Cysteine Peptidase Expression in Trichomonas vaginalis Isolates Displaying High- and Low-Virulence Phenotypes

Abstract

In the present study, we identified and characterized the cysteine peptidase (CP) profiles of Trichomonas vaginalis isolates exhibiting high- and low-virulence phenotypes using a combination of two-dimensional SDS-PAGE (2DE), tandem mass spectrometry (MS/MS), and data mining. Seven of the eight CPs identified belong to Clan CA, family C1, cathepsin L-like CP, and one belongs to Clan CD, family C13, asparaginyl endopeptidase-like CP. Quantitative and qualitative differences in CP expression were detected between the isolates. BLAST analysis followed by CLUSTAL alignment of amino acid sequences of differentially expressed CPs showed identity or high homology to previously described CP cDNA clones CP1, CP3, CP4, and to a secreted CP fraction of 30 kDa involved in apoptosis of vaginal epithelial cells. One- and two-dimensional-substrate gel analyses revealed the differential CP profiles between the isolates, indicating that the combination of zymography with 2DE and MS/MS might be a powerful experimental approach to map and identify active peptidases in T. vaginalis. Toxicity exerted upon HeLa cells by high- and low-virulence isolates was 98.3% and 31%, respectively. Pretreatment of parasites with specific Clan CA papain-like CP inhibitor l-3-carboxy-2,3-trans-epoxypropionyl-leucylamido(4-guanidino)butane (E-64) drastically reduced the cytotoxic effect to 21.7% and 0.8%, respectively, suggesting that T. vaginalis papain-like CPs are the main factors involved in the cellular damage.

Immunoproteomics of the active degradome to identify biomarkers for Trichomonas vaginalis

“…Most of the experimental MW and p I values of the identified proteins estimated with the PDQuest software were different when compared with the theoretical values obtained by the MASCOT search results. However, the multiple alignments of the identified CP sequences with those reported in the literature 20, 27, 37–41 showed small differences, possible due to the variability among the trichomonad isolates used. The localization of the identified peptides by the MS analysis is also marked (Supporting Information Fig.…”

Cysteine Peptidases, Secreted by Trichomonas gallinae, Are Involved in the Cytopathogenic Effects on a Permanent Chicken Liver Cell Culture

“…Even though such a result indicates that the detected spots represent the same protein baring different modifications, the lack of complete genomic sequence of T. gallinae leaves the debate open. It should be added that in comparison to proteomics studies analyzing peptidases in T. vaginalis [47], [52], in which cell lysates were used for investigation, here, only secreted peptidases were examined. Considering much lower amount of starting material it is no surprise that only single peptides were identified for most of the spots and obtained data did not enable clear distinction whether the analysed spots represent the same or different proteins.…”

Mechanisms ofTritrichomonas foetusPathogenicity in Cats with Insights from Venereal Trichomonosis

“…The cytotoxic effects of cysteine proteases include degradation of mucus, complement and immunoglobulin, and destruction of extracellular matrix (fibronectin, several types of collagen, and laminin) 43, 72, 73, 84, 86, 87. Cysteine proteases therefore can function as epithelial cell detaching factors and can mediate direct cytotoxic effects on vaginal and uterine epithelial cells by induction of apoptosis72, 73, 84, 88, 89, 90 (Fig 1D).…”