Inhalation of anthrax causes fatal bacteremia, indicating a meager host immune response. We previously showed that anthrax lethal toxin (LT) paralyzes neutrophils, a major component of innate immunity. Here, we have found that LT also inhibits actin-based motility of the intracellular pathogen Listeria monocytogenes. LT inhibition of actin assembly is mediated by blockade of Hsp27 phosphorylation, and can be reproduced by treating cells with the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580. Nonphosphorylated Hsp27 inhibits Listeria actin-based motility in cell extracts, and binds to and sequesters purified actin monomers. Phosphorylation of Hsp27 reverses these effects. RNA interference knockdown of Hsp27 blocks LT inhibition of Listeria actin-based motility. Rescue with wild-type Hsp27 accelerates Listeria speed in knockdown cells, whereas introduction of Hsp27 mutants incapable of phosphorylation or dephosphorylation causes slowing down. We propose that Hsp27 facilitates actin-based motility through a phosphorylation cycle that shuttles actin monomers to regions of new actin filament assembly. Our findings provide a previously unappreciated mechanism for LT virulence, and emphasize a central role for p38 MAP kinase-mediated phosphorylation of Hsp27 in actin-based motility and innate immunity.

Bacillus anthracis causes high-level bacteremia, strongly suggesting paralysis of the innate immune system. We have examined the effects of anthrax lethal toxin (LT) on human neutrophil chemotaxis, a process that requires actin filament assembly. Polymorphonuclear neutrophils (PMNs) treated with a sublethal concentration of LT (50 ng/mL) for 2 h demonstrated insignificant apoptosis or necrosis. However, this same concentration slowed human PMN formylmethionylleucylphenylalanine (FMLP)-stimulated chemokinesis by >60%, markedly reduced polar morphology, and rendered PMNs incapable of responding to a chemotactic gradient. These changes were accompanied by a >50% reduction in FMLP-induced actin filament assembly. One hour of exposure to LT failed to impair polarity or actin assembly, and the effects of LT were independent of mitogen-activated protein kinase kinase 1 inhibition. We conclude that 2 h of exposure to LT markedly impairs PMN actin assembly, and reductions in actin filament content are accompanied by a profound paralysis of PMN chemotaxis.

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...

Recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors triggers an innate immune response to colonizing or invading bacteria. Conversely, many bacteria have evolved mechanisms to dampen this response by downregulating the synthesis of such PAMPs. We have previously demonstrated that Pseudomonas aeruginosa growing in mucopurulent human respiratory mucus from cystic fibrosis patients represses the expression of its flagellin, a potent stimulant of the innate immune response. Here we demonstrate that this phenomenon occurs in response to the presence of neutrophil elastase in such mucus. Nonpurulent mucus from animals had no such repressive effect. Furthermore, lysed neutrophils from human blood reproduced the flagellin-repressive effect ex mucus and, significantly, had no effect on the viability of this organism. Neutrophil elastase, a component of the innate host defense system, has been described to be bactericidal for gram-negative bacteria and to degrade bacterial virulence factors. Thus, the resistance of P. aeruginosa to the bactericidal effect of neutrophil elastase, as well as this organism's ability to sense this enzyme's presence and downregulate the synthesis of a PAMP, may be the key factors in allowing P. aeruginosa to colonize the lungs. These findings demonstrate the dynamic nature of this bacterium's response to host defenses that ensures its success as a colonizer and also highlights the dual nature of defense molecules that confer advantages and disadvantages to both hosts and pathogens.Pseudomonas aeruginosa is an opportunistic pathogen of humans that infects immunocompromised patients, patients with cystic fibrosis (CF), and patients who are maintained on ventilators. CF is of particular interest in regard to P. aeruginosa because it is one of the few diseases where this organism persists in the lungs as a chronic colonizer before ultimately causing the death of such patients. Several plausible hypotheses have been proposed to explain the basis of the predilection of these patients for Pseudomonas infection, including the failure of defensin-mediated lung defense to clear this organism (32), increased adherence of bacteria to epithelial cells (1), and the failure of uptake of P. aeruginosa by airway cells due to defective CFTR-mediated uptake (11). A number of studies have shown that, in addition to these mechanisms that are believed to result from the underlying genetic defect, P. aeruginosa undergoes numerous changes during chronic colonization, which are believed to allow it to persist in the lungs despite a normal immune system. Chronically colonizing strains demonstrate numerous genetic and phenotypic changes, including alterations in the structure of their lipopolysaccharides (LPS) (9), conversion to a mucoid phenotype (7), and sometimes loss of their flagellum and pili (18). More recently, a longitudinal study has more precisely defined many of the genetic changes that may occur during prolonged colonization (23, 31).In addition to the genetic chan...