Mycobacterial strategies for developing antimicrobial resistance and survival in non-replicative persistent state
Application of antibiotics to susceptible bacterial culture generates a minor population of persisters that remain susceptible to antibiotics but can endure them for extended periods. Recently, antibiotic persisters (APs) of mycobacteria were reported to experience oxidative stress and develop resistance when treated with lethal doses of ciprofloxacin or rifampicin. However, the mechanisms driving the de novo emergence of resistance remained unclear. Our findings show that mycobacterial APs activate the SOS response causing up-regulation of the error-prone DNA polymerase DnaE2. The sustained expression of dnaE2 in APs results in the rapid evolution of resistance to antibiotics. Inhibition of RecA (regulator of SOS response) by an anti-parasitic drug suramin, decreases the conversion rate of persisters to resistors in a diverse group of bacteria. Our study highlights suramin's novel application as a broad-spectrum agent in combating the development of drug resistance.
Depletion of intracellular iron by a sequential passage in iron-free medium results in non-replicative persistence (NRP) in mycobacteria. Supplementation of iron to iron-deprived NRP culture resulted in regrowth. RNA-seq analysis of Mycobacterium smegmatis cultures from NRP and regrowth conditions revealed the upregulation of mycobacterial protein turnover pathway in both conditions. Proteomic analysis identified enzymes of the TCA cycle, ATP synthesis, and amino acid metabolism to be the targets of degradation during iron starvation. Deletion of the protein turnover pathway resulted in reduced bacterial survival in the NRP state and delayed the recovery upon iron supplementation. Further, dysregulation of the protein turnover pathway by conditional overexpression of ferredoxin affected bacterial viability during iron starvation. Our initial finding suggests that selective degradation of proteins during iron starvation could result in the metabolic reprogramming of bacteria leading to the establishment and stabilization of the NRP state in mycobacteria.