were restored (70%) by adding 40 mM exogenous putrescine to DAB-treated parasites (bar DP). C) Total protein extract from <i>T. vaginalis</i> grown in normal media (N)(lane 1); DAB-treated parasites (D)(lane 2); DAB-treated trichomonads transferred into exogenous putrescine media (DP)(lane 3); DAB-treated parasites transferred into normal medium (DN)(lane 4) and trichomonads grown in normal medium transferred to medium with 40 mM exogenous putrescine (NP)(lane 5) were blotted onto nitrocellulose membranes and incubated with anti-TvCP39 and anti-α-tubulin (loading control) antibodies. Arrowheads indicate the immunodetected protein for each antibody employed.</p

The <i>tvcp39</i> mRNA and protein stabilities are regulated by putrescine.

<p>A) RNAm levels of <i>tvcp39</i> by semi-quantitative RT-PCR analysis using total RNA from parasites treated with actinomycin D and grown in normal culture media (N); or DAB-treated parasites (D); or DAB-treated trichomonads transferred into 40 mM exogenous putrescine medium (DP); or DAB-treated trichomonads transferred to normal medium (DN); or trichomonads grown in normal medium and transferred into 40 mM exogenous putrescine medium (NP). Samples were taken at 0, 1, 3, 6, 8, 12 and 24 h for amplification of 110 pb of <i>tvcp39</i> mRNA and 112 bp of <i>β-tubulin</i> mRNA (<i>β-tub)</i>(loading control). Arrowheads indicate the amplification products obtained. B) Transcriptional blockade using actinomycin D. Trichomonads grown in normal medium (N), DAB-treated trichomonads (D), DAB-treated trichomonads transferred into 40 mM exogenous putrescine medium (DP), DAB-treated trichomonads transferred into normal medium (DN), and trichomonads grown in normal medium and transferred into an exogenous putrescine medium (NP) were treated with actinomycin D. Samples taken at several times (0, 1, 2, 6, 8, 12, and 24 h) were use to amplified the <i>tvcp39</i> mRNA which was quantified by densitometric analysis and normalized. Bars represent the mean of each sample and the standard errors were included. C) Blockage of protein synthesis by cycloheximide. Trichomonads were treated with 10 µg of cycloheximide and grown in normal culture media (N); or DAB-treated parasites (D); or DAB-treated trichomonads transferred into 40 mM exogenous putrescine medium (DP). Samples were taken at several times for Western blot analysis using anti-TvCP39 (dilution 1: 1000) and α-tubulin (dilution 1∶100) antibodies. Arrowheads indicate the TvCP39 and α-tubulin proteins. D) Densitometric analysis of the samples described in C. The bands corresponded to TvCP39 were quantified and normalized to α tubulin. Bars represent the mean of three biological triplicates.</p

Phylogenies of single-copy genes.

<p>Phylogenetic trees were inferred from the DNA sequences for each of the three single copy genes (A) CRN (N = 94), (B) PMS1 (N = 94), and (C) Mlh1a (N = 94), and from the concatenation of all the three genes (N = 94; D). Blue dots indicate nodes where bootstrap support is ≥50. The clustering of type 1 (green) and type 2 (red) isolates supports the two-type population structure. Instances of unlike clustering resulting in non-homologous tree topology provide possible evidence for cases of recombination.</p

Prevalence of <i>T. vaginalis</i> in women attending New York City STD clinics.

<p>The number of vaginal swabs collected at each clinic is shown, followed by the number of swabs found to be positive for <i>T. vaginalis</i> by wet mount, <i>in vitro</i> culture and PCR. NA: technician not available at clinic for wet-mount diagnosis during duration of sample collection.</p>*<p>n represents the number of samples diagnosed as positive via wet mount, and N represents the number of samples tested by wet mount. Type 1 parasites are more likely to be infected with the <i>T. vaginalis</i> virus TVV and are more susceptible to metronidazole, compared to type 2 parasites.</p

Linkage disequilibrium varies between type.

<p>(A) Pairwise linkage disequilibrium (LD) matrix for MS genotypes of type 1 (N = 95) and 2 (N = 93). Significant LD is highlighted in red. Type 2, with 42 cases of paired locus LD, shows a greater amount of LD in comparison to type 1 with 15 cases. (B) Heat maps indicating degree of LD between SNPs in single copy genes. Gene boundaries are indicated by black lines. The number of pairwise comparisons varies between the two types due to the difference in the number of polymorphisms analyzed, but a greater amount of pairwise LD in type 2 in comparison to type 1 is indicated.</p

Type 1-like parasites appear to have given rise to type 2 parasites.

<p>(<b>A</b>) Phylogenetic tree of the evolutionary relationship of type 1 (green, N = 37) and type 2 (red, N = 57) single copy gene protein sequences, with <i>Pentatrichomonas hominis</i> and <i>Tritrichomonas foetus</i> protein sequences as outgroups. Protein sequences for CRN, PMS1 and Mlh1a were concatenated for each isolate and the tree was bootstrapped 1000 times. BioNJ methods were used for tree reconstruction. Blue dots indicate nodes where bootstrap support is ≥50. (<b>B</b>) The allelic richness by type was calculated with ADZE using microsatellite data for all isolates. Type 1 (green, N = 76) shows greater allelic richness than type 2 (red, N = 64), supporting the former as the ancestral clade and the latter as derived.</p

Two cluster population structure of <i>T. vaginalis</i>.

<p>A Bayesian clustering model implemented in STRUCTURE 2.2 indicates a two-cluster population structure for <i>T. vaginalis</i> (N = 188). The green cluster (type 1, (N = 95)) and red cluster (type 2, (N = 93) are found in each geographical location, with the exception of Southern Africa where all samples are type 1, and Mexico, which has a statistically significant over-representation of type 2. PNG: Papua New Guinea. Isolates included in each geographical location can be found in <b><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001573#pntd.0001573.s006" target="_blank">Table S1</a></b>.</p

Unfolding profiles of TvTIM1 and I45G mutant monitored by Intrinsic (black squares) and ANS (red circles) fluorescence

<p>. The conditions are as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141747#pone.0141747.g004" target="_blank">Fig 4</a>. The protein concentration was 50 μg ml^-1. Data are normalized for ease of comparison.</p