Mechanisms of NK Cell-Macrophage Bacillus anthracis Crosstalk: A Balance between Stimulation by Spores and Differential Disruption by Toxins

NK cells are important immune effectors for preventing microbial invasion and dissemination, through natural cytotoxicity and cytokine secretion. Bacillus anthracis spores can efficiently drive IFN-γ production by NK cells. The present study provides insights into the mechanisms of cytokine and cellular signaling that underlie the process of NK-cell activation by B. anthracis and the bacterial strategies to subvert and evade this response. Infection with non-toxigenic encapsulated B. anthracis induced recruitment of NK cells and macrophages into the mouse draining lymph node. Production of edema (ET) or lethal (LT) toxin during infection impaired this cellular recruitment. NK cell depletion led to accelerated systemic bacterial dissemination. IFN-γ production by NK cells in response to B. anthracis spores was: i) contact-dependent through RAE-1-NKG2D interaction with macrophages; ii) IL-12, IL-18, and IL-15-dependent, where IL-12 played a key role and regulated both NK cell and macrophage activation; and iii) required IL-18 for only an initial short time window. B. anthracis toxins subverted both NK cell essential functions. ET and LT disrupted IFN-γ production through different mechanisms. LT acted both on macrophages and NK cells, whereas ET mainly affected macrophages and did not alter NK cell capacity of IFN-γ secretion. In contrast, ET and LT inhibited the natural cytotoxicity function of NK cells, both in vitro and in vivo. The subverting action of ET thus led to dissociation in NK cell function and blocked natural cytotoxicity without affecting IFN-γ secretion. The high efficiency of this process stresses the impact that this toxin may exert in anthrax pathogenesis, and highlights a potential usefulness for controlling excessive cytotoxic responses in immunopathological diseases. Our findings therefore exemplify the delicate balance between bacterial stimulation and evasion strategies. This highlights the potential implication of the crosstalk between host innate defences and B. anthracis in initial anthrax control mechanisms.     

The Bacillus anthracis Protein MprF Is Required for Synthesis of Lysylphosphatidylglycerols and for Resistance to Cationic Antimicrobial Peptides

During inhalational anthrax, Bacillus anthracis survives and replicates in alveolar macrophages, followed by rapid invasion into the host's bloodstream, where it multiplies to cause heavy bacteremia. B. anthracis must therefore defend itself from host immune functions encountered during both the intracellular and the extracellular stages of anthrax infection. In both of these niches, cationic antimicrobial peptides are an essential component of the host's innate immune response that targets B. anthracis. However, the genetic determinants of B. anthracis contributing to resistance to these peptides are largely unknown. Here we generated Tn917 transposon mutants in the ΔANR strain (pXO1⁻ pXO2⁻) of B. anthracis and screened them for altered protamine susceptibility. A protamine-sensitive mutant identified carried the transposon inserted in the BA1486 gene encoding a putative membrane protein homologous to MprF known in several gram-positive pathogens. A mutant strain with the BAS1375 gene (the orthologue of BA1486) deleted in the Sterne 34F2 strain (pXO1⁺ pXO2⁻) of B. anthracis exhibited hypersusceptibility not only to protamine but also to α-helical cathelicidin LL-37 and β-sheet defensin human neutrophil peptide 1 compared to the wild-type Sterne strain. Analysis of membrane lipids using isotopic labeling demonstrated that the BAS1375 deletion mutant is unable to synthesize lysinylated phosphatidylglycerols, and this defect is rescued by genetic complementation. Further, we determined the structures of these lysylphosphatidylglycerols by using various mass spectrometric analyses. These results demonstrate that in B. anthracis a functional MprF is required for the biosynthesis of lysylphosphatidylglycerols, which is critical for resistance to cationic antimicrobial peptides.     

