Computer-Intensive Statistical Procedures

The overall goal of this review is to summarize the current body of knowledge about the structure and function of major proteins of Bacillus anthracis and/or similar spore-forming organisms. B. anthracis is a key spore-forming biological threat agent, as well as human and animal Gram-positive bacterial pathogen. The structural information described here is limited to approximately the last 5 years. This information is then related to the role of the selected proteins in pathogenesis and in the possible development of novel vaccine and/or other antimicrobial agents against spore-forming organisms, including anthrax, a disease caused by B. anthracis.

Among spore-forming bacteria, Bacillus and Clostridium species are the predominant spore-forming bacilli that cause serious diseases. The biochemical properties and mechanism of catalysis of the novel spore germination protease that degrades small, acid-soluble proteins protecting DNA against damage, a cofactor independent phosphoglycerate mutase, NAD⁺ synthetase, and the three know B. anthracis toxins, protective antigen, lethal factor, and edema factor are described. The studies described in this work review and unify selected information critical for the prevention of microbial diseases such as anthrax. A strategy for the structure-guided development of new prophylactic and therapeutic agents is discussed.     

Bacillus species proteins involved in spore formation and degradation: from identification in the genome,to sequence analysis,and determination of function and structure

The members of Bacillus species are Gram-positive, ubiquitous spore-forming bacilli. Several genomic sequences have been made available during recent years, including Bacillus subtilis, a model organism among this genus, Bacillus anthracis, and their analyses provided a wealth of information about spore-forming bacteria. Some members of this species can cause serious diseases in livestock and humans. An important pathogen in this group of organisms is B. anthracis, which is the causative agent of anthrax. A summary of the B. subtilis genome information, based on the publicly released sequence, that allowed for the identification and characterization of new and novel proteins of this organism as well as similar proteins from other members of Bacillus species is provided. The primary goal for this work is to present a review of the genome sequence-identified genes that encode proteins involved in the sporulation, germination, and outgrowth processes. These three processes are essential for spore development and later its transformation into a vegetative cell. Additionally, for a few selected examples of the protein products of the identified genes, the application of bioinformatics and modeling tools is illustrated in order to determine their likely structure and function. Two three-dimensional models of the structures of such proteins, PrfA endonuclease and phosphatase PhoE, are presented together with the structure-based functional conclusions. The review of such studies provides an example of how the genomic sequence can be utilized in order to elucidate the structure and function of proteins, in particular proteins of the Bacillus species. Because only a limited number of proteins of Bacillus species organisms are involved in the synthesis and degradation of spores and have been characterized to date, this genome-based analysis may provide new insights into the developmental processes of bacterial organism.     

A novel FtsZ-like protein is involved in replication of the anthrax toxin-encoding pXO1 plasmid in Bacillus anthracis

Plasmid pXO1 encodes the tripartite anthrax toxin, which is the major virulence factor of Bacillus anthracis. In spite of the important role of pXO1 in anthrax pathogenesis, very little is known about its replication and maintenance in B. anthracis. We cloned a 5-kb region of the pXO1 plasmid into an Escherichia coli vector and showed that this plasmid can replicate when introduced into B. anthracis. Mutational analysis showed that open reading frame 45 (repX) of pXO1 was required for the replication of the miniplasmid in B. anthracis. Interestingly, repX showed limited homology to bacterial FtsZ proteins that are involved in cell division. A mutation in the predicted GTP binding domain of RepX abolished its replication activity. Genes almost identical to repX are contained on several megaplasmids in members of the Bacillus cereus group, including a B. cereus strain that causes an anthrax-like disease. Our results identify a novel group of FtsZ-related initiator proteins that are required for the replication of virulence plasmids in B. anthracis and possibly in related organisms. Such replication proteins may provide novel drug targets for the elimination of plasmids encoding the anthrax toxin and other virulence factors.     

