Functional Similarity of Prd-Containing Virulence Regulators In Bacillus Anthracis

<p><em>Bacillus anthracis </em>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; <em>lef, cya, </em>and <em>pag</em>, 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 <em>capBCADE</em>, and culture conditions that enhance AtxA activity result in <em>capBCADE</em> transcription in strains lacking <em>acpA</em> and <em>acpB</em>. 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 <em>lacZ</em> genes largely mirrored RNA-Seq data showing AtxA alone had activity on P<em>lef-lacZ</em>, and AcpA and AcpB had more activity than AtxA on P<em>capB-lacZ</em>. Studies to test the effect of AtxA levels on virulence and sporulation used <em>atxA </em>mutants. A mutant that overexpressed <em>atxA</em> and exhibited elevated AtxA and toxin levels <em>in vitro</em>, was not increased for virulence in a murine anthrax infection model. AtxA levels also affected sporulation efficiency. Culture of <em>B. anthracis </em>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 <em>Bacillus cereus, </em>AtxA2, had less activity than AtxA from <em>B. anthracis</em> potentially due to reduced dimer formation. The results provided in this dissertation increase our knowledge of virulence gene expression in <em>B. anthracis</em>, while advancing our understanding of this newly-discovered class of transcriptional regulators.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2018

Authors dc:creator

• Raynor, Malik
• <p>https://orcid.org/0000-0002-2324-1662</p>

Contributors dc:contributor

• Theresa M. Koehler, Ph.D.
• Kevin A. Morano, Ph.D.
• Peter J. Christie, Ph.D.

• Bacillus anthracis
• virulence
• pathogenesis
• toxin
• capsule
• bacteria
• microbiology
• gene regulation
• Bacteriology
• Biochemistry
• Molecular Biology
• Molecular Genetics
• Pathogenic Microbiology

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/857

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1903