Author ORCID Identifier

https://orcid.org/0000-0002-2324-1662

Date of Graduation

5-2018

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

Kevin A. Morano, Ph.D.

Committee Member

Peter J. Christie, Ph.D.

Committee Member

Hung Ton-That, Ph.D.

Committee Member

Eric J. Wagner, Ph.D.

Abstract

Bacillus anthracis produces three regulators, AtxA, AcpA, and AcpB, that control virulence gene expression and are members of an emerging class of regulators termed “PCVRs” (Phosphoenolpyruvate-dependent phosphotransferase regulation Domain-Containing Virulence Regulators). AtxA controls expression of the toxin genes; lef, cya, and pag, and is the master virulence regulator and archetype PCVR. AcpA and AcpB are less well studied. AcpA and AcpB independently positively control transcription of the capsule biosynthetic operon capBCADE, and culture conditions that enhance AtxA activity result in capBCADE transcription in strains lacking acpA and acpB. RNA-Seq was used to assess the regulons of the paralogs in strains producing individual PCVRs at native levels. Plasmid- and chromosome-borne genes were PCVR-controlled, with AtxA, AcpA, and AcpB having a ≥4-fold effect on transcript levels of 145, 130, and 49 genes respectively. Several genes were co-regulated by two or three PCVRs. Results from transcriptional reporters of PCVR-regulated promoters fused to promoterless lacZ genes largely mirrored RNA-Seq data showing AtxA alone had activity on Plef-lacZ, and AcpA and AcpB had more activity than AtxA on PcapB-lacZ. Studies to test the effect of AtxA levels on virulence and sporulation used atxA mutants. A mutant that overexpressed atxA and exhibited elevated AtxA and toxin levels in vitro, was not increased for virulence in a murine anthrax infection model. AtxA levels also affected sporulation efficiency. Culture of B. anthracis in medium containing bicarbonate and elevated carbon dioxide increased PCVR activity compared to culture in ambient air in medium lacking bicarbonate. However, neither the solubility nor stability of the regulators was affected by carbon dioxide concentration. AcpA and AcpB form homomultimers and multimerization was dependent on the EIIB-like domains, as shown previously for AtxA. Heteromultimers of AtxA-AcpA were detected and in co-expression experiments, AcpA activity was reduced by increased levels of AtxA. An AtxA orthologue in Bacillus cereus, AtxA2, had less activity than AtxA from B. anthracis potentially due to reduced dimer formation. The results provided in this dissertation increase our knowledge of virulence gene expression in B. anthracis, while advancing our understanding of this newly-discovered class of transcriptional regulators.

Keywords

Bacillus anthracis, virulence, pathogenesis, toxin, capsule, bacteria, microbiology, gene regulation

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