Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) in humans, is responsible for approximately three million deaths worldwide every year and estimated to infect one-third of the world's population. The re-emergence of this disease, caused by the lack of drug compliance, the appearance of multiple-drug-resistant (MDR) strains, and the AIDS epidemic, urge the development of new drugs with activity against MDR strains that could reduce the duration and complexity of current therapies. Acyl-CoA carboxylases (ACCase) have crucial roles in fatty acid metabolism in most living organisms and have been proposed as a good target for the development of a new class of antibacterial agents. In M. tuberculosis, six putative ACCases are predicted from the genome sequence and two of them, ACCase4 and ACCase6, are proposed to be involved in mycolic acid biosynthesis. This complex lipid is one of the main components of the M. tuberculosis cell envelope and has been associated with the survival and pathogenicity of this bacterium. The objective of the proposed research is to characterize ACCases4 and 6 from M. tuberculosis at the genetic, biochemical, and structural levels and understand, by using M. smegmatis as a working model, their physiological role and relevance in mycolic acid biosynthesis and in the survival of these microorganisms. Additionally, we will characterize in vivo and in vitro, putative ACCase inhibitors identified by in silico docking of chemical libraries, and use them to validate these enzyme complexes as target for the future development of antimycobacterial drugs. As a long term goal we will also put emphasis in understanding the molecular bases that determine the substrate specificity of each complex. For this, we will carry on structure based mutagenesis of the carboxyltransferase subunits (2) of the essential complex followed by the biochemical characterization of the mutated enzyme subunits.

Public Health Relevance

Mycobacterium tuberculosis causes more human deaths that any other single infectious organism with an estimated eight million new tuberculosis cases and two million fatalities each year. Given the current backdrop of emerging multi-drug resistant tuberculosis (MDR-TB), TB treatment is entering a new and challenging era where effective control requires the identification of new drugs and novel drug targets. In this project, we propose the characterization of two acyl-CoA carboxylase (ACCase) complexes of M. tuberculosis involved in the biosynthesis of mycolic acids, complex lipids present in the bacterial cell envelope which are essential for the pathogen survival and pathogenicity. We expect that the biochemical, physiological and structural characterization of these enzyme complexes will help us to achieve a more long-term goal of this project, the identification of ACCase inhibitors that could eventually be developed into new antimycobacterial drugs.

Agency
National Institute of Health (NIH)
Institute
Fogarty International Center (FIC)
Type
Small Research Grants (R03)
Project #
5R03TW007982-03
Application #
7821278
Study Section
International and Cooperative Projects - 1 Study Section (ICP1)
Program Officer
Sina, Barbara J
Project Start
2008-08-01
Project End
2011-04-30
Budget Start
2010-05-01
Budget End
2011-04-30
Support Year
3
Fiscal Year
2010
Total Cost
$31,914
Indirect Cost
Name
Institute of Molecular /Cellular Biology
Department
Type
DUNS #
974701257
City
Rosario
State
Country
Argentina
Zip Code
2000
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Salzman, V; Mondino, S; Sala, C et al. (2010) Transcriptional regulation of lipid homeostasis in mycobacteria. Mol Microbiol 78:64-77
Arabolaza, Ana; Shillito, Mary Elizabeth; Lin, Ting-Wan et al. (2010) Crystal structures and mutational analyses of acyl-CoA carboxylase beta subunit of Streptomyces coelicolor. Biochemistry 49:7367-76
Kurth, Daniel G; Gago, Gabriela M; de la Iglesia, Agustina et al. (2009) ACCase 6 is the essential acetyl-CoA carboxylase involved in fatty acid and mycolic acid biosynthesis in mycobacteria. Microbiology 155:2664-75