Meeting Presentation

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

Charlotte Toulouse, Kristina Metesch, Jens Pfannstiel, Julia Steuber
Victor J. DiRita, Editor
DOI: 10.1128/JB.00761-17

ABSTRACT

The electrogenic, sodium ion-translocating NADH:quinone oxidoreductase (NQR) from Vibrio cholerae 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 V. cholerae 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 nqr 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 nqr deletion strain represent a coordinated response which counteracts a drop in transmembrane voltage that challenges V. cholerae in its different habitats.

IMPORTANCE 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. Vibrio cholerae 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.

INTRODUCTION

Vibrio cholerae is a facultative anaerobic, Gram-negative bacterium which faces very different habitats, from low to high salinity, in either free-floating or biofilm-associated lifestyles (1). V. cholerae strains carrying virulence factors such as the cholera toxin cause cholera in humans, a severe diarrheal disease (2). The signals and networks which ultimately lead to the production of virulence factors in V. cholerae in the human intestine have been studied intensively, with ToxT being the most important regulator of the virulence regulatory cascade (3). There is growing evidence that in addition to signals from the environment, the metabolism of a pathogen also determines its virulence (4–6). In V. cholerae, there is a specific link between the production of virulence factors (cholera toxin, or CTX, and toxin coregulated pilus, or TCP) and the activity of its electrogenic NADH dehydrogenase, the Na+-translocating NADH:quinone oxidoreductase (NQR) of the respiratory chain (7, 8). The NQR oxidizes NADH generated from the catabolic breakdown of nutrients under reduction of quinone, thereby delivering electrons into the respiratory chain. This exergonic redox reaction is coupled to the endergonic transport of Na+ across the cytoplasmic membrane. The NQR is a membrane-bound flavo-iron sulfur protein complex composed of six subunits (9). The enzyme is of central importance for V. cholerae, since the electrochemical Na+ gradient generated by the NQR drives many other cellular processes, such as flagellar rotation, exchange of cations, and expulsion of antibiotics (10–12).

Comparison of the growth behavior of V. cholerae with that of the corresponding nqr deletion strain on mixed amino acids at high salt concentrations revealed that lack of the NQR did not affect initial growth rates but diminished final growth yield (6, 13). However, the NQR was mandatory for growth under alkaline, hypoosmotic conditions, and it was demonstrated that the in vivo function of NQR is to maintain the transmembrane voltage rather than to establish a chemical Na+ gradient (13). In a suckling mouse model of infection, the persistence of V. cholerae depended on the NQR (14). Moreover, biofilm formation of Vibrio cholerae was affected by NQR activity (15). Thus, the NQR is an important catabolic enzyme in V. cholerae that cannot be fully replaced in terms of function by the nonelectrogenic NADH:quinone oxidoreductase (NDH2, VC1890), which is also found in V. cholerae. Since NQR is not structurally related to the respiratory complex I (NADH:Q oxidoreductase) of mitochondria, and since many pathogens depend on the NQR as an electrogenic NADH dehydrogenase (16), the NQR is considered a nontraditional target for antibiotics (17). A synthetic furanone, which was inspired by the natural NQR inhibitor korormicin, prevented intracellular proliferation of Chlamydia trachomatis, which relies on NQR for respiration (18). These findings support the idea that the NQR, a catabolic enzyme, is druggable.

Here, we study the impact of the deletion of NQR on V. cholerae by a comparative proteomic approach, using a label-free method combined with nano-liquid chromatography electrospray ionization-mass spectrometry (LC-ESI-MS) (19, 20). Previous transcriptomic and metabolomic studies of a V. cholerae Δnqr strain revealed changes in metabolic flow through the tricarboxylic acid (TCA) cycle, virulence factor production, and purine metabolism compared to those of the reference strain. In addition, lack of NQR resulted in increased accumulation of acetate in the medium (21). Our proteome-based results confirm downregulation of the oxidative branch of the TCA cycle observed previously (6, 21) and reveal additional changes in the proteome of the V. cholerae Δnqr strain. The lack of NQR strongly shifts nitrogen metabolism and triggers uptake and degradation of nucleosides. Most unexpectedly, the Vibrio cholerae Δnqr strain exhibits a strong downregulation of proteins participating in copper detoxification, leading to increased copper sensitivity. The implications of our findings for survival of V. cholerae under conditions of impaired respiration in different environments are discussed.

