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Neutrophils are innate immune response cells designed to kill invading microorganisms. One of the mechanisms neutrophils use to kill bacteria is generation of damaging reactive oxygen species (ROS) via the respiratory burst. However, during enteric salmonellosis, neutrophil-derived ROS actually facilitates Salmonella expansion and survival in the gut. This seeming paradox led us to hypothesize that Salmonella may possess mechanisms to influence the neutrophil respiratory burst. In this work, we used an in vitro Salmonella-neutrophil co-culture model to examine the impact of enteric infection relevant virulence factors on the respiratory burst of human neutrophils. We report that neutrophils primed with granulocyte-macrophage colony stimulating factor and suspended in serum containing complement produce a robust respiratory burst when stimulated with viable STm. The magnitude of the respiratory burst increases when STm are grown under conditions to induce the expression of the type-3 secretion system-1. STm mutants lacking the type-3 secretion system-1 induce less neutrophil ROS than the virulent WT. In addition, we demonstrate that flagellar motility is a significant agonist of the neutrophil respiratory burst. Together our data demonstrate that both the type-3 secretion system-1 and flagellar motility, which are established virulence factors in enteric salmonellosis, also appear to directly influence the magnitude of the neutrophil respiratory burst in response to STm in vitro.

Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking its retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate, but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules.