Showing papers on "Bacillus anthracis published in 2010"
TL;DR: The results indicate that toxin secretion in B. anthracis is, at least, partially vesicle-associated, thus allowing concentrated delivery of toxin components to target host cells, a mechanism that may increase toxin potency.
Abstract: Extracellular vesicle production is a ubiquitous process in Gram-negative bacteria, but little is known about such process in Gram-positive bacteria. We report the isolation of extracellular vesicles from the supernatants of Bacillus anthracis, a Gram-positive bacillus that is a powerful agent for biological warfare. B. anthracis vesicles formed at the outer layer of the bacterial cell had double-membrane spheres and ranged from 50 to 150 nm in diameter. Immunoelectron microscopy with mAbs to protective antigen, lethal factor, edema toxin, and anthrolysin revealed toxin components and anthrolysin in vesicles, with some vesicles containing more than one toxin component. Toxin-containing vesicles were also visualized inside B. anthracis-infected macrophages. ELISA and immunoblot analysis of vesicle preparations confirmed the presence of B. anthracis toxin components. A mAb to protective antigen protected macrophages against vesicles from an anthrolysin-deficient strain, but not against vesicles from Sterne 34F2 and Sterne δT strains, consistent with the notion that vesicles delivered both toxin and anthrolysin to host cells. Vesicles were immunogenic in BALB/c mice, which produced a robust IgM response to toxin components. Furthermore, vesicle-immunized mice lived significantly longer than controls after B. anthracis challenge. Our results indicate that toxin secretion in B. anthracis is, at least, partially vesicle-associated, thus allowing concentrated delivery of toxin components to target host cells, a mechanism that may increase toxin potency. Our observations may have important implications for the design of vaccines, for passive antibody strategies, and provide a previously unexplored system for studying secretory pathways in Gram-positive bacteria.
306 citations
TL;DR: The sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features is described.
Abstract: Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as “B. cereus variety (var.) anthracis”.
153 citations
TL;DR: Data presented here support a model whereby LT-mediated activation of Nlrp1b and subsequent lysis of macrophages is not a mechanism used by B. anthracis to promote virulence, but rather a protective host-mediated innate immune response.
Abstract: Pathogenesis of Bacillus anthracis is associated with the production of lethal toxin (LT), which activates the murine Nalp1b/Nlrp1b inflammasome and induces caspase-1–dependent pyroptotic death in macrophages and dendritic cells. In this study, we investigated the effect of allelic variation of Nlrp1b on the outcome of LT challenge and infection by B. anthracis spores. Nlrp1b allelic variation did not alter the kinetics or pathology of end-stage disease induced by purified LT, suggesting that, in contrast to previous reports, macrophage lysis does not contribute directly to LT-mediated pathology. However, animals expressing a LT-sensitive allele of Nlrp1b showed an early inflammatory response to LT and increased resistance to infection by B. anthracis . Data presented here support a model whereby LT-mediated activation of Nlrp1b and subsequent lysis of macrophages is not a mechanism used by B. anthracis to promote virulence, but rather a protective host-mediated innate immune response.
142 citations
TL;DR: Computer analysis revealed that pathogen proteins preferentially interact with human proteins that are hubs and bottlenecks in the human PPI network, and computed modules of human-pathogen PPIs that are conserved amongst the three networks.
Abstract: Background
Bacillus anthracis, Francisella tularensis, and Yersinia pestis are bacterial pathogens that can cause anthrax, lethal acute pneumonic disease, and bubonic plague, respectively, and are listed as NIAID Category A priority pathogens for possible use as biological weapons. However, the interactions between human proteins and proteins in these bacteria remain poorly characterized leading to an incomplete understanding of their pathogenesis and mechanisms of immune evasion.
135 citations
TL;DR: An inverse relationship between murine macrophage sensitivity to lethal toxin and mouse susceptibility to spore infection is confirmed, and roles for Nlrp1bS, caspase-1, and IL-1β in countering anthrax infection are established.