RESULTS AND DISCUSSION

Deletion of the NQR induces global changes on proteome level of V. cholerae O395N1.Approximately 2,000 proteins were identified by nanoscale liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-tandem MS [MS/MS]) analysis in the V. cholerae reference or the nqr deletion strain. Among these, 84 proteins differed in abundance by a factor of ≥2 (false discovery rate [FDR] of ≤0.05) in the nqr mutant (Fig. 1). As expected, three peripheral subunits of the NQR (NqrA, NqrC, and NqrF) were detected in the reference strain only. We did not detect the membrane-bound NqrB, NqrD, and NqrE subunits in cell lysates from the reference strain, but it should be noted that peptides from hydrophobic, membrane-bound proteins were only rarely detected despite the use of SDS as the extraction detergent.

FIG 1
FIG 1

Comparative proteome analysis of the V. cholerae reference and Δnqr strains. (A) The volcano plot shows the distribution of proteome data by plotting the fold change (log2 ratio) of LFQ (label-free quantification) intensities between the V. cholerae Δnqr and reference strains versus the statistical significance (−log10 P value). Proteins increased in abundance are shown in blue, and proteins decreased in abundance are in green, whereas not differentially abundant proteins are illustrated as gray dots. Proteins were set as differentially abundant with an FDR (false discovery rate) below 0.05 (indicated by the black line). (B) Distribution and amount of 84 differentially abundant proteins in the V. cholerae Δnqr strain compared to the reference strain regarding their different functions. Protein functions were annotated according to the KEGG database algorithm. Proteins without annotation are classified as unknown function (Table 1 provides functional assignments based on further sequence analyses).

Proteins were categorized by their function. A first assignment relied on the KEGG database. The largest proportion of differentially abundant proteins involved proteins from metabolism (30 proteins) and transport systems (23 proteins) (Fig. 1). Remarkably, synthesis of virulence factors was clearly related to NQR function, with 2 proteins of the differentially abundant proteins belonging to this class. Protein translation was also affected in the nqr deletion strain, with a share of 4 (ribosome biogenesis) and 3 (transcription factors) differentially abundant proteins each. A large number of differentially abundant proteins (19 proteins) could not be assigned to a function by KEGG. We therefore searched for homologous sequences with known functions using the BLAST tool and selected genomes from Gram-negative bacteria. This led to additional assignments of functions to proteins which are summarized in Table 1.

View this table:
TABLE 1

Proteins of V. cholerae O395N1 Δnqr strain compared to the reference strain that were increased or decreased in abundancea

Since the redox Na+ pump is not operating, the V. cholerae Δnqr strain suffers from a decreased transmembrane voltage which impairs ATP synthesis by oxidative phosphorylation (13). Obviously, many of the differentially regulated proteins have a function in counteracting this deficiency, leading to increased ATP synthesis by substrate-level phosphorylation reactions. However, these reactions come to a halt when NAD+ becomes limiting for critical reaction steps like, e.g., the oxidation of glyceraldehyde 3-phosphate (GAP) by GAP dehydrogenase (GAPDH) (Fig. 2). Below we propose, based on the proteomic data, strategies of the V. cholerae Δnqr strain to overcome this limitation, like a shift toward the reductive branch of the citric acid cycle and the formation and excretion of reduced products like cadaverine or succinate (Fig. 2). We speculate that a shift to fermentation despite oxic conditions occurs which requires adaptations in the V. cholerae Δnqr strain, like improved repair of the pyruvate-formate lyase, the key enzyme for ATP synthesis via acetate kinase, and storing redox equivalents as cadaverine (Fig. 2). Furthermore, we suggest that cadaverine formation depletes the V. cholerae Δnqr strain for ammonium. Correspondingly, the strain showed alterations in the abundance of proteins involved in amino acid metabolism. Besides a modulation of virulence factor production, the mutant strain was also impaired in copper homeostasis (Table 1 and Fig. 3). In the following sections, the proteome changes in the V. cholerae Δnqr strain are described in more detail.