Sensor domains encoded in Bacillus anthracis virulence plasmids prevent sporulation by hijacking a sporulation sensor histidine kinase

Anthrax toxin and capsule, determinants for successful infection by Bacillus anthracis, are encoded on the virulence plasmids pXO1 and pXO2, respectively. Each of these plasmids also encodes proteins that are highly homologous to the signal sensor domain of a chromosomally encoded major sporulation sensor histidine kinase (BA2291) in this organism. B. anthracis Sterne overexpressing the plasmid pXO2-61-encoded signal sensor domain exhibited a significant decrease in sporulation that was suppressed by the deletion of the BA2291 gene. Expression of the sensor domains from the pXO1-118 and pXO2-61 genes in Bacillus subtilis strains carrying the B. anthracis sporulation sensor kinase BA2291 gene resulted in BA2291-dependent inhibition of sporulation. These results indicate that sporulation sensor kinase BA2291 is converted from an activator to an inhibitor of sporulation in its native host by the virulence plasmid-encoded signal sensor domains. We speculate that activation of these signal sensor domains contributes to the initiation of B. anthracis sporulation in the bloodstream of its infected host, a salient characteristic in the virulence of this organism, and provides an additional role for the virulence plasmids in anthrax pathogenesis.     

Germination and Amplification of Anthrax Spores by Soil-Dwelling Amoebas

While anthrax is typically associated with bioterrorism, in many parts of the world the anthrax bacillus (Bacillus anthracis) is endemic in soils, where it causes sporadic disease in livestock. These soils are typically rich in organic matter and calcium that promote survival of resilient B. anthracis spores. Outbreaks of anthrax tend to occur in warm weather following rains that are believed to concentrate spores in low-lying areas where runoff collects. It has been concluded that elevated spore concentrations are not the result of vegetative growth as B. anthracis competes poorly against indigenous bacteria. Here, we test an alternative hypothesis in which amoebas, common in moist soils and pools of standing water, serve as amplifiers of B. anthracis spores by enabling germination and intracellular multiplication. Under simulated environmental conditions, we show that B. anthracis germinates and multiplies within Acanthamoeba castellanii. The growth kinetics of a fully virulent B. anthracis Ames strain (containing both the pX01 and pX02 virulence plasmids) and vaccine strain Sterne (containing only pX01) inoculated as spores in coculture with A. castellanii showed a nearly 50-fold increase in spore numbers after 72 h. In contrast, the plasmidless strain 9131 showed little growth, demonstrating that plasmid pX01 is essential for growth within A. castellanii. Electron and time-lapse fluorescence microscopy revealed that spores germinate within amoebal phagosomes, vegetative bacilli undergo multiplication, and, following demise of the amoebas, bacilli sporulate in the extracellular milieu. This analysis supports our hypothesis that amoebas contribute to the persistence and amplification of B. anthracis in natural environments.     

Bacillus Anthracis Endospores Regulate Ornithine Decarboxylase and Inducible Nitric Oxide Synthase Through ERK1/2 and p38 Mitogen-Activated Protein Kinases

Interactions between Bacillus anthracis (B. anthracis) and host cells are of particular interest given the implications of anthrax as a biological weapon. Inhaled B. anthracis endospores encounter alveolar macrophages as the first line of defense in the innate immune response. Yet, the consequences of this interaction remain unclear. We have demonstrated that B. anthracis uses arginase, inherent in the endospores, to reduce the ability of macrophages to produce nitric oxide (^?NO) from inducible nitric oxide synthase (NOS2) by competing for l-arginine, producing l-ornithine at the expense of ^?NO. In the current study, we used genetically engineered B. anthracis endospores to evaluate the contribution of germination and the lethal toxin (LT) in mediating signaling pathways responsible for the induction of NOS2 and ornithine decarboxylase (ODC), which is the rate-limiting enzyme in the conversion of l-ornithine into polyamines. We found that induction of NOS2 and ODC expression in macrophages exposed to B. anthracis occurs through the activation of p38 and ERK1/2 MAP kinases, respectively. Optimal induction of NOS2 was observed following exposure to germination-competent endospores, whereas ODC induction occurred irrespective of the endospores’ germination capabilities and was more prominent in macrophages exposed to endospores lacking LT. Our findings suggest that activation of kinase signaling cascades that determine macrophage defense responses against B. anthracis infection occurs through distinct mechanisms.     

