A Caenorhabditis elegans nck-1 and filamentous actin-regulating protein pathway mediates a key cellular defense against bacterial pore-forming proteins

Abstract

Pore-forming proteins (PFPs) comprise the largest single class of bacterial protein virulence factors and are expressed by many human and animal bacterial pathogens. Cells that are attacked by these virulence factors activate epithelial intrinsic cellular defenses (or INCEDs) to prevent the attendant cellular damage, cellular dysfunction, osmotic lysis, and organismal death. Several conserved PFP INCEDs have been identified using the nematode Caenorhabditis elegans and the nematicidal PFP Cry5B, including mitogen-activated protein kinase (MAPK) signaling pathways. Here we demonstrate that the gene nck-1, which has homologs from Drosophila to humans and links cell signaling with localized F-actin polymerization, is required for INCED against small-pore PFPs in C. elegans. Reduction/loss of nck-1 function results in C. elegans hypersensitivity to PFP attack, a hallmark of a gene required for INCEDs against PFPs. This requirement for nck-1-mediated INCED functions cell-autonomously in the intestine and is specific to PFPs but not other tested stresses. Genetic interaction experiments indicate that nck-1-mediated INCED against PFP attack is independent of the major MAPK PFP INCED pathways. Proteomics and cell biological and genetic studies further indicate that nck-1 functions with F-actin cytoskeleton modifying genes like arp2/3, erm-1, and dbn-1 and that nck-1/arp2/3 promote pore repair at the membrane surface and protect against PFP attack independent of p38 MAPK. Consistent with these findings, PFP attack causes significant changes in the amount of actin cytoskeletal proteins and in total amounts of F-actin in the target tissue, the intestine. nck-1 mutant animals appear to have lower F-actin levels than wild-type C. elegans. Studies on nck-1 and other F-actin regulating proteins have uncovered a new and important role of this pathway and the actin cytoskeleton in PFP INCED and protecting an intestinal epithelium in vivo against PFP attack. Author SummaryThe mechanism of action for a significant number of bacterial protein toxins is the formation of pores in the membrane of target cells. Host cells contain programmed defenses against such attacks. Here we use the model system of the roundworm Caenorhabditis elegans and crystal proteins produced by Bacillus thuringiensis to demonstrate a new defense pathway mediated by the activity of the NCK-1 protein. Consistent with its known function in mammalian cells, the NCK-1-mediated defense involves various actin-interacting proteins and affects the kinetics of pore repair. This pathway is novel in its independence from MAPK signaling. Furthermore, the NCK-1 activity is found to be generally needed for defense against multiple pore-forming proteins and yet is specific to defense against such proteins but not against other environmental stressors.