Project description:The spore forming pathogen Bacillus anthracis is the etiologic agent of anthrax in humans and animals. It cycles through infected hosts as vegetative cells and is eventually introduced into the environment where it generates an endospore resistant to many harsh conditions. The endospores are subsequently ingested by the next host to begin the next cycle. Outbreaks of anthrax occur regularly worldwide in wildlife and livestock, and the potential for human infection exists whenever humans encounter infected animals. It is also possible to encounter intentional releases of anthrax spores, as was the case in October 2001. Consequently, it is important to be able to rapidly establish the provenance of infectious strains of B. anthracis. Here, we compare protein expression in seven low-passage wild isolates and four laboratory strains of B. anthracis grown under identical conditions using LC-MS/MS proteomic analysis. Of the 1,023 total identified proteins, 96 had significant abundance differences between wild and laboratory strains. Of those, 28 proteins directly related to sporulation were upregulated in wild isolates, with expression driven by Spo0A, CodY, and AbrB/ScoC. In addition, we observed evidence of changes in cell division and fatty acid biosynthesis between the two classes of strains, despite being grown under identical experimental conditions. These results suggest wild B. anthracis cells are more highly tuned to sporulate than their laboratory cousins, and this difference should be exploited as a method to differentiate between laboratory adapted cultures and low passage wild strains isolated during an anthrax outbreak. This knowledge should distinguish between intentional releases and exposure to strains in nature providing a basis for the type of response by public health officials and investigators.
Project description:The aim of the study was to carry out a CGH study utilizing a set of 39 diverse Bacillus isolates. Thirty four B. cereus and five B. anthracis strains and isolates were chosen so as to represent different lineages based on previous characterizations, including MLEE and MLST (Helgason, Okstad et al. 2000; Helgason, Tourasse et al. 2004). They represent the spectrum of B. cereus phenotypic diversity by including soil, dairy and periodontal isolates in addition to virulent B. anthracis strains.
Project description:The goal of this project was to screen soil samples for bacteria that may harbor B. anthracis virulence-associated genes (VAGs). There is currently no information about the prevalence of these types of organisms in the environment. Due to increased environmental monitoring of select agents by programs such as BioWatch and biodetection systems in place at the United States Post Offices and Department of State locations, it has become critical that we not only better understand the natural range of B. anthracis but also how widespread B. anthracis virulence genes are in environmental communities. Naturally occurring isolates containing the B. anthracis virulence genes could generate false-positive results in tests that detect the anthrax toxins, capsule or their associated genes. Understanding the true diversity and pathogenic potential of Bacillus spp. and particularly the B. cereus group is crucial not only in terms of understanding data from environmental monitoring but also diagnosing patients with clinical presentations similar to anthrax in the future. Severe and fatal disease caused by strains similar to B. anthracis could unnecessarily initiate emergency responses if anthrax was incorrectly suspected. Conversely, these strains may be used as bioterror agents requiring science-based responses; presently our limited understanding of these organisms does not permit data-driven decision making. We have investigated 700 aerobic sporoform soil isolates obtained from two areas in the Southwest of the US. Soil samples from the first site had been taken from public access land approximately 50 meters across from the work site of a fatal pneumonia case in a welding factory. This took place in year 2003 when B. cereus was isolated from a metal worker. The second site was targeted because of a recent case involving a deceased mule suspected to have died of a B. anthracis infection. Soil samples were initially analyzed at the CDC. Isolates were obtained by heating the soil at 65 degrees Celcius for 30 minutes followed by plating on agar media. All isolates were screened by PCR for the presence of B. anthracis genomic traits such as toxin genes (cya, lef and pag) as well as chromosomal markers. All isolates were also tested for their hemolytic activity as well as phage sensitivity.
Project description:Bacillus anthracis is a spore-forming bacterium that causes devastating infections and has been used as a bioterror agent. This pathogen can survive hostile environments through the signaling activity of two-component systems, which couple environmental sensing with transcriptional activation to initiate a coordinated response to stress. In this work, we describe the identification of a two-component system, EdsRS, which mediates the B. anthracis response to the antimicrobial compound targocil. Targocil is a cell envelope-targeting compound that is toxic to B. anthracis at high concentrations. Exposure to targocil causes damage to the cellular barrier and activates EdsRS to induce expression of a previously uncharacterized cardiolipin synthase, which we have named ClsT. Both EdsRS and ClsT are required for protection against targocil-dependent damage. Induction of clsT by EdsRS during targocil treatment results in an increase in cardiolipin levels, which protects B. anthracis from envelope damage. Together, these results reveal a two-component system signaling response to an envelope-targeting antimicrobial induces production of a phospholipid associated with stabilization of the membrane. Cardiolipin is then used to repair envelope damage and promote B. anthracis viability.
Project description:Bacillus anthracis causes anthrax infections in mammals. Large-scale mortality resulting from the intentional release of B. anthracis spores represents a potential bioterrorism threat. Inhalational anthrax almost invariably proceeds to fatal systemic infection, characterized by massive bacteremia. A better understanding of host-pathogen interactions is urgently needed for effective treatment of this lethal disease. However, virulence mechanisms used by B. anthracis to survive and multiply in human blood are not completely understood. Identification of genes that are differentially expressed during the growth of B. anthracis in human serum can elucidate how this pathogen successfully colonizes the bloodstream. We compared the transcriptional profile of B. anthracis growing in heat-inactivated human serum to that in LB medium. Genes involved in the biosynthesis of purines, certain amino acids and riboflavin and lipid metabolism, genes encoding ABC transporters, respiratory enzymes and several genes with hypothetical function were identified as being upregulated during growth in serum.
Project description:One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. This Series contains the NimbleGen array data only (no next-generation sequencing data). B. anthracis DNA was spiked at 6 different concentrations (1, 10, 100, 1000, 10000 and 100000 genome copies) into 1 ng of background nucleic acids extracted either from a soil sample or from an aerosol (air filter) sample. Two replicates of each combination of B. anthracis copy number and background sample were analyzed.
Project description:One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing.