A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells

Abstract

Trichomonas vaginalis is an extracellular eukaryotic parasite that causes the most common, non-viral sexually transmitted infection worldwide. Although disease burden is high, molecular mechanisms underlying T. vaginalis pathogenesis are poorly understood. Here, we identify a family of putative T. vaginalis rhomboid proteases and demonstrate catalytic activity for two, TvROM1 and TvROM3, using a heterologous cell cleavage assay. The two T. vaginalis intramembrane serine proteases display different subcellular localization and substrate specificities. TvROM1 is a cell surface membrane protein and cleaves atypical model rhomboid protease substrates, whereas TvROM3 appears to localize to the Golgi apparatus and recognizes a typical model substrate. To identify TvROM substrates, we interrogated the T. vaginalis surface proteome using both quantitative proteomic and bioinformatic approaches. Of the nine candidates identified, TVAG_166850 and TVAG_280090 were shown to be cleaved by TvROM1. Comparison of amino acid residues surrounding the predicted cleavage sites of TvROM1 substrates revealed a preference for small amino acids in the predicted transmembrane domain. Over-expression of TvROM1 increased attachment to and cytolysis of host ectocervical cells. Similarly, mutations that block the cleavage of a TvROM1 substrate lead to its accumulation on the cell surface and increased parasite adherence to host cells. Together, these data indicate a role for TvROM1 and its substrate(s) in modulating attachment to and lysis of host cells, which are key processes in T. vaginalis pathogenesis.

Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease

“…A number of native and model rhomboid substrates are single‐pass membrane proteins (Urban et al , ; Strisovsky et al , ; Riestra et al , ; Johnson et al , ; Saita et al , ). The cleavage of the transmembrane core of MgtE by YqgP described here adds to growing evidence that rhomboid proteases do engage in cleaving polytopic membrane proteins under some conditions (Erez & Bibi, ), although the features of transmembrane domains that they recognise still need to be properly structurally understood.…”

Auranofin inactivates Trichomonas vaginalis thioredoxin reductase and is effective against trichomonads in vitro and in vivo

“…The PCR product was cloned into the Master-Neo-(HA)2 expression vector [13], was confirmed by sequencing and was transfected into T. vaginalis G3 as described previously [14]. Expression was analysed by indirect immunofluorescence with antibodies against the haemagglutinin (HA) tag or against hydrogenosomal HSP70 as a marker of hydrogenosomes [15].…”

Why cells need intramembrane proteases – a mechanistic perspective

“…This ‘recognition motif’ can be located in the transmembrane domain or reside in the immediate juxtamembrane region , and it determines the k cat of the reaction . Although a particular motif has been identified in several model substrates and is indeed recognized by several rhomboid proteases , it would be naïve to assume that it will be universal as is sometimes incorrectly interpreted . The rhomboid protease family is large and diverse, and it is expected that there will be differences in recognition motif preferences across the family .…”