FIG 2
FIG 2

V. cholerae Δnqr strain reveals strong changes in abundance of proteins involved in carbon metabolism. Proteins increased in abundance are marked in green, and proteins decreased in abundance are in red. GAP (glyceraldehyde 3-phosphate)- and pyruvate-forming enzymes were increased, whereas enzymes of the oxidative branch of the TCA cycle (gray) were decreased in abundance. Underlined metabolic products were increased in the Δnqr strain according to reference 21. In the inner membrane (IM), NQR and NDH2 (alternative NADH dehydrogenase) catalyze NADH oxidation and quinone (Q) reduction, and SDH (succinate dehydrogenase) or FRD (fumarate reductase) reacts with quinone or quinole (QH2), respectively. The dicarboxylate transporters Dct and DcuB participate in transport of succinate. OmpK, a nucleoside-specific channel-forming protein, is embedded in the outer membrane (OM).

FIG 3
FIG 3

Effect of Cu2+ on growth of the V. cholerae reference or the nqr deletion strain. V. cholerae O395N1 (WT; upper) and its corresponding NQR mutant (Δnqr strain; lower) were grown on mixed amino acids (left) or minimal medium with glucose (right) supplemented with the indicated concentrations of CuSO4. Error bars of every second data point were omitted for clarity.

Fueling the GAPDH reaction for increased substrate level phosphorylation.Higher abundancy of GAPDH (VCA0843, regulation factor [RF] of +2.5) detected in the proteome of the nqr deletion strain is supposed to promote the formation of phosphoenolpyruvate, the phosphate donor to ADP in the central energy-generating step of glycolysis. To increase the amount of GAP, the V. cholerae Δnqr strain exhibited increased levels of enzymes for the degradation of thymidine, namely, thymidine phosphorylase (DeoA; VC2349, RF of +2.9) and phosphopentomutase (DeoB; VC2348, RF of +10.6). Notably, the abundance of the outer membrane porin OmpK (VC2305, RF of +2.2), which is responsible for the import of the nucleoside thymidine, was also enhanced (Fig. 2). Increasing the amount of glycerol kinase (VCA0744, RF of +3.4) is also expected to enhance GAP formation in the V. cholerae Δnqr strain (Fig. 2 and Table 1).

Metabolism of pyruvate and acetate.The enzyme tryptophanase (TnaA; VCA0161, RF of +13.4), which catalyzes the formation of indole from l-tryptophan, showed highly increased abundance in the V. cholerae Δnqr strain (Fig. 2). TnaA produces indole, pyruvate, and ammonia from tryptophan. Also, in the nqr deletion strain, increased abundance of N-acetylneuraminate (NANA) lyase (VC1776, RF of +2.8) and l-serine dehydratase (VC1300, RF of +2.3) is predicted to enhance production of pyruvate by degradation of NANA and serine, respectively (Fig. 2 and Table 1). The increased amount of these three enzymes suggests that more pyruvate is available for downstream reactions such as conversion to formate and acetyl coenzyme A (acetyl-CoA) by pyruvate formate lyase (PFL), which is not changed in abundance. Formation of acetyl-CoA in the V. cholerae Δnqr strain would be advantageous, since this energy-rich compound can be converted to acetyl phosphate, which reacts with ADP to form ATP (Fig. 2). The PFL is the key enzyme of mixed-acid fermentation performed by many enterobacteria (22, 23). PFL is an oxygen-sensitive enzyme which requires repair of its FeS centers by GrcA (VC2361, RF of +4) (24), a protein which was increased in the V. cholerae Δnqr strain compared to that in the reference strain (Table 1 and Fig. 2). We assume that the large amount of GrcA increased the amount of active PFL, although its overall abundancy in the reference and the nqr deletion strains was similar. In summary, we predict that the V. cholerae Δnqr strain is able to retrieve ATP from the conversion of pyruvate, bypassing the formation of NADH during oxidation of pyruvate to CO2 (Fig. 2) by the pyruvate dehydrogenase complex (not changed in abundance).

