scispace - formally typeset
Search or ask a question

Showing papers on "Bacillus anthracis published in 2007"


Journal ArticleDOI
23 May 2007-PLOS ONE
TL;DR: Findings indicate humans have played an important role in the evolution of anthrax by increasing the proliferation and dispersal of this now global disease.
Abstract: Anthrax, caused by the bacterium Bacillus anthracis, is a disease of historical and current importance that is found throughout the world. The basis of its historical transmission is anecdotal and its true global population structure has remained largely cryptic. Seven diverse B. anthracis strains were whole-genome sequenced to identify rare single nucleotide polymorphisms (SNPs), followed by phylogenetic reconstruction of these characters onto an evolutionary model. This analysis identified SNPs that define the major clonal lineages within the species. These SNPs, in concert with 15 variable number tandem repeat (VNTR) markers, were used to subtype a collection of 1,033 B. anthracis isolates from 42 countries to create an extensive genotype data set. These analyses subdivided the isolates into three previously recognized major lineages (A, B, and C), with further subdivision into 12 clonal sub-lineages or sub-groups and, finally, 221 unique MLVA15 genotypes. This rare genomic variation was used to document the evolutionary progression of B. anthracis and to establish global patterns of diversity. Isolates in the A lineage are widely dispersed globally, whereas the B and C lineages occur on more restricted spatial scales. Molecular clock models based upon genome-wide synonymous substitutions indicate there was a massive radiation of the A lineage that occurred in the mid-Holocene (3,064-6,127 ybp). On more recent temporal scales, the global population structure of B. anthracis reflects colonial-era importation of specific genotypes from the Old World into the New World, as well as the repeated industrial importation of diverse genotypes into developed countries via spore-contaminated animal products. These findings indicate humans have played an important role in the evolution of anthrax by increasing the proliferation and dispersal of this now global disease. Finally, the value of global genotypic analysis for investigating bioterrorist-mediated outbreaks of anthrax is demonstrated.

350 citations


Journal ArticleDOI
TL;DR: It is speculated that to understand the taxonomic relationship within this group of bacteria, special attention should be devoted also to the ecology and the population genetics of these species.
Abstract: Three species of the Bacillus cereus group (Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis) have a marked impact on human activity. Bacillus cereus and B. anthracis are important p...

235 citations


Journal ArticleDOI
TL;DR: To determine the size distribution of the spores of Bacillus anthracis, and compare its size with other Bacillus species grown and sporulated under similar conditions.
Abstract: Aims: To determine the size distribution of the spores of Bacillus anthracis, and compare its size with other Bacillus species grown and sporulated under similar conditions. Methods and Results: Spores from several Bacillus species, including seven strains of B. anthracis and six close neighbours, were prepared and studied using identical media, protocols and instruments. Here, we report the spore length and diameter distributions, as determined by transmission electron microscopy (TEM). We calculated the aspect ratio and volume of each spore. All the studied strains of B. anthracis had similar diameter (mean range between 0·81 ± 0·08 μm and 0·86 ± 0·08 μm). The mean lengths of the spores from different B. anthracis strains fell into two significantly different groups: one with mean spore lengths 1·26 ± 0·13 μm or shorter, and another group of strains with mean spore lengths between 1·49 and 1·67 μm. The strains of B. anthracis that were significantly shorter also sporulated with higher yield at relatively lower temperature. The grouping of B. anthracis strains by size and sporulation temperature did not correlate with their respective virulence. Conclusions: The spores of Bacillus subtilis and Bacillus atrophaeus (previously named Bacillus globigii), two commonly used simulants of B. anthracis, were considerably smaller in length, diameter and volume than all the B. anthracis spores studied. Although rarely used as simulants, the spores of Bacillus cereus and Bacillus thuringiensis had dimensions similar to those of B. anthracis. Significance and Impact of the Study: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefence against B. anthracis. The data presented here should help in the selection of simulants that better resemble the properties of B. anthracis, and thus, more accurately represent the performance of collectors, detectors and other countermeasures against this threat agent.

206 citations


Journal ArticleDOI
01 May 2007-Medicine
TL;DR: It is shown that antibiotics only improve survival if administered early in the course of anthrax infection, and vaccination that leads to the formation of antibodies to protective antigen is anti-inflammatory and beneficial in averting shock and improving survival.