Identification of Bacillus anthracis proteins associated with germination and early outgrowth by proteomic profiling of anthrax spores

The use of anthrax spores as a bioweapon has spurred efforts aimed at identifying key proteins expressed in Bacillus anthracis. Because spore germination and outgrowth occur prior to and are required for disease manifestations, blocking germination and early outgrowth with novel vaccines or inhibitors targeting critical B. anthracis germination and outgrowth-associated factors is a promising strategy in mitigating bioterror. By screening 587 paired protein spots that were isolated from dormant and germinating anthrax spores, respectively, we identified 10 spore proteins with statistically significant germination-associated increases and decreases. It is likely that proteins whose levels change during germination may play key roles in the germination and outgrowth processes, and they should be listed as priority targets for development of prophylactic and therapeutic agents against anthrax. The 31 new proteins identified in this study also complement an emerging proteomic database of B. anthracis.     

Poly-γ-d-glutamic acid and protective antigen conjugate vaccines induce functional antibodies against the protective antigen and capsule of Bacillus anthracis in guinea-pigs and rabbits

Anthrax is a lethal infectious disease caused by the spore-forming Bacillus anthracis . The two major virulence factors of B. anthracis are exotoxin and the poly-γ- d -glutamic acid (PGA) capsule. The three components of the exotoxin, protective antigen (PA), lethal factor and edema factor act in a binary combination, which results in massive edema and organ failure in the progress of anthrax disease. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Because PA can elicit a protective immune response, it has been a target of the anthrax vaccine. In addition to PA, efforts have been made to include PGA as a component of the anthrax vaccine. In this study, we report that PA–PGA conjugates induce expressions of anti-PA, anti-PGA and toxin-neutralizing antibodies in guinea-pigs and completely protect guinea-pigs against a 50 × LD₅₀ challenge with fully virulent B. anthracis spores. Polyclonal rabbit antisera produced against either PA or ovalbumin conjugated to a PGA-15mer offer a partial passive protection to guinea-pigs against B. anthracis infection, indicating that anti-PGA antibodies play a protective role. Our results demonstrate that PA–PGA conjugate vaccines are effective in the guinea-pig model, in addition to the previously reported mouse model.     

Bacillus Species Proteins Involved in Spore Formation and Degradation: From Identification in the Genome,to Sequence Analysis,and Determination of Function and Structure

The members of Bacillus species are Gram-positive, ubiquitous spore-forming bacilli. Several genomic sequences have been made available during recent years, including Bacillus subtilis, a model organism among this genus, Bacillus anthracis, and their analyses provided a wealth of information about spore-forming bacteria. Some members of this species can cause serious diseases in livestock and humans. An important pathogen in this group of organisms is B. anthracis, which is the causative agent of anthrax. A summary of the B. subtilis genome information, based on the publicly released sequence, that allowed for the identification and characterization of new and novel proteins of this organism as well as similar proteins from other members of Bacillus species is provided. The primary goal for this work is to present a review of the genome sequence-identified genes that encode proteins involved in the sporulation, germination, and outgrowth processes. These three processes are essential for spore development and later its transformation into a vegetative cell. Additionally, for a few selected examples of the protein products of the identified genes, the application of bioinformatics and modeling tools is illustrated in order to determine their likely structure and function. Two three-dimensional models of the structures of such proteins, PrfA endonuclease and phosphatase PhoE, are presented together with the structure-based functional conclusions. The review of such studies provides an example of how the genomic sequence can be utilized in order to elucidate the structure and function of proteins, in particular proteins of the Bacillus species. Because only a limited number of proteins of Bacillus species organisms are involved in the synthesis and degradation of spores and have been characterized to date, this genome-based analysis may provide new insights into the developmental processes of bacterial organism.     

Antigen delivery by attenuated Bacillus anthracis: new prospects in veterinary vaccines

This report summarizes the recent investigations on the use of Bacillus anthracis as a live vector for delivery of antigens. Recombinant strains were constructed by engineering the current live Sterne vaccine. This vaccine, used to prevent anthrax in cattle, causes side-effects due to anthrax toxin activities. Bacteria producing a genetically detoxified toxin factor were devoid of lethal effects and were as protective as the Sterne strain against experimental anthrax. Moreover, B. anthracis expressing a foreign antigen controlled by an in vivo inducible promoter were able to generate either antibody or cellular protective responses against heterologous diseases.     

