Interpretation of the results is complicated by the fact that the replicative and metabolic state of the bacteria during the persistent infection phase remains unclear. (14K) GUID:?E3C0660F-1589-46B3-AA32-874A32684A6C Figure S4: Effect of pH and temperature on PptT activity. PptT (200 nM) was incubated for 3 hours with the BCG and show that it is required for persistence of BCG in both infected macrophages and immunodeficient mice. We generated a conditional expression mutant of 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. Author Summary and of and its close relative Lycopene BCG in both macrophages and the mouse model. Our findings demonstrate that PptT plays a key role in multiplication and persistence of the tubercle bacillus and is therefore an attractive target for drug discovery. We also developed an assay that promises to be a powerful tool for high-throughput screening of PptT inhibitors. Introduction 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). Long-term 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 now starting to pose a serious public health problem [1]. Moreover has a highly lipid-rich hydrophobic cell wall with a low permeability that contributes to its intrinsic drug resistance [7], [8]. This envelope contains lipids with unusual Rabbit polyclonal to ANG4 structures, including mycolic acids which are very long-chain fatty acids found in all mycobacteria, and a number of extractable lipids containing methyl-branched fatty acids Lycopene that contribute to pathogenicity [9]C[12]. 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 virulence [17]. Thus, PptT plays a major role in the biology of and related pathogenic mycobacteria, being required for the synthesis of components that are needed for growth and others involved in virulence (Figure 1B). PptT is therefore a potential target for drug development. To test whether PptT is essential for the viability of strains of the complex, we generated a conditional knockout mutant in BCG, using a TetR-controlled gene expression system [16], [18]. We found that the expression of was required to sustain BCG growth assay amenable to high-throughput screening is an asset that facilitates the search for potential inhibitors and their improvement. In this study, we addressed these various Lycopene points for PptT and demonstrate that it fulfills all the requirements for a clinically relevant drug target. Open in a separate window Figure 1 Role of PptT Lycopene in growth and biochemical characterization of conditional mutants of BCG and expression mutant of BCG, named PMM99, based on the use of a TetR/expression regulation system [16]. We generated a similar mutant, named PMM168, in H37Rv using the same strategy as for the construction of PMM99 (Figure S1 and [16]). Both mutants grew normally on 7H11 plates supplemented with anhydrotetracycline (ATc; 100 ng/ml for the BCG mutant and 300 ng/ml for the mutant) but were unable to grow on plates in the absence of ATc, in contrast to the wild-type strain (Figure 2A and [16]) indicating that expression of is required for growth on solid medium. Note that the concentration of ATc required for the mutant was higher than for the BCG mutant. Open in a separate window Figure 2 Effect of PptT depletion on the growth of BCG and of H37Rv wild-type (WT) and the PMM168 mutant strain were grown in.