STUDY OF Salmonella Typhimurium ECOTIN GENE IN INTERACTION OF BACTERIA WITH GUT PROTEASES AND INTRACELLULAR LIFESTYLE IN MACROPHAGES

SAPOSNIK LM; CORIA LM; PASQUEVICH KA; CASSATARO J

Congreso; CONGRESO CONJUNTO SAIB-SAMIGE 2021; 2021

Salmonella Typhimurium is a common pathogen associated to the development of acute diarrhea. The most usual way of infection is by eating contaminated food. There, Salmonella encounters the first line of defense in the lumen of our gastrointestinal tract (GI), where microorganisms, antigens and food are degraded in a nonspecific fashion by pH and gastric, pancreatic and biliary secretions. How protease inhibitors present in Salmonella’s genome might contribute to survival in the gut proteolytic environment, stablish colonization and develop diarrhea is poorly understood. Ecotin is a gene present in many bacteria species encoding a protein, which has been shown to inhibit a wide range of proteases. In this work, we studied the growth of Salmonella Typhimurium wild type and ecotin knock-out strain (∆ecotin) in presence of porcine pancreatin, we found that after incubation the replication of the ∆ecotin was attenuated when compared with the wild type strain. As pancreatin composition represents a mixture of proteases, we aimed to study them individually. We found that after incubation with porcine elastase the replication of ∆ecotin was attenuated. In both cases the complementation in trans with a plasmid encoding the ecotin gene restored the phenotype observed in the ∆ecotin to the wild type strain. Other important sources of proteases are the different cell types that Salmonella encounters while travelling the GI to finally establish the colonization, within these, macrophages are a preferential niche for the pathogen. Thus, we studied invasion and replication of the different strains in J774 murine macrophages. We found no differences in invasion but 4 h after the bacterial uptake, the replication of ∆ecotin was attenuated when compared to the wild type strain. This replication defect was also seen when doing a competitive 1:1 assay between ∆ecotin and the wild type strain in J774 murine macrophages. Taking all into account, these results indicate that ecotin may contribute to defending the bacteria against proteases in the GI tract and helping in the initial infection steps of macrophages.