How GBPs attack cytosol-invading bacteria

• Applicant: Dr. Felix Scharte
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 538254340

Caspases-4/5/11 (CASP-4/5/11) are cytosolic sensors for bacterial lipopolysaccharide (LPS), which, upon activation, cause pyroptotic cell death through cleavage of gasdermin-D. How caspases gain access to LPS embedded in the membranes of cytosol-invading bacteria remains unclear. Only recently, guanylate-binding proteins (GBPs) have been identified to assemble on the surface of Gram-negative bacteria into polyvalent signalling platforms required for activation of CASP 4. GBPs are interferon-inducible GTPases that function against bacterial, protozoan and viral pathogens. GBP recruitment to Gram-negative bacteria is dependent on prenylation that helps to anchor the protein to the bacterial outer membrane. GBP coat formation and activation of CASP-4 are hierarchically controlled: GBP1 senses the invader, is essential to initiate platform assembly and recruits all other GBPs; GBP2 and GBP4 control CASP-4 recruitment while GBP3 governs CASP-4 activation. Although GBP1 can bind LPS directly, GBP recruitment to bacteria does not represent a simple diffusion-limited binding event but rather proceeds through the formation of intermediate GBP-assemblies (unpublished data), suggesting the existence of further proteins controlling the process and possibly even a ‘loading machinery’. Many open questions regarding this pathway remain: ‘What is the composition of GBP-assemblies?’, ‘What cofactors are involved in GBP-assembly formation?’, ‘How is CASP-4 delivered to and activated at the bacterial GBP coat?’ and ‘Which post-translational mechanisms regulate GBP functions?’. The aims of my project are therefore to investigate how the GBP-coat forms on cytosol-invading bacteria and to identify the machinery required for its formation and the activation of CASP-4. In part 1 I will determine the molecular determinants of GBP coat formation, focussing on candidates possibly involved in the processing and regulation of GBPs. This includes RCE1 and ICMT, responsible for C-terminal processing and priming of GTPases to associate with membranes, and RNF213, the E3 ligase responsible for LPS ubiquitylation. In part 2 I will use CRISPR technology to identify novel genes required for GBP coat formation and bacteria-induced pyroptosis. With my project I will elucidate the process of GBP recruitment to cytosolic bacteria and CASP 4-dependent induction of pyroptosis which will provide substantial new knowledge about GBP-related cell-autonomous immunity and unravel possible targets for regulation of inflammation and sepsis.

• DFG Programme: WBP Fellowship
• International Connection: United Kingdom
• Host: Dr. Felix Randow