A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells
Fig 5
Screening of the T. vaginalis surface proteome with a parasite search motif identifies an additional TvROM1 substrate.
(A) Graphical representation of the amino acids found in the predicted cleavage site of 20 parasite rhomboid protease substrates and the canonical Drosophila rhomboid substrate Spitz. The height of an amino acid indicates its relative frequency. Residue colors demark the properties of their side chains: small = black (A and G), basic = blue (K), aliphatic = orange (L, I, and V), green = uncharged, polar (Y, T, S, and N), nonpolar, nonaliphatic = purple (F and P). The predicted cleavage site is marked by a red arrow. The most common amino acids in these proteins were used to generate a “parasite search motif” (shown below). (B) Flow chart of the approach taken to identify putative TvROM1 substrates by searching the T. vaginalis surface proteome published by de Miguel et al. 2010 with the parasite search motif. (C) The accession numbers and predicted TM domain of the five putative surface proteome substrates are shown. The parasite search motif is indicated by blue font. Capital letters indicate amino acids of the predicted TM domain, lower case letters denote amino acids found outside the predicted TM domain. (D) The TM domain of putative substrate TVAG_280090 is cleaved by TvROM1. A GFP-EBA-175 chimeric protein that has its TM domain replaced with that of TVAG_280090 was co-expressed with TvROM1 or TvROM1 catalytic His to Ala mutant (mut) in the HEK293 heterologous cell cleavage assay. Negative control lacked co-transfection with a TvROM (Negative-lane 1). Western blot analyses of whole cell lysates confirm expression of the TvROM1 wt and mut proteins (top panel) and the full length substrate (filled arrowhead, middle panel). Analyses of conditioned media from the co-transfectants show a GFP-tagged cleavage product in the media (red arrowhead, bottom panel) of TvROM1 co-transfectants (TvROM1) and not in the TvROM1 catalytic His to Ala mutant (TvROM1 mut). The remaining putative substrates were not cleaved by TvROM1 or TvROM3 (see S4 Fig).