제1저자 김화영(미생물학교실, BK21)
공동교신저자 윤상선(미생물학교실, BK21)
J Biol Chem. 2018 Apr 13(15):5679-5694. doi: 10.1074/jbc.RA117.000383. Epub 2018 Feb 23.
Guanosine tetra- and pentaphosphate increase antibiotic tolerance by reducing reactive oxygen species production in Vibrio cholerae.
Kim HY1,2, Go J1,2, Lee KM1,2, Oh YT3,4, Yoon SS5,2.
1From the Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, and.2the Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine, Seoul 03722, Korea and.3From the Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, and firstname.lastname@example.org Freshwater Bioresources Utilization Division, Nakdonggang National Institute of Biological Resources, SangJu 37242, Korea.5From the Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, and email@example.com.
The pathogen Vibrio cholerae is the causative agent of cholera. Emergence of antibiotic-resistant V. cholerae strains is increasing, but the underlying mechanisms remain unclear. Herein, we report that the stringent response regulator and stress alarmone guanosine tetra- and pentaphosphate ((p)ppGpp) significantly contributes to antibiotic tolerance in V. cholerae We found that N16961, a pandemic V. cholerae strain, and its isogenic (p)ppGpp-overexpressing mutant ΔrelAΔspoT are both more antibiotic-resistant than (p)ppGpp0 (ΔrelAΔrelVΔspoT) and ΔdksA mutants, which cannot produce or utilize (p)ppGpp, respectively. We also found that additional disruption of the aconitase B-encoding and tricarboxylic acid (TCA) cycle gene acnB in the (p)ppGpp0 mutant increases its antibiotic tolerance. Moreover, expression of TCA cycle genes, including acnB, was increased in (p)ppGpp0, but not in the antibiotic-resistant ΔrelAΔspoT mutant, suggesting that (p)ppGpp suppresses TCA cycle activity, thereby entailing antibiotic resistance. Importantly, when grown anaerobically or incubated with an iron chelator, the (p)ppGpp0 mutant became antibiotic-tolerant, suggesting that reactive oxygen species (ROS) are involved in antibiotic-mediated bacterial killing. Consistent with that hypothesis, tetracycline treatment markedly increased ROS production in the antibiotic-susceptible mutants. Interestingly, expression of the Fe(III) ABC transporter substrate-binding protein FbpA was increased 10-fold in (p)ppGpp0, and fbpA gene deletion restored viability of tetracycline-exposed (p)ppGpp0 cells. Of note, FbpA expression was repressed in the (p)ppGpp-accumulating mutant, resulting in a reduction of intracellular free iron, required for the ROS-generating Fenton reaction. Our results indicate that (p)ppGpp-mediated suppression of central metabolism and iron uptake reduces antibiotic-induced oxidative stress in V. cholerae.