Dataset Information

Effect of over expressing protective antigen on global gene transcription in Bacillus anthracis BH500.

ABSTRACT: Protective antigen (PA) of Bacillus anthracis is being considered as a vaccine candidate against anthrax and its production has been explored in several heterologous host systems. Since the systems tested introduced adverse issues such as inclusion body formation and endotoxin contamination, the production from B. anthracis is considered as a preferred method. The present study examines the effect of PA expression on the metabolism of B. anthracis producing strain, BH500, by comparing it with a control strain carrying an empty plasmid. The strains were grown in a bioreactor and RNA-seq analysis of the producing and non-producing strain was conducted. Among the observed differences, the strain expressing rPA had increased transcription of sigL, the gene encoding RNA polymerase ?54, sigB, the general stress transcription factor gene and its regulators rsbW and rsbV, as well as the global regulatory repressor ctsR. There were also decreased expression of intracellular heat stress related genes such as groL, groES, hslO, dnaJ, and dnaK and increased expression of extracellular chaperons csaA and prsA2. Also, major central metabolism genes belonging to TCA, glycolysis, PPP, and amino acids biosynthesis were up-regulated in the PA-producing strain during the lag phase and down-regulated in the log and late-log phases, which was associated with decreased specific growth rates. The information obtained from this study may guide genetic modification of B. anthracis to improve PA production.

SUBMITTER: Sharma AK

PROVIDER: S-EPMC6208434 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Effect of over expressing protective antigen on global gene transcription in Bacillus anthracis BH500.

Sharma Ashish K AK Leppla Stephen H SH Pomerantsev Andrei P AP Shiloach Joseph J

Scientific reports 20181031 1

Protective antigen (PA) of Bacillus anthracis is being considered as a vaccine candidate against anthrax and its production has been explored in several heterologous host systems. Since the systems tested introduced adverse issues such as inclusion body formation and endotoxin contamination, the production from B. anthracis is considered as a preferred method. The present study examines the effect of PA expression on the metabolism of B. anthracis producing strain, BH500, by comparing it with a ...[more]

PMID: 30382110

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Project description:BACKGROUND: Anthrax is caused by Bacillus anthracis that produce two exotoxins, lethal toxin and edema toxin. The lethal toxin is composed of the lethal factor (LF) complexed with the cell binding protective antigen (PA83, 83 kDa). Likewise, the edema factor (EF) binds to the PA83 to form the edema toxin. Once PA83 is bound to the host cell surface, a furin-like protease cleaves the full-length, inactive protein into 63 kDa and 20 kDa antigens (PA63 and PA20). PA63 forms a heptamer and is internalized via receptor mediated endocytosis forming a protease-stable pore, which allows EF and LF to enter the cell and exert their toxic effects.Both proteolytically cleaved protective antigens (PA63 and PA20 fragments) are found in the blood of infected animals. The 63 kDa protective antigen PA63 fragment has been thoroughly studied while little is known about the PA20. METHODS: In this study we examined the role of PA20 using high throughput gene expression analysis of human peripheral blood mononuclear cells (PBMC) exposed to the PA20. We constructed a PA mutant in which a Factor Xa proteolytic recognition site was genetically engineered into the protective antigen PA83 to obtain PA20 using limited digestion of this recombinant PA83 with trypsin. RESULTS: Global gene expression response studies indicated modulation of various immune functions and showed gene patterns indicative of apoptosis via the Fas pathway in a subset of the lymphoid cells. This finding was extended to include observations of increased Caspase-3 enzymatic activity and the identification of increases in the population of apoptotic, but not necrotic cells, based on differential staining methods. We identified a list of approximately 40 inflammatory mediators and heat-shock proteins that were altered similarly upon exposure of PBMC to either rPA20 or B. anthracis spores/vegetative cells. CONCLUSION: This study shows that the PA20 has an effect on human peripheral blood leukocytes and can induce apoptosis in the absence of other PA components.

Dataset Information

Effect of over expressing protective antigen on global gene transcription in Bacillus anthracis BH500.

Publications

Effect of over expressing protective antigen on global gene transcription in Bacillus anthracis BH500.

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