A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells
Fig 4
Use of quantitative proteomics identifies a putative TvROM1 substrate.
(A) Flow chart of the approach taken to identify putative substrates for TvROM1 using quantitative proteomics of cell supernatants from HA-TvROM1 T. vaginalis transfectants treated with vehicle or 50 μM 3,4-DCI serine protease inhibitor. (B) The profile of proteins identified in two independent mass spectrometry experiments and the magnitude of change on a log2 scale, errors bars denote the standard error. Proteins that decreased with 3,4-DCI treatment have log2(DCI/DMSO) ratios<0, and those that increased are >0. (C) The predicted TM domains of the five putative substrates identified in A, and the percent decrease in protein levels with 3,4-DCI vs. DMSO vehicle treatment is shown. Capital letters indicate amino acids of the predicted TM domain, lowercase letters denote amino acids found outside the predicted TM domain. (D) The TM domain of TVAG_166850 is cleaved by TvROM1. A fusion protein composed of GFP-P. falciparum EBA-175 and the TVAG_166850 TM domain was co-expressed with HA-TvROM1 (lane 2) or a TvROM1 catalytic His to Ala mutant (lane 3) using the HEK293 heterologous cell cleavage assay. Negative control lacked co-transfection with a TvROM (lane 1). Western blot analysis of whole cell lysates (top and middle panels) and media (bottom panel) was performed as described in text and Fig 2 legend. The expression of TvROM1 proteins and the substrate (filled arrowhead) is confirmed in top and middle panels, respectively. Bottom panel shows release of the cleaved substrate (open, red arrowhead) specifically in TvROM1 co-transfectants. The remaining putative substrates were not cleaved by TvROM1 or TvROM3 (see S3 Fig).