The Bacillus anthracis toxin anthrolysin O modulates human macrophage and neutrophil function

Bacillus anthracis (BA), a large Gram-positive bacillus capable of producing heat resistant endospores, is the cause of the disease anthrax. Because of the high mortalitly associated with the disease and its threat as an agent of bioterror, elucidation of new virulence factors produced by the bacterium has been a focus in the anthrax field. Here we describe the characterization of Anthrolysin O (ALO), a new BA virulence factor and its lethal and sub-lethal effects on human neutrophils and macrophages. A member of the cholesterol-dependent cytolysin (CDC) family of toxins, recombinant ALO (rALO) or native ALO secreted by viable BA, is lethal to human primary polymorphonuclear leukocytes (PMNs), monocytes, monocyte-derived macrophages (MDMs), lymphocytes, THP-1 monocytic human cell line and ME-180, Detroit 562, and A549 epithelial cells. ALO cytotoxicity is dose- and time-dependent and susceptibility to ALO-mediated lysis differs between cell types. Differential cell sensitivity to ALO-mediated lysis is independent of the amount of membrane cholesterol in different cell types and Toll-like receptor (TLR4) expression. However, ALO-mediated cytotoxicity is dependent on the presence of extracellular calcium. In addition, the viability of monocytes and hMDMs was assayed in the presence of vegetative Sterne strains 7702 (ALO+), UT231 (ALO-), and a complemented strain expressing ALO, UT231(pUTE544), and was dependent upon the expression of ALO. Cytotoxicity of rALO is seen as low as 0.070 nM in the absence of serum. All direct cytotoxic activity is inhibited by the addition of cholesterol or serum concentration as low as 10%, which is characteristic of CDCs. Although ALO causes hemolysis and cytotoxicity, at sublethal concentrations ALO modulates human neutrophil and macrophage function. We describe that full-length ALO and a truncated non-lytic ALO devoid of domain 4 ([delta]D4 ALO) both inhibit human PMN and RAW 264.7 macrophage chemotaxis towards IL-8 and fMLP (formyl-Met-Leu-Phe). For the first time we show that a CDC, ALO, inhibits chemotaxis towards fMLP by preventing F-actin polymerization. Inhibition of p38 MAPK with SKF86002 reveals that ALO-induced inhibition of actin polymerization is dependent upon ALO's ability to induce phosphorylation of p38 MAPK. ALO stimulates human neutrophil degranulation of myeloperoxidase (MPO) and elastase by inducing a calcium flux, which requires stimulation of PKC. [delta]D4 ALO does not cause release of MPO. Treating human neutrophils with ALO 30 min prior to stimulation with PMA (phorbol myristate acetate) results in priming of the oxidative burst. ALO alone does not directly induce a neutrophil oxidative burst. The results of the present work suggest that ALO at sub-lytic, non-lethal concentrations has the ability to alter human PMN and macrophage function through pore-independent and pore-dependent mechanisms. ALO produces a continuum of lethal and non-lethal effects depending on the toxin's specific concentration. Non-lytic toxoids, including those lacking domain 4 or tryptophan mutants in this region, will be useful tools in future studies. These toxoids can be used to delineate whether results observed in cells are the result of non-lytic micropore formation, which induces calcium fluxes and/or stress responses, versus those caused by non-pore forming mechanisms, likely including stimulation of cells through TLR4, as ALO stimulates mouse bone-marrow derived macrophage TLR4. Stimulation of human macrophages and neutrophils by rALO shows that rALO stimulates TLR4 differently than LPS, including faster NFkB activation and decreased cytokine production, likely due to its ability, unlike LPS, to form micropores. Future studies will focus on stimulation of human TLR4 and resulting effects on phagocyte function as well as signaling pathways affected by calcium flux. CDC stimulation of cells should be looked at as multifaceted, resulting in unique signal transduction cascades induced by multiple signals. Taken together, our data support the idea that ALO, a multi-functional toxin, may contribute to the pathogenesis of BA by altering neutrophil and macrophage antibacterial activities by several previously unidentified mechanisms.

Topic: Bacillus and Francisella bacterial research
Subfield: Molecular Biology
Field: Biochemistry, Genetics and Molecular Biology
Domain: Life Sciences