Detecting extracellular enteropathogens by intracellular sensors

Abstract

Enteropathogenic E. coli (EPEC) and Enterohaemmorhagic E. coli (EHEC) are extracellular Gram-negative enteric pathogens that cause diarrhoeal disease in humans. They cause actin polymerisation downstream of intimin-induced clustering of Type-3 Secretion System effector Tir (Translocated intimin receptor), leading to the formation of pedestals. Guanylate Binding Protein 1 (GBP1) and caspase-4 are cytosolic lipopolysaccharide (LPS) sensors involved in detecting and responding to cytosolic Gram-negative pathogen infections. However, the role of GBP1 and caspase-4 in epithelial cells upon extracellular pathogen infection remains unknown. In this study, we investigate the mechanisms involved in detecting EPEC and EHEC infections in intestinal epithelial cells by the intracellular sensors GBP1 and caspase- 4. We show that epithelial cells recognise extracellular pathogen infection in a GBP1 and caspase-4-dependent manner. This mechanism is gasdermin-D-dependent and NLRP3-caspase-1-independent. We observe recruitment of caspase-4 to actin pedestals at sites of infection in a GBP1-dependent manner and determine that GBP1 is essential in intestinal epithelial cells for full pyroptotic response to extracellular enteropathogens. We demonstrate that the recruitment of cytosolic sensors GBP1 and caspase-4 to sites of extracellular pathogen infections in epithelial cells is LPS-independent and requires only actin polymerisation triggered by Tir-clustering at the cell membrane. Our study broadens the scope of GBP1 as a cytosolic immune sensor and provides evidence for its role in LPS-independent pathogen sensing mechanisms. The observation that GBP1 is involved in the detection of extracellular pathogens is of consequence to future studies that may investigate the involvement of cytosolic sensors in immune responses to other pathogens.