RESUMO
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is a leading cause of infectious disease mortality. The salicylic acid derived small molecule siderophores known as mycobactins are essential in vivo for iron acquisition of Mtb where iron is restricted in the host. Herein, we synthesize and explore the mechanism of action of polyfluorinated salicylic acid derivates, which were previously reported to possess potent antimycobacterial activity. We hypothesized fluorinated salicylic acid derivates may inhibit mycobactin biosynthesis through initial bioactivation and conversion to downstream metabolites that block late steps in assembly of the mycobactins. Enzymatic studies demonstrated that some of the fluorinated salicylic acid derivatives compounds were readily activated by the bifunctional adenylating enzyme MbtA, responsible for incorporation of salicylic acid into the mycobactin biosynthetic pathway; however, they did not inhibit mycobactin biosynthesis as confirmed by LS-MS/MS using an authentic synthetic mycobactin standard. Further mechanistic analysis of the most active derivative (Sal-4) using an MbtA-overexpressing Mtb strain as well as complementation studies with iron and salicylic acid revealed Sal-4 cannot be antagonized by overexpression of MbtA or through supplementation with iron or salicylic acid. Taken together, our results indicate the observed antimycobacterial activity of polyfluorinated salicylic acid derivative is independent of mycobactin biosynthesis.
Assuntos
Mycobacterium tuberculosis , Sideróforos , Sideróforos/metabolismo , Mycobacterium tuberculosis/metabolismo , Ácido Salicílico/farmacologia , Ácido Salicílico/metabolismo , Espectrometria de Massas em Tandem , Ferro/metabolismoRESUMO
Doxycycline, an FDA-approved tetracycline, is used in tuberculosis in vivo models for the temporal control of mycobacterial gene expression. In these models, animals are infected with recombinant Mycobacterium tuberculosis carrying genes of interest under transcriptional control of the doxycycline-responsive TetR-tetO unit. To minimize fluctuations of plasma levels, doxycycline is usually administered in the diet. However, tissue penetration studies to identify the minimum doxycycline content in food achieving complete repression of TetR-controlled genes in tuberculosis (TB)-infected organs and lesions have not been conducted. Here, we first determined the tetracycline concentrations required to achieve silencing of M. tuberculosis target genes in vitro Next, we measured doxycycline concentrations in plasma, major organs, and lung lesions in TB-infected mice and rabbits and compared these values to silencing concentrations measured in vitro We found that 2,000 ppm doxycycline supplemented in mouse and rabbit feed is sufficient to reach target concentrations in TB lesions. In rabbit chow, the calcium content had to be reduced 5-fold to minimize chelation of doxycycline and deliver adequate oral bioavailability. Clearance kinetics from major organs and lung lesions revealed that doxycycline levels fall below concentrations that repress tet promoters within 7 to 14 days after doxycycline is removed from the diet. In summary, we have shown that 2,000 ppm doxycycline supplemented in standard mouse diet and in low-calcium rabbit diet delivers concentrations adequate to achieve full repression of tet promoters in infected tissues of mice and rabbits.
