Date of Award

2018-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Hugues Ouellet

Abstract

Mycobacterium tuberculosis (Mtb) is a serious health issue arising from the increasing incidence of multi-drug resistant (MDR-TB) strains that are becoming more prevalent in high-risk countries, especially here in the Texas/Mexico border. Confirmed cases and outbreaks in schools and hospitals are more frequent in this region due to the high-volume pedestrian traffic between the two cities, and the lack of a modernized health system in Mexico. Identification of new Mtb metabolic pathways for development of novel antitubercular drugs is urgently needed. The goal of this current work investigates two Mtb fatty acyl-CoA ligase enzymes' (FadD17A1 and FadD19A1) to explore whether removal of these genes would affect Mtb's ability to grow on cholesterol using M. smegmatis as a model. It has been previously shown that FadD19A1 is essential for degradation of steroids with C24 branched side-chains in vivo [1]. Our initial hypoThesis was that the utilization of host cholesterol reshapes Mtb's metabolism as an adaptive mechanism for survival in macrophages and that these two acyl-CoA ligases (FadD17A1 and FadD19A1) play an essential role in the cholesterol degradation process. We have been able to show that slight redundancy exists in these enzymes' ability to turn over all acid intermediates of cholestenone tested in vitro including 23,24-bisnor-chol-1,4-dienic acid (C22), (23,24-bisnor-5α-cholenic acid-3-one (C22), 5-cholenic acid-3β-ol (C24), 4-cholenic acid-3β-ol (C24), as well as 25-R and 25-S isomers of 3-oxo-4-cholestenoic acid (C27). The 25-S isomer of 3-oxo-4-cholestenoic acid (C27) seems to be the best substrate for activity with FadD19A1, while 4-cholenic acid-3β-ol (C24) appears to be the best substrate for FadD17A1. We have also been able to show that both FadD17A1 and FadD19A1 can act on C5-C18 saturated fatty acids but have a stronger affinity for mid-chain saturated fatty acids, and increased activity with longer chain fatty acids can be accomplished by introducing one and two degrees of unsaturation. Our next goal was to evaluate the effect of removal of both acyl-CoA ligase enzymes' and determine the effect on growth utilizing various media, as well as metabolic profiling of mutant strains. A slight growth deficiency was obtained with our mutant strains after the log phase only when utilizing a more complex media with multiple carbon sources. These data support the initial hypoThesis of this study which predicts important roles for the FadDs in the reshaping of Mtb's metabolism from glucose to cholesterol. Profiling data demonstrated significant accumulation of cholestenoic acid (4-CHOL) in mutant strains deficient in fadD19A2 as well as 4-CHOL-26OOMe and 4-CHOL-26OOEt in all mutant strains deficient in any combination of the FadD enzymes. Detection of methylated C and D ring metabolite CD-26OOMe was the only accumulated CD ring metabolite detected in the fadD mutant strains compared to wt. Accumulation of 4-cholenic acid (4-C) and detection of 23,24-Bisnor-4-Cholenic acid (4-BNC) was only possible when fadD mutant strains were also deficient for expression of KstD. Detection of methylated acid intermediates including 4-CHOL-26OOMe, 4-C-24OOMe, and 4-BNC-22OOMe were now also detectable and higher in mutant strains, deficient for expression of KstD as well, compared to wt. As expected, C and D ring intermediates among these kstd/fadD mutants was lower including CD-26OOMe, CD-24OOMe and CD-22OOMe compared to wt. This data indicates esterification mechanisms as well as subsequent opening of rings A and B as compensatory mechanisms when side-chain degradation is blocked. Use of a propionate reporter construct allowed us to visualize decreased levels of propionate among the mutant strains when grown in the presence of cholest-4-en-3-one, supporting the compensatory mechanisms above. Acid-fast staining of our mutant strains illustrated that all our mutants, except for the Î?fadD19A2 and Î?fadD19A2/fadD19B1 mutants, appear to have lost their acid-fast phenotype. Together, our data demonstrate that targeting of enzymes acting on the side chain degradation of cholesterol will not lead to production of toxic intermediates and bacteriostatic effects, but will instead lead to altered endogenous propionate production and subsequent altered lipid composition of the cell wall. These results provide promising avenues for continued investigation of ligase enzymes in this pathway for development of novel therapeutics capable of targeting the synThesis of many virulence lipids that could be used to help control the proliferation of drug resistant tuberculosis.

Language

en

Provenance

Received from ProQuest

File Size

129 pages

File Format

application/pdf

Rights Holder

Johnathan Salim Abou-Fadel

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