Interferon-inducible CXC chemokines directly contribute to host defense against inhalational anthrax in a murine model of infection

Chemokines have been found to exert direct, defensin-like antimicrobial activity in vitro, suggesting that, in addition to orchestrating cellular accumulation and activation, chemokines may contribute directly to the innate host response against infection. No observations have been made, however, demonstrating direct chemokine-mediated promotion of host defense in vivo. Here, we show that the murine interferon-inducible CXC chemokines CXCL9, CXCL10, and CXCL11 each exert direct antimicrobial effects in vitro against Bacillus anthracis Sterne strain spores and bacilli including disruptions in spore germination and marked reductions in spore and bacilli viability as assessed using CFU determination and a fluorometric assay of metabolic activity. Similar chemokine-mediated antimicrobial activity was also observed against fully virulent Ames strain spores and encapsulated bacilli. Moreover, antibody-mediated neutralization of these CXC chemokines in vivo was found to significantly increase host susceptibility to pulmonary B. anthracis infection in a murine model of inhalational anthrax with disease progression characterized by systemic bacterial dissemination, toxemia, and host death. Neutralization of the shared chemokine receptor CXCR3, responsible for mediating cellular recruitment in response to CXCL9, CXCL10, and CXCL11, was not found to increase host susceptibility to inhalational anthrax. Taken together, our data demonstrate a novel, receptor-independent antimicrobial role for the interferon-inducible CXC chemokines in pulmonary innate immunity in vivo. These data also support an immunomodulatory approach for effectively treating and/or preventing pulmonary B. anthracis infection, as well as infections caused by pathogenic and potentially, multi-drug resistant bacteria including other spore-forming organisms.     

Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins.

Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic "A-B" paradigm for protein toxins. The binary toxins produced by B. anthracis, B. cereus, C. botulinum, C. difficile, C. perfringens, and C. spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects. The "B" components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution. Following release of a 20-kDa N-terminal peptide, the activated "B" components form homoheptameric rings that subsequently dock with an "A" component(s) on the cell surface. By following an acidified endosomal route and translocation into the cytosol, "A" molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases. Recently, B. anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B. anthracis. This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine. The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria.     

Genotyping of Bacillus cereus strains by microarray-based resequencing

The ability to distinguish microbial pathogens from closely related but nonpathogenic strains is key to understanding the population biology of these organisms. In this regard, Bacillus anthracis, the bacterium that causes inhalational anthrax, is of interest because it is closely related and often difficult to distinguish from other members of the B. cereus group that can cause diverse diseases. We employed custom-designed resequencing arrays (RAs) based on the genome sequence of Bacillus anthracis to generate 422 kb of genomic sequence from a panel of 41 Bacillus cereus sensu lato strains. Here we show that RAs represent a "one reaction" genotyping technology with the ability to discriminate between highly similar B. anthracis isolates and more divergent strains of the B. cereus s.l. Clade 1. Our data show that RAs can be an efficient genotyping technology for pre-screening the genetic diversity of large strain collections to selected the best candidates for whole genome sequencing.     

Bacillus anthracis secretes proteins that mediate heme acquisition from hemoglobin

Acquisition of iron is necessary for the replication of nearly all bacterial pathogens; however, iron of vertebrate hosts is mostly sequestered by heme and bound to hemoglobin within red blood cells. In Bacillus anthracis, the spore-forming agent of anthrax, the mechanisms of iron scavenging from hemoglobin are unknown. We report here that B. anthracis secretes IsdX1 and IsdX2, two NEAT domain proteins, to remove heme from hemoglobin, thereby retrieving iron for bacterial growth. Unlike other Gram-positive bacteria, which rely on cell wall anchored Isd proteins for heme scavenging, B. anthracis seems to have also evolved NEAT domain proteins in the extracellular milieu and in the bacterial envelope to provide for the passage of heme.     