162 citations


Journal ArticleDOI
TL;DR: It is demonstrated that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that p XO1 and pXo2 define the B. anthracis species.
Abstract: The plasmids of the members of the Bacillus cereus sensu lato group of organisms are essential in defining the phenotypic traits associated with pathogenesis and ecology. For example, Bacillus anthracis contains two plasmids, pXO1 and pXO2, encoding toxin production and encapsulation, respectively, that define this species pathogenic potential, whereas the presence of a Bt toxin-encoding plasmid defines Bacillus thuringiensis isolates. In this study the plasmids from B. cereus isolates that produce emetic toxin or are linked to periodontal disease were sequenced and analyzed. Two periodontal isolates examined contained almost identical ∼272-kb plasmids, named pPER272. The emetic toxin-producing isolate contained one ∼270-kb plasmid, named pCER270, encoding the cereulide biosynthesis gene cluster. Comparative sequence analyses of these B. cereus plasmids revealed a high degree of sequence similarity to the B. anthracis pXO1 plasmid, especially in a putative replication region. These plasmids form a newly defined group of pXO1-like plasmids. However, these novel plasmids do not contain the pXO1 pathogenicity island, which in each instance is replaced by plasmid specific DNA. Plasmids pCER270 and pPER272 share regions that are not found in any other pXO1-like plasmids. Evolutionary studies suggest that these plasmids are more closely related to each other than to other identified B. cereus plasmids. Screening of a population of B. cereus group isolates revealed that pXO1-like plasmids are more often found in association with clinical isolates. This study demonstrates that the pXO1-like plasmids may define pathogenic B. cereus isolates in the same way that pXO1 and pXO2 define the B. anthracis species.

155 citations


Journal ArticleDOI
TL;DR: In this paper, the authors used the genetic algorithm for rule-set prediction modeling system to model the ecological niche for Bacillus anthracis in the contiguous United States using wildlife and livestock outbreaks and several environmental variables.
Abstract: The ecology and distribution of Bacillus anthracis is poorly understood despite continued anthrax outbreaks in wildlife and livestock throughout the United States. Little work is available to define the potential environments that may lead to prolonged spore survival and subsequent outbreaks. This study used the genetic algorithm for rule-set prediction modeling system to model the ecological niche for B. anthracis in the contiguous United States using wildlife and livestock outbreaks and several environmental variables. The modeled niche is defined by a narrow range of normalized difference vegetation index, precipitation, and elevation, with the geographic distribution heavily concentrated in a narrow corridor from southwest Texas northward into the Dakotas and Minnesota. Because disease control programs rely on vaccination and carcass disposal, and vaccination in wildlife remains untenable, understanding the distribution of B. anthracis plays an important role in efforts to prevent/eradicate the disease. Likewise, these results potentially aid in differentiating endemic/natural outbreaks from industrial-contamination related outbreaks or bioterrorist attacks.

150 citations


Journal ArticleDOI
TL;DR: This work determined the roles of several B. anthracis orthologues of Bacillus subtilis coat protein genes in spore assembly and virulence and found that SpoIVA has a critical role in directing the assembly of the coat and exosporium to an area around the forespore.
Abstract: Bacillus spp. and Clostridium spp. form a specialized cell type, called a spore, during a multistep differentiation process that is initiated in response to starvation. Spores are protected by a morphologically complex protein coat. The Bacillus anthracis coat is of particular interest because the spore is the infective particle of anthrax. We determined the roles of several B. anthracis orthologues of Bacillus subtilis coat protein genes in spore assembly and virulence. One of these, cotE, has a striking function in B. anthracis: it guides the assembly of the exosporium, an outer structure encasing B. anthracis but not B. subtilis spores. However, CotE has only a modest role in coat protein assembly, in contrast to the B. subtilis orthologue. cotE mutant spores are fully virulent in animal models, indicating that the exosporium is dispensable for infection, at least in the context of a cotE mutation. This has implications for both the pathophysiology of the disease and next-generation therapeutics. CotH, which directs the assembly of an important subset of coat proteins in B. subtilis, also directs coat protein deposition in B. anthracis. Additionally, however, in B. anthracis, CotH effects germination; in its absence, more spores germinate than in the wild type. We also found that SpoIVA has a critical role in directing the assembly of the coat and exosporium to an area around the forespore. This function is very similar to that of the B. subtilis orthologue, which directs the assembly of the coat to the forespore. These results show that while B. anthracis and B. subtilis rely on a core of conserved morphogenetic proteins to guide coat formation, these proteins may also be important for species-specific differences in coat morphology. We further hypothesize that variations in conserved morphogenetic coat proteins may play roles in taxonomic variation among species.

