The Influence Of The Pep-Pts And Other Metabolic Systems On The Master Virulence Regulator Atxa And Toxin Gene Expression In Bacillus Anthracis

Author

Naomi Bier-Reizes, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical SciencesFollow

Author ORCID Identifier

0000-0002-4918-5814

Date of Graduation

8-2019

Document Type

Dissertation (PhD)

Program Affiliation

Microbiology and Molecular Genetics

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Theresa M. Koehler, Ph.D.

Committee Member

Zheng (Jake) Chen, Ph.D.

Committee Member

Nicholas DeLay, Ph.D.

Committee Member

Heidi B. Kaplan, Ph.D.

Committee Member

Hung Ton-That, Ph.D.

Abstract

AtxA, the master virulence gene regulator in Bacillus anthracis, is a PRD-containing virulence regulator (PCVR) as indicated by the crystal structure, post-translational modifications, and activity of the protein. PCVRs are transcriptional regulators, named for the regulatory domains subject to phosphorylation by the phosphoenolpyruvate phosphotransferase system (PEP-PTS), termed PTS Regulatory Domains (PRD), and for their impact on virulence gene expression. Generally, the phosphorylation of a PCVR regulates protein activity and multimerization. AtxA is phosphorylated at two histidine residues - one in each of its two PRDs. Phosphorylation at position 199 allows for AtxA to positively affect expression of virulence genes, whereas phosphorylation at position 379 prevents or destabilizes dimerization, and therefore ablates activity of AtxA. Interestingly, current data from experiments employing physiological, genetic, and biochemical approaches do not support the predominant model of PCVR function in which the PTS proteins HPr and EI are responsible for phosphorylation of AtxA. Rather, we have determined that HPr and EI are required for transcription of the atxA gene. Assessment of atxA transcript levels using a transcriptional reporter assay revealed that transcript levels were 2.5-fold lower in a mutant lacking HPr and EI compared to the parent strain, and that this change is enough to affect toxin production. Mutants harboring HPr proteins altered for phosphotransfer activity were unable to restore atxA transcription to wild-type levels, suggesting that phosphotransfer activity of HPr and EI is important in the regulation of atxA gene production. In a mouse model of late-stage anthrax disease the PTS double mutant (HPr^-EI^-) was attenuated for virulence. Virulence was restored by expressing atxA from an alternative, PTS-independent promoter region indicating the PTS influences virulence through control of atxA expression. These findings are intriguing; the proteins (HPr and EI) that were hypothesized to regulate protein (AtxA) activity are in fact involved in regulating transcription of the gene encoding AtxA. HPr is a phosphotransfer protein and is not predicted to bind DNA. Thus, we hypothesize that HPr transfers a phosphate to a downstream transcriptional regulator to influence atxA gene transcription. Future studies aim to identify the transcriptional regulator involved in this process.

Keywords

PTS, bacterial sugar phosphotransferase system, AtxA, Bacillus anthracis