Identification and characterization of clinical Bacillus spp. isolates phenotypically similar to Bacillus anthracis

Bacillus anthracis, the etiological agent of anthrax, is a gram-positive, spore-forming rod, with colonies exhibiting a unique ground-glass appearance, and lacking hemolysis and motility. In addition to these phenotypes, several others traits are characteristic of B. anthracis such as susceptibility to gamma phage, the presence of two virulence plasmids (pX01 and pX02), and specific cell wall and capsular antigens that are commonly detected by direct fluorescent-antibody assays. We report on the identification and characterization of 14 Bacillus megaterium and four Bacillus sp. clinical isolates that are nonhemolytic, nonmotile, and produce a capsule antigenically similar to B. anthracis. This work furthers our understanding of Bacillus diversity and the limitations of the assays and phenotypes that are used to differentiate species in this genus. Further work is necessary to understand whether these strains are opportunistic pathogens or just contaminates.     

A viral nanoparticle with dual function as an anthrax antitoxin and vaccine

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.     

Bacillus anthracis toxins inhibit human neutrophil NADPH oxidase activity

Bacillus anthracis, the causative agent of anthrax, is a Gram-positive, spore-forming bacterium. B. anthracis virulence is ascribed mainly to a secreted tripartite AB-type toxin composed of three proteins designated protective Ag (PA), lethal factor, and edema factor. PA assembles with the enzymatic portions of the toxin, the metalloprotease lethal factor, and/or the adenylate cyclase edema factor, to generate lethal toxin (LTx) and edema toxin (ETx), respectively. These toxins enter cells through the interaction of PA with specific cell surface receptors. The anthrax toxins act to suppress innate immune responses and, given the importance of human neutrophils in innate immunity, they are likely relevant targets of the anthrax toxin. We have investigated in detail the effects of B. anthracis toxin on superoxide production by primary human neutrophils. Both LTx and ETx exhibit distinct inhibitory effects on fMLP (and C5a) receptor-mediated superoxide production, but have no effect on PMA nonreceptor-dependent superoxide production. These inhibitory effects cannot be accounted for by induction of neutrophil death, or by changes in stimulatory receptor levels. Analysis of NADPH oxidase regulation using whole cell and cell-free systems suggests that the toxins do not exert direct effects on NADPH oxidase components, but rather act via their respective effects, inhibition of MAPK signaling (LTx), and elevation of intracellular cAMP (ETx), to inhibit upstream signaling components mediating NADPH oxidase assembly and/or activation. Our results demonstrate that anthrax toxins effectively suppress human neutrophil-mediated innate immunity by inhibiting their ability to generate superoxide for bacterial killing.     

Surface protein IsdC and Sortase B are required for heme-iron scavenging of Bacillus anthracis

Bacillus anthracis, the spore-forming agent of anthrax, requires iron for growth and is capable of scavenging heme-iron during infection. We show here that the B. anthracis iron-regulated surface determinants (isd) locus encompasses isdC, specifying a heme-iron binding surface protein. Anchoring of IsdC to the cell wall envelopes of vegetative bacilli requires srtB, which encodes sortase B. Purified sortase B cleaves IsdC between the threonine and the glycine of its NPKTG motif sorting signal. B. anthracis variants lacking either isdC or srtB display defects in heme-iron scavenging, suggesting that IsdC binding to heme-iron in the cell wall envelope contributes to bacterial uptake of heme.     