147 citations


Journal ArticleDOI
Benjamin E. Turk1
TL;DR: Recent progress is reviewed in elucidating the mechanisms by which LF and EF influence host signalling and thereby contribute to disease.
Abstract: Infectious microbes face an unwelcoming environment in their mammalian hosts, which have evolved elaborate multicelluar systems for recognition and elimination of invading pathogens. A common strategy used by pathogenic bacteria to establish infection is to secrete protein factors that block intracellular signalling pathways essential for host defence. Some of these proteins also act as toxins, directly causing pathology associated with disease. Bacillus anthracis, the bacterium that causes anthrax, secretes two plasmid-encoded enzymes, LF (lethal factor) and EF (oedema factor), that are delivered into host cells by a third bacterial protein, PA (protective antigen). The two toxins act on a variety of cell types, disabling the immune system and inevitably killing the host. LF is an extraordinarily selective metalloproteinase that site-specifically cleaves MKKs (mitogen-activated protein kinase kinases). Cleavage of MKKs by LF prevents them from activating their downstream MAPK (mitogen-activated protein kinase) substrates by disrupting a critical docking interaction. Blockade of MAPK signalling functionally impairs cells of both the innate and adaptive immune systems and induces cell death in macrophages. EF is an adenylate cyclase that is activated by calmodulin through a non-canonical mechanism. EF causes sustained and potent activation of host cAMP-dependent signalling pathways, which disables phagocytes. Here I review recent progress in elucidating the mechanisms by which LF and EF influence host signalling and thereby contribute to disease.

145 citations


Journal ArticleDOI
TL;DR: The results indicate that NE can effectively adjuvant rPA for intranasal immunization, which potentially could lead to a needle-free anthrax vaccine requiring fewer doses and having fewer side effects than the currently available human vaccine.
Abstract: The currently available commercial human anthrax vaccine requires multiple injections for efficacy and has side effects due to its alum adjuvant. These factors limit its utility when immunizing exposed populations in emergent situations. We evaluated a novel mucosal adjuvant that consists of a nontoxic, water-in-oil nanoemulsion (NE). This material does not contain a proinflammatory component but penetrates mucosal surfaces to load antigens into dendritic cells. Mice and guinea pigs were intranasally immunized with recombinant Bacillus anthracis protective antigen (rPA) mixed in NE as an adjuvant. rPA-NE immunization was effective in inducing both serum anti-PA immunoglobulin G (IgG) and bronchial anti-PA IgA and IgG antibodies after either one or two mucosal administrations. Serum anti-PA IgG2a and IgG2b antibodies and PA-specific cytokine induction after immunization indicate a Th1-polarized immune response. rPA-NE immunization also produced high titers of lethal-toxin-neutralizing serum antibodies in both mice and guinea pigs. Guinea pigs nasally immunized with rPA-NE vaccine were protected against an intradermal challenge with ∼1,000 times the 50% lethal dose (∼1,000× LD50) of B. anthracis Ames strain spores (1.38 × 103 spores), which killed control animals within 96 h. Nasal immunization also resulted in 70% and 40% survival rates against intranasal challenge with 10× LD50 and 100× LD50 (1.2 × 106 and 1.2 × 107) Ames strain spores. Our results indicate that NE can effectively adjuvant rPA for intranasal immunization. This potentially could lead to a needle-free anthrax vaccine requiring fewer doses and having fewer side effects than the currently available human vaccine.

