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: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. Eighty-four query strains were investigated in this study, with each query strain hybridized against the reference strain, Sterne. Two dye-swap experiments were performed with seventeen strains, for a total of four hybridizations per query strain. The other strains have a single dye experiment, for a total of two hybridizations per query strain. Each 70mer oligo spotted on the B. cereus species microarray is spotted once. Positive controls on the array consist of oligos designed from the sequenced reference genome, Sterne, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana.

Project description:Bacillus anthracis is a gram-positive, aerobic, spore-forming, rod-shaped bacterium which has recently been used as an agent of bioterrorism. Because there is a significant delay between the initial contact of the spore with the host and clinical evidence of disease, there appears to be temporary containment of the pathogen by the innate immune system. Contact with the human alveolar macrophage (HAM) plays a key role in the innate immune response to B. anthracis spores. Therefore, the early macrophage response to anthrax exposure is important in understanding the pathogenesis of this disease. The majority of genes modulated by spores were upregulated, and a lesser number were downregulated. The data was subjected to Ingenuity Pathway analysis, the Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis, and the Promoter Analysis and Interaction Network Toolset (PAINT). Among the upregulated genes, we identified a group of chemokine ligands, apoptosis genes and, interestingly, keratin filament genes. Central hubs regulating the activated genes were TNF-ï¡, NF-ÎºB and their ligands/receptors. Other activated genes included IL-1ï¡ and IL-18. RNA for these, and several additional cytokines including IL-6, IL-1ï¢, IP-10 and GM-CSF, were differentially expressed from 1.6- to 27-fold. The microarray cytokine data is consistent with our previously published findings which demonstrated that there was 4- to 43-fold induction of these cytokines at the transcriptional and translational levels as determined by RNase protection assays and ELISA. The PAINT analysis revealed that the majority of the genes affected by spores contain the binding site for c-Rel, a member of the NF-ÎºB family of transcription factors. Other transcription regulatory elements contained in many of the upregulated genes were c-Myb, CP2, Barbie Box, E2F and CRE-BP1. This study is the first detailed microarray analysis to describe the HAM response to B. anthracis. Experiment Overall Design: In this paper, we exposed HAM to B. anthracis Sterne spores at an MOI of 1 for 6 hours. RNA was extracted from HAM and analyzed by the Affymetrix Human Genome U133 Plus 2.0 Array. The transcriptional profile of Bacillus anthracis spore-treated HAM was compared with mock infected cells, and differentially expressed genes were identified.

Project description:Bacillus anthracis is a gram-positive, aerobic, spore-forming, rod-shaped bacterium which has recently been used as an agent of bioterrorism. Because there is a significant delay between the initial contact of the spore with the host and clinical evidence of disease, there appears to be temporary containment of the pathogen by the innate immune system. Contact with the human alveolar macrophage (HAM) plays a key role in the innate immune response to B. anthracis spores. Therefore, the early macrophage response to anthrax exposure is important in understanding the pathogenesis of this disease. The majority of genes modulated by spores were upregulated, and a lesser number were downregulated. The data was subjected to Ingenuity Pathway analysis, the Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis, and the Promoter Analysis and Interaction Network Toolset (PAINT). Among the upregulated genes, we identified a group of chemokine ligands, apoptosis genes and, interestingly, keratin filament genes. Central hubs regulating the activated genes were TNF-alpha, NF-κB and their ligands/receptors. Other activated genes included IL-1alpha and IL-18. RNA for these, and several additional cytokines including IL-6, IL-1gamma, IP-10 and GM-CSF, were differentially expressed from 1.6- to 27-fold. The microarray cytokine data is consistent with our previously published findings which demonstrated that there was 4- to 43-fold induction of these cytokines at the transcriptional and translational levels as determined by RNase protection assays and ELISA. The PAINT analysis revealed that the majority of the genes affected by spores contain the binding site for c-Rel, a member of the NF-κB family of transcription factors. Other transcription regulatory elements contained in many of the upregulated genes were c-Myb, CP2, Barbie Box, E2F and CRE-BP1. This study is the first detailed microarray analysis to describe the HAM response to B. anthracis.

Project description:Purpose: Small regulatory RNAs (sRNAs) are short transcripts that base-pair to mRNA targets or interact with regulatory proteins. sRNA function has been extensively studied in Gram-negative bacteria; comparatively less is known about sRNAs in Firmicutes. Here we investigate two sRNAs encoded by the virulence plasmid pXO1 of Bacillus anthracis, the causative agent of anthrax. We designated the sRNAs as as “XrrA” and “XrrB” (for pXO1-encoded regulatory RNA). We constructed deletion mutants ∆xrrA, ∆xrrB, and ∆xrrA∆xrrB in an Ames strain background and compared gene expression of these mutants to that of the Ames-derived parent strain using RNA-seq. Methods: Total RNA from Ames strains UTA37 (Parent), UTA38 (∆xrrB), UTA39 (∆xrrA), UTA41 (∆xrrA∆xrrB) was isolated from cultures grown in CA-CO2 in triplicate, using saturated phenol: chloroform extraction. One microgram of pure and quality-checked RNA from each sample was subjected to Illumina sequencing by synthesis using a NextSeq550 sequencer. Sequencing by synthesis was performed to generate 75bp paired-end reads. Two 130M-read sequencing runs (Run1 and Run2) were performed and an average of 31M reads per sample were obtained. Results: All bioinformatic analysis was performed using the publicly available Galaxy web resource (https://usegalaxy.org/). Comparison of the transcriptomes of a virulent Ames-derived strain to isogenic sRNA-null mutants revealed multiple 4.0- to >100-fold differences in gene expression. Deletion of xrrA resulted in 50 transcripts showing a ≥ 4.0-fold change in expression compared to the parent strain. In contrast, deletion of xrrB led to one transcript having a ≥ 4.0-fold change in expression compared to the parent strain. In the ∆xrrA∆xrrB mutant, levels of 116 transcripts were affected with a fold-change of ≥ 4.0. Many sRNA-regulated targets were chromosome genes associated with branched-chain amino acid metabolism, proteolysis, and transmembrane transport. Conclusions: In this work, we provide experimental evidence for sRNA-mediated regulation in the mammalian pathogen B. anthracis. XrrA and XrrB are the first regulatory RNAs examined in B. anthracis and are shown here to have multiple effects on gene expression in this pathogen. The data suggest distinct regulatory roles for XrrA, as well as some functional overlap between XrrA and XrrB.

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Identification of Bacillus anthracis genes differentially expressed in human serum

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