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Gene expression profiling of human alveolar macrophages infected by B. anthracis spores

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE14390 | GEO | 2009/09/07

SECONDARY ACCESSION(S): PRJNA111363

REPOSITORIES: GEO

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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:Bacterial spores play an important role in disease initiation, transmission and persistence. The outermost spore layer, the exosporium, is important as it is the first point of contact between the spore and the environment and may be involved in spore adherence, protection and germination. Clostridium sordellii is a highly lethal, spore forming pathogen that causes soft-tissue infections, enteritis and toxic-shock syndrome. Despite the importance of C. sordellii spores in disease, spore proteins from this bacterium have not been defined or interrogated functionally. In this study, we identified the C. sordellii outer spore proteome and two of the identified proteins, CSA and CSB, were characterised using a genetic and phenotypic approach. Both proteins were essential for the correct formation and positioning of the C. sordellii spore coat and exosporium. The absence of CSA reduced sporulation levels and increased spore sensitivity to heat, sodium hydroxide and hydrochloric acid. By comparison, CSB was required for normal levels of spore adherence to cervical, but not vaginal, cells, with csb mutant spores having increased adherence properties. The establishment of a mouse infection model of the gastrointestinal tract for C. sordellii allowed the role of CSA and CSB to be interrogated in an infected host. Following the oral administration of spores to mice, the wild-type strain efficiently colonized the gastrointestinal tract, with the peak of bacterial numbers occurring at one day post-infection. Colonization was reduced by two logs at four days post-infection. By comparison, mice infected with the csb mutant did not show a reduction in bacterial numbers. The absence of CSB therefore allows the csb mutant to persist within the gastrointestinal tract. We conclude that C. sordellii outer spore proteins are important for the structural and functional integrity of spores, and for colonization and persistence during infection. Finally, Clostridium difficile, Bacillus cereus and Bacillus anthracis encode proteins with homology to CSA and CSB but these bacteria produce spores that are structurally dissimilar to those of C. sordellii, and the function of the proteins in these hosts is different to that in C. sordellii. These findings suggest that, despite their homology, spore proteins can have variable functions in different bacterial species which highlights the necessity of studying each spore protein in the cognate species from which it originates.

Project description:Description Bacillus subtilis forms highly resistant, metabolically inactive spores upon nutrient limitation. These endospores pose challenges to the food and medical sectors. Spores reactivate their metabolism upon contact with germinants through germination and outgrowth, and then develop into vegetative cells. However, the mechanism of the activation of the molecular machinery that triggers spore germination and outgrowth is unclear. To gain further insight into spore germination and outgrowth, the transcriptome and proteome changes during Bacillus subtilis spore conversion to vegetative cells were analyzed. The transcriptome analysis also allowed us to trace the different functional groups of genes expressed during this conversion. For each time-point sampled, the change in the spore proteome was quantitatively monitored relative to the reference proteome of 15N metabolically labelled vegetative cells. Of the quantified proteins, 60 percent are common to vegetative cells and spores, indicating that spores have a minimal set of proteins sufficient for the resumption of metabolism upon completion of germination. The shared proteins thus represent the most basic survival kit for spore-based life. Until the phase transition, defined as the completion of germination, we observed no significant change in the proteome or the transcriptome. Our analysis identified 34 abundant mRNA transcripts in the dormant spores, at least 31 of which were rapidly degraded after the phase transition. We observed 3152 differentially expressed genes, and demonstrated with our mass spectrometry analyses the differential expression of 323 proteins. Our data show that 173 proteins from dormant spores, both proteins unique to spores and proteins shared with vegetative cells, are lost after completion of germination. Further analysis is required to functionally interpret the observed protein loss. The observed diverse timing of the synthesis of different protein sets reveals a putative core-strategy of the revival of life starting from the B. subtilis spore.

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Gene expression profiling of human alveolar macrophages infected by B. anthracis spores

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