Date of Award

1-1-2021

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (xx, 394 pages) : illustrations.

Dissertation/Thesis Chair

Kathleen A McDonough

Committee Members

Paul S Masters, Nicholas J Mantis, Joseph T Wade, Guangchun Bai

Keywords

central carbon metabolism, cyclic-AMP signaling, Mycobacterium tuberculosis, phosphodiesterase, signal transduction, Cyclic nucleotide phosphodiesterases, Cyclic adenylic acid, Mycobacteria, Tuberculosis, Cellular signal transduction

Subject Categories

Microbiology | Molecular Biology

Abstract

Phosphodiesterases (PDEs) are integral components of 3’,5’-cyclic adenosine monophosphate (cAMP) signaling pathways by degrading cAMP to modulate the concentration, duration, and localization of the cAMP signal which maintains the specificity of cAMP pathways. The human pathogen, Mycobacterium tuberculosis (Mtb) has a unique cAMP network architecture with at least 15 adenylyl cyclases (ACs) that generate cAMP, but only one characterized PDE, Rv0805, which is found exclusively in pathogenic mycobacteria. Rv0805 can influence Mtb cAMP levels but the absence of Rv0805 orthologs in non-pathogenic mycobacteria and apparent separation of Rv0805 from cAMP directed roles led to numerous questions surrounding PDE function in and beyond cAMP level maintenance. A better understanding of the role of Rv0805 may elucidate novel pathways utilized by Mtb during infection. Rv0805 facilitated growth of Mtb during macrophage infection, indicating a role for Rv0805 in Mtb pathogenesis. In vitro growth assays were then used to further deconstruct the role of Rv0805 in the Mtb model organism, M. bovis Bacillus Calmette-Guerin (BCG). BCG growth on propionate, which yields a toxic product of fatty acid metabolism, propionyl-CoA, was facilitated by Rv0805. The addition of vitamin B12 (B12), which activates a separate propionate utilization pathway, rescued the growth of BCGΔRv0805 on propionate containing media, indicating that Rv0805 mitigated propionate-associated toxicity in BCG. The importance of Rv0805 for bacterial growth was highlighted in an in vitro condition that combined glycerol with propionate (GP). Suppressor mutations of BCGΔRv0805 were found within an acetate and potentially propionate related pathway, Pta-AckA, and Rv0806c. These data highlighted Rv0805 as a novel regulator of bacterial metabolism, especially in the presence of propionate. Rv0805 cyclic nucleotide monophosphate (cNMP) catalytic activity facilitated growth and regulated cAMP levels of BCG grown on GP. A BCGΔRv0805 pta frameshift suppressor mutant grown on GP had lower levels of cAMP than its parental strain, consistent with excess cAMP as the basis of the Rv0805 dependent growth defect. However, overexpression of a putative mycobacterial PDE, Rv1339, that is conserved amongst mycobacteria, only marginally improved growth of BCGΔRv0805 on GP, despite lowering global cAMP levels. Additionally, the presence of an Rv0805 localization domain was also required for Rv0805 function, indicating that Rv0805 may regulate cAMP or other cNMP substrates within a specific location. Taken together, this work provides novel insight into the regulation of Mtb’s complex cAMP regulatory network by highlighting that multiple PDEs are involved in cAMP homeostasis within TB complex mycobacteria. Rv0805 was found to be an important factor of pathogenic mycobacterial physiology through detoxifying propionyl-CoA. The importance of Rv0805 for bacterial growth was largely separate from its role in regulating total cAMP levels. However, the cNMP hydrolytic activity of Rv0805 was still crucial, suggesting an alternative role of Rv0805 may be to regulate local cAMP or cNMP substrates and when this regulation is perturbed global changes in metabolism and gene expression occur. This study clarified the role of Rv0805 by demonstrating cNMP activity is a dominant function of this PDE, uncovered a novel PDE, and suggests that PDEs regulate separate cNMP pathways within TB complex mycobacteria. Additionally, this study lays the groundwork for future studies to elucidate the key members and substrates of these PDE gated pathways.

Download

Share

COinS