Inhalation anthrax results in high-grade bacteremia and is accompanied by a delay in the rise of the peripheral polymorphonuclear neutrophil (PMN) count and a paucity of PMNs in the infected pleural fluid and mediastinum. Edema toxin (ET) is one of the major Bacillus anthracis virulence factors and consists of the adenylate cyclase edema factor (EF) and protective antigen (PA). Relatively low concentrations of ET (100 to 500 ng/ml of PA and EF) significantly impair human PMN chemokinesis, chemotaxis, and ability to polarize. These changes are accompanied by a reduction in chemoattractant-stimulated PMN actin assembly. ET also causes a significant decrease in Listeria monocytogenes intracellular actin-based motility within HeLa cells. These defects in actin assembly are accompanied by a >50-fold increase in intracellular cyclic AMP and a >4-fold increase in the phosphorylation of protein kinase A. We have previously shown that anthrax lethal toxin ( Inhalation anthrax can lead to sepsis and death within days if not diagnosed early and treated effectively (21). Epidemiological analyses of the anthrax bioterrorist attacks in 2001 indicated a mean duration of 4.5 days between exposure and symptom onset in the six inhalation anthrax cases for whom the exposure dates could be determined. Analyses of the clinical findings from 10 of the 11 inhalation anthrax cases revealed normal or minimally elevated peripheral polymorphonuclear neutrophil (PMN) counts at the time of hospital admission, despite high-level Bacillus anthracis bacteremia (22). Furthermore, heavily infected pleural fluid demonstrated a paucity of white blood cells. In the fatal cases, mediastinal infection was associated with marked edema and hemorrhage but minimal infiltration by acute inflammatory cells (16). Similarly, experimental inhalation anthrax in monkeys was associated with edema and hemorrhage of the mediastinum and pulmonary interstitium, with absent or modest infiltration by neutrophils (37). These findings suggest impaired delivery of neutrophils to the sites of infection during the early stages of systemic B. anthracis infection.B. anthracis produces three exotoxins, protective antigen (PA), edema factor (EF) and lethal factor (LF), that account for many of the clinical manifestations of this deadly pathogen. PA binds to the widely distributed host cell receptors and then self-associates into heptamers and ushers LF and EF into the cytoplasm of cells (4). The anthrax toxins have been termed AB toxins, PA combined with LF being called lethal toxin (LT), and PA combined with EF termed edema toxin (ET). LF is a Zn 2ϩ -dependent metalloprotease that cleaves mitogenactivated protein kinase kinases (12). EF is a calcium calmodulin-dependent adenylate cyclase, an enzyme that converts ATP to cyclic AMP (cAMP) and pyrophosphate (17) and increases intracellular cAMP levels (26).Neutrophils constitute the first line of defense against bacterial infections. These phagocytic cells are able to quickly crawl, or chemotax, to the site of infection, and def...

BackgroundAnthrax lethal toxin (LT), produced by the Gram-positive bacterium Bacillus anthracis, is a highly effective zinc dependent metalloprotease that cleaves the N-terminus of mitogen-activated protein kinase kinases (MAPKK or MEKs) and is known to play a role in impairing the host immune system during an inhalation anthrax infection. Here, we present the transcriptional responses of LT treated human monocytes in order to further elucidate the mechanisms of LT inhibition on the host immune system.ResultsWestern Blot analysis demonstrated cleavage of endogenous MEK1 and MEK3 when human monocytes were treated with 500 ng/mL LT for four hours, proving their susceptibility to anthrax lethal toxin. Furthermore, staining with annexin V and propidium iodide revealed that LT treatment did not induce human peripheral monocyte apoptosis or necrosis. Using Affymetrix Human Genome U133 Plus 2.0 Arrays, we identified over 820 probe sets differentially regulated after LT treatment at the p <0.001 significance level, interrupting the normal transduction of over 60 known pathways. As expected, the MAPKK signaling pathway was most drastically affected by LT, but numerous genes outside the well-recognized pathways were also influenced by LT including the IL-18 signaling pathway, Toll-like receptor pathway and the IFN alpha signaling pathway. Multiple genes involved in actin regulation, signal transduction, transcriptional regulation and cytokine signaling were identified after treatment with anthrax LT.ConclusionWe conclude LT directly targets human peripheral monocytes and causes multiple aberrant gene responses that would be expected to be associated with defects in human monocyte’s normal signaling transduction pathways and function. This study provides further insights into the mechanisms associated with the host immune system collapse during an anthrax infection, and suggests that anthrax LT may have additional downstream targets outside the well-known MAPK pathway.

Hemorrhage is a prominent clinical manifestation of systemic anthrax. Therefore, we have examined the effects of anthrax lethal and edema toxins on human platelets. We find that anthrax lethal toxin fails to cleave its target, mitogen-activated protein kinase 1, and anthrax edema toxin fails to increase intracellular cyclic adenosine monophosphate. Surface expression of toxin receptors tumor endothelial marker 8 and capillary morphogenesis gene 2, as well as coreceptor low density lipoprotein receptor-related protein 6 (LRP6), are markedly reduced, preventing toxin binding to platelets. Our studies suggest that the hemorrhagic clinical manifestations of systemic anthrax are unlikely to be caused by the direct binding and entry of anthrax toxins into human platelets.