Omp19 of Brucella abortus is a broad spectrum protease inhibitor from I38 family

KARINA A. PASQUEVICH; MARIANELA V. CARABAJAL; MARÍA L. DARRIBA; ANDRÉS E. IBAÑEZ; LAURA BRUNO; LORENA M. CORIA; JULIANA CASSATARO

Congreso; 9th General Meeting of the International Proteolysis Society 2015; 2015

International Proteolysis Society

In our laboratory we are working with a Brucella abortus protein called Omp19. BLAST, Pfam and MEROPS sequence analysis indicated that Omp19 has some degree of identity with bacterial protease inhibitors of the family I38, such as inh from Erwinia chrysanthemi or aprin from Pseudomonas aeruginosa. Using casein-Bodipy substrate we tested Omp19´s ability to inhibit different proteases. U-Omp19 inhibited the aspartic protease pepsin, serin proteases (pancreatic elastase, trypsin and α-chimotrypsin), and cystein proteases (cathepsin L, B and S). Also it partially inhibited the activity of carboxypeptidase B but could not inhibit carboxypeptidase A. Stability studies showed that U-Omp19 retained its full protease inhibitor activity when previously exposed to a broad pH (2-8) or temperature (25-100°C) range. Kinetic analysis of the inhibition using specific fluorogenic substrates demonstrated that Omp19 inhibited trypsin, α-chymotrypsin, pancreatic elastase in a mixed noncompetitive manner with inhibition constants in the micromolar range and alpha values between 1.8 and 3.4, while inhibited cathepsin L by a competitive mechanism.Omp19 partially inhibited the proteolytic activity of stomach and intestine extracts as well as of microsomal content from macrophages. Moreover in vivo experiments showed that oral delivery of Omp19 inhibited the activity of pepsin at the stomach and trypsin, α-chymotrypsin, pancreatic elastase at the duodenum. Moreover, Omp19 was able to inhibit cathepsin L within dendritic cell lysosomes.All together these results indicate that Omp19 of B. abortus is a broad spectrum, pH- and thermal-stable protease inhibitor from I38 family. Although a Brucella cognate protease for this inhibitor wasn´t found yet, further work of our laboratory showed that these activities may play a role in Brucella´s ability to survive the bactericidal activity of gastrointestinal proteases during oral infection and in Omp19´s adjuvant activity.