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A Bacillus anthracis RNA binding protein post-transcriptionally regulates two component signaling through RNA turnover [RIP-Seq]

ABSTRACT: Formaldehyde cross-linking RNA immunoprecipitation coupled with illumina sequencing (fRIP-seq) was performed to pinpoint the direct RNA targets of KrrA using a FLAG-tagged KrrA construct that is driven by a constitutive promoter Plgt (pOS1.PlgtkrrA-FLAG).

ORGANISM(S): Bacillus anthracis

PROVIDER: GSE193211 | GEO | 2022/01/10

REPOSITORIES: GEO

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Project description:BackgroundAnthrax 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.MethodsIn 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.ResultsGlobal 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.ConclusionThis study shows that the PA20 has an effect on human peripheral blood leukocytes and can induce apoptosis in the absence of other PA components.

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A Bacillus anthracis RNA binding protein post-transcriptionally regulates two component signaling through RNA turnover [RIP-Seq]

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