Host defenses against bacterial pore-forming toxins

Abstract

Pore-forming toxins (PFTs), the most common bacterial toxins, contribute to infection by perforating host cell membranes. Excessive use and lack of new development of antibiotics are causing increasing numbers of drug-resistant bacteria, like methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis. A review of primary literature shows PFTs may form a viable target for new antibiotics, warranting their further investigation. This thesis describes three studies on host responses to PFTs, using Caenorhabditis elegans as a model for mammalian pathogenesis. The first part describes a large-scale study of genes involved in protection against PFTs. A genome-wide RNAi screen identifies 106 genes important for defense. Interactome analysis reveals a third of these to function in a single defense network, involving the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase (JNK). Microarray analyses show that JNK but not p38 is a master regulator of transcriptional PFT responses. The transcription factor AP-1 is found to function downstream of JNK in protecting against PFTs. AP-1’s protective effect extends to mammalian cells, making it the first known transcription factor broadly required for metazoan cells in PFT defense. The second study focuses on host cell membrane repair after PFT attack. Using fluorescent markers, it is found that PFTs trigger increased endocytosis. Loss of RAB-5 or RAB-11, key regulators of vesicle trafficking, results in PFT hypersensitivity and deficient PFT-induced endocytosis. Via diffusion of a fluorescent dye into cells through PFT pores, the presence of pores at the plasma membrane can be determined in vivo. A short PFT pulse initially perforates C. elegans intestinal cells, but after sufficient recovery time the pores are removed. Loss of RAB-5 or RAB-11 inhibits this membrane repair. Electron microscopy shows that PFTs cause intestinal cells to shed microvilli from the cell surface, which requires RAB-11. This work directly correlates vesicle trafficking and membrane repair, proves their in vivo relevance in PFT defense, and identifies the involvement of RAB-5 and RAB-11. The third study shows that neuronal pathways regulate PFT responses. PFTs are found to inhibit feeding in C. elegans. Animals lacking the neuronally expressed Go? homolog goa-1 show constitutive feeding on PFTs and PFT hypersensitivity. Serotonin (5-HT), a neurotransmitter that controls feeding, also causes constitutive feeding on PFT and hypersensitivity. 5-HT’s effects are evolutionary conserved, as it induces similar effects in Spodoptera frugiperda. Although analogous to loss of goa-1, genetic analyses suggest that 5-HT’s effects function through an independent mechanism. Loss of goa-1 causes hypersensitivity to bacterial infection due to increased neurosecretion, and mutation of unc-31 causes resistance through decreased neurosecretion. This correlation does not exist for PFT defense, as loss of unc-31 leads to PFT hypersensitivity, which is rescued by reconstituting unc-31 to the worm’s nervous system. Based on literature and further experimental evidence, goa-1 and unc-31 are concluded to function independently. This work indentifies the first molecular PFT-defense pathways that function outside the tissue that is under direct attack by the PFT, and sheds light on the genetics underlying feeding inhibition by PFTs in insects and nematodes.