137 citations


Journal ArticleDOI
TL;DR: It is reported that B. anthracis spores germinate and establish infections at the initial site of inoculation in both inhalational and cutaneous infections without needing to be transported to draining lymph nodes, and that inhaled spores establish initial infection in nasal-associated lymphoid tissues.
Abstract: Bacillus anthracis causes three forms of anthrax: inhalational, gastrointestinal, and cutaneous. Anthrax is characterized by both toxemia, which is caused by secretion of immunomodulating toxins (lethal toxin and edema toxin), and septicemia, which is associated with bacterial encapsulation. Here we report that, contrary to the current view of B. anthracis pathogenesis, B. anthracis spores germinate and establish infections at the initial site of inoculation in both inhalational and cutaneous infections without needing to be transported to draining lymph nodes, and that inhaled spores establish initial infection in nasal-associated lymphoid tissues. Furthermore, we found that Peyer's patches in the mouse intestine are the primary site of bacterial growth after intragastric inoculation, thus establishing an animal model of gastrointestinal anthrax. All routes of infection progressed to the draining lymph nodes, spleen, lungs, and ultimately the blood. These discoveries were made possible through the development of a novel dynamic mouse model of B. anthracis infection using bioluminescent non-toxinogenic capsulated bacteria that can be visualized within the mouse in real-time, and demonstrate the value of in vivo imaging in the analysis of B. anthracis infection. Our data imply that previously unrecognized portals of bacterial entry demand more intensive investigation, and will significantly transform the current perception of inhalational, gastrointestinal, and cutaneous B. anthracis pathogenesis.

132 citations


Journal ArticleDOI
TL;DR: Six SNPs were found to be highly specific for the Ames strain, the strain used in the 2001 bioterrorist attacks in the United States, and this evolutionary and genomics-based approach provides an effective means for the discovery of strain-specific SNPs in B. anthracis.
Abstract: Highly precise diagnostics and forensic assays can be developed through a combination of evolutionary analysis and the exhaustive examination of genomic sequences. In Bacillus anthracis, whole-genome sequencing efforts revealed ca. 3,500 single-nucleotide polymorphisms (SNPs) among eight different strains and evolutionary analysis provides the identification of canonical SNPs. We have previously shown that SNPs are highly evolutionarily stable, and the clonal nature of B. anthracis makes them ideal signatures for subtyping this pathogen. Here we identified SNPs that define the lineage of B. anthracis that contains the Ames strain, the strain used in the 2001 bioterrorist attacks in the United States. Sequencing and real-time PCR were used to validate these SNPs across B. anthracis strains, including (i) 88 globally and genetically diverse isolates; (ii) isolates that were shown to be genetic relatives of the Ames strain by multiple-locus variable number tandem repeat analysis (MLVA); and (iii) several different lab stocks of the Ames strain, including a clinical isolate from the 2001 letter attack. Six SNPs were found to be highly specific for the Ames strain; four on the chromosome, one on the pX01 plasmid, and one on the pX02 plasmid. All six SNPs differentiated the B. anthracis Ames strain from the 88 unique B. anthracis strains, while five of the six separated Ames from its close genetic relatives. The use of these SNPs coupled with real-time PCR allows specific and sensitive (<100 fg of template DNA) identification of the Ames strain. This evolutionary and genomics-based approach provides an effective means for the discovery of strain-specific SNPs in B. anthracis.

Journal ArticleDOI
TL;DR: Based on results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.
Abstract: The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-μg dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] of B. anthracis Sterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected with B. anthracis Sterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.

Journal ArticleDOI
TL;DR: It is demonstrated that 49 of the 64 abundantly expressed proteins from B. anthracis-infected animals are immunogenic, and a list of 84 in vivo-expressed immunogens for future evaluation for vaccine development, diagnostics, and/or therapeutic intervention is generated.
Abstract: In a previous comparative proteomic study of Bacillus anthracis examining the influence of the virulence plasmids and of various growth conditions on the composition of the bacterial secretome, we identified 64 abundantly expressed proteins (T. Chitlaru, O. Gat, Y. Gozlan, N. Ariel, and A. Shafferman, J. Bacteriol. 188:3551-3571, 2006). Using a battery of sera from B. anthracis-infected animals, in the present study we demonstrated that 49 of these proteins are immunogenic. Thirty-eight B. anthracis immunogens are documented in this study for the first time. The relative immunogenicities of the 49 secreted proteins appear to span a >10,000-fold range. The proteins eliciting the highest humoral response in the course of infection include, in addition to the well-established immunogens protective antigen (PA), Sap, and EA1, GroEL (BA0267), AhpC (BA0345), MntA (BA3189), HtrA (BA3660), 2,3-cyclic nucleotide diesterase (BA4346), collagen adhesin (BAS5205), an alanine amidase (BA0898), and an endopeptidase (BA1952), as well as three proteins having unknown functions (BA0796, BA0799, and BA0307). Of these 14 highly potent secreted immunogens, 11 are known to be associated with virulence and pathogenicity in B. anthracis or in other bacterial pathogens. Combining the results reported here with the results of a similar study of the membranal proteome of B. anthracis (T. Chitlaru, N. Ariel, A. Zvi, M. Lion, B. Velan, A. Shafferman, and E. Elhanany, Proteomics 4:677-691, 2004) and the results obtained in a functional genomic search for immunogens (O. Gat, H. Grosfeld, N. Ariel, I. Inbar, G. Zaide, Y. Broder, A. Zvi, T. Chitlaru, Z. Altboum, D. Stein, S. Cohen, and A. Shafferman, Infect. Immun. 74:3987-4001, 2006), we generated a list of 84 in vivo-expressed immunogens for future evaluation for vaccine development, diagnostics, and/or therapeutic intervention. In a preliminary study, the efficacies of eight immunogens following DNA immunization of guinea pigs were compared to the efficacy of a PA DNA vaccine. All eight immunogens induced specific high antibody titers comparable to the titers elicited by PA; however, unlike PA, none of them provided protection against a lethal challenge (50 50% lethal doses) of virulent B. anthracis strain Vollum spores.

