Protein Palmitoylation Plays an Important Role in Trichomonas vaginalis Adherence - PubMed

Palmitoylation of TvTSP8 might be contributing to regulate T. vaginalis aggregation. A, ABE assay using TvTSP8-HA overexpressing parasites treated with 100 μ

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2-BP (right panel) or negative control treated with vehicle (DMSO) only. 2 mg of protein lysate was labeled with HDPD-biotin and incubated with Neutravidin-Agarose. After washes, 25% of the elution of each sample was loaded on the gel. NH2OH-; without hydroxylamine, NH2OH+; with hydroxylamine. A monoclonal anti-hemmaglutinin (HA) antibody was used to detect HA tagged TvTSP8. B, TvTSP8-HA overexpressing parasites were grown until reach 10⁶cells/ml, followed by treatment with 50 or 100 μ

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of 2-BP for 4 h. Illustrative pictures were taken at 4 h. C, Quantification of the number of parasite aggregates per fields. 30 fields were analyzed in three independent experiments. Data are expressed as mean fold change related to mock-treated control parasites ± the standard error of the mean (S.E.). ANOVA followed by Tukey's post hoc test were used to determine significant differences. **p < 0,005, ****p < 0,0001. D, Ability to form clumps of TvTSP9 full-length (TVAG_287570) transfected parasites. A representative picture is shown. E, Cells expressing C-terminal HA-tagged versions of TvTSP8 protein treated with 100 μ

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of 2-BP or DMSO (vehicle) were stained for immunofluorescence microscopy using a mouse anti-HA antibody. The nucleus (blue) was also stained with 4′,6′-diamidino-2-phenylindole. F, TvTSP8-HA transfected parasites were incubated with 100 μ

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2BP or DMSO for 16 h, lysed and separated by optiprep density gradient centrifugation. Fractions were collected from the top of the gradient. Equal volume aliquots from each fraction were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted using anti-HA or anti-TCTP antibodies. Fractions 1–3 represent the low-density membrane fractions (L), whereas 7–9 the high-density fractions (H).