Bacillus anthracis multiplication, persistence, and genetic exchange in the rhizosphere of grass plants

Bacillus anthracis, the causative agent of anthrax, is known for its rapid proliferation and dissemination in mammalian hosts. In contrast, little information exists regarding the lifestyle of this important pathogen outside of the host. Considering that Bacillus species, including close relatives of B. anthracis, are saprophytic soil organisms, we investigated the capacity of B. anthracis spores to germinate in the rhizosphere and to establish populations of vegetative cells that could support horizontal gene transfer in the soil. Using a simple grass plant-soil model system, we show that B. anthracis strains germinate on and around roots, growing in characteristic long filaments. From 2 to 4 days postinoculation, approximately one-half of the B. anthracis CFU recovered from soil containing grass seedlings arose from heat-sensitive organisms, while B. anthracis CFU retrieved from soil without plants consisted of primarily heat-resistant spores. Co-inoculation of the plant-soil system with spores of a fertile B. anthracis strain carrying the tetracycline resistance plasmid pBC16 and a selectable B. anthracis recipient strain resulted in transfer of pBC16 from the donor to the recipient as early as 3 days postinoculation. Our findings demonstrate that B. anthracis can survive as a saprophyte outside of the host. The data suggest that horizontal gene transfer in the rhizosphere of grass plants may play a role in the evolution of the Bacillus cereus group species.     

Genetic diversity in the protective antigen gene of Bacillus anthracis

Bacillus anthracis is a gram-positive spore-forming bacterium that causes the disease anthrax. The anthrax toxin contains three components, including the protective antigen (PA), which binds to eucaryotic cell surface receptors and mediates the transport of toxins into the cell. In this study, the entire 2,294-nucleotide protective antigen gene (pag) was sequenced from 26 of the most diverse B. anthracis strains to identify potential variation in the toxin and to further our understanding of B. anthracis evolution. Five point mutations, three synonymous and two missense, were identified. These differences correspond to six different haploid types, which translate into three different amino acid sequences. The two amino acid changes were shown to be located in an area near a highly antigenic region critical to lethal factor binding. Nested primers were used to amplify and sequence this same region of pag from necropsy samples taken from victims of the 1979 Sverdlovsk incident. This investigation uncovered five different alleles among the strains present in the tissues, including two not seen in the 26-sample survey. One of these two alleles included a novel missense mutation, again located just adjacent to the highly antigenic region. Phylogenetic (cladistic) analysis of the pag corresponded with previous strain grouping based on chromosomal variation, suggesting that plasmid evolution in B. anthracis has occurred with little or no horizontal transfer between the different strains.     

Quantitative immunofluorescence studies of the serology of Bacillus anthracis spores.

A fluorescein-conjugated antibody against formalin-inactivated spores of Bacillus anthracis Vollum reacted only weakly with a variety of Bacillus species in microfluorometric immunofluorescence assays. A conjugated antibody against spores of B. anthracis Sterne showed little affinity for spores of several B. anthracis isolates including B. anthracis Vollum, indicating that more than one anthrax spore serotype exists.     

Retrocyclins kill bacilli and germinating spores of Bacillus anthracis and inactivate anthrax lethal toxin

Theta-defensins are cyclic octadecapeptides encoded by the modified alpha-defensin genes of certain nonhuman primates. The recent demonstration that human alpha-defensins could prevent deleterious effects of anthrax lethal toxin in vitro and in vivo led us to examine the effects of theta-defensins on Bacillus anthracis (Sterne). We tested rhesus theta-defensins 1-3, retrocyclins 1-3, and several analogues of RC-1. Low concentrations of theta-defensins not only killed vegetative cells of B. anthracis (Sterne) and rendered their germinating spores nonviable, they also inactivated the enzymatic activity of anthrax lethal factor and protected murine RAW-264.7 cells from lethal toxin, a mixture of lethal factor and protective antigen. Structure-function studies indicated that the cyclic backbone, intramolecular tri-disulfide ladder, and arginine residues of theta-defensins contributed substantially to these protective effects. Surface plasmon resonance studies showed that retrocyclins bound the lethal factor rapidly and with high affinity. Retrocyclin-mediated inhibition of the enzymatic activity of lethal factor increased substantially if the enzyme and peptide were preincubated before substrate was added. The temporal discrepancy between the rapidity of binding and the slowly progressive extent of lethal factor inhibition suggest that post-binding events, perhaps in situ oligomerization, contribute to the antitoxic properties of retrocyclins. Overall, these findings suggest that theta-defensins provide molecular templates that could be used to create novel agents effective against B. anthracis and its toxins.