Abstract: Bacillus anthracis infects hosts as a spore, germinates, and disseminates in its vegetative form. Production of anthrax lethal and edema toxins following bacterial outgrowth results in host death. Macrophages of inbred mouse strains are either sensitive or resistant to lethal toxin depending on whether they express the lethal toxin responsive or non-responsive alleles of the inflammasome sensor Nlrp1b (Nlrp1bS/S or Nlrp1bR/R, respectively). In this study, Nlrp1b was shown to affect mouse susceptibility to infection. Inbred and congenic mice harboring macrophage-sensitizing Nlrp1bS/S alleles (which allow activation of caspase-1 and IL-1β release in response to anthrax lethal toxin challenge) effectively controlled bacterial growth and dissemination when compared to mice having Nlrp1bR/R alleles (which cannot activate caspase-1 in response to toxin). Nlrp1bS-mediated resistance to infection was not dependent on the route of infection and was observed when bacteria were introduced by either subcutaneous or intravenous routes. Resistance did not occur through alterations in spore germination, as vegetative bacteria were also killed in Nlrp1bS/S mice. Resistance to infection required the actions of both caspase-1 and IL-1β as Nlrp1bS/S mice deleted of caspase-1 or the IL-1 receptor, or treated with the Il-1 receptor antagonist anakinra, were sensitized to infection. Comparison of circulating neutrophil levels and IL-1β responses in Nlrp1bS/S,Nlrp1bR/R and IL-1 receptor knockout mice implicated Nlrp1b and IL-1 signaling in control of neutrophil responses to anthrax infection. Neutrophil depletion experiments verified the importance of this cell type in resistance to B. anthracis infection. These data confirm an inverse relationship between murine macrophage sensitivity to lethal toxin and mouse susceptibility to spore infection, and establish roles for Nlrp1bS, caspase-1, and IL-1β in countering anthrax infection.
115 citations
TL;DR: B. thuringiensis provided the best overall fit as a non-pathogenic surrogate for B. anthracis and is suggested for focusing on this surrogate in future experiments of spore fate and transport modelling.
Abstract: Bacillus anthracis, the causative agent of anthrax, is a proven biological weapon. In order to study this threat, a number of experimental surrogates have been used over the past 70 years. However, not all surrogates are appropriate for B. anthracis, especially when investigating transport, fate and survival. Although B. atrophaeus has been widely used as a B. anthracis surrogate, the two species do not always behave identically in transport and survival models. Therefore, we devised a scheme to identify a more appropriate surrogate for B. anthracis. Our selection criteria included risk of use (pathogenicity), phylogenetic relationship, morphology and comparative survivability when challenged with biocides. Although our knowledge of certain parameters remains incomplete, especially with regards to comparisons of spore longevity under natural conditions, we found that B. thuringiensis provided the best overall fit as a non-pathogenic surrogate for B. anthracis. Thus, we suggest focusing on this surrogate in future experiments of spore fate and transport modelling.
113 citations
TL;DR: The crystal structure of the core of a lethal toxin complex to 3.1-Å resolution is reported; the structure contains a PA octamer bound to four LF PA-binding domains (LFN), and provides insight into the mechanism of translocation-coupled protein unfolding.
Abstract: The anthrax lethal toxin protein transporter consists of protective antigen (PA) and lethal factor (LF), with LF unfolding for translocation into the host cell. Structural and functional analyses now indicate how each of four LFs unfolds and binds into amphipathic clefts on the surface of the PA octomer.
112 citations
TL;DR: Ratiometric fluorescent detection of anthrax spores can be achieved through the displacement of a different sensitizer by DPA, and lanthanide (Ln3+)-based luminescent detection of DPA has been most promising owing to the unique photophysical properties of Ln3-DPA chelates.
Abstract: Anthrax is an acute disease, concurrently a potential biological warfare agent caused by Bacillus Anthracis. The accurate, rapid, sensitive, and selective detection of Bacillus spores plays a vital role in order to prevent a biological attack or outbreak of disease. Bacterial spores contain a main core cell which is enclosed by protective layers. As a major component of these protective layers, bacterial spores contain up to 1 M dipicolinic acid (DPA), accounting for 5−15 % of the dry mass of the bacterial spore. Hence, DPA is a convenient biomarker for these spores. In recent years a number of biological and chemical detection methods for Bacillus Anthracis spores have been investigated. Biological methods are based on polymerase chain reactions and immunoassays. Important chemical methods employ vibrational spectroscopy (FT-IR, Raman and SERS) and photoluminescence. Among them, lanthanide (Ln3+)-based luminescent detection of DPA has been most promising owing to the unique photophysical properties of Ln3+-DPA chelates, including their bright luminescence upon sensitization by DPA, the long luminescence lifetimes compared to free Ln3+, and the concomitantly high luminescence enhancement ratio upon coordination of DPA to the Ln3+ center. Besides the use of DPA itself as a sensitizer, ratiometric fluorescent detection of anthrax spores can be achieved through the displacement of a different sensitizer by DPA.
100 citations
TL;DR: Drosophila melanogaster is used to identify the Rab11/Sec15 exocyst, which acts at the last step of endocytic recycling, as a novel target of both EF and LF, which may contribute to toxin-dependent barrier disruption and vascular dysfunction during B. anthracis infection.
Abstract: Bacillus anthracis is the causative agent of anthrax in humans and other mammals(1,2). In lethal systemic anthrax, proliferating bacilli secrete large quantities of the toxins lethal factor (LF) and oedema factor (EF), leading to widespread vascular leakage and shock. Whereas host targets of LF (mitogen-activated protein-kinase kinases) and EF (cAMP-dependent processes)(3) have been implicated in the initial phase of anthrax(1,2), less is understood about toxin action during the final stage of infection. Here we use Drosophila melanogaster to identify the Rab11/Sec15 exocyst, which acts at the last step of endocytic recycling, as a novel target of both EF and LF. EF reduces levels of apically localized Rab11 and indirectly blocks vesicle formation by its binding partner and effector Sec15 (Sec15-GFP), whereas LF acts more directly to reduce Sec15-GFP vesicles. Convergent effects of EF and LF on Rab11/Sec15 inhibit expression of and signalling by the Notch ligand Delta and reduce DE-cadherin levels at adherens junctions. In human endothelial cells, the two toxins act in a conserved fashion to block formation of Sec15 vesicles, inhibit Notch signalling, and reduce cadherin expression at adherens junctions. This coordinated disruption of the Rab11/Sec15 exocyst by anthrax toxins may contribute to toxin-dependent barrier disruption and vascular dysfunction during B. anthracis infection.
90 citations
TL;DR: This work demonstrates that anthrax toxin uptake through CMG2 and the resulting impairment of myeloid cells are essential to anthrax infection.
87 citations
TL;DR: The data suggest that toxin-mediated lethality in rats as well as macrophage sensitivity in this animal model are controlled by a single locus on chromosome 10 that is likely to be the inflammasome NLR sensor, Nlrp1.
Abstract: Anthrax lethal toxin (LT) is a bipartite protease-containing toxin and a key virulence determinant of Bacillus anthracis In mice, LT causes the rapid lysis of macrophages isolated from certain inbred strains, but the correlation between murine macrophage sensitivity and mouse strain susceptibility to toxin challenge is poor In rats, LT induces a rapid death in as little as 37 minutes through unknown mechanisms We used a recombinant inbred (RI) rat panel of 19 strains generated from LT-sensitive and LT-resistant progenitors to map LT sensitivity in rats to a locus on chromosome 10 that includes the inflammasome NOD-like receptor (NLR) sensor, Nlrp1 This gene is the closest rat homolog of mouse Nlrp1b, which was previously shown to control murine macrophage sensitivity to LT An absolute correlation between in vitro macrophage sensitivity to LT-induced lysis and animal susceptibility to the toxin was found for the 19 RI strains and 12 additional rat strains Sequencing Nlrp1 from these strains identified five polymorphic alleles Polymorphisms within the N-terminal 100 amino acids of the Nlrp1 protein were perfectly correlated with LT sensitivity These data suggest that toxin-mediated lethality in rats as well as macrophage sensitivity in this animal model are controlled by a single locus on chromosome 10 that is likely to be the inflammasome NLR sensor, Nlrp1
TL;DR: Current challenges and various improvements associated with anthrax agent detection are discussed, including improved laboratory methods for B. anthracis identification.
TL;DR: Basic characteristics of the bacterium and its products, key aspects of pathogenesis, and the pathologic changes commonly observed in each animal model species are focused on.
Abstract: Anthrax is a lethal disease caused by the bacterium Bacillus anthracis. There are three principal forms of the disease in humans-cutaneous, gastrointestinal, and inhalational-depending on the route of exposure. Of these, inhalational anthrax is the most dangerous; it is rapidly fatal; and it has been used as a deadly biological warfare agent in the last decade. Suitable animal models of inhalational anthrax have been utilized to study pathogenesis of disease, investigate bacterial characteristics such as virulence, and test effectiveness of vaccines and therapeutics. To date, mice, guinea pigs, rabbits, and nonhuman primates are the principal animal species used to study inhalational anthrax. Mice are valuable in studying early pathogenesis and bacterial characteristics. Few pathologic changes occur in the mouse models but may include marked bacteremia and lymphocyte destruction in the spleen and mediastinal lymph nodes. Rabbits and guinea pigs rapidly develop fulminate systemic disease, and pathologic findings often include necrotizing lymphadenitis; splenitis; pneumonia; vasculitis; and hemorrhage, congestion, and edema in multiple tissues. Nonhuman primates consistently develop the full range of classic lesions of human inhalational anthrax, including meningitis; lymphadenitis; splenitis; mediastinitis; pneumonia; vasculitis; and hemorrhage, congestion, and edema in multiple tissues. This review focuses on basic characteristics of the bacterium and its products, key aspects of pathogenesis, and the pathologic changes commonly observed in each animal model species.
TL;DR: Specific virulence factors adapted to the insect intestine might exist in B. thuringiensis and B. entomophila, which suggests a co-evolution between host and pathogens and supports the close links between B. cereus and more distant links to their relative B. anthracis.
TL;DR: Results suggest that BslA functions as the surface adhesin of the anthrax pathogen B. anthracis strain Ames, and that few vegetative forms could be recovered from internal organs of animals infected with the bslA mutant.
Abstract: Microbial pathogens use adhesive surface proteins to bind to and interact with host tissues, events that are universal for the pathogenesis of infectious diseases. A surface adhesin of Bacillus anthracis, the causative agent of anthrax, required to mediate these steps has not been discovered. Previous work identified BslA, an S-layer protein, to be necessary and sufficient for adhesion of the anthrax vaccine strain, Bacillus anthracis Sterne, to host cells. Here we asked whether encapsulated bacilli require BslA for anthrax pathogenesis in guinea pigs. Compared with the highly virulent parent strain B. anthracis Ames, bslA mutants displayed a dramatic increase in the lethal dose and in mean time-to-death. Whereas all tissues of animals infected with B. anthracis Ames contained high numbers of bacilli, only few vegetative forms could be recovered from internal organs of animals infected with the bslA mutant. Surface display of BslA occurred at the poles of encapsulated bacilli and enabled the binding of vegetative forms to host cells. Together these results suggest that BslA functions as the surface adhesin of the anthrax pathogen B. anthracis strain Ames.
TL;DR: The presence of a prominent moonlighting enzyme, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the surface and in the extracellular medium of Bacillus anthracis and human plasminogen predominantly interacted with the rGapA isoform at physiological concentrations and the interaction was found to be lysine dependent.
