Abstract
The cell envelope of Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, contains lipids with unusual structures. These lipids play a key role in both virulence and resistance to the various hostile environments encountered by the bacteria during infection. They are synthesized by complex enzymatic systems, including type-I polyketide synthases and type-I and -II fatty acid synthases, which require a post-translational modification to become active. This modification consists of the covalent attachment of the 4′-phosphopantetheine moiety of Coenzyme A catalyzed by phosphopantetheinyl transferases (PPTases). PptT, one of the two PPTases produced by mycobacteria, is involved in post-translational modification of various type-I polyketide synthases required for the formation of both mycolic acids and lipid virulence factors in mycobacteria. Here we identify PptT as a new target for anti-tuberculosis drugs; we address all the critical issues of target validation to demonstrate that PptT can be used to search for new drugs. We confirm that PptT is essential for the growth of M. bovis BCG in vitro and show that it is required for persistence of M. bovis BCG in both infected macrophages and immunodeficient mice. We generated a conditional expression mutant of M. tuberculosis, in which the expression of the pptT gene is tightly regulated by tetracycline derivatives. We used this construct to demonstrate that PptT is required for the replication and survival of the tubercle bacillus during the acute and chronic phases of infection in mice. Finally, we developed a robust and miniaturized assay based on scintillation proximity assay technology to search for inhibitors of PPTases, and especially of PptT, by high-throughput screening. Our various findings indicate that PptT meets the key criteria for being a therapeutic target for the treatment of mycobacterial infections.
Highlights
The standard therapy for the treatment of tuberculosis, a disease still responsible for more than 1.5 million deaths and 8 million new cases per year, includes several antibiotics that must be taken for several months
This envelope contains lipids with unusual structures, including mycolic acids which are very long-chain fatty acids found in all mycobacteria, and a number of extractable lipids containing methylbranched fatty acids that contribute to pathogenicity [9,10,11,12]
The envelope has very high lipid content and contains lipids with unusual structures. Some of these lipids are synthesized by complex enzymatic systems that can only become functional after post-translational modification by a 49phosphopantetheinyl transferase named PptT
Summary
The standard therapy for the treatment of tuberculosis, a disease still responsible for more than 1.5 million deaths and 8 million new cases per year, includes several antibiotics that must be taken for several months (http://www.who.int/tb/dots/treatment) Longterm use of these drugs can cause serious side-effects especially in patients with immunodeficiency disorders and favors the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) mutants which are starting to pose a serious public health problem [1]. The synthesis of most of these lipids involves large multifunctional enzymes named polyketide synthases (PKS) and two fatty acid synthase (FAS) systems [9], [13] These enzymes are converted from inactive apo-forms to the functional holo-forms by the covalent attachment of a 49-phosphopantetheine (P-pant) group [14], [15].
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