Guanosine tetra- and pentaphosphate increase antibiotic tolerance by reducing reactive oxygen species production in Vibrio cholerae

Young Taek Oh,Kang-Mu Lee,Hwa Young Kim,Sang Sun Yoon,Junhyeok Go

doi:10.1074/jbc.ra117.000383

Abstract

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.

Highlights

The pathogen Vibrio cholerae is the causative agent of cholera
Cholera is characterized by cholera toxin (CT)-induced profuse watery diarrhea
Mortality includes victims who fail to receive immediate interventions and young patients with immature stomach function [82]. These cases apparently need prompt antibiotic treatment to reduce the volume of diarrhea and kill the causative pathogen, V. cholerae

Summary

Results

SR regulates bacterial responses to overcome unfavorable growth conditions in V. cholerae. Consistent with previous results, sharper decreases in bacterial viability were detected when using exponential-phase cells (Fig. S2, A and C) Together, these results demonstrated that deletion of the acnB gene conferred a survival advantage to V. cholerae in the presence of antibiotic stresses. Antibiotic-susceptible (p)ppGpp0 and ⌬dksA mutants maintained their regular curve-shaped morphotype even at the stationary phase (Fig. 3C and Fig. S3) These results clearly suggest that bacterial cells with reduced size are probably more resistant to antibiotic treatment. Of particular interest is that disruption of the acnB gene resulted in shorter and thinner morphotypes, a phenotype observed under (p)ppGpp-accumulating conditions (Fig. 3C and Fig. S3) Together, these results demonstrated that (i) cell shape changes could be induced upon intracellular (p)ppGpp accumulation or metabolic alterations by acnB gene mutation and (ii) such changes are closely related to bacterial responses to antibiotic treatment

Other TCA cycle mutants also exhibit increased antibiotic tolerance

Discussion

Bacterial strains and growth conditions

Relevant characteristic

Antibiotic tolerance assay

Gene name

Scanning electron microscopy analysis

ROS measurement

Findings

EPR analysis