RESUMO
Tuberculous sputum contains multipleMycobacterium tuberculosispopulations with different requirements for isolationin vitro These include cells that form colonies on solid media (plateableM. tuberculosis), cells requiring standard liquid medium for growth (nonplateableM. tuberculosis), and cells requiring supplementation of liquid medium with culture supernatant (SN) for growth (SN-dependentM. tuberculosis). Here, we describe protocols for the cryopreservation and direct assessment of antimicrobial tolerance of theseM. tuberculosispopulations within sputum. Our results show that first-line drugs achieved only modest bactericidal effects on all three populations over 7 days (1 to 2.5 log10reductions), and SN-dependentM. tuberculosiswas more tolerant to streptomycin and isoniazid than the plateable and nonplateableM. tuberculosisstrains. Susceptibility of plateableM. tuberculosisto bactericidal drugs was significantly increased after passagein vitro; thus, tolerance observed in the sputum samples from the population groups was likely associated with mycobacterial adaptation to the host environment at some time prior to expectoration. Our findings support the use of a simpleex vivosystem for testing drug efficacies against mycobacteria that have phenotypically adapted during tuberculosis infection.
Assuntos
Adaptação Fisiológica , Antituberculosos/farmacologia , Bioensaio , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Estreptomicina/farmacologia , Criopreservação , Farmacorresistência Bacteriana/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/fisiologia , Fenótipo , Escarro/microbiologia , Tuberculose Pulmonar/microbiologiaRESUMO
We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria.
Assuntos
Acil Coenzima A/metabolismo , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Mycobacterium/metabolismo , Acil Coenzima A/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação/genética , DNA Bacteriano/genética , Polarização de Fluorescência , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/genética , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Mycobacterium/genética , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Ácido Oleico/farmacologia , Palmitoil Coenzima A/química , Palmitoil Coenzima A/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Antimicrobials targeting cell wall biosynthesis are generally considered inactive against nonreplicating bacteria. Paradoxically, we found that under nonpermissive growth conditions, exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, RaaS (for regulator of antimicrobial-assisted survival), encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial death under nonpermissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination therapies for persistent infectious diseases, such as tuberculosis.
