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Environmental regulation of toxin production in Bacillus anthracis.

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Názov: Environmental regulation of toxin production in Bacillus anthracis.
Autori: Bothra, Ankur, Pomerantsev, Andrei, Schwarz, Benjamin, Mondal, Anupam, Bohrnsen, Eric, Sangwan, Nitika, Stromberg, Kaitlin A., Moayeri, Mahtab, Ma, Qian, Fattah, Rasem, Ganesan, Sundar, Bosio, Catharine M., Leppla, Stephen H.
Zdroj: PLoS Pathogens; 12/1/2025, Vol. 21 Issue 12, p1-25, 25p
Abstrakt: Pathogenic Bacillus anthracis strains carry two plasmids - pX01, which encodes a tripartite protein exotoxin complex (PA, LF, and EF); and pX02, which encodes a poly-D-gamma-glutamic acid capsule. A multidomain transcription factor, AtxA, regulates the expression of these virulence genes. AtxA has two DNA-binding Helix-Turn-Helix (HTH) domains, two phosphoenolpyruvate: carbohydrate phosphotransferase system regulatory domains (PRD1 and PRD2), and a putative EIIB domain (a component of PTS sugar transport EII-complexes). Previous studies showed that glucose and CO2 increase AtxA-dependent toxin gene transcription, along with histidine phosphorylation of PRD1 and PRD2. Our transcriptional profiling of virulence factors, PA secretion, and fluorescent reporter strain analyses confirms a synergistic effect of glucose and CO2 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. Intracellular imaging using FLIM confirms a physical interaction of PtsG and AtxA through EIIB domain of AtxA. Using phosphomimetic and phosphoablative mutants of AtxA, we confirmed that the physical interaction of PtsG and AtxA is essential for AtxA activity. Finally, the synergy between glucose and CO2 was targeted by deleting pyruvate carboxylase Pyc (Δpyc), which regulates anaplerosis. This deletion confirms that Pyc stimulates the level of phosphoenolpyruvate (PEP) and increases the phosphorelay in glucose-PTS to enhance AtxA activity. Therefore, we propose that a histidine-phosphorelay from PEP regulates AtxA via PTS enzymatic activity, impacting AtxA activity through physical interaction of AtxA and PtsG. Finally, we propose AtxA as an integral component of the glucose-PTS, where transcriptional activity of AtxA is regulated by environmental signals including glucose and CO2. Author summary: Bacillus anthracis, the bacterium that causes anthrax, produces potent toxins that are central to its virulence. How environmental signals like glucose and carbon dioxide (CO₂) control toxin production has remained unclear. Here, we reveal a key mechanism by which the master virulence regulator, AtxA, integrates environmental and metabolic cues to control toxin gene expression. Using transcriptomics, metabolomics, and protein-protein interaction analyses, we show that AtxA directly interacts with the glucose-specific phosphotransferase system (PTS), specifically with the glucose permease PtsG. This interaction, mediated through AtxA's EIIB domain, is essential for its transcriptional activity and toxin production. Furthermore, pyruvate carboxylase (Pyc) enhances phosphoenolpyruvate (PEP) availability, strengthening PTS-dependent signaling and boosting AtxA activation under glucose and CO₂ conditions. Disruption of ptsG significantly reduced toxin expression and bacterial virulence in mice. Together, our findings uncover how B. anthracis senses and responds to host-like environments to regulate toxin production, revealing an important link between metabolism and virulence. [ABSTRACT FROM AUTHOR]
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Databáza: Complementary Index
Popis
Abstrakt:Pathogenic Bacillus anthracis strains carry two plasmids - pX01, which encodes a tripartite protein exotoxin complex (PA, LF, and EF); and pX02, which encodes a poly-D-gamma-glutamic acid capsule. A multidomain transcription factor, AtxA, regulates the expression of these virulence genes. AtxA has two DNA-binding Helix-Turn-Helix (HTH) domains, two phosphoenolpyruvate: carbohydrate phosphotransferase system regulatory domains (PRD1 and PRD2), and a putative EIIB domain (a component of PTS sugar transport EII-complexes). Previous studies showed that glucose and CO<subscript>2</subscript> increase AtxA-dependent toxin gene transcription, along with histidine phosphorylation of PRD1 and PRD2. Our transcriptional profiling of virulence factors, PA secretion, and fluorescent reporter strain analyses confirms a synergistic effect of glucose and CO<subscript>2</subscript> 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. Intracellular imaging using FLIM confirms a physical interaction of PtsG and AtxA through EIIB domain of AtxA. Using phosphomimetic and phosphoablative mutants of AtxA, we confirmed that the physical interaction of PtsG and AtxA is essential for AtxA activity. Finally, the synergy between glucose and CO<subscript>2</subscript> was targeted by deleting pyruvate carboxylase Pyc (Δpyc), which regulates anaplerosis. This deletion confirms that Pyc stimulates the level of phosphoenolpyruvate (PEP) and increases the phosphorelay in glucose-PTS to enhance AtxA activity. Therefore, we propose that a histidine-phosphorelay from PEP regulates AtxA via PTS enzymatic activity, impacting AtxA activity through physical interaction of AtxA and PtsG. Finally, we propose AtxA as an integral component of the glucose-PTS, where transcriptional activity of AtxA is regulated by environmental signals including glucose and CO<subscript>2</subscript>. Author summary: Bacillus anthracis, the bacterium that causes anthrax, produces potent toxins that are central to its virulence. How environmental signals like glucose and carbon dioxide (CO₂) control toxin production has remained unclear. Here, we reveal a key mechanism by which the master virulence regulator, AtxA, integrates environmental and metabolic cues to control toxin gene expression. Using transcriptomics, metabolomics, and protein-protein interaction analyses, we show that AtxA directly interacts with the glucose-specific phosphotransferase system (PTS), specifically with the glucose permease PtsG. This interaction, mediated through AtxA's EIIB domain, is essential for its transcriptional activity and toxin production. Furthermore, pyruvate carboxylase (Pyc) enhances phosphoenolpyruvate (PEP) availability, strengthening PTS-dependent signaling and boosting AtxA activation under glucose and CO₂ conditions. Disruption of ptsG significantly reduced toxin expression and bacterial virulence in mice. Together, our findings uncover how B. anthracis senses and responds to host-like environments to regulate toxin production, revealing an important link between metabolism and virulence. [ABSTRACT FROM AUTHOR]
ISSN:15537366
DOI:10.1371/journal.ppat.1013587