TL;DR: Analyzing acid-solubilized, purified carbohydrates by mass spectrometry and NMR spectroscopy, secondary cell wall polysaccharide (SCWP) is identified as the ligand of S-layer proteins and suggests that tagO-mediated assembly of linkage units tethers pyruvylated SCWP to the B. anthracis envelope, thereby enabling S- layer assembly and providing for the pathogenesis of anthrax infections.
TL;DR: The results suggested that the ANTXR2-mediated cytoplasmic delivery of LF was enhanced by CTSB-dependent autophagic flux.
TL;DR: Improvement of this improved expression vaccine strategy reduced the number of inoculations and length of the boosting period, leading to animal protection via efficacious bacterial adjuvanticity and safe oral delivery of this vaccine to mucosal immune cells, including dendritic cells.
Abstract: Aim: Induction of protective immunity against pathogenic microbes, including Bacillus anthracis, requires efficient vaccines that potentiate antibody avidity and increase T-cell longevity. We recently reported that the delivery of targeted B. anthracis protective antigen (PA) genetically fused to a DC-binding peptide (DCpep) by Lactobacillus acidophilus induced mucosal and systemic immunity against B. anthracis challenge in mice. Materials & methods: Improvement of this oral vaccine strategy was attempted by use of the high copy and genetically stable q-replicating vector, pTRKH2, for expression of the targeted PA fusion protein in Lactobacillus gasseri, a common human commensal microbe, to vaccinate animals against anthrax Sterne infection. Results: Oral application of L. gasseri expressing the PA–DCpep fusion proteins elicited robust PA-neutralizing antibody and T-cell mediated immune responses against anthrax Sterne challenge, resulting in complete animal survival. Collectively, this improved expressio...
TL;DR: Evidence is presented that a B. anthracis S-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC, which suggests that the IsD system can receive heme from multiple inputs and may reflect an adaptation of B. Anthracis to changing iron reservoirs during an infection.
Abstract: The sequestration of iron by mammalian hosts represents a significant obstacle to the establishment of a bacterial infection. In response, pathogenic bacteria have evolved mechanisms to acquire iron from host heme. Bacillus anthracis, the causative agent of anthrax, utilizes secreted hemophores to scavenge heme from host hemoglobin, thereby facilitating iron acquisition from extracellular heme pools and delivery to iron-regulated surface determinant (Isd) proteins covalently attached to the cell wall. However, several Gram-positive pathogens, including B. anthracis, contain genes that encode near iron transporter (NEAT) proteins that are genomically distant from the genetically linked Isd locus. NEAT domains are protein modules that partake in several functions related to heme transport, including binding heme and hemoglobin. This finding raises interesting questions concerning the relative role of these NEAT proteins, relative to hemophores and the Isd system, in iron uptake. Here, we present evidence that a B. anthracis S-layer homology (SLH) protein harboring a NEAT domain binds and directionally transfers heme to the Isd system via the cell wall protein IsdC. This finding suggests that the Isd system can receive heme from multiple inputs and may reflect an adaptation of B. anthracis to changing iron reservoirs during an infection. Understanding the mechanism of heme uptake in pathogenic bacteria is important for the development of novel therapeutics to prevent and treat bacterial infections.
TL;DR: This review will focus on recent discoveries in siderophore biosynthesis and biology in this group, which contains numerous human pathogens, most notably the causative agent of anthrax, Bacillus anthracis.
Abstract: Recent observations have shed light on some of the endogenous iron-acquisition mechanisms of members of the Bacillus cereus sensu lato group. In particular, pathogens in the B. cereus group use siderophores with both unique chemical structures and biological roles. This review will focus on recent discoveries in siderophore biosynthesis and biology in this group, which contains numerous human pathogens, most notably the causative agent of anthrax, Bacillus anthracis.
TL;DR: It is concluded that PA may form an octameric oligomeric state as a means to produce a more stable and active LT complex that could circulate freely in the blood.
