GBPs in inflammasome activation and host defense to infection

PROJECT SUMMARY/ABSTRACT Inflammasomes alert the mammalian immune system to the presence of infection and tissue damage. These cytosolic protein complexes detect danger signals or microbial products released by a wide variety of intracellular pathogens. In the case of bacterial pathogens, a number of prokaryotic signatures are recognized including the major cell-wall constituent of most Gram-negative species, lipopolysaccharide (LPS). Detection of LPS inside host cells activates a “non-canonical“ inflammasome pathway where caspase-4 (formerly caspase-11 in mice) act as upstream sensors to stimulate inflammasome complex assembly and processing of the pore-forming protein, Gasdermin D (Gsdmd), further downstream. Gsdmd pores release protective cytokines and contribute to a lytic form of cell death termed pyroptosis that may help eliminated infected host cells. How these sequential events are co-ordinated and the host factors involved remains a major question in the field of innate immunity and host defense. Here, we focus on members of a new 65-73kDa immune GTPase family termed Guanylate- Binding Proteins (GBPs) that control distinct steps in the non-canonical pathway. Our recent published and unpublished results suggest specific GBPs target cytosolic bacteria to help liberate LPS for caspase-4 detection whereas other GBP family members control Gsdmd trafficking to the plasma membrane further downstream and facilitate bacterial killing. Thus GBPs offer a unique opportunity to dissect how this process unfolds. In Aim 1, we will test the respective contributions of 4 human GBPs (GBPs1-4) and their closest mouse GBP orthologs (Gbps1-3) to caspase-4-dependent immunity against Gram-negative Salmonella typhimurium (Stm) infection in vitro and in vivo. CRISPR-Cas9 deleted human and mouse cells as well as newly-created Gbp1-/-, Gbp2-/-, Gbp3- /- and tissue-specific Gbp∆chr.3H1 mice will be infected with Stm variants designed to interfere with the recruitment of GBPs onto the bacterial surface, their responsiveness to LPS, or their ability to facilitate apolipoprotein L (APOL)-dependent bacteriolysis. Thereafter, we will dissect the molecular and structural mechanisms enlisted by these GBPs to confer their intracellular functions as part of Aim 2. Here gene-deficient macrophages and epithelia will be complemented with GBP mutants harboring distinct biochemical lesions to reveal how GBPs direct the inflammasome core machinery to LPS-positive bacteria for controlling downstream events such as Gsdmd trafficking to the plasma membrane or APOL-mediated bacterial killing. Native cryo-electron tomography studies will also examine the GBP defense complex on the bacterial outer membrane that serves as a platform for inflammasome assembly. Collectively, our proposal examines a new set of host factors that act at different stages within the non-canonical signaling cascade as part of a unique functional hierarchy, helping choreograph these events with major implications for the treatment of sepsis and Gram-negative bacterial infections.