Control of The Master Virulence Regulatory Gene Atxa In Bacillus Anthracis

<p>Transcription of the <em>Bacillus anthracis</em> structural genes for the anthrax toxin proteins and biosynthetic operon for capsule are positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of <em>atxA</em> in virulence factor expression, a <em>B. anthracis</em> <em>atxA</em>-null mutant is avirulent in a murine model for anthrax. In batch culture, multiple signals impact <em>atxA </em>transcript levels, and the timing and steady state level of <em>atxA </em>expression is critical for optimal toxin and capsule synthesis. Despite the apparent complex control of <em>atxA</em> transcription, only one <em>trans</em>-acting protein, the transition state regulator AbrB, has been demonstrated to directly interact with the <em>atxA</em> promoter. The AbrB-binding site has been described, but additional <em>cis</em>-acting control sequences have not been defined. Using transcriptional <em>lacZ</em> fusions, electrophoretic mobility shift assays, and Western blot analysis, the <em>cis</em>-acting elements and <em>trans</em>-acting factors involved in regulation of <em>atxA</em> in <em>B. anthracis </em>strains containing either both virulence plasmids, pXO1 and pXO2, or only one plasmid, pXO1, were studied. This work demonstrates that <em>atxA</em> transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA, and an A+T-rich upstream element (UP-element) for RNA polymerase (RNAP). In addition, the data show that a <em>trans</em>-acting protein(s) other than AbrB negatively impacts <em>atxA</em> transcription when it binds specifically to a 9-bp palindrome within <em>atxA</em> promoter sequences located downstream of P1. Mutation of the palindrome prevents binding of the <em>trans</em>-acting protein(s) and results in a corresponding increase in AtxA and anthrax toxin production in a strain- and culture-dependent manner.</p> <p>The identity of the <em>trans</em>-acting repressor protein(s) remains elusive; however, phenotypes associated with mutation of the repressor binding site have revealed that the <em>trans</em>-acting repressor protein(s) indirectly controls <em>B. anthracis</em> development. Mutation of the repressor binding site results in misregulation and overexpression of AtxA in conditions conducive for development, leading to a marked sporulation defect that is both <em>atxA</em>- and <em>pXO2-61</em>-dependent. pXO2-61 is homologous to the sensor domain of sporulation sensor histidine kinases and is proposed to titrate an activating signal away from the sporulation phosphorelay when overexpressed by AtxA. These results indicate that AtxA is not only a master virulence regulator, but also a modulator of proper <em>B. anthracis</em> development. Also demonstrated in this work is the impact of the developmental regulators AbrB, Spo0A, and SigH on <em>atxA</em> expression and anthrax toxin production in a genetically incomplete (pXO1+, pXO2-) and genetically complete (pXO1+, pXO2+) strain background. AtxA and anthrax toxin production resulting from deletion of the developmental regulators are strain-dependent suggesting that factors on pXO2 are involved in control of <em>atxA</em>. The only developmental deletion mutant that resulted in a prominent and consistent strain-independent increase in AtxA protein levels was an <em>abrB</em>-null mutant. As a result of increased AtxA levels, there is early and increased production of anthrax toxins in an <em>abrB</em>-null mutant. In addition, the <em>abrB</em>-null mutant exhibited an increase in virulence in a murine model for anthrax. In contrast, virulence of the <em>atxA</em> promoter mutant was unaffected in a murine model for anthrax despite the production of 5-fold more AtxA than the <em>abrB</em>-null mutant. These results imply that AtxA is not the only factor impacting pathogenesis in an <em>abrB</em>-null mutant. Overall, this work highlights the complex regulatory network that governs expression of <em>atxA</em> and provides an additional role for AtxA in <em>B. anthracis</em> development.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2012

Author dc:creator

• Dale, Jennifer L

Contributors dc:contributor

• Theresa M. Koehler, PhD
• William Margolin, PhD
• Peter J. Christie, PhD

• Pathogenic Microbiology

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/259

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1276