Journal ArticleDOI
TL;DR: Data are provided that provide compelling evidence that AsbA performs the penultimate step in the biosynthesis of petrobactin, involving condensation of 3,4-dihydroxybenzoyl spermidine with citrate to form 3, 4- dihydroxbenzoysl s permidinyl citrate, and reveal that AsBB catalyzes condensing of a second molecule to form the mature siderophore.
Abstract: The asbABCDEF gene cluster from Bacillus anthracis is responsible for biosynthesis of petrobactin, a catecholate siderophore that functions in both iron acquisition and virulence in a murine model of anthrax. We initiated studies to determine the biosynthetic details of petrobactin assembly based on mutational analysis of the asb operon, identification of accumulated intermediates, and addition of exogenous siderophores to asb mutant strains. As a starting point, in-frame deletions of each of the genes in the asb locus (asbABCDEF) were constructed. The individual mutations resulted in complete abrogation of petrobactin biosynthesis when strains were grown on iron-depleted medium. However, in vitro analysis showed that each asb mutant grew to a very limited extent as vegetative cells in iron-depleted medium. In contrast, none of the B. anthracis asb mutant strains were able to outgrow from spores under the same culture conditions. Provision of exogenous petrobactin was able to rescue the growth defect in each asb mutant strain. Taken together, these data provide compelling evidence that AsbA performs the penultimate step in the biosynthesis of petrobactin, involving condensation of 3,4-dihydroxybenzoyl spermidine with citrate to form 3,4-dihydroxybenzoyl spermidinyl citrate. As a final step, the data reveal that AsbB catalyzes condensation of a second molecule of 3,4-dihydroxybenzoyl spermidine with 3,4-dihydroxybenzoyl spermidinyl citrate to form the mature siderophore. This work sets the stage for detailed biochemical studies with this unique acyl carrier protein-dependent, nonribosomal peptide synthetase-independent biosynthetic system.

Journal ArticleDOI
TL;DR: An anti-LF single-chain variable fragment (scFv) that originated from an immunized macaque and was obtained by phage display is described, demonstrating that the approach can generate antibody fragments suitable for prophylaxis and therapeutics.
Abstract: The anthrax lethal toxin (LT) consists of two subunits, the protective antigen (PA) and the lethal factor (LF), and is essential for anthrax pathogenesis. Several recombinant antibodies directed against PA and intended for medical use have been obtained, but none against LF, despite the recommendations of anthrax experts. Here we describe an anti-LF single-chain variable fragment (scFv) that originated from an immunized macaque (Macaca fascicularis) and was obtained by phage display. Panning of the library of 1.8 x 10(8) clones allowed the isolation of 2LF, a high-affinity (equilibrium dissociation constant, 1.02 nM) scFv, which is highly neutralizing in the standardized in vitro assay (50% inhibitory concentration, 1.20 +/- 0.06 nM) and in an in vivo assay. The scFv neutralizes anthrax LT by inhibiting the formation of the LF-PA complex. The genes encoding 2LF are very similar to those of human immunoglobulin germ line genes, sharing substantial (84.2%) identity with their most similar, germinally encoded counterparts; this feature favors medical applications. These results, and others formerly published, demonstrate that our approach can generate antibody fragments suitable for prophylaxis and therapeutics.

