Staphylococcus aureus ATP Synthase Promotes Biofilm Persistence by Influencing Innate Immunity

Abstract

<i>Staphylococcus aureus</i> is a major cause of prosthetic joint infection (PJI), which is characterized by biofilm formation. <i>S. aureus</i> biofilm skews the host immune response toward an anti-inflammatory profile by the increased recruitment of myeloid-derived suppressor cells (MDSCs) that attenuate macrophage proinflammatory activity, leading to chronic infection. A screen of the Nebraska Transposon Mutant Library identified several hits in the ATP synthase operon that elicited a heightened inflammatory response in macrophages and MDSCs, including <i>atpA</i>, which encodes the alpha subunit of ATP synthase. An <i>atpA</i> transposon mutant (Δ<i>atpA</i>) had altered growth kinetics under both planktonic and biofilm conditions, along with a diffuse biofilm architecture that was permissive for leukocyte infiltration, as observed by confocal laser scanning microscopy. Coculture of MDSCs and macrophages with Δ<i>atpA</i> biofilm elicited significant increases in the proinflammatory cytokines interleukin 12p70 (IL-12p70), tumor necrosis factor alpha (TNF-α), and IL-6. This was attributed to increased leukocyte survival resulting from less toxin and protease production by Δ<i>atpA</i> biofilm as determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The enhanced inflammatory response elicited by Δ<i>atpA</i> biofilm was cell lysis-dependent since it was negated by polyanethole sodium sulfanate treatment or deletion of the major autolysin, Atl. In a mouse model of PJI, Δ<i>atpA</i>-infected mice had decreased MDSCs concomitant with increased monocyte/macrophage infiltrates and proinflammatory cytokine production, which resulted in biofilm clearance. These studies identify <i>S. aureus</i> ATP synthase as an important factor in influencing the immune response during biofilm-associated infection and bacterial persistence.<b>IMPORTANCE</b> Medical device-associated biofilm infections are a therapeutic challenge based on their antibiotic tolerance and ability to evade immune-mediated clearance. The virulence determinants responsible for bacterial biofilm to induce a maladaptive immune response remain largely unknown. This study identified a critical role for <i>S. aureus</i> ATP synthase in influencing the host immune response to biofilm infection. An <i>S. aureus</i> ATP synthase alpha subunit mutant (Δ<i>atpA</i>) elicited heightened proinflammatory cytokine production by leukocytes <i>in vitro</i> and <i>in vivo</i>, which coincided with improved biofilm clearance in a mouse model of prosthetic joint infection. The ability of <i>S. aureus</i> Δ<i>atpA</i> to augment host proinflammatory responses was cell lysis-dependent, as inhibition of bacterial lysis by polyanethole sodium sulfanate or a Δ<i>atpA</i>Δ<i>atl</i> biofilm did not elicit heightened cytokine production. These studies reveal a critical role for AtpA in shaping the host immune response to <i>S. aureus</i> biofilm.