lesser, c. | bioRxiv Science

bioRxiv2026

Mechanisms of mucosal immunity to oral Shigella infection in a physiological mouse model

Shigella flexneri causes bacillary dysentery, a diarrheal disease responsible for significant global morbidity and mortality. Despite extensive efforts, there is no licensed Shigella vaccine, and due to the lack of tractable and physiological models, mechanisms of adaptive immunity to Shigella are poorly understood. Here, we establish a mouse model that permits mechanistic dissection of adaptive immunity to a physiological oral challenge with Shigella. We find primary Shigella infection confers robust cross-serotype protection against secondary challenge, in a manner strictly dependent on the adaptive immune compartment. Shigella infection induces Shigella-specific CD4+ and CD8+ T cells, but only CD4+ T cells are required for protection. CD4+ T cells produce IFN{gamma} upon secondary challenge, and help B cells produce Shigella-specific IgA. Neither anti-Shigella antibodies nor IFN{gamma} are individually required for immunity to Shigella, but loss of both eliminates protective immunity. Collectively, our results demonstrate that CD4+ T cells orchestrate antibody and cytokine defense against shigellosis.

immunology↗

bioRxiv2022

GBP2 aggregates LPS and activates the caspase-4 inflammasome independent of the bacterial encapsulation factor GBP1

Sensing and killing of intracellular bacterial pathogens are important features of cell-autonomous immunity. The cytokine gamma-interferon (IFN{gamma}) enhances cell-autonomous immunity through upregulation of interferon stimulated genes such as guanylate binding proteins (GBPs). GBPs promote defense against Gram-negative cytosolic bacteria in part through the induction of an inflammatory cell death pathway called pyroptosis. To activate pyroptosis, GBPs facilitate caspase-4 sensing of the Gram-negative bacterial outer membrane component lipopolysaccharide (LPS). There are seven human GBP paralogs and it is unclear how each GBP contributes to LPS sensing and pyroptosis induction. GBP1 forms a multimeric microcapsule on the surface of cytosolic bacteria through direct interactions with LPS and recruits caspase-4 to bacteria, a process deemed essential for caspase-4 activation. In contrast to GBP1, closely related paralog GBP2 is unable to bind bacteria on its own but requires GBP1 for direct bacterial binding. Unexpectedly, we find that GBP2 overexpression can restore Gram-negative-induced pyroptosis in GBP1KO cells, without GBP2 binding to the bacterial surface. A mutant of GBP1 that lacks the triple arginine motif required for microcapsule formation also rescues pyroptosis in GBP1KO cells, showing that binding to bacteria is dispensable for GBPs to promote pyroptosis. Instead, we find that GBP2, like GBP1, directly binds and aggregates free LPS through protein polymerization. This provides a novel mechanistic framework for non-canonical inflammasome activation where GBP1 or GBP2 assemble cytosol-contaminating LPS into a protein-LPS interface for caspase-4 activation as part of a coordinated host response to Gram-negative bacterial infections. Significance StatementSensing Gram-negative bacterial lipopolysaccharide by human caspase-4 is critical for host defense to intracellular Gram-negative bacterial pathogens. Human guanylate binding proteins (GBPs) facilitate caspase-4 activation in response to Gram-negative infections by a poorly understood mechanism. The prevailing model suggests GBP1 binding to bacteria and consequential recruitment of caspase-4 to the bacterial surface are essential for triggering this host response. Here, we show GBP1 binding to bacteria is dispensable for caspase-4 activation and identify GBP2 as an additional lipopolysaccharide-binding protein that can functionally replace GBP1. We demonstrate that GBP1 and GBP2 share the ability to form lipopolysaccharide-protein complexes, which, we propose, allow caspase-4 activation. Our study provides a new mechanistic framework for cytosolic LPS sensing.

immunology↗