Study of toll-like receptor-dependent immune cell activation and death induced by a pore-forming toxin, Vibrio cholerae cytolysin

Abstract

Vibrio cholerae cytolysin (VCC) is a pore-forming toxin (PFT) secreted by the cholera pathogen, V. cholerae. Being a PFT, VCC forms pores in membrane lipid bilayer, and damages the cell membranes. VCC shows diverse pathophysiological effects that include enterotoxicity, hemolytic activity, and cytotoxicity. Accordingly, VCC is considered as a potent virulence factor of V. cholerae. Apart from its membrane-damaging PFT functionality, VCC can also act as a Pathogen-Associated Molecular Pattern (PAMP) and can activate the host immune cells. Moreover, damaged cell membrane fragments harboring the VCC oligomeric assembly, upon VCC-mediated cell lysis, can act as the potential Damage-Associated Molecular Patterns (DAMPs) to further modulate the host immune system. However, the precise role of VCC in the pathogenesis process of V. cholerae, particularly in terms of its ability to modulate the immune cell functions, remains under-explored. In this direction, the present work explores the crucial role of VCC as a PAMP/DAMP in modulating the activation and death responses in the immune cells. Our study reveals that VCC activates the immune cells, such as dendritic cells and macrophages, leading to the pro-inflammatory cytokine production. Toll-like receptors (TLRs) are the key initiators of an inflammatory response. Notably, while VCC is recognized by the TLR2/6 heterodimer in various innate immune cells, our study shows that VCC activates dendritic cells via recognition through a novel TLR1/4 heterodimer. We demonstrate that the differential expression of TLRs in different cell types is the major factor responsible for this differential VCC recognition. Our study further reveals that the TLR1/4/MyD88 signalling pathway plays crucial roles in the VCC-mediated inflammatory responses in vivo. Upon TLR recognition, VCC activates intracellular signalling pathways involving MAPKs such as JNK and p38, leading to the activation of transcription factors NF-κB and AP-1. Additionally, we find that the reactive oxygen species (ROS), induced by VCC, contribute to these pro-inflammatory responses. Finally, our study demonstrates that VCC induces mortality in mice via the TLR1/4/MyD88 pathway, and it serves as the key toxin responsible for cell death following biofilm formation by V. cholerae on immune cells. Altogether, our study shows that VCC engages a novel TLR heterodimer assembly to activate and kill the host innate immune cells.