Convergent evolution of diverse Bacillus anthracis outbreak strains toward altered surface oligosaccharides that modulate anthrax pathogenesis

Bacillus anthracis, a spore-forming gram-positive bacterium, causes anthrax. The external surface of the exosporium is coated with glycosylated proteins. The sugar additions are capped with the unique monosaccharide anthrose. The West African Group (WAG) B. anthracis have mutations rendering them anthrose deficient. Through genome sequencing, we identified 2 different large chromosomal deletions within the anthrose biosynthetic operon of B. anthracis strains from Chile and Poland. In silico analysis identified an anthrose-deficient strain in the anthrax outbreak among European heroin users. Anthrose-deficient strains are no longer restricted to West Africa so the role of anthrose in physiology and pathogenesis was investigated in B. anthracis Sterne. Loss of anthrose delayed spore germination and enhanced sporulation. Spores without anthrose were phagocytized at higher rates than spores with anthrose, indicating that anthrose may serve an antiphagocytic function on the spore surface. The anthrose mutant had half the LD₅₀ and decreased time to death (TTD) of wild type and complement B. anthracis Sterne in the A/J mouse model. Following infection, anthrose mutant bacteria were more abundant in the spleen, indicating enhanced dissemination of Sterne anthrose mutant. At low sample sizes in the A/J mouse model, the mortality of ΔantC-infected mice challenged by intranasal or subcutaneous routes was 20% greater than wild type. Competitive index (CI) studies indicated that spores without anthrose disseminated to organs more extensively than a complemented mutant. Death process modeling using mouse mortality dynamics suggested that larger sample sizes would lead to significantly higher deaths in anthrose-negative infected animals. The model was tested by infecting Galleria mellonella with spores and confirmed the anthrose mutant was significantly more lethal. Vaccination studies in the A/J mouse model showed that the human vaccine protected against high-dose challenges of the nonencapsulated Sterne-based anthrose mutant. This work begins to identify the physiologic and pathogenic consequences of convergent anthrose mutations in B. anthracis.

A study of the spontaneous loss of the spore coat monosaccharide anthrose suggests that convergent evolution in several anthrax strains towards increased pathogenicity could exacerbate global human and animal anthrax disease.     

The role of anthrolysin O in gut epithelial barrier disruption during Bacillus anthracis infection

Gastrointestinal (GI) anthrax, caused by the bacterial infection of Bacillus anthracis, posts a significant bioterrorism threat by its relatively high mortality rate in humans. Different from inhalational anthrax by the route of infection, accumulating evidence indicates the bypass of vegetative bacteria across GI epithelium is required to initiate GI anthrax. Previously, we reported that purified anthrolysin O (ALO), instead of tripartite anthrax edema and lethal toxins, is capable of disrupting gut epithelial tight junctions and barrier function in cultured cells. Here, we show that ALO can disrupt intestinal tissue barrier function in an ex vivo mouse model. To explore the effects of ALO in a cell culture model of B. anthracis infection, we showed that anthrax bacteria can effectively reduce the monolayer integrity of human Caco-2 brush-border expressor (C2BBE) cells based on the reduced transepithelial resistance and the increased leakage of fluorescent dye. This disruption is likely caused by tight junction dysfunction observed by the reorganization of the tight junction protein occludin. Consequently, we observe significant passage of vegetative anthrax bacteria across C2BBE cells. This barrier disruption and bacterial crossover requires ALO since ALO-deficient B. anthracis strains fail to induce monolayer dysfunction and allow the passage of anthrax bacteria. Together these findings point to a pivotal role for ALO within the establishment of GI anthrax infection and the initial bypass of the epithelial barrier.     

