Activation induced by pore-forming bacterial toxins

Pore-forming bacterial toxins are considered to be the principal virulence factors contributing to severe inflammatory diseases in humans and animals. The pore-forming toxins are released from Gram-negative bacteria as soluble monomeric proteins, which then assemble into oligomeric pores on their target cell membranes. These toxins induce inflammation through their unique pore-forming ability and concomitant cytolysis of their host cells. However, the actions of these toxins are concentration-dependent, that is, high concentrations of toxins cause cytolysis, whereas sub-lytic concentrations of toxin cause a cellular activation that generates various proinflammatory mediators. Most studies have focused on the mechanisms of pore formation by these toxins, and little effort has been given to exploring the mechanisms underlying their induction of cellular activation cascades.The nuclear factor (NF)-κB family of transcription factors is involved in cellular activation and inflammatory responses in various cell types. Several proinflammatory cytokines, such as tumour necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-8, are known to be regulated by NF-κB. NF-κB exists in the cytoplasm as an inactive heterodimer consisting of p50 and p65 subunits in complex with an inhibitory protein, inhibitor of κB (IκB); IκB binds NF-κB in the cytoplasm and prevents its translocation to the nucleus. NF-κB activation leads to phosphorylation, ubiquitination and degradation of IκB, resulting in nuclear translocation of NF-κB, which subsequently binds to specific promoter and/or enhancer sequences for expression of target genes.Dragneva et al. 1xSubcytocidal attack by staphylococcal alpha-toxin activates NF-κB and induces interleukin-8 production. Dragneva, Y. et al. Infect. Immun. 2001; 69: 2630–2635Crossref | PubMed | Scopus (45)See all References1 recently demonstrated that, at sub-lytic concentrations, staphylococcal alpha-toxin activates NF-κB translocation in human myelomonocytic (THP-1) leukemia cells and ECV304 cells. A mutant staphylococcal strain that produces a biologically inactive toxin did not cause any of these effects, further demonstrating that these effects were caused by native toxin. IL-8 production was measured as a functional consequence of NF-κB translocation, and it was found that the native, but not inactive, toxin induced the production of significant amounts of IL-8. Dragneva et al. compared native toxin-induced expression of IL-8 in NF-κB (p50/65) double knockout cells with induction in normal cells, and demonstrated that IL-8 expression was significantly lower in knockout cells. In a related study, Hsuan et al. 2xMannheimia haemolytica leukotoxin and endotoxin induced cytokine gene expression in bovine alveolar macrophages requires NF-κB activation and intracellular calcium elevation. Hsuan, S.L. et al. Microb. Pathog. 1999; 26: 263–273Crossref | PubMed | Scopus (40)See all References2 also demonstrated that, at sub-lytic concentrations, a pore-forming toxin of Mannheimia haemolytica induces translocation of NF-κB and expression of TNF-α, IL-1β and IL-8 in bovine alveolar macrophages. This study has also shown that elevation of intracellular calcium is a prerequisite for translocation of NF-κB and expression of cytokine genes. In this context, Uhlen et al. 3xAlpha-hemolysin of uropathogenic E. coli induces Ca2+ oscillations in renal epithelial cells. Uhlen, P. et al. Nature. 2000; 405: 694–697Crossref | PubMed | Scopus (143)See all References3 have shown that sub-lytic concentrations of Escherichia coli alpha-haemolysin cause intracellular calcium oscillations and subsequent IL-6 and IL-8 production in renal epithelial cells. Although this study did not show NF-κB translocation by the E. coli toxin, it is not unreasonable to assume that NF-κB is involved in IL-6 and IL-8 production in renal cells.Collectively, these studies support the hypothesis that the ability of sub-lytic concentrations of pore-forming toxins to induce NF-κB translocation and cytokine production, leading to inflammation, is probably crucial for their position as key virulence factors. Host cell activation induced by sub-lytic concentrations of these toxins might be equally as important an event as pore formation in the pathogenesis of inflammation and the eventual severe, uncontrolled inflammatory response, which generates proinflammatory mediators and subsequently damages tissue in vivo. However, construction of isogenic bacterial mutants that produce biologically active toxins able to induce cellular activation of host cells without causing transmembrane pores will be crucial if we are to understand the contribution of activation cascades to the inflammatory process.