A Dual Role for the Bacillus anthracis Master Virulence Regulator AtxA: Control of Sporulation and Anthrax Toxin Production

Abstract

Bacillus anthracis is an endemic soil bacterium that exhibits two different lifestyles. In the soil environment, B. anthracis undergoes a cycle of saprophytic growth, sporulation, and germination. In mammalian hosts, the pathogenic lifestyle of B. anthracis is spore germination followed by vegetative cell replication, but cells do not sporulate. During infection, and in specific culture conditions, transcription of the structural genes for the anthrax toxin proteins and the biosynthetic operon for capsule synthesis is positively controlled by the regulatory protein AtxA. A critical role for the atxA gene in B. anthracis virulence has been established. Here we report an inverse relationship between toxin production and sporulation that is linked to AtxA levels. During culture in conditions favoring sporulation, B. anthracis produces little to no AtxA. When B. anthracis is cultured in conditions favoring toxin gene expression, AtxA is expressed at relatively high levels and sporulation rate and efficiency are reduced. We found that a mutation within the atxA promoter region resulting in AtxA over-expression leads to a marked sporulation defect. The sporulation phenotype of the mutant is dependent upon pXO2-0075, an atxA-regulated open reading frame located on virulence plasmid pXO2. The predicted amino acid sequence of the pXO2-0075 protein has similarity to the sensor domain of sporulation sensor histidine kinases. It was shown previously that pXO2-0075 overexpression suppresses sporulation. We have designated pXO2-0075 "skiA" for "sporulation kinase inhibitor." Our results indicate that in addition to serving as a positive regulator of virulence gene expression, AtxA modulates B. anthracis development.

Keywords: Bacillus; anthrax; biological; development; sporulation; toxins; transcription factors.

FIGURE 1

Toxin production and sporulation are inversely related. (A) Growth curve and heat-resistant CFU determination of Ames cultured in sporulation (PA-air; hashed line/diamonds) and toxin-inducing (CACO₃ +5% CO₂; solid line/squares) conditions. Production of (B) LF and (C) AtxA in sporulation and toxin-inducing conditions. Cell-free supernatants for LF and cell lysates for AtxA production were obtained from early exponential (2 h), transition (4 h), and stationary (7 h) phases of growth and subjected to Western blot analysis using rabbit α-LF, and α-AtxA antibody. Protein loads were normalized to OD₆₀₀. The data are representative of three separate experiments.

FIGURE 2

Phase contrast microscopy showing sporulation. (A) Cultures in PA-air, or (B) CACO₃ +5% CO₂. ΔskiA is equivalent to ΔpXO2-0075. The data are representative of three separate experiments.

FIGURE 3

Spore quantitation, AtxA protein abundance, and skiA transcript levels in PA-air. (A) Heat-resistant CFU/ml of parent and mutant derivatives. (B) AtxA protein levels in parent and mutant strain backgrounds. Culture samples were obtained during transition (T4) and stationary (T7) phases of growth, and subjected to Western blot analysis using rabbit α-AtxA antibody. Protein loads were determined based on OD₆₀₀ values and normalized to cross-reactive products from α-AtxA or α-RNAP-β blots. These data are representative of three separate experiments. Non-detectable (ND) levels of AtxA are denoted. (C) RT-qPCR of skiA transcripts at 4 and 7 h, respectively, normalized to the parent control. These data represent average values of detectable transcripts from three independent cultures. Asterisks denote p-values ≤ 0.05 relative to parent.

FIGURE 4

Spore quantitation, AtxA protein abundance, and skiA transcript levels in CACO₃ + 5% CO₂. (A) Heat-resistant CFU/ml of parent and mutant derivatives. (B) AtxA protein levels in parent and mutant strain backgrounds. Culture samples were obtained during transition (T4) and stationary (T7) phases of growth, and subjected to Western blot analysis using rabbit α-AtxA antibody. Protein loads were determined based on OD₆₀₀ values and normalized to Ponceau S stained membranes. These data are representative of three separate experiments. (C) RT-qPCR of skiA transcripts at 4 and 7 h, respectively, normalized to the parent control. These data represent average values of detectable transcripts from three independent cultures. Asterisks denote p-values ≤ 0.05 relative to parent.

FIGURE 5

Capsule production of B. anthracis parent and mutant strains in toxin-inducing conditions (CACO₃ + 5% CO₂). Qualitative analysis of capsule production using India ink exclusion assays. These data are representative of three separate experiments.

FIGURE 6

Model for regulation of atxA gene expression in toxin-inducing and sporulation conditions. The developmental regulators AbrB, Spo0A, and SigH regulate atxA transcription in a condition-dependent manner. In toxin-inducing conditions, AbrB binds to a region upstream of P1 to actively repress atxA expression, whereas in sporulation conditions, AbrB plays a minor role in control of atxA. In toxin-inducing conditions, atxA positively controls skiA transcription. In sporulation conditions, the atxA repressor protein (referred to as ‘Repressor’ in the model) interacts with a palindromic sequence located downstream of P1 (Dale et al., 2012). Activity of the repressor is down-regulated by SkiA. BA2291 is a sensor histidine kinase that indirectly activates Spo0A in sporulation conditions, and acts as a phosphatase in toxin-inducing conditions. SkiA acts as a phosphate sink in a BA2291-dependent manner in toxin-inducing conditions. Additional signals impacting atxA expression are carbohydrate availability, temperature, and redox potential. Thick lines denote important trans-acting factors or signals controlling atxA expression in the given culture condition. Thin lines denote minimal impact. Hashed lines indicate suggested or indirect (BA2291) functions. Curved lines/arrows represent indirect effects on atxA transcription.