The fate of acetate.What is the fate of acetate in the V. cholerae Δnqr strain? Excretion and reutilization of acetate (the so-called acetate switch) first described for Escherichia coli (25) were previously observed in the V. cholerae reference strain studied here (6, 21). The authors also demonstrated that the V. cholerae Δnqr strain exhibited increased acetate production compared to the reference strain during growth on mixed amino acids at 30°C and concluded that in the absence of NQR, the acetate switch is broken (6). In accord with these findings, we observed during growth on glucose strong decrease in abundance of acetyl-CoA synthase (ACS; VC0298, RF of −4.9) in the nqr deletion strain (Table 1 and Fig. 2). Of note, ACS influences virulence of V. cholerae in the Drosophila melanogaster model of infection (5). In the V. cholerae Δnqr strain, the decreased amount of aconitate hydratase (or aconitase; VC1338, RF of −2.4) is expected to result in diminished NADH formation by the oxidative branch of the TCA cycle, which would save NAD+ for glycolysis and substrate-level phosphorylation. Aconitase (VC1338, RF of −2.4) also represents a critical enzyme for the glyoxylate shunt with downstream enzymes isocitrate lyase (VC0736, RF of −2.3) and malate synthase (VC0734, RF of −2). By downregulation of the latter (Table 1), utilization of acetate as a carbon source is proposed to be impaired in the V. cholerae Δnqr strain.

Metabolism of cadaverine and succinate in the V. cholerae Δnqr strain.It was previously reported that cadaverine accumulates in the V. cholerae Δnqr strain (21), and cadA encoding the lysine decarboxylase was the most strongly upregulated gene in the nqr deletion strain in the transcriptomics study (21). This study also revealed very high expression of the lysine/cadaverine antiporter gene cadB. Cadaverine was increased in the nqr deletion mutant (21). In accord with this study, we observed high abundance of aspartate ammonia lyase (VC2698, RF of +5) and of lysine decarboxylase (VC0281, RF of +3.4), whereas CadB was not detected, probably because of its hydrophobic nature. In the following we speculate why the increased abundance of these proteins is beneficial for the V. cholerae Δnqr strain. We assume that the diminished NADH formation in the oxidative branch of the TCA cycle in the nqr deletion strain is not sufficient to maintain NAD+ at a critical threshold concentration for glycolysis. To remove reducing equivalents, the metabolism of fumarate and succinate, two metabolites from the reductive branch of the TCA cycle, was altered in the nqr deletion strain. By increasing the amount of aspartate ammonia lyase (VC2698, RF of +5), we expect increased production of aspartate from fumarate. Aspartate is the precursor of lysine (Fig. 2), and lysine concentration was presumably kept low due to the large amount of lysine decarboxylase (VC0281, RF of +3.4) present in the V. cholerae Δnqr strain. By this prognosticated continuous withdrawal of lysine, the reaction chain leading from aspartate to lysine is stimulated, including two redox reactions involving the oxidation of NADPH. Transhydrogenase reactions are expected to replenish the NADPH pool under oxidation of NADH and formation of NAD+, which again enters glycolysis in the V. cholerae Δnqr strain. The strong upregulation of the aspartate ammonia lyase (VC2698, RF of +5) completes the assumed pathway, leading to increased cadaverine production and excretion in the V. cholerae Δnqr strain.

