ECOTIN FROM SALMONELLA TYPHIMURIUM PROTECTS BACTERIA FROM GUT LUMEN AND INTRACELLULAR PROTEASES

SAPOSNIK, LUCAS; CORIA, LORENA; BRUNO L; GUAIMAS, FRANCISCO; PANDOLFI, JULIETA; URGA M. E.; SABBIONE FLORENCIA; MCCLELLAND MICHAEL; TREVANI ANALIA; KARINA PASQUEVICH; CASSATARO J.

Cordoba

Congreso; XVIII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL; 2023

Salmonella Typhimurium causes acute diarrhea upon oral infection in humans. The harsh and proteolytic environment found in the gastrointestinal tract is the first obstacle these bacteria encounter after infection. In the gut, Salmonella triggers an inflammatory response mainly sustained by polymorphonuclear leukocytes that translocate from the lamina propria to the lumen. Following colonization macrophages take up the bacteria and act as a replicative niche. How S. Typhimurium survives the gut and intracellular proteases is poorly understood. Our current hypothesis is that Salmonella synthesizes protease inhibitors to hijack the host’s proteolytic defense system and evade its responses against infection. To test our hypothesis, we studied the ecotin gene, which is widely spread in bacteria encoding a protein that has been shown to inhibit a wide range of proteases. In this work, we assessed the effect of porcine pancreatin - a mixture of several pancreatic digestive proteases - on S. Typhimuriim wild type (wt) and ecotin knock-out strain (∆ecotin). We found that after incubation of Salmonella with pancreatin, the bacterial loads of ∆ecotin were significantly lower than those of the wt strain. Following the physiopathology of Salmonella, we tested the ability of bacteria to establish infection in the murine model of Salmonella induced colitis. After 24 h of infection the gut inflammation triggered by ∆ecotin infection was attenuated in comparison to the one elicited by the wt strain. Also, the bacterial loads in the gut epithelium were significantly lower in ∆ecotin infected mice than in the wt infected mice. As neutrophils are a hallmark for Salmonella’s infection and their microbicide activity is well characterized against a wide range of pathogens, we tested the ability of wt and ∆ecotin strains to survive when incubated with human purified neutrophils. Results indicated that ∆ecotin is less resistant than the wt strain to the protease mediated microbicide activity of neutrophils. Also, the ∆ecotin strain showed an attenuated survival capacity to the action of purified neutrophil granules or purified neutrophil extracellular traps in comparison to the wt strain. After the initial steps of the infection, macrophages are an important replication niche for Salmonella. Considering the intracellular proteases of macrophages and their bactericide ability, we wondered if ecotin could help the bacteria to establish intracellular replication. While no differences in bacterial invasion to J774 murine macrophages cell line were found, ∆ecotin strain had a lower replication rate at 4 h post invasion than the wt strain. Altogether these results highlight the importance of ecotin as a virulence factor of Salmonella. In conclusion, Ecotin helps bacteria to survive against the host proteolytic activity allowing the bacteria to establish the gut colonization and the further development of the disease.