Cysteine peptidase expression in Trichomonas vaginalis isolates displaying high- and low-virulence phenotypes.
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.
De Jesus
Jose Batista
JB
Departamento de Ciências Naturais, Universidade Federal de São João del Rei, São João del Rei, MG, Brazil. jbj@ioc.fiocruz.br
Cuervo
Patrícia
P
Britto
Constança
C
Sabóia-Vahia
Leonardo
L
Costa E Silva-Filho
Fernando
F
Borges-Veloso
Andre
A
Barreiros Petrópolis
Débora
D
Cupolillo
Elisa
E
Barbosa Domont
Gilberto
G
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014246:Trichomonas vaginalis / Q000201:enzymology* / Q000472:pathogenicity
D015800:Protozoan Proteins; D003546:Cysteine Endopeptidases