MECHANISMS OF COLONIZATION RESISTANCE TO SHIGELLA FLEXNERI

Abstract

Shigella flexneri is an enteric bacterial pathogen responsible for shigellosis. Components of the commensal microbiota promote colonization resistance in mice and antibiotic treatment is required for susceptibility to S. flexneri infection. The mechanisms of resistance to colonization by S. flexneri have not been defined in the streptomycin-treated mouse model of infection. We hypothesize that a consortium of anaerobes cultured from healthy mice and serially passaged to select for streptomycin-resistant bacteria can confer protection against S. flexneri infection in streptomycin-treated mice. A close relative of Shigella, E. coli, uses nitrogen respiration to overcome colonization resistance in the streptomycin-treated mouse. We hypothesize that S. flexneri similarly uses nitrogen respiration to overcome colonization resistance and that a mutant for nitrate reductase activity is impaired in the ability to colonize streptomycin-treated mice. Intracellular carbon metabolism through the phosphotransacetylase-acetate kinase pathway has been demonstrated by S. flexneri during in vitro infection of cells in tissue culture. We hypothesize that this form of intracellular carbon metabolism also contributes to the ability of S. flexneri to colonize. Consortia of commensal bacteria were cultured from healthy mice under selective conditions and characterized by 16S rRNA sequencing. Proton Nuclear Magnetic Resonance Spectroscopy was used to demonstrate the production of a short-chain fatty acid by the cultured bacteria that has previously been implicated in counteracting Shigella-mediated virulence. The cultured consortia of bacteria did not confer protection in the murine model of S. flexneri infection, but the selective culturing method repeatedly enriched for specific genera of bacteria and yielded 16S rRNA profiles distinct from the gut of the healthy mouse. Nitrogen respiration and carbon metabolism pathways were investigated as potential mechanisms used by S. flexneri to promote colonization. The importance of the phosphotransacetylase-acetate kinase carbon metabolism pathway in intracellular proliferation of S. flexneri was confirmed in vitro, while nitrate reductase activity was dispensable for colonization of streptomycin-treated mice. By addressing the role of commensal bacteria, as well as nitrogen respiration and intracellular carbon metabolism, this work advances our understanding of the factors allowing colonization by S. flexneri and sets the stage for future study.