The Salmonella phage shock protein system is required for defense against host antimicrobial peptides

Abstract Macrophages are professional phagocytes that play a major role in engulfing and eliminating invading pathogens. Some intracellular pathogens, such as Salmonella enterica serovar Typhimurium, exploit macrophages as niches for their replication, which requires precise and dynamic modulation of bacterial gene expression in order to resist the hostile intracellular environment. Here, we present a comprehensive analysis of the global transcriptome of S. Typhimurium across four stages of infection of primary macrophages. Our results revealed a profound change in early-stage gene expression dominated by pathways linked to metabolic processes required for Salmonella adaptation to the proinflammatory conditions of the macrophage. We identified the phage shock protein (Psp) system to be highly expressed in intracellular S. Typhimurium, with sustained high expression over the course of infection. We determined that the Psp system is regulated by the virulence-associated two-component system SsrA-SsrB, which coordinates its expression with critical bacterial functions required for immune evasion and intracellular survival. Functional assays demonstrated that the Psp system mediates resistance to host antimicrobial peptides, including cathelicidin-related antimicrobial peptide (CRAMP), which we demonstrate supports bacterial persistence in host tissues and survival within macrophages. Our findings establish the Psp system as a new and critical adaptive mechanism for evading host immune defenses and highlight the utility of temporal transcriptomics in unraveling the genetic strategies employed by S. Typhimurium during macrophage infection. Author summary Salmonella enterica is an important global pathogen that infects a wide range of mammalian hosts, requiring it to survive in diverse and hostile environments. A key aspect of Salmonella pathogenesis is its ability to reside within host immune cells like macrophages, where it must rapidly adapt to intracellular conditions. To do so, the bacteria must ensure correct spatiotemporal expression of virulence genes to maximise fitness in each environment. To better understand how Salmonella modulates its gene expression during infection of host cells, we defined its transcriptome at four distinct stages of primary murine macrophage infection. Our findings reveal that the first stage of early infection is dominated by changes in gene expression of metabolic circuits. Furthermore, we identified the phage-shock protein (Psp) system as highly expressed within intracellular Salmonella throughout the course of infection as a result of regulatory evolution that coordinates its expression with virulence genes. We showed that this system is required for bacterial survival within macrophages and host tissues by mediating resistance to host cationic antimicrobial peptides. These findings highlight the dynamic nature of Salmonella’s transcriptional response during macrophage infection and uncover a previously unknown function of the Psp system as an adaptive mechanism for evading host immune defenses.