Although the classical antibiotic spectinomycin is a potent bacterial protein synthesis inhibitor, poor antimycobacterial activity limits its clinical application for treating tuberculosis. Using structure-based design, we generated a new semisynthetic series of spectinomycin analogs with selective ribosomal inhibition and excellent narrow-spectrum antitubercular activity. In multiple murine infection models, these spectinamides were well tolerated, significantly reduced lung mycobacterial burden and increased survival. In vitro studies demonstrated a lack of cross resistance with existing tuberculosis therapeutics, activity against multidrug-resistant (MDR) and extensively drug-resistant tuberculosis and an excellent pharmacological profile. Key to their potent antitubercular properties was their structural modification to evade the Rv1258c efflux pump, which is upregulated in MDR strains and is implicated in macrophage-induced drug tolerance. The antitubercular efficacy of spectinamides demonstrates that synthetic modifications to classical antibiotics can overcome the challenge of intrinsic efflux pump-mediated resistance and expands opportunities for target-based tuberculosis drug discovery.

The Mycobacterium bovis P55 gene, located downstream from the gene that encodes the immunogenic lipoprotein P27, has been characterized. The gene was identical to the open reading frame of the Rv1410c gene in the genome of Mycobacterium tuberculosis H37Rv, annotated as a probable drug efflux protein. Genes similar to P55 were present in all species of the M. tuberculosis complex and other mycobacteria such as Mycobacterium leprae and Mycobacterium avium . By Western blotting, P55 was located in the membrane fraction of M. bovis . When transformed into Mycobacterium smegmatis after cloning, P55 conferred aminoglycoside and tetracycline resistance. The levels of resistance to streptomycin and tetracycline conferred by P55 were decreased in the presence of the protonophore carbonyl cyanide m -chlorophenylhydrazone and the pump inhibitors verapamil and reserpine. M. smegmatis cells expressing the plasmid-encoded P55 accumulated less tetracycline than the control cells. We conclude that P55 is a membrane protein implicated in aminoglycoside and tetracycline efflux in mycobacteria.

Bacterial efflux pumps have traditionally been studied as low-level drug resistance determinants. Recent insights have suggested that efflux systems are often involved with fundamental cellular physiological processes, suggesting that drug extrusion may be a secondary function. In Mycobacterium tuberculosis , little is known about the physiological or drug resistance roles of efflux pumps. Using Mycobacterium bovis BCG as a model system, we showed that deletion of the Rv1410c gene encoding the P55 efflux pump made the strain more susceptible to a range of toxic compounds, including rifampin (rifampicin) and clofazimine, which are first- and second-line antituberculosis drugs. The efflux pump inhibitors carbonyl cyanide m -chlorophenylhydrazone (CCCP) and valinomycin inhibited the P55-determined drug resistance, suggesting the active export of the compounds by use of the transmembrane proton and electrochemical gradients as sources of energy. In addition, the P55 efflux pump mutant was more susceptible to redox compounds and displayed increased intracellular redox potential, suggesting an essential role of the efflux pump in detoxification processes coupled to oxidative balance within the cell. Finally, cells that lacked the p55 gene displayed smaller colony sizes and had a growth defect in liquid culture. This, together with an increased susceptibility to the cell wall-targeting compounds bacitracin and vancomycin, suggested that P55 is needed for proper cell wall assembly and normal growth in vitro. Thus, P55 plays a fundamental role in oxidative stress responses and in vitro cell growth, in addition to contributing to intrinsic antibiotic resistance. Inhibitors of the P55 efflux pump could help to improve current treatments for tuberculosis.

Tuberculosis is one of the major causes of infection across the world. The emergence of multi-, extensively- and totally drug-resistant strains of Mycobacterium tuberculosis contributes to the lack of therapeutic options available. The mechanisms associated with this resistance could involve mutations in genes coding for target proteins, decreased permeability, increased efflux and so on. Resistance mediated by efflux systems has become more relevant, since these systems help the bacteria to extrude antibiotics until relevant mutations emerge and become established in the population. Therefore, compounds that inhibit these transport systems are of major importance and have been studied in the last few years. Not only do these compounds act on the bacterial efflux systems but they have also been explored for their dual role as boosters of the macrophage-infected cells. The search for novel compounds or combinations of adjuvant compounds and antibiotics to treat mycobacterial multidrug-resistant infections has become a major goal in the treatment of these diseases.