The largest superfamily of bacterial virulence factors is pore-forming toxins (PFTs). PFTs are secreted by both pathogenic and non-pathogenic bacteria. PFTs sometimes kill or induce pro-pathogen signaling in mammalian cells, all primarily through plasma membrane perforation, though the parameters that determine these outcomes are unclear. Membrane binding, calcium influx, pore size, and membrane repair are factors that influence PFT cytotoxicity. To test the contribution of membrane binding to cytotoxicity and repair, we compared the closely related, similarly-sized PFTs Perfringolysin O (PFO) from Clostridium perfringens and Streptolysin O (SLO) from Streptococcus pyogenes. Cell death kinetics for PFO and SLO were different because PFO increased in cytotoxicity over time. We introduced known L3 loop mutations that swap binding affinity between toxins and measured hemolytic activity, nucleated cell death kinetics and membrane repair using viability assays, and live cell imaging. Altered hemolytic activity was directly proportional to toxin binding affinity. In contrast, L3 loop alterations reduced nucleated cell death, and they had limited effects on cytotoxicity kinetics and membrane repair. This suggests other toxin structural features, like oligomerization, drives these parameters. Overall, these findings suggest that repair mechanisms and toxin oligomerization add constraints beyond membrane binding on toxin evolution and activity against nucleated cells.

Repair of plasma membranes damaged by bacterial pore-forming toxins, such as streptolysin O or perfringolysin O, during septic cardiomyopathy or necrotizing soft tissue infections is mediated by several protein families. However, the activation of these proteins downstream of ion influx is poorly understood. Here, we demonstrate that following membrane perforation by bacterial cholesterol-dependent cytolysins, calcium influx activates mixed lineage kinase 3 independently of protein kinase C or ceramide generation. Mixed lineage kinase 3 uncouples mitogen-activated kinase kinase (MEK) and extracellular-regulated kinase (ERK) signaling. MEK signals via an ERK-independent pathway to promote rapid annexin A2 membrane recruitment and enhance microvesicle shedding. This pathway accounted for 70% of all calcium ion-dependent repair responses to streptolysin O and perfringolysin O, but only 50% of repair to intermedilysin. We conclude that mixed lineage kinase signaling via MEK coordinates microvesicle shedding, which is critical for cellular survival against cholesterol-dependent cytolysins.

Cholesterol-dependent cytolysins (CDCs) are key virulence factors involved in many lethal bacterial infections, including pneumonia, necrotizing soft tissue infections, bacterial meningitis, and miscarriage. Host responses to these diseases involve myeloid cells, especially macrophages. Macrophages use several systems to detect and respond to cholesterol-dependent cytolysins, including membrane repair, mitogen-activated protein (MAP) kinase signaling, phagocytosis, cytokine production, and activation of the adaptive immune system. However, CDCs also promote immune evasion by silencing and/or destroying myeloid cells. While there are many common themes between the various CDCs, each CDC also possesses specific features to optimally benefit the pathogen producing it. This review highlights host responses to CDC pathogenesis with a focus on macrophages. Due to their robust plasticity, macrophages play key roles in the outcome of bacterial infections. Understanding the unique features and differences within the common theme of CDCs bolsters new tools for research and therapy.