Anthrolysin O and Other Gram-positive Cytolysins Are Toll-like Receptor 4 Agonists

Abstract

Exposure of bone marrow–derived macrophages (BMDMs) to low concentrations of Bacillus anthracis lethal toxin (LT), whose catalytic subunit is lethal factor (LF), results in induction of a robust apoptotic response dependent on activation of Toll-like receptor (TLR)4. A similar TLR4-dependent apoptotic response is observed when BMDMs are infected with live B. anthracis (Sterne strain). However, TLR4 is considered to be a specific signaling receptor for lipopolysaccharide (LPS), a typical product of gram-negative bacteria, whereas B. anthracis is gram-positive. To understand how B. anthracis can activate TLR4, we analyzed its culture supernatants and found them to contain a potent TLR4-stimulating activity that can also induce apoptosis in macrophages in which the antiapoptotic p38 MAP kinase (whose activation is prevented by LF) was inhibited. Purification of this activity suggested it consists of anthrolysin O (ALO), a member of the cholesterol-dependent cytolysin (CDC) family. We show that recombinant ALO can activate TLR4 in a manner independent of LPS contamination and, together with LT, can induce macrophage apoptosis. We also provide genetic evidence that ALO is required for induction of macrophage apoptosis in response to infection with live B. anthracis and that other CDC family members share the ability to activate TLR4.

Group A Streptococcus Activates Type I Interferon Production and MyD88-dependent Signaling without Involvement of TLR2, TLR4, and TLR9

“…The IFN production did not require the presence of the GAS-encoded cytolysins SLO and SLS. This finding is surprising, since during infections with other Gram-positive bacteria, either the cytolysin itself or cytolysin-mediated cytoplasmic escape of bacteria from phagocytic vesicles was implicated in triggering IFN production (12,(23)(24)(25). However, the requirement for IRF3 in the IFN production suggested that other aspects of the GAS-induced IFN production resembled the well established TBK1/IRF3 signaling pathway.…”

“…In this regard, GAS-derived streptolysins are different from several other cytolysins that have been reported to induce IFN-␤ synthesis through activation of TLR4 (25). Another proposed mechanism of IFN-␤ induction by Gram-positive bacteria (e.g.…”

“…Cytolysins are thought to enable cytoplasmic escape of phagocytosed bacteria. In addition, cytolysins were also shown to directly stimulate IFN production (25). The GAS genome contains genes for two cytolysins.…”

“…Other TLRs frequently engaged by bacteria are TLR4 and TLR9. TLR4 is the receptor for lipopolysaccharide of Gramnegative bacteria, but it has been shown to recognize also cell wall components of mycobacteria and several cytolysins of Gram-positive bacteria (25,43,44). TLR9, a CpG DNA receptor localized in endosomal and lysosomal membranes of macrophages and, most prominently, dendritic cells, can bind DNA from both Gram-positive and Gram-negative species (35,45).…”

Bacterial Virulence Factors: Secreted for Survival

“…Furthermore, in combination with lethal factor, anthrolysin O is known to induce apoptosis in intoxicated cells (Fig. 2c) [67]. The structure, function and physiological relevance of anthrolysin O has been well elucidated during the last decade, however there are no studies investigating the specificity of various drug molecules to inhibit the anthrolysin O, till now.…”