A New Murine Model for Gastrointestinal Anthrax Infection

The scientific community has been restricted by the lack of a practical and informative animal model of gastrointestinal infection with vegetative Bacillus anthracis. We herein report the development of a murine model of gastrointestinal anthrax infection by gavage of vegetative Sterne strain of Bacillus anthracis into the complement-deficient A/J mouse strain. Mice infected in this manner developed lethal infections in a dose-dependent manner and died 30 h-5 d following gavage. Histological findings were consistent with penetration and growth of the bacilli within the intestinal villi, with subsequent dissemination into major organs including the spleen, liver, kidney and lung. Blood cultures confirmed anthrax bacteremia in all moribund animals, with approximately 1/3 showing co-infection with commensal enteric organisms. However, no evidence of immune activation was observed during infection. Time-course experiments revealed early compromise of the intestinal epithelium, characterized by villus blunting and ulceration in the ileum and jejunum. A decrease in body temperature was most predictive of near-term lethality. Antibiotic treatment of infected animals 24 h following high-dose bacterial gavage protected all animals, demonstrating the utility of this animal model in evaluating potential therapeutics.     

Human Transferrin Confers Serum Resistance against Bacillus anthracis

The innate immune system in humans consists of both cellular and humoral components that collaborate to eradicate invading bacteria from the body. Here, we discover that the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, does not grow in human serum. Fractionation of serum by gel filtration chromatography led to the identification of human transferrin as the inhibiting factor. Purified transferrin blocks growth of both the fully virulent encapsulated B. anthracis Ames and the non-encapsulated Sterne strain. Growth inhibition was also observed in serum of wild-type mice but not of hypotransferrinemic mice that only have ∼1% circulating transferrin levels. We were able to definitely assign the bacteriostatic activity of transferrin to its iron-binding function: neither iron-saturated transferrin nor a recombinant transferrin mutant unable to bind iron could inhibit growth of B. anthracis. Additional iron could restore bacterial growth in human serum. The observation that other important Gram-positive pathogens are not inhibited by transferrin suggests they have evolved effective mechanisms to circumvent serum iron deprivation. These findings provide a better understanding of human host defense mechanisms against anthrax and provide a mechanistic basis for the antimicrobial activity of human transferrin.     

Interactions between Bacillus anthracis and Plants May Promote Anthrax Transmission

Environmental reservoirs are essential in the maintenance and transmission of anthrax but are poorly characterized. The anthrax agent, Bacillus anthracis was long considered an obligate pathogen that is dormant and passively transmitted in the environment. However, a growing number of laboratory studies indicate that, like some of its close relatives, B. anthracis has some activity outside of its vertebrate hosts. Here we show in the field that B. anthracis has significant interactions with a grass that could promote anthrax spore transmission to grazing hosts. Using a local, virulent strain of B. anthracis, we performed a field experiment in an enclosure within a grassland savanna. We found that B. anthracis increased the rate of establishment of a native grass (Enneapogon desvauxii) by 50% and that grass seeds exposed to blood reached heights that were 45% taller than controls. Further we detected significant effects of E. desvauxii, B. anthracis, and their interaction on soil bacterial taxa richness and community composition. We did not find any evidence for multiplication or increased longevity of B. anthracis in bulk soil associated with grass compared to controls. Instead interactions between B. anthracis and plants may result in increased host grazing and subsequently increased transmission to hosts.     

Monoclonal antibody against the poly-γ-d-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge

Background

The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT.

Methods

To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K_d values of 0.8 μM and 2.6 μM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed.

Results

Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen.

General significance

Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.     

