Metabolic Reprogramming of Vibrio cholerae Impaired in Respiratory NADH Oxidation Is Accompanied by Increased Copper Sensitivity

Abstract

The electrogenic, sodium ion-translocating NADH:quinone oxidoreductase (NQR) from is frequent in pathogenic bacteria and a potential target for antibiotics. NQR couples the oxidation of NADH to the formation of a sodium motive force (SMF) and therefore drives important processes, such as flagellar rotation, substrate uptake, and energy-dissipating cation-proton antiport. We performed a quantitative proteome analysis of O395N1 compared to its variant lacking the NQR using minimal medium with glucose as the carbon source. We found 84 proteins (regulation factor of ≥2) to be changed in abundance. The loss of NQR resulted in a decrease in the abundance of enzymes of the oxidative branch of the tricarboxylic acid (TCA) cycle and an increase in abundance of virulence factors AcfC and TcpA. Most unexpected, the copper resistance proteins CopA, CopG, and CueR were decreased in the deletion strain. As a consequence, the mutant exhibited diminished resistance to copper compared to the reference strain, as confirmed in growth studies using either glucose or mixed amino acids as carbon sources. We propose that the observed adaptations of the deletion strain represent a coordinated response which counteracts a drop in transmembrane voltage that challenges in its different habitats. The importance of the central metabolism for bacterial virulence has raised interest in studying catabolic enzymes not present in the host, such as NQR, as putative targets for antibiotics. lacking the NQR, which is studied here, is a model to estimate the impact of specific NQR inhibitors on the phenotype of a pathogen. Our comparative proteomic study provides a framework to evaluate the chances of success of compounds directed against NQR with respect to their bacteriostatic or bactericidal action.

Impact of Na + -Translocating NADH:Quinone Oxidoreductase on Iron Uptake and nqrM Expression in Vibrio cholerae

“…1). While the cytosolic Nqr subunits and ApbE were detected in the proteome of wt V. cholerae (18,19), the putative Fe-S maturation factor NqrM, encoded by the gene located downstream of the nqr operon, was not detected. NqrM is a rather small protein, with only 77 amino acids, which does not have many tryptic cleavage sites.…”

“…The V. cholerae genome has been completely sequenced (19), and iron transport systems are encoded on both of its chromosomes. In our recent proteome study (18), we compared the abundance of proteins in V. cholerae O395N1 (reference strain) with that in its mutant Δnqr derivative strain, where the six structural genes nqrABCDEF of the operon encoding the membrane-bound NQR respiratory complex are deleted (20). About 2,000 proteins were identified in each strain, and most of the iron transporters were present in both the wt and Δnqr strain in similar abundances.…”

“…Effect of iron limitation on growth of V. cholerae wild type, the nqr deletion strain, and a mutant defective in ubiquinone biosynthesis. The V. cholerae cells used for the proteome analysis were grown in a glucose minimal medium without added transition metals (18). We first asked if these cells had suffered from iron limitation, which resembles the situation in the human host when iron is restricted.…”

“…A V. cholerae mutant strain lacking the structural nqrABCDEF genes is impaired in the generation of a transmembrane potential (17), but it is able to grow by shifting metabolism from respiratory toward fermentative ATP formation (18). A comparative, quantitative proteome study of the V. cholerae reference (wild type [wt]) and the nqr deletion strains revealed a 2.7-fold increase in abundance of the predicted Fe 2ϩ transporter (FeoB) in the nqr mutant strain (18). Here, we study the impact of iron on growth and expression of feoB in the V. cholerae reference strain and nqr deletion strain and on a mutant defective in ubiquinone biosynthesis, the ΔubiC strain.…”

Genetic dissection of the fermentative and respiratory contributions supportingVibrio choleraehypoxic growth

“…In addition to this core NO 3 − reduction machinery, we identified two proteins that have not previously been implicated in NO 3 − respiration, including NqrF, a critical subunit of the Na+-translocating NADH:quinone dehydrogenase Nqr. In V. cholerae , Na + -Nqr has been reported to regulate bacterial motility, metabolism (30), resistance against heavy metals (31), and virulence (32–34). Deletion of Na + -Nqr in V. cholerae impaired membrane potential and delayed NO 3 − reduction to NO 2 − relative to the WT strain, suggesting that Na + -Nqr is required for maintenance of the ETC under NO 3 − respiratory conditions.…”