Low Shear Modeled Microgravity Induces Unexpected Motility Phenotypes in Salmonella Typhimurium

Bacteria routinely exhibit unexpected phenotypic and molecular changes in response to spaceflight and spaceflight-analogue conditions, yet the mechanisms by which they sense and respond to these low fluid shear environments are not fully elucidated. We previously demonstrated that spaceflight and low shear modeled microgravity (LSMMG) altered motility and chemotaxis gene expression in Salmonella enterica serovar Typhimurium (S. Typhimurium), raising the possibility that flagella mediate responses of the pathogen to these environments. Herein, we investigated whether LSMMG culture alters S. Typhimurium motility and examined the role of flagella in regulating pathogenesis-associated stress and infection phenotypes. LSMMG enhanced the swimming motility of wild-type S. Typhimurium relative to 1xg controls; a trend which persisted even in the absence of the global stress response regulators Hfq and RpoS. This finding was unexpected, as {Delta}hfq mutants are typically defective for motility under conventional culture conditions. Motility was also observed in the flagella-deficient {Delta}flhDC mutant following LSMMG and 1xg culture, although the relative motility pattern differed relative to wild-type. Collectively, these results indicate that flagella contribute to LSMMG-enhanced motility, but are not strictly required under these conditions. Conditioned supernatant exchange demonstrated that LSMMG-induced motility changes are cell-intrinsic rather than mediated by extracellular factors. While flagella were dispensable for many pathogenesis-related phenotypes tested, their deletion selectively altered the magnitude of LSMMG-associated thermal stress and intracellular survival in human intestinal epithelial cells. Together, these findings demonstrate that motility and pathogenesis-related responses in S. Typhimurium are governed by multiple regulatory pathways that differentially respond to LSMMG and 1xg conditions. IMPORTANCESpaceflight and spaceflight-analogue conditions alter bacterial physiology in unexpected ways that are important for pathogenesis, yet the mechanisms by which bacteria sense and respond to low fluid shear environments remain incompletely understood. This study shows that low shear modeled microgravity (LSMMG) enhances Salmonella Typhimurium motility and produces unexpected motility phenotypes in mutants lacking Hfq or the flagellar master regulator FlhDC. These findings indicate that flagellar biosynthesis contributes to LSMMG-enhanced motility but is not strictly required for motility under these conditions. We also suggest that flagella influence the magnitude of selected stress and infection phenotypes rather than serving as an absolute requirement for LSMMG responsiveness. Together, these results highlight the complexity of bacterial mechanotransduction under simulated microgravity conditions and advances our understanding of how a foodborne pathogen adapts to physiological low fluid shear environments encountered both in space and during terrestrial infection of the intestinal tract.