Global Functional Analyses of Cellular Responses to Pore-Forming Toxins

Abstract

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (∼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs.

Author Summary

The plasma membrane surrounds cells and protects their interior from the environment and from attack by disease-causing agents like bacteria and viruses. Bacteria that cause disease have discovered that an effective way to attack cells is to secrete proteins (pore-forming toxins) that breach, i.e., form holes in, the plasma membrane. How cells deal with and survive this kind of attack is poorly understood. Here, we report on the first large-scale study of the genes and mRNA transcripts that respond to pore-forming toxin attack in cells. We find that a remarkable portion, >0.5%, of the cell's genome protects it against pore-forming toxins. These data led us to look more closely at mitogen-activated protein kinase pathways as regulators of pore-forming toxin defenses. We find that half of the PFT-induced protective response is controlled by a single, conserved signaling pathway in cells, which controls a complex array of downstream targets and which protects against both large pore and small pore toxins. Our results indicate that defense against pore-forming toxins is a very ancient defense that utilizes a much more complex and extensive response in cells than previously demonstrated.