Characterization of the role of a putative fatty acyl-CoA synthetase, fadD5, in Mycobacterium tuberculosis

Mycobacterium tuberculosis, the agent of tuberculosis, causes nearly two million deaths per year and latently infects one-third of the world's population. One key adaptation that Mycobacterium tuberculosis established to survive in its human host is a reliance on lipids as a carbon and energy source. M. tuberculosis H37Rv has 36 fadD genes annotated in the genome as putative fatty acyl-CoA synthetases, which are enzymes that activate fatty acids for further metabolism. One such gene, fadD5 (Rv0166), is located within the mce1 operon, a cluster of genes associated with M. tuberculosis persistence. The second chapter of this dissertation focuses on the resulting effects of disrupting the putative fatty acid binding site of fadD5 in H37Rv M. tuberculosis. No significant differences were found in the growth of the mutant and wild-type strains in vitro in nutrient-rich broth or in activated RAW264.7 cells. However, the fadD5 mutant was diminished in growth in minimal medium containing a long-chain fatty acid, mycolic acid. In the third chapter, the fadD5 mutant is shown to have a lipid profile unique from those of the wild-type M. tuberculosis and the mce1 mutant strains. The fourth chapter discusses the attenuated phenotype of the fadD5 mutant strain in the mouse model of tuberculosis. C57BL/6 mice infected with the fadD5 mutant survived significantly longer than those infected with wild-type, and the mutant never attained the plateau phase of infection in the mouse lungs typically seen with wild- type. The steady-state infection phase was maintained for up to 168 days at a level one to two logs less than that shown by wild-type. These observations raise a rather intriguing possibility that FadD5 may serve to utilize mycolic acids as a nutrient source for the long-term survival in vivo of the tubercle bacilli.