TL;DR: It is reported that integrin β1, another cell surface von Willebrand A domain protein, can mediate and potentiate anthrax toxin endocytosis and serve independently as a low-affinity PA receptor.
Abstract: To kill macrophages, the lethal factor component of Bacillus anthracis toxin binds to a carrier protein (PA), which then interacts with the CMG2 receptor protein on the cell surface and is endocytosed into the cytoplasm. CMG2, as well as TEM8, a second PA receptor not present on macrophages, contain a von Willebrand A domain that is crucial for toxin binding. Here we report that integrin β1, another cell surface von Willebrand A domain protein, can mediate and potentiate anthrax toxin endocytosis. By using microarray-based analysis to globally correlate gene expression profiles with toxin sensitivity, we associated toxin effects with the integrin-activating proteins osteopontin and CD44. Further study showed that PA binds to α4β1– and α5β1–integrin complexes, leading to their conjoint endocytosis, and also interacts—weakly relative to CMG2 but comparably to TEM8—with purified α5β1 complex in vitro. Monoclonal antibody directed against β1-integrin or its α integrin partners reduced PA/integrin endocytosis and anthrax toxin lethality, and hyaluronic acid—which interferes with CD44-mediated integrin activation—had similar effects. Remarkably, whereas deficiency of CMG2 protected macrophages from rapid killing by large toxin doses (>50 ng/mL), by 24 h the toxin-treated cells were dead. Such late killing of CMG2-deficient cells by high dose toxin as well as the late death observed during exposure of CMG2-producing macrophages to low-dose toxin (<1 ng/mL), was dependent on integrin function. Effects of inactivating both CMG2 and integrin were synergistic. Collectively, our findings argue strongly that β1-integrin can both potentiate CMG2-mediated endocytosis and serve independently as a low-affinity PA receptor.
TL;DR: The first two enzymes for the BSH biosynthetic pathway in B. anthracis are characterized, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 Å resolution, identifies several active-site interactions important for the specific recognition of l- malate, but not other α-hydroxy acids, as the acceptor substrate.
Abstract: Bacillithiol (Cys-GlcN-malate, BSH) has recently been identified as a novel low-molecular weight thiol in Bacillus anthracis, Staphylococcus aureus, and several other Gram-positive bacteria lacking glutathione and mycothiol. We have now characterized the first two enzymes for the BSH biosynthetic pathway in B. anthracis, which combine to produce α-d-glucosaminyl l-malate (GlcN-malate) from UDP-GlcNAc and l-malate. The structure of the GlcNAc-malate intermediate has been determined, as have the kinetic parameters for the BaBshA glycosyltransferase (→GlcNAc-malate) and the BaBshB deacetylase (→GlcN-malate). BSH is one of only two natural products reported to contain a malyl glycoside, and the crystal structure of the BaBshA-UDP-malate ternary complex, determined in this work at 3.3 A resolution, identifies several active-site interactions important for the specific recognition of l-malate, but not other α-hydroxy acids, as the acceptor substrate. In sharp contrast to the structures reported for the GlcNAc-1-d-myo-inositol-3-phosphate synthase (MshA) apo and ternary complex forms, there is no major conformational change observed in the structures of the corresponding BaBshA forms. A mutant strain of B. anthracis deficient in the BshA glycosyltransferase fails to produce BSH, as predicted. This B. anthracis bshA locus (BA1558) has been identified in a transposon-site hybridization study as required for growth, sporulation, or germination [Day, W. A., Jr., Rasmussen, S. L., Carpenter, B. M., Peterson, S. N., and Friedlander, A. M. (2007) J. Bacteriol. 189, 3296-3301], suggesting that the biosynthesis of BSH could represent a target for the development of novel antimicrobials with broad-spectrum activity against Gram-positive pathogens like B. anthracis. The metabolites that function in thiol redox buffering and homeostasis in Bacillus are not well understood, and we present a composite picture based on this and other recent work.
TL;DR: It is demonstrated that electroporation mediated delivery enhanced the immunogenicity of DNA vaccines in nonhuman primates over 100-fold as compared to conventional intramuscular injection.