Bacillus anthracis Peptidoglycan Stimulates an Inflammatory Response in Monocytes through the p38 Mitogen-Activated Protein Kinase Pathway

We hypothesized that the peptidoglycan component of B. anthracis may play a critical role in morbidity and mortality associated with inhalation anthrax. To explore this issue, we purified the peptidoglycan component of the bacterial cell wall and studied the response of human peripheral blood cells. The purified B. anthracis peptidoglycan was free of non-covalently bound protein but contained a complex set of amino acids probably arising from the stem peptide. The peptidoglycan contained a polysaccharide that was removed by mild acid treatment, and the biological activity remained with the peptidoglycan and not the polysaccharide. The biological activity of the peptidoglycan was sensitive to lysozyme but not other hydrolytic enzymes, showing that the activity resides in the peptidoglycan component and not bacterial DNA, RNA or protein. B. anthracis peptidoglycan stimulated monocytes to produce primarily TNFα; neutrophils and lymphocytes did not respond. Peptidoglycan stimulated monocyte p38 mitogen-activated protein kinase and p38 activity was required for TNFα production by the cells. We conclude that peptidoglycan in B. anthracis is biologically active, that it stimulates a proinflammatory response in monocytes, and uses the p38 kinase signal transduction pathway to do so. Given the high bacterial burden in pulmonary anthrax, these findings suggest that the inflammatory events associated with peptidoglycan may play an important role in anthrax pathogenesis.     

Characterization of AmiBA2446, a Novel Bacteriolytic Enzyme Active against Bacillus Species

There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis, the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls. In this work, we performed in silico analysis of the genome of Bacillus anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446. This enzyme exists as a homodimer, as determined by size exclusion studies. It possesses N-acetylmuramoyl-l-alanine amidase activity, as determined from liquid chromatography-mass spectrometry (LC-MS) analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 was an autolysin of bacterial origin. We characterized the effects of enzyme concentration and phase of bacterial growth on bactericidal activity and observed close to a 5-log reduction in the viability of cells of Bacillus cereus 4342, a surrogate for B. anthracis. We further tested the bactericidal activity of AmiBA2446 against various Bacillus species and demonstrated significant activity against B. anthracis and B. cereus strains. We also demonstrated activity against B. anthracis spores after pretreatment with germinants. AmiBA2446 enzyme was also stable in solution, retaining its activity after 4 months of storage at room temperature.     

Penetration of the Blood-Brain Barrier by Bacillus anthracis Requires the pXO1-Encoded BslA Protein

Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis. Human infection occurs after the ingestion, inhalation, or cutaneous inoculation of B. anthracis spores. The subsequent progression of the disease is largely mediated by two native virulence plasmids, pXO1 and pXO2, and is characterized by septicemia, toxemia, and meningitis. In order to produce meningitis, blood-borne bacteria must interact with and breach the blood-brain barrier (BBB) that is composed of a specialized layer of brain microvascular endothelial cells (BMEC). We have recently shown that B. anthracis Sterne is capable of penetrating the BBB in vitro and in vivo, establishing the classic signs of meningitis; however, the molecular mechanisms underlying the central nervous system (CNS) tropism are not known. Here, we show that attachment to and invasion of human BMEC by B. anthracis Sterne is mediated by the pXO1 plasmid and an encoded envelope factor, BslA. The results of studies using complementation analysis, recombinant BslA protein, and heterologous expression demonstrate that BslA is both necessary and sufficient to promote adherence to brain endothelium. Furthermore, mice injected with the BslA-deficient strain exhibited a significant decrease in the frequency of brain infection compared to mice injected with the parental strain. In addition, BslA contributed to BBB breakdown by disrupting tight junction protein ZO-1. Our results identify the pXO1-encoded BslA adhesin as a critical mediator of CNS entry and offer new insights into the pathogenesis of anthrax meningitis.Bacillus anthracis, the etiologic agent of anthrax, is a gram-positive spore-forming bacterium that is commonly found in soil (29). The bacterium can infect animals and humans by ingestion, inhalation, or cutaneous inoculation of B. anthracis spores (8). Spores are taken up by resident macrophages that migrate to the lymph nodes (15). Here, the spores germinate into vegetative bacteria, multiply, and then disseminate throughout the host, causing septicemia and toxemia (8). Systemic disease can be complicated by the onset of a fulminant and rapidly fatal hemorrhagic meningitis and meningoencephalitis (27). Anthrax meningitis is associated with a high mortality rate despite intensive antibiotic therapy (24). Biopsy studies after an outbreak of inhalational anthrax and experimental studies of inhalational infection in rhesus monkeys demonstrated the presence of bacilli in the central nervous system (CNS) and pathologies consistent with suppurative and hemorrhagic meningitis in the majority of cases (1, 12). The intentional release of B. anthracis spores (19) during the 2001 bioterrorism event resulted in a case of meningitis (19), necessitating a need for a better understanding of the pathogenesis of anthrax meningitis and CNS infection.To cause meningitis, blood-borne bacteria must interact with and breach the blood-brain barrier (BBB). The majority of the BBB is anatomically represented by the cerebral microvascular endothelium; brain microvascular endothelial cells (BMEC) are joined by tight junctions and display a paucity of pinocytosis, thereby effectively limiting the passage of substances and maintaining the CNS microenvironment (4, 5). Despite its highly restrictive nature, certain bacterial pathogens are still able to penetrate the BBB and gain entry into the CNS. The presence of bacilli in the brains of patients (1, 24) and in experimental models of anthrax infection (42, 44) suggests that vegetative B. anthracis cells are able to cross the BBB to initiate meningeal inflammation and the classic pathology associated with meningitis.B. anthracis harbors two large virulence plasmids, pXO1 and pXO2 (8), which are required for full virulence, as strains lacking these plasmids are attenuated in animal models of infection (29). B. anthracis Sterne (pXO1⁺ pXO2⁻) has been utilized as a vaccine strain (41) but is still widely used in both in vitro and in vivo studies of anthrax infection since it causes lethal disease in mouse models of infection (46). Despite the crucial roles of pXO1 and pXO2 in anthrax disease pathogenesis, very few plasmid-encoded factors have been characterized. The best described are the antiphagocytic polyglutamyl capsule, encoded by biosynthetic enzymes on pXO2, and the anthrax toxin complex comprised of protective antigen, lethal factor (LF), and edema factor (EF), encoded by pXO1 (8, 29). Sequence analysis of the pXO1 plasmid revealed that the majority of plasmid-encoded factors, ∼70%, were of unknown function (31). More recently, in silico analysis identified novel pXO1-encoded proteins with immunogenic potential and relevance for pathogenesis. These included factors with putative adherent and invasive properties (2). Interestingly, two of the immunoreactive proteins were predicted surface layer (S-layer) proteins (2), one of which, B. anthracis S-layer protein A (BslA, pXO1-90), has recently been described and shown to mediate adherence of the vegetative form to host cells (20).Using in vitro and in vivo model systems, we have recently shown that B. anthracis Sterne adheres to and invades brain endothelium (44). This interaction was partially dependent on the pXO1-encoded anthrax toxins; however, the molecular mechanisms that contribute to B. anthracis penetration of the BBB are currently unknown. In this study, we investigate the role of pXO1 in B. anthracis Sterne''s interaction with brain endothelium and identify the encoded BslA adhesin as a critical mediator for BBB attachment and penetration during the pathogenesis of anthrax meningitis.     

Anthrax toxin receptor 2 mediates Bacillus anthracis killing of macrophages following spore challenge

Initiation of inhalation anthrax is believed to involve phagocytosis of Bacillus anthracis spores by alveolar macrophages, followed by spore germination within the phagolysosome. In order to establish a systemic infection, it is predicted that bacilli then escape from the macrophage and replicate extracellularly. Mechanisms utilized by B. anthracis to escape from the macrophage are not well characterized, but a role for anthrax toxin has been proposed. Here we report the isolation of an anthrax toxin-resistant cell line (R3D) following chemical mutagenesis of toxin-sensitive RAW 264.7 murine macrophage cells. Both R3D and RAW 264.7 cells phagocytize spores of a B. anthracis Sterne strain. However, RAW 264.7 cells are killed following spore challenge, whereas R3D cells survive. Resistance to toxin and spore challenge correlates with loss of expression of anthrax toxin receptor 2 (ANTXR2/CMG-2). When R3D cells are complemented with cDNA encoding either murine ANTXR2 or human anthrax toxin receptor 1 (ANTXR1/TEM-8), toxin and spore challenge susceptibility are restored, indicating that over-expression of either ANTXR can confer susceptibility to anthrax spore challenge. Taken together, these results indicate that anthrax toxin expression by the germinated spore enables B. anthracis killing of the macrophage from within.     