Thus, while part of the fumarate is predicted to be used for cadaverine formation under oxidation of NADPH, fumarate may also be reduced by quinol produced by the nonelectrogenic NADH:quinone oxidoreductase (NDH2; not changed in abundance), which is known to be active in the V. cholerae Δnqr strain (26) (Fig. 2). This reaction is catalyzed by the membrane-bound succinate dehydrogenase/fumarate reductase (SDH/FRD; not changed in abundance), a central enzyme of the reductive branch of the TCA cycle. Together, NDH-2 and FRD are assumed to convert NADH plus fumarate to NAD+ plus succinate. In the V. cholerae Δnqr strain, we observed an increased abundance of DcuB (VCA0205, RF of +4.7), the exporter of succinate (27). In parallel, the abundance of the succinate uptake system Dct (VC1929, RF of −4.6) (28) was diminished. This would favor net succinate export in the nqr deletion strain and again could be a means to deplete the cell of reducing equivalents stored in organic compounds.

A link between ammonium and cadaverine metabolism.We hypothesize that cadaverine represents a fermentation product in the aerobically grown V. cholerae Δnqr strain and expect a withdrawal of nitrogen from important anabolic reactions. With 18.7 mM NH4Cl, the N source in the minimal medium used here was limiting (29). Nitrogen limitation in the V. cholerae Δnqr strain was also evident from decreased abundance of enzymes participating in the degradation of amino acids (Table 1). All five participating enzymes of the tyrosine degradation pathway could be identified, and three of them revealed decreased protein levels in the V. cholerae Δnqr strain (hydroxypentylpyruvate dioxygenase, VC1344, RF of −3.8; homogentisate 1,2-dioxygenase, VC1345, RF of −2; and fumarylacetoacetase, VC1346, RF of −2.3). All four enzymes of the histidine degradation pathway were detected (Table 1). Among them, the histidine ammonia lyase (VC1202, RF of −2.2), the urocanate hydratase (VC1203, RF of −2.3), and the imidazolone proprionase (VC1205, RF of −2.3) exhibited decreased expression in the V. cholerae Δnqr strain. The arginine deiminase (VC0423, RF of −3.4) was also less abundant (Table 1). This shift in N-metabolism most likely was due to the activity of GlnB (nitrogen regulatory protein P-II; VC0606, RF of +7.4), which was strongly increased in abundance in the V. cholerae Δnqr strain.