Journal ArticleDOI
TL;DR: Fatal cases in 2 metalworkers and the associated investigation were described and anthrax toxin genes were identified in B. cereus isolates from both patients using polymerase chain reaction.
Abstract: Bacillus cereus pneumonia is unusual in nonimmunocompromised hosts. We describe fatal cases in 2 metalworkers and the associated investigation. Anthrax toxin genes were identified in B. cereus isolates from both patients using polymerase chain reaction. Finding anthrax toxin genes in non-Bacillus anthracis isolates has, to our knowledge, only been reported once previously.

Journal ArticleDOI
TL;DR: It is suggested that BclA acts as a shield to not only reduce the ease with which spores germinate but also change the surface properties of the spore, which, in turn, may impede the interaction with host matrix substances.
Abstract: Bacillus collagen-like protein of anthracis (BclA) is the immunodominant glycoprotein on the exosporium of Bacillus anthracis spores. Here, we sought to assess the impact of BclA on spore germination in vitro and in vivo, surface charge, and interaction with host matrix proteins. For that purpose, we constructed a markerless bclA null mutant in B. anthracis Sterne strain 34F2. The growth and sporulation rates of the ΔbclA and parent strains were nearly indistinguishable, but germination of mutant spores occurred more rapidly than that of wild-type spores in vitro and was more complete by 60 min. Additionally, the mean time to death of A/J mice inoculated subcutaneously or intranasally with mutant spores was lower than that for the wild-type spores even though the 50% lethal doses of the two strains were similar. We speculated that these in vitro and in vivo differences between mutant and wild-type spores might reflect the ease of access of germinants to their receptors in the absence of BclA. We also compared the hydrophobic and adhesive properties of ΔbclA and wild-type spores. The ΔbclA spores were markedly less water repellent than wild-type spores, and, probably as a consequence, the extracellular matrix proteins laminin and fibronectin bound significantly better to mutant than to wild-type spores. These studies suggest that BclA acts as a shield to not only reduce the ease with which spores germinate but also change the surface properties of the spore, which, in turn, may impede the interaction of the spore with host matrix substances.

Journal ArticleDOI
TL;DR: It is shown that CapD, a γ-polyglutamic acid depolymerase encoded on the B. anthracis capsule plasmid, degraded purified capsule and removed the capsule from the surface of anthrax bacilli.
Abstract: The poly-γ-d-glutamic acid capsule confers antiphagocytic properties on Bacillus anthracis and is essential for virulence. In this study, we showed that CapD, a γ-polyglutamic acid depolymerase encoded on the B. anthracis capsule plasmid, degraded purified capsule and removed the capsule from the surface of anthrax bacilli. Treatment with CapD induced macrophage phagocytosis of encapsulated B. anthracis and enabled human neutrophils to kill encapsulated organisms. A second glutamylase, PghP, a γ-polyglutamic acid hydrolase encoded by Bacillus subtilis bacteriophage ΦNIT1, had minimal activity in degrading B. anthracis capsule, no effect on macrophage phagocytosis, and only minimal enhancement of neutrophil killing. Thus, the levels of both phagocytosis and killing corresponded to the degree of enzyme-mediated capsule degradation. The use of enzymes to degrade the capsule and enable phagocytic killing of B. anthracis offers a new approach to the therapy of anthrax.

Journal ArticleDOI
04 Jan 2007-Vaccine
TL;DR: It is shown that recombinant spores can be used to confer protective immunity in a murine model using an in vitro toxin neutralization assay and a challenge experiment with the latter showing protection to 100 median lethal dose of B. anthracis spores.

Journal ArticleDOI
20 Apr 2007-Vaccine
TL;DR: This work separately engineered and expressed domain 4 of PA (PAD4) and domain 1 of lethal factor (LFD1) as fusions to lichenase (LicKM), a thermostable enzyme from Clostridium thermocellum, and transiently expressed these fusions in Nicotiana benthamiana.

Journal ArticleDOI
TL;DR: It is discovered that rabbit IgG antibodies elicited by Bacillus anthracis spores specifically recognize a tetrasaccharide chain that decorates the outermost surfaces of the B. anthracIS exosporium.
Abstract: Using photogenerated glycan arrays, we characterized a large panel of synthetic carbohydrates for their antigenic reactivities with pathogen-specific antibodies. We discovered that rabbit IgG antibodies elicited by Bacillus anthracis spores specifically recognize a tetrasaccharide chain that decorates the outermost surfaces of the B. anthracis exosporium. Since this sugar moiety is highly specific for the spores of B. anthracis, it appears to be a key biomarker for detection of B. anthracis spores and development of novel vaccines that target anthrax spores.

