A SALMONELLA TYPHIMURIUM PROTEASE INHIBITOR PROTECTS BACTERIA FROM NEUTROPHIL KILLING

SAPOSNIK, LUCAS; PASQUEVICH K; CORIA, LORENA; BRUNO L; SABBIONE FLORENCIA; TREVANI ANALIA; CASSATARO JULIANA

Cordoba

Congreso; XVII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL; 2022

Salmonella Typhimurium is a common pathogen, which causes acute diarrhea upon oral infection in humans. In the mouse model of gastroenteritis and after reaching the intestinal epithelium, this bacterium triggers an inflammatory response mainly sustained by polymorphonuclear leukocytes that translocate from the lamina propria to the gut lumen. This inflammation, far from killing the pathogen, creates a suitable niche for the bacteria to replicate and colonize the gut. In this environment, the neutrophil respiratory burst generates electron acceptors that can be used by Salmonella in its respiratory chain. In addition, the neutrophil elastase imparts a shift in the microbiota composition to further help the pathogen colonization. How Salmonella survives against neutrophil proteolytic activity is poorly understood. For this reason, we decided to study how the protease inhibitors present in the Salmonella genome could assist bacteria to survive the neutrophil response. The ecotin protein from Salmonella is a protease inhibitor, with homologues in many bacteria. We performed a 1:1 competitive assay between the wild type (WT) and the ecotin knock-out strain (∆ecotin) in the mouse model of gastroenteritis to study the bacterial loads in the cecum and spleen. We found that 72 h post infection the ∆ecotin strain was attenuated in the cecum of infected animals, while no difference was observed in the dissemination to the spleen. Then, we studied Salmonella-neutrophil interaction using purified human neutrophils. We studied the survival of Salmonella Typhimurium WT and ∆ecotin in presence of neutrophils with and without DPI to inhibit the respiratory burst and focus on the proteolytic activity. We found that after incubation the survival of ∆ecotin strain was lower when compared with the WT strain. As neutrophils can kill bacteria by phagocytosis or by releasing primary and azurophilic granules and NETs, we aimed to study these contributions individually to better understand the neutrophil-Salmonella interaction. We found no differences in the intracellular survival of ∆ecotin when compared to the WT strain. However, when facing bacteria with neutrophil granules, we found a decrease in survival for the ∆ecotin compared to the wild type strain. In the assays performed, a complementation in trans with a plasmid expressing the ecotin protein restored the phenotype observed to the level of the WT strain. In conclusion, these results suggest that ecotin may contribute to establish the infection in the inflamed gut of mice and defend the bacteria against luminal neutrophils.