TL;DR: Supernatants from cultures of growing bacteria and constituents of the cell wall could serve as germinants of dormant B. subtilis and B. anthracis spores, and the presence of these molecules in the milieu as germination signals might be physiologically relevant.
Abstract: Certain Gram-positive bacteria—as exemplified by Bacillus anthracis, the causative agent of anthrax—can produce dormant and environmentally resistant spores under conditions of nutritional limitation. These spores exit from dormancy via the process of germination that is triggered by exposure to specific molecules.[1] While the precise chemical nature of these molecules, known as germinants, varies according to the organism, they are typically nutrients.[2] Recently, we reported that supernatants from cultures of growing bacteria and constituents of the cell wall could serve as germinants of dormant B. subtilis and B. anthracis spores.[3] Since fragments of the cell wall are released in the course of bacterial growth, the presence of these molecules in the milieu as germination signals might be physiologically relevant.
TL;DR: The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity, and the application of B. thuringiensis spores as internal controls further reduces false negative results.
Abstract: Background
Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples.
TL;DR: The cooperative action of all four GSLEs in vivo was investigated by combining in-frame deletion mutations to generate all possible double, triple, and quadruple GSLE mutant strains, greatly simplifying decontamination measures.
Abstract: Bacterial spores remain dormant and highly resistant to environmental stress until they germinate. Completion of germination requires the degradation of spore cortex peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four GSLEs: CwlJ1, CwlJ2, SleB, and SleL. In this study, the cooperative action of all four GSLEs in vivo was investigated by combining in-frame deletion mutations to generate all possible double, triple, and quadruple GSLE mutant strains. Analyses of mutant strains during spore germination and outgrowth combined observations of optical density loss, colony-producing ability, and quantitative identification of spore cortex fragments. The lytic transglycosylase SleB alone can facilitate enough digestion to allow full spore viability and generates a variety of small and large cortex fragments. CwlJ1 is also sufficient to allow completion of nutrient-triggered germination independently and is a major factor in Ca2+-dipicolinic acid (DPA)-triggered germination, but its enzymatic activity remains unidentified because its products are large and not readily released from the spore9s integuments. CwlJ2 contributes the least to overall cortex digestion but plays a subsidiary role in Ca2+-DPA-induced germination. SleL is an N-acetylglucosaminidase that plays the major role in hydrolyzing the large products of other GSLEs into small, rapidly released muropeptides. As the roles of these enzymes in cortex degradation become clearer, they will be targets for methods to stimulate premature germination of B. anthracis spores, greatly simplifying decontamination measures.
TL;DR: The role of insects in spreading anthrax infection, although not major, cannot be neglected from an epidemiological point of view and it is suggested that fly control should be considered as part of anthrax control programs.
Abstract: Anthrax is a disease of human beings and animals caused by the encapsulated, spore-forming, Bacillus anthracis. The potential role of insects in the spread of B. anthracis to humans and domestic animals during an anthrax outbreak has been confirmed by many studies. Among insect vectors, the house fly Musca domestica is considered a potential agent for disease transmission. In this study, laboratory-bred specimens of Musca domestica were infected by feeding on anthrax-infected rabbit carcass or anthrax contaminated blood, and the presence of anthrax spores in their spots (faeces and vomitus) was microbiologically monitored. It was also evaluated if the anthrax spores were able to germinate and replicate in the gut content of insects. These results confirmed the role of insects in spreading anthrax infection. This role, although not major, given the huge size of fly populations often associated with anthrax epidemics in domestic animals, cannot be neglected from an epidemiological point of view and suggest that fly control should be considered as part of anthrax control programs.
TL;DR: Investigating the surface adhesive properties of Bacillus subtilis and Bacillus anthracis spores indicated that both species were monopolar with a preponderance of electron-donating potential and mutant spores missing outer layers had different surface thermodynamic and transport properties as compared to wild type spores.