IFN-γ—Driven Activation Restricts Systemic Infection by a Minimal Effector Strain of Salmonella Typhimurium and Is Overcome by T3SS Effectors 2258032

Abstract

Abstract Introduction Salmonella enterica serovar Typhimurium (STm) employs a type III secretion system (T3SS) to translocate effector proteins that enable intracellular survival and replication. Through targeted mutagenesis and phenotypically guided genome reassembly, we identified a minimal set of SPI-2—encoded effectors sufficient for intracellular replication in vitro (STmθ). Although this effector module is conserved across S. enterica serovars, these genes alone are insufficient to promote systemic virulence in vivo without the addition of an accessory T3SS effector locus, suggesting the presence of host immune barriers that restrict STm θ dissemination. Methods To define immune barriers limiting STm θ in vivo, we used intraperitoneal (IP) infection models in mice deficient in MyD88 or interferon-γ (IFN-γ) signaling. We developed an STm-specific anti-LPS antibody to track bacterial association with immune subsets by CyTOF. To mechanistically dissect this restriction, we modeled infection in bone marrow—derived macrophages (BMDMs) with or without IFN-γ pre-treatment. Results Survival and bacterial burden analyses revealed that IFN-γ is the dominant host pathway restricting systemic dissemination of STm θ, while MyD88 signaling plays a route-dependent role, limiting infection of T3SS-deleted strain (effectorless) after intragastric (IG) but not IP challenge. High-dimensional CyTOF profiling demonstrated IFN-γ—dependent expansion and occupancy of immune subsets with STm LPS that restrict STm θ. IFN-γ pre-treatment reduced STm θ replication in BMDMs but had little effect on strains expressing additional T3SS effectors, indicating resistance to macrophage-intrinsic IFN-γ—mediated restriction Conclusion These findings identify IFN-γ as a central barrier to STm θ dissemination and reveal route-specific roles for MyD88 signaling in limiting bacterial pathogenesis. This work provides a framework to define how additional accessory T3SS effector subvert innate immune defences to enable systemic infection. Funding Source NIH, The Welch foundation, Burroughs Wellcome Fund Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)