Fig 1.
Subcellular localization of HA-tagged TvROMs in T. vaginalis transfectants.
Fluorescence microscopy images of indirect immunofluorescence assays (IFA) performed on T. vaginalis exogenously expressing N-terminal hemagglutinin (HA)-tagged TvROMs 1–3. (A-C) HA-TvROM1 transfectants were reacted with membrane impermeable biotin (EZ-Link-Sulfo-NHS-SS-Biotin) and IFA was then performed using formaldehyde fixation and staining with rabbit anti-HA (A-red) and a mouse anti-Biotin (B-green) antibodies. (C) Merge shows co-localization of HA-TvROM1 with the biotin-labeled T. vaginalis cell surface, scale bar = 10 μm. IFA images of HA-TvROM2 (D & E) and HA-TvROM3 (F & G) using a mouse anti-HA antibody (green) and nuclear staining using 4’-6’-diamidino-2-phenylindole (DAPI-blue). HA-TvROM2 and HA-TvROM3 show localization in a line structure adjacent to the nucleus, scale bar = 5 μm. Two juxtanuclear structures of different sizes can be observed in early and late dividing cells in HA-TvROM2 transfectants (D, see arrows). Phase images are shown on the right.
Fig 2.
TvROM catalytic activity analyses.
The activity of T. vaginalis rhomboid proteases were tested by co-transfecting the proteases with known model rhomboid substrates in a heterologous cell cleavage assay using HEK293 cells. Proteases were HA tagged and substrates contained an N-terminal GFP tag to allow detection. (A, B, D and E) Whole cell lysates (WCL) and conditioned media (CM) collected from co-transfectants was analyzed by Western blot analyses [39]. Top panels: rhomboid protease detected in WCL using an anti-HA antibody; middle panels: full-length (FL, filled arrowheads) and cleaved substrates (open arrowheads) detected in WCL using an anti-GFP antibody; bottom panels: cleaved substrate fragments detected in CM using an anti-GFP antibody (open, red arrowheads). The substrates tested were (A) APP+7 residues of the Drosophila melanogaster Spitz protein encompassing the rhomboid protease cleavage site (APP+Spi7), (B) Plasmodium falciparum EBA-175, (D) human EphB3, and (E) Providencia stuartii TatA. The positive control HA-tagged rhomboid protease (lane 2) used for cleavage of Spitz (A), human EphB3 (D), and TatA (E) was DmRho1; the positive control protease for cleavage of EBA-175 (B) was HA-tagged PfROM4 (lane 2). Negative controls (lane 1) were transfected with only substrates. TvROM1/TvROM3 = wild type protease; TvROM1 mut/TvROM3 mut = protease with the catalytic histidine mutated to alanine. TvROM3 was found to cleave only the Spitz TM domain segment of APP+Spi7 (A middle panel, lane 5) whereas TvROM1 does not cleave the Spitz sequence (A bottom panel, lane 3) but does cleave the other 3 substrates (B, D, and E bottom panels, lane 3). (F) Summary of the cleavage data shown in panels A, B, D, and E. (C) The location of EBA-175 cleavage by PfROM4 (control; top panel) and TvROM1 (bottom panel) determined by subjecting the immunoprecipitated cleavage fragment to MALDI-TOF analysis. Red arrows indicate that both proteases cleave the substrate between the two small amino acids alanine (A) and glycine (G).
Fig 3.
Serine protease activity and TvROM1 contribute to T. vaginalis attachment and lysis of ectocervical cells.
