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The mycobacterium tuberculosis outer membrane channel protein CpnT confers susceptibility to toxic molecules
Publication . Danilchanka, Olga; Pires, David; Anes, Elsa; Niederweis, Michael
Mycobacterium tuberculosis, the causative agent of tuberculosis, is protected from toxic solutes by an effective outer membrane permeability barrier. Recently, we showed that the outer membrane channel protein CpnT is required for efficient nutrient uptake by M. tuberculosis and Mycobacterium bovis BCG. In this study, we found that the cpnT mutant of M. bovis BCG is more resistant than the wild type to a large number of drugs and antibiotics, including rifampin, ethambutol, clarithromycin, tetracycline, and ampicillin, by 8- to 32-fold. Furthermore, the cpnT mutant of M. bovis BCG was 100-fold more resistant to nitric oxide, a major bactericidal agent required to control M. tuberculosis infections in mice. Thus, CpnT constitutes the first outer membrane susceptibility factor in slow-growing mycobacteria. The dual functions of CpnT in uptake of nutrients and mediating susceptibility to toxic molecules are reflected in macrophage infection experiments: while loss of CpnT was detrimental for M. bovis BCG in macrophages that enable bacterial replication, presumably due to inadequate nutrient uptake, it conferred a survival advantage in macrophages that mount a strong bactericidal response. Importantly, the cpnT gene showed a significantly higher density of nonsynonymous mutations in drug-resistant clinical M. tuberculosis strains, indicating that CpnT is under selective pressure in human tuberculosis and/or during chemotherapy. Our results indicate that the CpnT channel constitutes an outer membrane gateway controlling the influx of nutrients and toxic molecules into slow-growing mycobacteria. This study revealed that reducing protein-mediated outer membrane permeability might constitute a new drug resistance mechanism in slow-growing mycobacteria.
Esters of pyrazinoic acid are active against pyrazinamide-resistant strains of Mycobacterium tuberculosis and other naturally resistant mycobacteria in vitro and ex vivo within macrophages
Publication . Pires, David; Valente, Emília; Simões, Marta Filipa; Carmo, Nuno; Testa, Bernard; Constantino, Luis; Anes, Elsa
Pyrazinamide (PZA) is active against major Mycobacterium tuberculosis species (M. tuberculosis, M. africanum, and M. microti) but not against M. bovis and M. avium. The latter two are mycobacterial species involved in human and cattle tuberculosis and in HIV coinfections, respectively. PZA is a first-line agent for the treatment of human tuberculosis and requires activation by a mycobacterial pyrazinamidase to form the active metabolite pyrazinoic acid (POA). As a result of this mechanism, resistance to PZA, as is often found in tuberculosis patients, is caused by point mutations in pyrazinamidase. In previous work, we have shown that POA esters and amides synthesized in our laboratory were stable in plasma (M. F. Simões, E. Valente, M. J. Gómez, E. Anes, and L. Constantino, Eur J Pharm Sci 37:257-263, 2009, http://dx.doi.org/10.1016/j.ejps.2009.02.012). Although the amides did not present significant activity, the esters were active against sensitive mycobacteria at concentrations 5- to 10-fold lower than those of PZA. Here, we report that these POA derivatives possess antibacterial efficacy in vitro and ex vivo against several species and strains of Mycobacterium with natural or acquired resistance to PZA, including M. bovis and M. avium. Our results indicate that the resistance probably was overcome by cleavage of the prodrugs into POA and a long-chain alcohol. Although it is not possible to rule out that the esters have intrinsic activity per se, we bring evidence here that long-chain fatty alcohols possess a significant antimycobacterial effect against PZA-resistant species and strains and are not mere inactive promoieties. These findings may lead to candidate dual drugs having enhanced activity against both PZA-susceptible and PZA-resistant isolates and being suitable for clinical development.
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Fundação para a Ciência e a Tecnologia
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3599-PPCDT
Número da atribuição
PTDC/BIA-BCM/102123/2008