Structure of the Bacillus anthracis Sortase A Enzyme Bound to Its Sorting Signal: A FLEXIBLE AMINO-TERMINAL APPENDAGE MODULATES SUBSTRATE ACCESS*

The endospore forming bacterium Bacillus anthracis causes lethal anthrax disease in humans and animals. The ability of this pathogen to replicate within macrophages is dependent upon the display of bacterial surface proteins attached to the cell wall by the B. anthracis Sortase A (^BaSrtA) enzyme. Previously, we discovered that the class A ^BaSrtA sortase contains a unique N-terminal appendage that wraps around the body of the protein to contact the active site of the enzyme. To gain insight into its function, we determined the NMR structure of ^BaSrtA bound to a LPXTG sorting signal analog. The structure, combined with dynamics, kinetics, and whole cell protein display data suggest that the N terminus modulates substrate access to the enzyme. We propose that it may increase the efficiency of protein display by reducing the unproductive hydrolytic cleavage of enzyme-protein covalent intermediates that form during the cell wall anchoring reaction. Notably, a key active site loop (β7/β8 loop) undergoes a disordered to ordered transition upon binding the sorting signal, potentially facilitating recognition of lipid II.     

UV Resistance of Bacillus anthracis Spores Revisited: Validation of Bacillus subtilis Spores as UV Surrogates for Spores of B. anthracis Sterne

Recent bioterrorism concerns have prompted renewed efforts towards understanding the biology of bacterial spore resistance to radiation with a special emphasis on the spores of Bacillus anthracis. A review of the literature revealed that B. anthracis Sterne spores may be three to four times more resistant to 254-nm-wavelength UV than are spores of commonly used indicator strains of Bacillus subtilis. To test this notion, B. anthracis Sterne spores were purified and their UV inactivation kinetics were determined in parallel with those of the spores of two indicator strains of B. subtilis, strains WN624 and ATCC 6633. When prepared and assayed under identical conditions, the spores of all three strains exhibited essentially identical UV inactivation kinetics. The data indicate that standard UV treatments that are effective against B. subtilis spores are likely also sufficient to inactivate B. anthracis spores and that the spores of standard B. subtilis strains could reliably be used as a biodosimetry model for the UV inactivation of B. anthracis spores.     

Bacillus anthracis physiology and genetics

Bacillus anthracis is a member of the Bacillus cereus group species (also known as the “group 1 bacilli”), a collection of Gram-positive spore-forming soil bacteria that are non-fastidious facultative anaerobes with very similar growth characteristics and natural genetic exchange systems. Despite their close physiology and genetics, the B. cereus group species exhibit certain species-specific phenotypes, some of which are related to pathogenicity. B. anthracis is the etiologic agent of anthrax. Vegetative cells of B. anthracis produce anthrax toxin proteins and a poly-d-glutamic acid capsule during infection of mammalian hosts and when cultured in conditions considered to mimic the host environment. The genes associated with toxin and capsule synthesis are located on the B. anthracis plasmids, pXO1 and pXO2, respectively. Although plasmid content is considered a defining feature of the species, pXO1- and pXO2-like plasmids have been identified in strains that more closely resemble other members of the B. cereus group. The developmental nature of B. anthracis and its pathogenic (mammalian host) and environmental (soil) lifestyles of make it an interesting model for study of niche-specific bacterial gene expression and physiology.