(A) Fluorescently labeled T. vaginalis incubated with ectocervical cell monolayers in the presence of increasing concentrations of the serine protease inhibitor 3,4-dichloroisocoumarin (3,4-DCI) followed by quantification of adhered parasites. The average fold change in attachment compared to vehicle control for four experiments each performed in triplicate is shown. Error bars denote the standard error, **p<0.01. (B) Parasites incubated with ectocervical cell monolayers in the presence of increasing 3,4-DCI followed by assessment of ectocervical cell lysis. The average fold change in cytolysis compared to vehicle control for three experiments performed in triplicate is shown. Error bars denote the standard error, **p<0.01. (C) Average fold difference in attachment of HA-TvROM1 transfectants compared to empty vector transfectants shown for four experiments each conducted in triplicate, with standard error shown as error bars, **p<0.01. (D) Average fold change in cytolysis of ectocervical cells by HA-TvROM1 transfectants compared to empty vector transfectants, results are from four experiments performed in triplicate. Standard error is shown as error bars, **p<0.01.
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).
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).
Fig 6.
Phenotypic analysis of predicted rhomboid cleavage site mutation in the putative substrate TVAG_166850.
(A) Predicted topology of the putative substrate TVAG_166850 using the Spoctopus TM prediction program, illustrated using the TOPO2 graphical representation program. TVAG_166850WT was tagged at the N-terminus with a GFP tag (green box). The predicted rhomboid cleavage site (scissors) and the predicted P1-P1’ cleavage site residues are highlighted in blue. The surrounding parasite search motif residues are highlighted in red. The predicted TM residues are shown below. A rhomboid cleavage site mutant was generated by mutating the predicted Ala-Gly P1-P1’ residues (underlined in sequence) to Phe-Phe residues to generate the GFP-TVAG_166850AG/FF mutant. (B) TvROM1 efficiently cleaves the wild type (wt) TVAG_166850 TM and not the TVAG_166850AG/FF mutant TM. Proteases and chimeras of GFP-EBA-175 with the TVAG_166850 wt TM or mut TM were co-transfected in the heterologous cell cleavage assay. Lanes 1–5 = co-transfection with wild type GFP-EBA-175-TVAG_166850WT TM; lanes 6–10 = co-transfection with GFP-EBA-175-TVAG_166850AG/FF mutant TM. Western blot analyses of whole cell lysates confirm expression of wt and mut rhomboid proteases (top panel) and the full length substrates (FL, middle panel). Analyses of conditioned media show a GFP-tagged cleavage product in the media (red, open arrowhead, bottom panel) of TvROM1 co-transfected with wild type TVAG_166850 (lane 4) which is reduced ~90% in mutant TVAG_166850AG/FF co-transfectants (lane 9). Co-transfection with mutant TvROM1 abolishes cleavage (lanes 5 & 10). Lanes 1 & 6 = no metalloprotease inhibitor BB-94, lane 2–5 & 7–10 = 10 μM metalloprotease inhibitor BB-94. Lanes 3 & 8 = co-transfection with TgROM5 (control). (C) The GFP-TVAG_166850WT and GFP-TVAG_166850AG/FF mutant proteins were exogenously expressed in T. vaginalis. Transfectants were stained with an anti-GFP antibody without permeabilization at 4°C to detect surface levels of the fusion protein quantified using flow cytometry. Three cultures (cultures #1–3) in three independent experiments were analyzed. Representative results from one experiment are shown. Top panel shows the GFP+ cell population, similar percentages of GFP+ cells were detected for the wt and mutant transfectants. Bottom histogram shows the fluorescence intensity distribution of the GFP+ population. GFP-TVAG_166850AG/FF mutant transfectants had at least three-fold higher mean fluorescence intensity (MFI) levels compared to wild type transfectants. (D) The ability of GFP-TVAG_166850WT and GFP-TVAG_166850AG/FF mutant transfectants to attach to ectocervical cells was compared to empty vector transfectants. Results show the average of three experiments, each conducted in triplicate. Exogenous expression of GFP-TVAG_166850WT leads to a statistically significant increase in attachment compared to empty vector transfectants (*p<0.05). Overexpression of the GFP-TVAG_166850AG/FF mutant leads to an even greater increase in attachment compared to empty vector (**p<0.01) and wildtype GFP-TVAG_166850WT transfectants (**p<0.01).