Macrolide resistance and susceptibility in Mycobacterium tuberculosis

Tuberculosis has been recognized as a major public health program worldwide, exacerbated greatly by the HIV pandemic. The length and complexity of antibiotic therapy for tuberculosis and the emergence of multi-drug resistant strains makes a compelling case for the development of new anti-TB drugs.; Macrolides, antibiotics of choice for respiratory pathogens, represent a potential class for the development of new anti TB drugs although existing macrolides lack sufficient potency. The main goal of this project was to elucidate the mechanisms involved in macrolide innate resistance in TB and to understand the structural features that are the basis of macrolide resistance and activity in TB. A better knowledge of innate resistance will allow the rational design of novel macrolides able to exert activity by eluding the resistance mechanisms. Specific aims included the identification of major membrane transporters and the role of putative modifying enzymes such as erythromycin esterases. The approach consisted of cloning the corresponding genes into E. coli , and expressing them into M. smegmatis, strain naturally sensitive to macrolides. Transposon knock-out for the genes of interest were studied as well. The extent of target modification by ribosome methyl-transferase and its inducibility of such event was studied by primer extension. The macrolide minimal inhibitory concentration (MIC) was measured to evaluate the role of each gene product in macrolide resistance.; In order to gain a better understanding of structure-activity relationships with respect to this organism and to identify new macrolides or ketolides with superior activity against M. tuberculosis more than 30 macrolides and ketolides with a variety of substitutions were evaluated.; Several macrolides and ketolides bearing various substitutions at the 6,9 and 11,12 positions had MICs of ≤4muM, which represents an improvement over first and second-generation macrolides. Macrolides that were both potent and selectively toxic for M. tuberculosis were evaluated for activity in TB infected macrophages and in mice. RU66252, an 11,12 carbazate derivative, demonstrated a dose-responsive activity in both assays.; Some of the knowledge acquired from these studies has already provided sufficient insight to develop new potent anti-TB macrolides for possible therapeutic use in the near future.