Response of <i>Staphylococcus aureus</i> physiology and Agr quorum sensing to low-shear modeled microgravity

<i>Staphylococcus aureus</i> is commonly isolated from astronauts returning from spaceflight. Previous analysis of omics data from <i>S. aureus</i> low Earth orbit cultures indicated significantly increased expression of the Agr quorum sensing system and its downstream targets in spaceflight samples compared to ground controls. In this current study, the rotary cell culture system (RCCS) was used to investigate the effect of low-shear modeled microgravity (LSMMG) on <i>S. aureus</i> physiology and Agr activity. When cultured in the same growth medium and temperature as the previous spaceflight experiment, <i>S. aureus</i> LSMMG cultures exhibited decreased <i>agr</i> expression and altered growth compared to normal gravity control cultures, which are typically oriented with oxygenation membrane on the bottom of the high aspect rotating vessel (HARV). When <i>S. aureus</i> was grown in an inverted gravity control orientation (oxygenation membrane on top of the HARV), reduced Agr activity was observed relative to both traditional control and LSMMG cultures, signifying that oxygen availability may affect the observed differences in Agr activity. Metabolite assays revealed increased lactate and decreased acetate excretion in both LSMMG and inverted control cultures. Secretomics analysis of LSMMG, control, and inverted control HARV culture supernatants corroborated these results, with inverted and LSMMG cultures exhibiting a decreased abundance of Agr-regulated virulence factors and an increased abundance of proteins expressed in low-oxygen conditions. Collectively, these studies suggest that the orientation of the HARV oxygenation membrane can affect <i>S. aureus</i> physiology and Agr quorum sensing in the RCCS, a variable that should be considered when interpreting data using this ground-based microgravity model.IMPORTANCE<i>S. aureus</i> is commonly isolated from astronauts returning from spaceflight and from surfaces within human-inhabited closed environments such as spacecraft. Astronaut health and immune function are significantly altered in spaceflight. Therefore, elucidating the effects of microgravity on <i>S. aureus</i> physiology is critical for assessing its pathogenic potential during long-term human space habitation. These results also highlight the necessity of eliminating potential confounding factors when comparing simulated microgravity model data with actual spaceflight experiments.