Saturday, November 23, 2024
11/23/2024
Human cells including epithelial cells are equipped with cell-autonomous host defense programs providing protection against intracellular pathogens. These host protective programs are controlled in a temporal and spatial manner and respond to extracellular cues including pro-inflammatory cytokines released by professional immune cells. Arguably, the most potent inducer of antibacterial cell-autonomous host defense is lymphocyte- derived interferon-gamma (IFN). IFN-receptor signaling in human epithelial cells induces the expression of hundreds of IFN-stimulated genes (ISGs) encoding antimicrobial proteins. While a few ISGs have been studied in much detail, our understanding of most ISGs and their functions in host defense is limited. We recently discovered a critical role for IFN-inducible guanylate binding proteins (GBPs) in cell-autonomous immunity to Gram-negative bacterial pathogens invading the host cell cytosol. Human GBP1 is a cytosolically localized member this GBP family and able to directly detect a central building block of the Gram-negative bacterial outer membrane, i.e. lipopolysaccharide (LPS). Our previous work showed that GBP1, through these LPS interactions, forms a multimeric microcapsule or coatomer surrounding Gram-negatives invading the host cell cytosol. However, GBP1 binding to the bacterial surface on its own is neither bactericidal nor bacteriostatic, implicating other GBP1-dependent co-factors required for sterilizing cell-autonomous immunity. Aim1 of our proposal will explore a novel GBP1-dependent pathway that results in bacterial ubiquitylation, a well-established ‘eat-me’ signal that marks cytosolic microbial invaders for destruction. Our studies are multidisciplinary and include biochemical approaches levering a novel in vitro GBP1-bacteria binding assay that we established. Using this assay, we will directly assess how GBP1 promotes the recruitment of IFN-inducible host ubiquitin E3 ligases to the surface of the Gram-negative cytosol-invading bacterial pathogen Shigella flexneri. S. flexneri is a human- adapted enteric pathogen that causes dysentery and diarrheal deaths, especially in children in low- and middle- income countries. Our preliminary data demonstrate that wildtype S. flexneri is resistant to GBP1-dependent and -independent host-driven bacterial ubiquitylation. Therefore, in our second aim we will identify and characterize a network of virulence factors used by S. flexneri to escape IFN-induced ubiquitylation and associated host defenses. To define the physiological relevance of these antagonistic relationships between antibacterial ISGs and counter-acting bacterial virulence mechanisms employed by S. flexneri, we will exploit a novel mouse model of intestinal S. flexneri infections. Using ISG-deficient mouse lines established in our lab and, reciprocally, infecting with S. flexneri mutants defective for virulence effectors targeting the same ISGs, we will determine the physiological importance of GBP- and other ISG-driven host defenses and their evasion by S. flexneri for bacterial pathogenesis and enteric disease.
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