SUMMARY Gram-negative bacteria generate an outer-membrane (OM) barrier to their environment. The PhoPQ-gene regulators coordinate OM-barrier remodeling mechanisms for bacterial resistance to innate-immune killing strategies. A screen for genes necessary for the PhoPQ-regulated OM-barrier of Salmonella Typhimurium has identified an inner-membrane (IM) protein, PbgA that binds glycerophospholipids known as cardiolipins and relies upon its periplasmic domain for their PhoPQ-regulated delivery to the OM. Purified PbgA forms tetramers in vitro and binds acidic cardiolipin head-groups using periplasmic arginines near the IM that are necessary for PbgA to function in vivo. The periplasmic-globular region of PbgA interacts with the OM in a PhoPQ-dependent manner suggesting PbgA forms a complex that may bridge the envelope to allow cardiolipin movement. Deleting portions of the periplasmic domain severely attenuates Salmonellae in their pathogenesis supporting that regulated delivery of cardiolipins to the OM is necessary for their survival within host tissues that activate PhoPQ.

Bacterial pathogens have evolved sophisticated mechanisms to sense and adapt to redox stress in nature and within the host. However, deciphering the redox environment encountered by intracellular pathogens in the mammalian cytosol is challenging, and that environment remains poorly understood. In this study, we assessed the contributions of the two redox-responsive, Spx-family transcriptional regulators to the virulence of Listeria monocytogenes, a Gram-positive facultative intracellular pathogen. Spx-family proteins are highly conserved in Firmicutes, and the L. monocytogenes genome contains two paralogues, spxA1 and spxA2. Here, we demonstrate that spxA1, but not spxA2, is required for the oxidative stress response and pathogenesis. SpxA1 function appeared to be conserved with the Bacillus subtilis homologue, and resistance to oxidative stress required the canonical CXXC redox-sensing motif. Remarkably, spxA1 was essential for aerobic growth, demonstrating that L. monocytogenes SpxA1 likely regulates a distinct set of genes. Although the ΔspxA1 mutant did not grow in the presence of oxygen in the laboratory, it was able to replicate in macrophages and colonize the spleens, but not the livers, of infected mice. These data suggest that the redox state of bacteria during infection differs significantly from that of bacteria growing in vitro. Further, the host cell cytosol may resemble an anaerobic environment, with tissue-specific variations in redox stress and oxygen concentration.

An imbalance of cellular oxidants and reductants causes redox stress, which must be rapidly detected to restore homeostasis. In bacteria, the Firmicutes encode conserved Spx‐family transcriptional regulators that modulate transcription in response to redox stress. SpxA1 is an Spx‐family orthologue in the intracellular pathogen Listeria monocytogenes that is essential for aerobic growth and pathogenesis. Here, we investigated the role of SpxA1 in growth and virulence by identifying genes regulated by SpxA1 in broth and during macrophage infection. We found SpxA1‐activated genes encoding heme biosynthesis enzymes and catalase ( kat ) were required for L. monocytogenes aerobic growth in rich medium. An Spx‐recognition motif previously defined in Bacillus subtilis was identified in the promoters of SpxA1‐activated genes and proved necessary for the proper activation of two genes, indicating this regulation by SpxA1 is likely direct. Together, these findings elucidated the mechanism of spxA1 essentiality in vitro and demonstrated that SpxA1 is required for basal expression of scavenging enzymes to combat redox stress generated in the presence of oxygen.