Oxidation of dCTP contributes to antibiotic lethality in stationary-phase mycobacteria

Abstract

Growing evidence shows that generation of reactive oxygen species (ROS) derived from antibiotic-induced metabolic perturbation contribute to antibiotic lethality. However, our knowledge of the mechanisms by which antibiotic-induced oxidative stress actually kills cells remains elusive. Here, we show that oxidation of dCTP underlies ROS-mediated antibiotic lethality via induction of DNA double-strand breaks (DSBs). Deletion of -encoded 5-OH-dCTP-specific pyrophosphohydrolase potentiates antibiotic killing of stationary-phase mycobacteria, but did not affect antibiotic efficacy in exponentially growing cultures. Critically, the effect of deletion on potentiating antibiotic killing is associated with antibiotic-induced ROS and accumulation of 5-OH-dCTP. Independent lines of evidence presented here indicate that the increased level of DSBs observed in the mutant is a dead-end event accounting for enhanced antibiotic killing. Moreover, we provided genetic evidence that 5-OH-dCTP is incorporated into genomic DNA via error-prone DNA polymerase DnaE2 and repair of 5-OH-dC lesions via the endonuclease Nth leads to the generation of lethal DSBs. This work provides a mechanistic view of ROS-mediated antibiotic lethality in stationary phase and may have broad implications not only with respect to antibiotic lethality but also to the mechanism of stress-induced mutagenesis in bacteria.

Arginine-deprivation–induced oxidative damage sterilizes Mycobacterium tuberculosis

“…This common response involves changes in iron and redox metabolism and accumulation of ROS causing oxidative damage of DNA, RNA, and proteins, ending in cell death (7). ROS-mediated oxidative damage is also a recognized rapid killing mechanism for Mtb (8)(9)(10)(11)(12)(13). The mycobactericidals isoniazid (INH) and vitamin C have been shown to induce rapid death of Mtb by ROS generation, leading to oxidative damage (8,9,13).…”

Putative TetR family transcriptional regulator Rv1019 ofMycobacterium tuberculosisis an auto-repressor and a negative regulator ofmfd-mazGoperon

“…Mycobacterial MazG is involved in DNA synthesis by the hydrolysis of dNTPs and elimination of mutagenic dNTPs generated by oxidative stresses (32–34). Deletion of mazG has shown to cause genomic instability in dormant Mtb (34), whereas antibiotic-induced oxidative stress reduced the survival of ΔmazG Mtb in activated macrophages (33). We found that downregulation of the expression of MSMEG_5422 , the orthologue of mazG, in M. smegmatis constitutively expressing Rv1019, made the bacteria more sensitive to H 2 O 2.…”

Antibiotic killing through oxidized nucleotides

“…In PNAS, Fan et al (14) explore whether different classes of bactericidal antibiotics used to treat Mycobacterium tuberculosis, the causative agent of tuberculosis, share a common killing cascade. They used bactericidal antibiotics of the aminoglycoside and fluoroquinolone classes that were also used in previous bactericidal antibiotic lethality studies in E. coli.…”

“…Fan et al (14) focus on the oxidized derivative of dCTP, 5-OH-dCTP, rather than on the oxidized derivative of guanine, 8-Oxo-G. The presence of four mutT-like genes in mycobacteria and the absence of the mutagenic signature associated with 8-Oxo-G (GC→TA) in mutational analysis studies suggest that mycobacteria are efficient in removing 8-Oxo-G from its genome.…”

“…Fan et al (14) show that the mazG mutant of M. tuberculosis and of Mycobacterium smegmatis is more sensitive than the respective wild-type strain to bactericidal antibiotics in stationary phase and during macrophage infection. This sensitivity occurs presumably because of the incorporation of 5-OH-dCTP into DNA.…”