Student Theses and Dissertations

Date of Award

2012

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

McKinney Laboratory

Abstract

Mycobacterium tuberculosis has been a human pathogen for the history of mankind, but we are only now beginning to understand how it is able to survive and persist indefinitely in the host. Understanding carbon metabolism of the pathogen during infection is key, not only as a source of potential drug targets, but also for elucidating the environment in vivo, so that drugs can be tested under relevant conditions. Studies have revealed that, during infection, M. tuberculosis relies on gluconeogenic carbon sources rather than sugars. Fatty acids, cholesterol, and amino acids have all been demonstrated as usable carbon sources in vitro and can all generate propionyl-CoA. The methylcitrate cycle, which, in M. tuberculosis, uses a bifunctional isocitrate lyase/methylisocitrate lyase (ICL/MCL), is one of the two routes for metabolism of propionyl-CoA. A mutant strain of M. tuberculosis lacking the ICL/MCL was rapidly cleared from the lungs of infected mice. However, the upstream enzymes of this pathway have been demonstrated to be dispensable for infection and survival in the mouse model. The methylmalonyl- CoA route of propionyl-CoA utilization can be activated in vitro by addition of the vitamin B12 cofactor of the methylmalonyl-CoA mutase. This route may buffer the loss of the methylcitrate cycle in vivo, depending on B12 availability or production in the host. The work here examines the relative use of the methylcitrate cycle and methylmalonyl-CoA pathways in M. tuberculosis and in the related, nonpathogenic species, M. smegmatis, using genetic mutants of either or both of the metabolic routes. It is shown here that, as for M. tuberculosis, M. smegmatis preferentially uses the methylcitrate cycle for growth on propionate. In the absence of the methylcitrate cycle M. smegmatis, in contrast to M. tuberculosis, can eventually endogenously activate the methylmalonyl-CoA pathway in vitro, presumably through B12 synthesis. In mutants of both species, lacking both pathways, the use of other carbon sources in the media is inhibited in the presence of propionate. This dominant inhibition implies the accumulation of toxic metabolites derived from the inability to metabolize propionate, as has been suggested by previous studies. To detect propionate-derived intermediates, metabolite analysis by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used in this study. Accumulation of these metabolites under propionate exposure was identified in a strain of M. smegmatis impaired in both metabolic routes, but not in the wild-type. These studies also revealed similar accumulation under glucose growth, where the mutant strain displayed a slight growth defect, and also under no-carbon conditions, where the mutant demonstrated a survival defect compared to wild-type. These findings suggest a role of the propionate pathways for endogenously derived propionyl-CoA as well as during starvationinduced amino acid and/or fatty acid mobilization. The M. tuberculosis mutant strains generated here were tested in the mouse infection model. The methylmalonyl-CoA mutase was found to be individually dispensable for growth in vivo. However, a strain with the additional deletion of the methylcitrate cycle was attenuated during the early stage of infection and caused less tissue pathology, even after the bacterial burden reached wild-type levels. While propionate metabolism may not be required per se for in vivo growth, the suggested accumulation of toxic intermediates, demonstrated here in M. smegmatis, may indicate a required role for ICL/MCL in M. tuberculosis for detoxification of propionyl-CoA in vivo.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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