Identification and characterization of a surface-associated, subtilisin-like serine protease inTrichomonas vaginalis

Abstract

SUMMARYTrichomonas vaginalisis a protozoan parasite causing trichomonosis, a sexually transmitted infection in humans. This parasite has numerous proteases, most of which are cysteine proteases that appear to be involved in adherence and cytotoxicity of host cells. In this report we identify and characterize a putative subtilisin-like serine protease (SUB1). Thesub1gene encodes a 101-kDa protein.In silicoanalyses predict signal and pro-peptides at the N-terminus, and a transmembrane helix at the carboxy-terminal region. Thesub1gene was found as single copy by Southern analysis, albeit additional serine protease related genes are annotated in theT. vaginalisgenome. The expression ofsub1could only be detected by RT-PCR and Ribonuclease Protection Assays, suggesting a low abundant mRNA. Thesub1gene transcription start site was correctly assigned by RPA. The transcript abundance was found to be modulated by the availability of iron in the growth medium. Antibodies raised to a specific SUB1 peptide recognized a single protein band (~82 kDa) in Western blots, possibly representing the mature form of the protein. Immunofluorescence showed SUB1 on the trichomonad surface, and in dispersed vesicles throughout the cytoplasm. A bioinformatic analysis of genes annotated as serine proteases in theT. vaginalisgenome is also presented. To our knowledge this is the first putative serine protease experimentally described forT. vaginalis.