Abstract 1498 Environmental Regulation of Toxin Production in Bacillus Anthracis

Pathogenic B. anthracis carry two plasmids: pX01, which encodes a tripartite protein exotoxin complex (PA, LF, and EF); and pX02, which encodes a poly-D-glutamic acid capsule. A multidomain transcription factor, AtxA, regulates the expression of these virulence genes. AtxA has two DNA-binding HTH domains, two PTS (Phosphoenolpyruvate Sugar Phosphotransferase System) regulatory domains (PRD1 and PRD2), and a putative EIIB domain (a component of PTS sugar transport EII-complexes). Earlier studies showed that glucose and carbon dioxide increase AtxA-dependent toxin gene transcription, as does histidine phosphorylation of PRD1 and PRD2 of AtxA. We propose that a histidine-phosphorelay originating from phosphoenolpyruvate (PEP) regulates AtxA via enzymatic activity in the PTS, and thus pathways regulating the intracellular concentration of PEP impact AtxA activity. The anaplerotic response in bacteria, activated under high carbon dioxide concentrations, is one mechanism for control of the concentrations of PEP and other TCA intermediates. Our analyses of transcriptional profiling of virulence factors, secretion of PA, and use of a fluorescent reporter strain suggest a synergistic effect of carbon dioxide and sugars on AtxA-dependent toxin production. Deletion of AtxA (ΔatxA) significantly reduced glucose uptake in bacteria, suggesting that AtxA may act within the glucose-PTS system. Mutation analysis of the EIIB domain of AtxA identified the cysteine at position 402 as essential for the transcriptional activity of AtxA. Deletion of glucose PTS permease ptsG (ΔptsG) significantly reduced the expression of PA, LF, and EF. Loss of ptsG also caused attenuation in a mouse model of infection. However, deletion of pyruvate carboxylase pyc (Δpyc), regulating anaplerosis, had minimal effect on toxin production. Biochemical and metabolic profiling of ΔatxA, ΔptsG, Δpyc, and ΔptsG/Δpyc strains showed a significant shift in the glycolytic and anaplerotic intermediates, implicating glucose as a primary stimulus for anthrax toxin production through AtxA. Subsequently, the environmental CO2 will stimulate the anaplerotic flux and alter the phosphorelay in glucose-PTS to enhance AtxA activity. Finally, we propose AtxA as an integral component of the glucose-PTS, where transcriptional activity of AtxA is regulated by environmental signals like glucose and CO2. This research was supported by the intramural research programs of the National Institute of Allergy and Infectious Diseases of National Institutes of Health.