Assuntos
Antibacterianos/farmacocinética , Doxiciclina/farmacocinética , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Tuberculose/metabolismo , Ração Animal , Animais , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Disponibilidade Biológica , Cálcio/farmacologia , Modelos Animais de Doenças , Doxiciclina/administração & dosagem , Doxiciclina/uso terapêutico , Feminino , Inativação Gênica , Pulmão/metabolismo , Camundongos , Coelhos , Resistência a Tetraciclina , Distribuição Tecidual/genética , TransgenesRESUMO
Chemotherapy for tuberculosis (TB) is lengthy and could benefit from synergistic adjuvant therapeutics that enhance current and novel drug regimens. To identify genetic determinants of intrinsic antibiotic susceptibility in Mycobacterium tuberculosis, we applied a chemical genetic interaction (CGI) profiling approach. We screened a saturated transposon mutant library and identified mutants that exhibit altered fitness in the presence of partially inhibitory concentrations of rifampin, ethambutol, isoniazid, vancomycin, and meropenem, antibiotics with diverse mechanisms of action. This screen identified the M. tuberculosis cell envelope to be a major determinant of antibiotic susceptibility but did not yield mutants whose increase in susceptibility was due to transposon insertions in genes encoding efflux pumps. Intrinsic antibiotic resistance determinants affecting resistance to multiple antibiotics included the peptidoglycan-arabinogalactan ligase Lcp1, the mycolic acid synthase MmaA4, the protein translocase SecA2, the mannosyltransferase PimE, the cell envelope-associated protease CaeA/Hip1, and FecB, a putative iron dicitrate-binding protein. Characterization of a deletion mutant confirmed FecB to be involved in the intrinsic resistance to every antibiotic analyzed. In contrast to its predicted function, FecB was dispensable for growth in low-iron medium and instead functioned as a critical mediator of envelope integrity.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Parede Celular/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/efeitos dos fármacos , Serina Proteases/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Etambutol/farmacologia , Galactanos/biossíntese , Perfilação da Expressão Gênica , Humanos , Bombas de Íon/deficiência , Bombas de Íon/genética , Isoniazida/farmacologia , Ligases/genética , Ligases/metabolismo , Manosiltransferases/genética , Manosiltransferases/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Meropeném , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Peptidoglicano/biossíntese , Rifampina/farmacologia , Serina Proteases/metabolismo , Tienamicinas/farmacologia , Vancomicina/farmacologiaRESUMO
In recent years, whole-cell-based screens for novel small molecule inhibitors active against Mycobacterium tuberculosis in culture followed by the whole-genome sequencing of spontaneous resistant mutants have identified multiple chemical scaffolds thought to kill the bacterium through the inactivation of the mycolic acid transporter, MmpL3. Consistent with the fact that MmpL3 is required for the formation of the mycobacterial outer membrane, we have conclusively shown in this study, using conditionally regulated knockdown mutants, that mmpL3 is required for the replication and viability of M. tuberculosis, both under standard laboratory growth conditions and during the acute and chronic phases of infection in mice. Speaking for the vulnerability of this target, silencing mmpL3 had a rapid bactericidal effect on actively replicating cells in vitro and reduced by 3 to 5 logs in less than 4 weeks the bacterial loads of acutely and chronically infected mouse lungs, respectively. Depletion of MmpL3 further rendered M. tuberculosis hypersusceptible to MmpL3 inhibitors. The exquisite vulnerability of MmpL3 at all stages of the infection establishes this transporter as an attractive new target with the potential to improve and shorten current drug-susceptible and drug-resistant tuberculosis chemotherapies.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Pulmão/efeitos dos fármacos , Proteínas de Membrana Transportadoras/genética , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológico , Animais , Carga Bacteriana/efeitos dos fármacos , Transporte Biológico , Ciprofloxacina/farmacologia , Modelos Animais de Doenças , Doxiciclina/farmacologia , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Isoniazida/farmacologia , Pulmão/microbiologia , Pulmão/patologia , Proteínas de Membrana Transportadoras/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Resistente a Múltiplos Medicamentos/patologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologiaRESUMO
Chemotherapeutic options to treat tuberculosis are severely restricted by the intrinsic resistance of Mycobacterium tuberculosis to the majority of clinically applied antibiotics. Such resistance is partially provided by the low permeability of their unique cell envelope. Here we describe a complementary system that coordinates resistance to drugs that have penetrated the envelope, allowing mycobacteria to tolerate diverse classes of antibiotics that inhibit cytoplasmic targets. This system depends on whiB7, a gene that pathogenic Mycobacterium shares with Streptomyces, a phylogenetically related genus known as the source of diverse antibiotics. In M. tuberculosis, whiB7 is induced by subinhibitory concentrations of antibiotics (erythromycin, tetracycline, and streptomycin) and whiB7 null mutants (Streptomyces and Mycobacterium) are hypersusceptible to antibiotics in vitro. M. tuberculosis is also antibiotic sensitive within a monocyte model system. In addition to antibiotics, whiB7 is induced by exposure to fatty acids that pathogenic Mycobacterium species may accumulate internally or encounter within eukaryotic hosts during infection. Gene expression profiling analyses demonstrate that whiB7 transcription determines drug resistance by activating expression of a regulon including genes involved in ribosomal protection and antibiotic efflux. Components of the whiB7 system may serve as attractive targets for the identification of inhibitors that render M. tuberculosis or multidrug-resistant derivatives more antibiotic-sensitive.