Bacillus anthracis virulence regulator AtxA: oligomeric state, function and CO₂‐signalling

Abstract

Summary AtxA, a unique regulatory protein of unknown molecular function, positively controls expression of the major virulence genes of Bacillus anthracis. The 475-amino acid sequence of AtxA reveals DNA-binding motifs and regions similar to proteins associated with the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). We used strains producing native and functional epitope-tagged AtxA proteins to examine protein-protein interactions in cell lysates and in solutions of purified protein. Co-affinity purification, non-denaturing poly-acrylamide gel electrophoresis, and bis(maleimido)hexane (BMH) cross-linking experiments revealed AtxA homo-multimers. Dimers were the most abundant species. BMH cross-links available cysteines within 13Å. To localize interaction sites, six AtxA mutants containing distinct Cys→Ser substitutions were tested for multimerization and cross-linking. All mutants multimerized, but one mutation, C402S, prevented cross-linking. Thus, BMH uses C402 to make the inter-molecular bond between AtxA proteins, but C402 is not required for protein-protein interaction. C402 is in a region bearing amino acid similarity to Enzyme IIB proteins of the PTS. The AtxA EIIB motif may function in protein oligomerization. Lastly, cultures grown with elevated CO2/bicarbonate exhibited increased AtxA dimer/monomer ratios and increased AtxA activity, relative to cultures grown without added CO2/bicarbonate, suggesting that this host-associated signal enhances AtxA function by shifting the dimer/monomer equilibrium toward the dimeric state.

cis -Acting Elements That Control Expression of the Master Virulence Regulatory Gene atxA in Bacillus anthracis

“…These results suggest that the expression and function of the trans-acting repressor protein(s) is not impacted by the CO 2 /bicarbonate signal. Notably, previous reports indicated that the CO 2 /bicarbonate signal enhances toxin and capsule gene expression by affecting AtxA protein function, rather than atxA expression (10,13,24).…”

“…An isogenic ANR-1 atxA-null mutant (UT374) was created using pUTE937 and the markerless temperature-sensitive integration system previously described by Pflughoeft et al (37). Detailed explanation of pUTE937 construction can be found in the study reported by Hammerstrom et al (24). Similar methods were used to create the isogenic ANR-1 atxA-up mutant (UT398).…”

“…Four-milliliter culture samples were centrifuged at 10,000 ϫ g for 10 min. Preparation of cell lysates for Western blot analysis of AtxA was performed according to the methods described by Hammerstrom et al (24). To assess lethal factor (LF) protein levels, 1 ml of corresponding supernatant was passed through a 0.2-m filter (Thermo Scientific) and applied to a nitrocellulose membrane, using vacuum blotting with a slot blot apparatus (Hoefer Scientific, San Francisco, CA).…”

“…The major virulence gene regulator of Bacillus anthracis, AtxA, positively affects transcription of the anthrax toxin and capsule biosynthetic genes (11,12,14,22,27,31,44,57,58). AtxA has motifs associated with DNA binding, phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domains (PRDs), and multimerization (24,26,56,58), but the precise molecular mechanism by which AtxA impacts transcription is not clear. Nevertheless, steady-state levels of AtxA are critical for optimal transcription of the anthrax toxin and capsule genes, and an atxA-null mutant is avirulent in a murine model for anthrax (8, 10-12, 23, 58).…”

Bacterial Virulence Factors: Secreted for Survival

“…The pathogenicity of Bacillus anthracis is governed by two large extrachromosomal plasmids namely pXO1 and pXO2. The plasmid pXO1 encodes protective antigen, lethal factor, edema factor and anthrax toxin activator A (AtxA), a central regulator for toxin synthesis [41]. These secreted toxin units enable the pathogen to establish a systemic infection in short duration of time.…”