Journal ArticleDOI
TL;DR: This method represents a highly specific and rapid diagnostic tool for early anthrax and has a potential additional role as a research tool for understanding toxemia and effects of medical countermeasures for anthrax.
Abstract: The lethal toxin produced during Bacillus anthracis infection is a complex of protective antigen, which localizes the toxin to the cell receptor, and lethal factor (LF), a zinc-dependent endoproteinase whose known targets include five members of the mitogen-activated protein kinase kinase (MAPKK) family of response regulators. We have developed a method for detecting functional LF in serum. Anti-LF murine monoclonal antibodies immobilized on magnetic protein G beads were used to capture and concentrate the LF from serum. The captured LF was exposed to an optimized MAPKK-based peptide substrate, which it hydrolyzed into two smaller peptides. The LF cleavage products were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and quantified by isotope dilution-MS. The entire analytical method can be performed in less than 4 h with detection of LF levels as low as 0.05 ng/mL. The method was used to quantify LF levels in serum from rhesus macaques infected with B. a...

Journal ArticleDOI
TL;DR: The research is based on the idea that compounds designed to block the pores can inhibit the action of virulence factors, and that the chances to find high affinity blocking agents increase if they have the same symmetry as the target pore.

Journal ArticleDOI
TL;DR: PlyB is described, which displays potent lytic activity against the Bacillus anthracis-like strain ATCC 4342 and represents a new class of anthrax lysin and a new defensive tool in the armament against anthrax-mediated bioterrorism.

Journal ArticleDOI
TL;DR: This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax.
Abstract: The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel antitoxins and vaccines that act rapidly and with minimal adverse effects. B. anthracis produces an AB-type toxin composed of the receptor-binding moiety protective antigen (PA) and the enzymatic moieties edema factor and lethal factor. PA is a key target for both antitoxin and vaccine development. We used the icosahedral insect virus Flock House virus as a platform to display 180 copies of the high affinity, PA-binding von Willebrand A domain of the ANTXR2 cellular receptor. The chimeric virus-like particles (VLPs) correctly displayed the receptor von Willebrand A domain on their surface and inhibited lethal toxin action in in vitro and in vivo models of anthrax intoxication. Moreover, VLPs complexed with PA elicited a potent toxin-neutralizing antibody response that protected rats from anthrax lethal toxin challenge after a single immunization without adjuvant. This recombinant VLP platform represents a novel and highly effective, dually-acting reagent for treatment and protection against anthrax.

Journal ArticleDOI
TL;DR: This study aims to compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp.
Abstract: Aims: To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. Methods and Results: We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. Conclusions: The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. Significance and Impact of the Study: The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants.

Journal ArticleDOI
TL;DR: Results suggest that one potential role of the BclA fibers may be to inhibit nonspecific interactions between B. anthracis spores with nonprofessional phagocytic cells and thus direct the spores towards uptake by macrophages during initiation of infection in mammals.
Abstract: Bacillus anthracis is the causative agent of anthrax, and the spore form of the bacterium represents the infectious particle introduced into a host. The spore is surrounded by an exosporium, a loose-fitting membrane composed of proteins and carbohydrates from which hair-like projections extend. These projections are composed mainly of BclA (Bacillus-collagen-like protein of B. anthracis). To date, exact roles of the exosporium structure and BclA protein remain undetermined. We examined differences in spore binding of wild-type Ames and a bclA mutant of B. anthracis to bronchial epithelial cells as well as to the following other epithelial cells: A549, CHO, and Caco-2 cells; the IMR-90 fibroblast line; and human umbilical vein vascular endothelium cells. The binding of wild-type Ames spores to bronchial epithelial cells appeared to be a dose-dependent, receptor-ligand-mediated event. There were similar findings for the bclA mutant, with an additional nonspecific binding component likely leading to significantly more adherence to all nonprofessional phagocytic cell types. In contrast, we detected no difference in adherence and uptake of spores by macrophages for either the wild-type Ames or the bclA mutant strain. These results suggest that one potential role of the BclA fibers may be to inhibit nonspecific interactions between B. anthracis spores with nonprofessional phagocytic cells and thus direct the spores towards uptake by macrophages during initiation of infection in mammals.

