RESUMEN
Bacterial persistence coupled with biofilm formation is directly associated with failure of antibiotic treatment of tuberculosis. We have now identified 4-(4,7-DiMethyl-1,2,3,4-tetrahydroNaphthalene-1-yl)Pentanoic acid (DMNP), a synthetic diterpene analogue, as a lead compound that was capable of suppressing persistence and eradicating biofilms in Mycobacterium smegmatis. By using two reciprocal experimental approaches - ΔrelMsm and ΔrelZ gene knockout mutations versus relMsm and relZ overexpression technique - we showed that both RelMsm and RelZ (p)ppGpp synthetases are plausible candidates for serving as targets for DMNP. In vitro, DMNP inhibited (p)ppGpp-synthesizing activity of purified RelMsm in a concentration-dependent manner. These findings, supplemented by molecular docking simulation, suggest that DMNP targets the structural sites shared by RelMsm, RelZ, and presumably by a few others as yet unidentified (p)ppGpp producers, thereby inhibiting persister cell formation and eradicating biofilms. Therefore, DMNP may serve as a promising lead for development of antimycobacterial drugs.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Diterpenos/farmacología , Ligasas/metabolismo , Mycobacterium smegmatis/enzimología , Antibacterianos/síntesis química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Sitios de Unión , Diterpenos/química , Diterpenos/metabolismo , Ligasas/antagonistas & inhibidores , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/fisiología , Estructura Terciaria de ProteínaRESUMEN
The extensively discussed idea of oxidative stress development under antibiotic treatment was confirmed using an antioxidant gene expression (soxRS-, oxyR-regulon) approach, including microaerobic cultivation conditions. The killing action of antibiotics and their ability to cause peroxide oxidative stress in Escherichia coli cells was comparable to a similar hydrogen peroxide capacity; therefore, the involvement of intracellular hydrogen peroxide production in the killing action of antibiotics seems plausible under conditions studied. The temporary increase of ATP/ADP (which returned to untreated levels in 10 min) and the intensification of respiration preceded the development of oxidative stress. The sharp rise in ATP/ADP was due to the accumulation of ATP with a slight increase in the ADP content. We proposed that ATP accumulation was not a result of increased respiration but was due to the inhibition of energy-consuming processes. The association of reactive oxygen species formation under antibiotic treatment with the inhibition of direct electron flow pathway along the respiratory chain, and a possible role of a sharp rise in ATP/ADP in this process is hypothesized.
Asunto(s)
Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacosRESUMEN
Bactericidal antibiotics (fluoroquinolones, aminoglycosides and cephalosporins) at their sublethal concentrations were able to produce hydroxyl radicals, hydrogen peroxide and superoxide anions (ROS) in Escherichia coli cells, which resulted in damage to proteins and DNA. The cells responded to oxidative stress by a 2-3-fold increase in cell polyamines (putrescine, spermidine) produced as a consequence of upregulation of ornithine decarboxylase (ODC). Relief of oxidative stress by cessation of culture aeration or addition of antioxidants substantially diminished or even completely abolished polyamine accumulation observed in response to antibiotics. Alternatively, inhibition of polyamine synthesis resulted in enhancement of oxidative stress in antibiotic-processed cells. When added to antibiotic-inhibited culture, polyamines reduced intracellular ROS production and thereby prevented damage to proteins and DNA. These effects eventually resulted in a substantial increase in cell viability, growth recovery and antibiotic resistance that were more strongly expressed in polyamine-deficient mutants.