Subcytocidal Attack by Staphylococcal Alpha-Toxin Activates NF-κB and Induces Interleukin-8 Production

Abstract

Formation of transmembrane pores by staphylococcal alpha-toxin can provoke a spectrum of events depending on target cell species and toxin dose, and in certain cases, repair of the lesions has been observed. Here, we report that transcriptional processes are activated as a response of cells to low toxin doses. Exposure of monocytic (THP-1) or epithelial (ECV304) cells to 40 to 160 ng/ml alpha-toxin provoked a drop in cellular ATP level that was followed by secretion of substantial amounts of interleukin-8 (IL-8). Cells transfected with constructs comprising the proximal IL-8 promoter fused to luciferase or to green fluorescent protein cDNA exhibited enhanced reporter gene expression following toxin treatment. Electrophoretic mobility shift and immunofluorescence assays demonstrated that IL-8 secretion was preceded by activation of NF-B. Transfection experiments conducted with p65/p50 double-deficient cells showed that activation of the IL-8 promoter/reporter by toxin was absolutely dependent on NF-B. In contrast, this transcription factor was not required for lesion repair. Attack of cells by low doses of a pore-forming toxin can lead to transcriptional gene activation, which is followed by production of mediators that may contribute to the initiation and propagation of inflammatory lesions.More than 300 publications dealing with pore-forming bacterial toxins have been published during the past decade, the majority focusing on structural aspects and mechanisms of pore formation (3, 6, 7). Biological consequences other than cell lysis have been described, e.g., secretory responses (4, 5), production of lipid mediators (11), interleukin-1␤ (IL-1␤) maturation (27), and programmed cell death (12). Killing of human keratinocytes by alpha-toxin is due to enhanced permeability for monovalent ions (27). Certain cells can repair a limited number of lesions (24). These findings all indicate that cells attacked by pore-forming toxins are not inevitably and instantly paralyzed and that they retain the capacity to mount active responses to membrane damage. In this investigation, we questioned whether cell damage by alpha-toxin might result in the activation and exploitation of transcriptional mechanisms. NF-B transcription factors are involved in the response to many types of stress. Proteins encoded by NF-B-regulated genes are devoted to intercellular signaling, cell adhesion, and other defense-related functions (9, 10). NF-B has also been implicated in the regulation of apoptosis (1,8). Biochemical features of NF-B include constitutive expression in the cytosol, rapid activation, and translocation into the nucleus.We report that alpha-toxin causes rapid activation of NF-B, which leads to the expression of IL-8 in monocytic THP-1 cells and in ECV304 cells. Transcriptional activation was also shown in 3T3 fibroblasts, where it was found that recovery to sublethal toxin attack was not dependent on activation of this transcription factor. MATERIALS AND METHODSWild type alpha-toxin and the nonlytic toxin mutant H35R were prepar...

Enhancement of bacterial virulence by antibody neutralization of immune-activating toxins

“…In fact, it appears to be a common theme that crosses bacterial species, as both Gram-positive and Gram-negative bacteria produces toxins that are also recognized by the host and activate innate immunity. 28 33,[35][36][37][38][39] Overall, a delicate balance needs to be struck in order for the innate immune system to function optimally, allowing a bacterial infection to be cleared. Ideally, the immune system needs to be activated to a level that allows efficient removal and/or killing of pathogens.…”

Response of corneal epithelial cells toStaphylococcus aureus

“…63,64 This permeability has been shown to have important cellular consequences in fibroblasts, endothelial, and lymphocytes, such as ATP depletion and stimulation of mitogen activated protein kinase (MAPK) signaling pathways. 65,66 It was also found that signaling could be blocked by extracellular ion chelators and high molecular weight dextrans; thus, it appears that osmosensing may constitute another tier of pathogen recognition. 65,66 The response of human corneal epithelial cells to stimulation by model toxigenic or non-toxigenic S. aureus was also strikingly similar to that of human vaginal epithelial cells exposed to an Since TLR-based recognition of Gram positive bacteria is a main sensing mechanism of mammalian cells, 18 we tested whether induction of CCL20 expression by HCECs and their primary cell counterparts is dependent on TLR2 or NOD2 as might be predicted.…”

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“…a-toxin exposure has been shown to be a strong neutrophil attractant to the site of infection. 80,81 Bacterial genomic CpG-DNA represents a PAMP which can activate innate immune cells such as macrophages and DCs to secrete cytokines. 82 The observation that S. aureus DNA induces production of cytokines such as TNFa, IL-12 and IFg 83 and can elicit an inflammatory response in a cutaneous mouse model 84,85 raises questions about a possible involvement of TLR9 in S. aureus infections.…”