Journal ArticleDOI
TL;DR: In this paper, the authors describe three major steps in infection: (i) an invasion phase in the lung, during which toxins have short-distance effects on lung phagocytes; (ii) a phase of bacillus proliferation in the mediastinal lymph nodes, with local effects of toxins; and (iii) a terminal, diffusion phase, characterized by a high blood bacterial load and by long distance effects of poisons, leading to host death.
Abstract: Summary Inhalational anthrax is a life-threatening infectious disease of considerable concern, especially as a potential bioterrorism agent. Progress is gradually being made towards understanding the mechanisms used by Bacillus anthracis to escape the immune system and to induce severe septicaemia associated with toxaemia and leading to death. Recent advances in fundamental research have revealed previously unsuspected roles for toxins in various cell types. We summarize here pathological data for animal models and macroscopic histological examination data from recent clinical records, which we link to the effects of toxins. We describe three major steps in infection: (i) an invasion phase in the lung, during which toxins have short-distance effects on lung phagocytes; (ii) a phase of bacillus proliferation in the mediastinal lymph nodes, with local effects of toxins; and (iii) a terminal, diffusion phase, characterized by a high blood bacterial load and by long-distance effects of toxins, leading to host death. The pathophysiology of inhalational anthrax thus involves interactions between toxins and various cell partners, throughout the course of infection.

Journal ArticleDOI
TL;DR: This study reports the first genome-wide analysis of B. anthracis gene expression during infection of host phagocytes and identifies metabolic pathways that appear to be important to the bacterium during intracellular growth, as well as individual genes that show significant induction in vivo.
Abstract: The interaction between Bacillus anthracis and the mammalian phagocyte is one of the central stages in the progression of inhalational anthrax, and it is commonly believed that the host cell plays a key role in facilitating germination and dissemination of inhaled B. anthracis spores. Given this, a detailed definition of the survival strategies used by B. anthracis within the phagocyte is critical for our understanding of anthrax. In this study, we report the first genome-wide analysis of B. anthracis gene expression during infection of host phagocytes. We developed a technique for specific isolation of bacterial RNA from within infected murine macrophages, and we used custom B. anthracis microarrays to characterize the expression patterns occurring within intracellular bacteria throughout infection of the host phagocyte. We found that B. anthracis adapts very quickly to the intracellular environment, and our analyses identified metabolic pathways that appear to be important to the bacterium during intracellular growth, as well as individual genes that show significant induction in vivo. We used quantitative reverse transcription-PCR to verify that the expression trends that we observed by microarray analysis were valid, and we chose one gene (GBAA1941, encoding a putative transcriptional regulator) for further characterization. A deletion strain missing this gene showed no phenotype in vitro but was significantly attenuated in a mouse model of inhalational anthrax, suggesting that the microarray data described here provide not only the first comprehensive view of how B. anthracis survives within the host cell but also a number of promising leads for further research in anthrax.

Journal ArticleDOI
TL;DR: In this article, the results of an investigation to detect Bacillus anthracis spores in liquids using microfabricated magnetoelastic particles (MEPs) have been presented.
Abstract: This paper presents the results of an investigation to detect Bacillus anthracis spores in liquids using microfabricated magnetoelastic particles (MEP). Free standing, magnetoelastic resonating particles, as small as 500 μm × 100 μm × 4 μm, have been made using microelectronics fabrication techniques. These magnetoelastic resonators feature wireless signal transmission and are suitable for use in air and liquids. Affinity-selected filamentous bacteriophage was employed as the bio-molecular recognition agent and was immobilized onto all surfaces of the resonators. This phage was selected from a landscape phage library specifically for binding B. anthracis spores, the causative agent for the disease anthrax. The biosensors were tested in B. anthracis spore solutions with concentrations from 103 to 108 cfu/ml. A detection limit of 103 cfu/ml, with a sensitivity of 6.5 kHz/decade was observed. Scanning electron microscopy (SEM) micrographs of the tested sensors were used for visual correlation of the sensors’ responses. To investigate specificity, the biosensors were exposed to mixed spore solutions containing B. anthracis Sterne, Bacillus cereus and Bacillus megaterium spore strains. The phage-coated biosensors were proven to have better binding preference to B. anthracis spores than other strains. The study on the binding kinetics have shown that the dissociation constant (Kd) and the binding valency for the sensor in B. anthracis spores solutions are 193 cfu/ml and 2.32.