Copper and iron homeostasis in the V. cholerae Δnqr strain.Copper is an essential transition metal and is a cofactor in proteins important for a variety of bacterial processes, like electron transport by the copper-dependent cytochrome c oxidase. However, surplus copper is also highly toxic for several reasons, such as the destruction of FeS clusters of dehydratases, accompanied by formation of reactive oxygen species and the ligation to thiolates (30, 31). Especially in its cuprous (Cu1+) state, copper is highly reactive and bacteria have developed multiple strategies to overcome copper toxicity. These include copper export, for example, by ATP-dependent Cu1+ transporters, oxidation of Cu1+ to less reactive Cu2+, and sequestration of copper by specific binding proteins. In V. cholerae, information about copper tolerance-related proteins is limited. V. cholerae contains neither the prominent CusABC copper efflux proteins nor CueO, a well-studied Cu1+ oxidase (30). V. cholerae harbors CopA, an ATP-dependent transport protein, pumping Cu1+ and also Ag+ from the cytoplasm to the periplasm (32, 33). The copG gene (VC2216) is located adjacent to copA (VC2215) and codes for an uncharacterized protein. In V. cholerae O395N1's close relative, V. cholerae O1 biovar El Tor strain FJ147, this gene (100% identity) is annotated as copG, coding for the copper amine oxidase CopG, and is proposed to be a periplasmic copper binding protein with copper detoxifying abilities, although its specific function is still unknown (32). Marrero et al. (32) proposed that CueR acts as a transcriptional regulator for CopA and CopG. CueR (VC0974, RF of −2.3), CopA (VC2215, RF of −3.5), and CopG (VC2216, RF of −9.1) showed decreased abundance in the V. cholerae Δnqr strain. We also detected the copper-associated protein CutC (VC0730) in both strains in similar amounts. Since the detoxifying proteins CopA and CopG, as well as their regulator, CueR, were decreased in abundance in the nqr deletion strain, we analyzed bacterial growth in the presence of copper on glucose minimal medium and on mixed amino acids. Addition of 120 μM CuSO4 was sufficient to completely inhibit the growth of both the reference and nqr deletion strains in minimal medium with glucose. At a critical threshold concentration of 60 μM CuSO4, the reference strain exhibited growth, whereas the V. cholerae Δnqr strain was no longer able to grow (Fig. 3). We repeated the experiments on LB medium. Unlike minimal medium, which is limited in trace metals and might lead to mismetallation of cellular proteins, LB medium contains trace metals in sufficient amounts but also includes chelating agents which decrease the biological availability of added metals (34). This is why LB medium is suited to analyze copper toxicity under conditions which are not limiting for trace elements. Indeed, the copper tolerance of both strains was much higher in LB medium than in minimal medium, and the reference strain was not diminished in growth up to 1 mM CuSO4. During exposure to 1.5 mM CuSO4, growth of the reference strain was significantly decreased in the late exponential phase (10 to 15 h) but not in the early exponential phase or in the stationary phase. In contrast, the final optical density of the V. cholerae Δnqr strain in the presence of 1.5 mM CuSO4 was already significantly diminished by approximately 50% compared to that of no CuSO4 addition (Fig. 3). These results demonstrate that a functional NQR is important to support copper detoxification in V. cholerae and suggest that CopA and CopG, probably under control by CueR, confer copper resistance to V. cholerae. In the following, we propose a role for NQR in copper detoxification. The NQR is a source for superoxide in the cytoplasm and in the periplasm of V. cholerae (26, 35). Superoxide reduces Cu2+ to Cu1+ (36), which initiates upon binding to CueR the transcription of copA and copG. CueR of E. coli is a Cu1+-specific responsive regulator (37). CueR itself is also proposed to be upregulated during copper exposure (36, 38). By providing superoxide as the reducing agent for the conversion of Cu2+ to Cu1+, the NQR might stimulate copper detoxification triggered by the Cu1+-CueR complex.

Besides copper detoxification, comparative proteomics also indicated a change in iron homeostasis in the V. cholerae Δnqr strain. A putative iron(III) ABC transporter, periplasmic iron compound-binding protein (VCA0685) (39), showed decreased abundance in the V. cholerae Δnqr strain with a regulation factor of −5.7. Periplasmic iron compound-binding proteins, such as FhuD in E. coli, are siderophore-binding components of the transporter operating under aerobic conditions (40). They are involved in the transport of iron(III) hydroxamates across the membrane (41). Under anoxic conditions, the ferrous iron transport protein B (FeoB) is responsible for iron(II) uptake in E. coli (42). FeoB (VC2077, RF of +2.7) is increased in abundance in the V. cholerae Δnqr strain assuming higher iron(II) uptake rates. Interestingly, bacterioferritin (ftn; VC0365, RF of +2.0) was increased in abundance in the V. cholerae Δnqr strain. Bacterioferritin is an iron storage protein also responsible for oxidation of iron(II) to iron(III) (43), assuming that in V. cholerae Δnqr mutants, increased iron levels are counteracted by larger amounts of bacterioferritin.

There is an apparent contradiction between our proteomic study performed with glucose minimal medium and the previously reported transcriptomic study using LB medium, although the very same V. cholerae strains were compared. A couple of copper-related genes were shown to be slightly (RF of ≤2) regulated in the transcriptomic study (21). The expression level of cutC was reported to be 1.65-fold downregulated, whereas copG was 2-fold increased in the early exponential growth phase and 1.5-fold increased in the mid-exponential growth phase of the V. cholerae Δnqr strain (21). The proteins involved in iron homeostasis were not found to be regulated in the transcriptomic study (21). In view of these major discrepancies, it seems that the availability of trace metals in the growth media differed too much to allow comparison of results obtained by proteomic and transcriptomic analyses.

