Study of a protease inhibitor and its role in oral Brucella abortus infection

CARAVAJAL MARIANELA; REY, DIEGO; PASQUEVICH KA; COMERCI, DJ; CASSATARO JULIANA

Mar del Plata

Congreso; LI Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología molecular; 2015

Brucellosis is aworld widespread zoonotic disease. Oral infection is one of the ways in whichthe disease is acquired. Previous results demonstrated that a purifiedrecombinant Brucella spp. proteincalled U-Omp19 is able to inhibit in vitro the activity of maingastrointestinal proteases. In this work, we performed kinetic assays usingtrypsin-, α-chymotrypsin- and pancreatic elastase-specific substrates anddemonstrated that U-Omp19 inhibits the three serin proteases by a mixednoncompetitive mechanism with Ki values in the μM range. To test if Omp19 inthe Brucella membrane protects itagainst the bactericidal activity of the main gut proteases, an Omp19-deficientstrain (Δomp19 Ba) and thecomplemented mutant (Δomp19-omp19 Ba)were generated. In contrast to WT Ba, Δomp19 Ba was susceptible to elastase bactericidal activity (P<0.001 vsWT Ba and ∆omp19-omp19 Ba). Trypsinand α-chymotrypsin did not kill any Ba strain. Finally, BALB/c mice wereintragastrically inoculated with 1x108 CFU of either Δomp19 Ba or the parental WT strain. Twentydays later there was a significant reduction in the CFU in thespleens of ∆omp19Ba in comparison with WT-inoculated mice (P<0,0001 vs WT Ba). Altogether ourresults indicate that Omp19 plays a role in Brucella´s ability to withstand theharsh environment of the gastrointestinal tract and establish a chronicinfection after oral infection.