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.

The need for early-on diagnostic tools to assess the folding and solubility of expressed protein constructs in vivo is of great interest when dealing with recalcitrant proteins. In this paper, we took advantage of the picomolar sensitivity of the bipartite GFP1-10/GFP11 system to investigate the solubility of the Mycobacterium tuberculosis 4'-phosphopantetheinyl transferase PptT, an enzyme essential for the viability of the tubercle bacillus. In vivo and in vitro complementation assays clearly showed the improved solubility of the full-length PptT compared to its N- and C-terminally truncated counterparts. However, initial attempts to purify the full-length enzyme overexpressed in Escherichia coli cells were hampered by aggregation issues overtime that caused the protein to precipitate within hours. The fact that the naturally occurring Coenzyme A and Mg(2+), essentials for PptT to carry out its function, could play a role in stabilizing the enzyme was confirmed using DSF experiments. In vitro activity assays were performed using the ACP substrate from the type I polyketide synthase PpsC from M. tuberculosis, a 2188 amino-acid enzyme that plays a major role in the virulence and pathogenicity of this microbial pathogen. We selected the most soluble and compact ACP fragment (2042-2188), identified by genetic selection of in-frame fragments from random library experiments, to monitor the transfer of the P-pant moiety from Coenzyme A onto a conserved serine residue of this ACP domain.