Virulence.The loss of NQR is associated not only with global metabolomics changes but also with altered virulence-related processes involving virulence factors (7), motility (21), colonization, and biofilm properties (15). In vivo, in a suckling mouse model the persistence of V. cholerae El Tor N16961 was diminished in a mutant lacking the NQR (14). In vitro, Häse and Mekalanos were the first to observe an effect of nqr expression on virulence gene regulation in V. cholerae O395 (7). Flagellar rotation is driven by the membrane potential, so the drop in transmembrane voltage in the V. cholerae Δnqr strain (13) will also contribute to its diminished motility on soft agar (21). Additionally, diminished transmembrane voltage was brought into context with reduced monolayer biofilm formation of Vibrio cholerae O139 (15). On the metabolomic or transcriptomic level the impact of nqr deletion was investigated during early- and mid-exponential growth phase on mixed amino acids and resulted in a downregulation of genes expressing components of the flagellum (21). This finding was confirmed here by comparative proteomics with cells grown on glucose: the flagellar L-ring protein FlgH (VC2194, RF of −3.5) was less abundant in the V. cholerae Δnqr strain (Table 1).

Motility and attachment capacities are critical for bacterial survival and colonization success. V. cholerae attaches to host cells via the toxin-coregulated pilus (TCP) and the accessory colonization factor (Acf) (44). TCP and cholera toxin (CT) are essential for infection, and their expression is regulated by the transcriptional regulator ToxT (7), which itself is under the control of the membrane-associated ToxR and TcpP transcriptional regulators. A lack of NQR was reported to result in upregulation of ToxT, CT, and TCP, but only during early exponential growth phase on mixed amino acids (8). During mid- and late exponential growth phases, the lack of NQR was reported to be accompanied by diminished CT production (8). Minato et al. (6, 8) proposed that the impaired aerobic respiration resulting from the loss of NQR leads to a change of TCA cycle intermediates and increased acetyl-CoA levels. Acetyl-CoA was proposed to act as an intracellular signal affecting toxT expression, especially in early exponential growth phase. Our study now extends these findings for the V. cholerae Δnqr strain in mid-exponential growth phase with glucose as the substrate. Here, TcpA (VC0828, RF of +4.1) and AcfC (VC0841, RF of +2.6) were clearly increased in abundance, whereas CT, as a soluble and mainly extracellular protein, could not be detected with our approach.

The V. cholerae El Tor biotype strain C6706, with a mutation in the methionine transcriptional regulator MetR, revealed strong defects during intestinal colonization in the suckling mouse model (45). We also note that MetR, a key player of the methionine metabolism, was increased in abundance (VC1706, RF of +2) in the V. cholerae Δnqr strain.

Conclusions.Deletion of the NQR, the first respiratory complex in the electron transfer chain of V. cholerae, resulted in a strong shift of the proteome of this pathogen, which was dominated by metabolic adaptations. In concert, these changes allowed initial growth rates as fast as those of the wild type. We speculate that this response reflects a regulatory switch to deal with decreased transmembrane voltage in hostile environments due to bacterial (46) or host (47) effector molecules. Such molecules can be proteins like defensins, released by the host (47), or small bacterial compounds like 4-hydroxyquinoline-N-oxide (HQNO), which disrupt the membrane potential of competing bacteria (46). Furthermore, the nqr deletion led to an unexpected copper sensitivity. In the host environment, the failure to excrete toxic copper could weaken the V. cholerae Δnqr strain in its response toward copper-mediated attack by immune cells (48), offering a rationale for diminished in vivo persistence of the mutant.

MATERIALS AND METHODS

Strains and cultivation.Vibrio cholerae O395N1 (ΔctxA) (49) was used as the reference strain and compared to its Vibrio cholerae O395N1 Δnqr derivative strain lacking the six nqrABCDEF genes (50). Strains were grown in the presence of 50 g liter−1 streptomycin at 37°C on mixed amino acids (LB; 10 g liter−1 tryptone, 5 g liter−1 yeast extract, 1 g liter−1 NaCl) or in phosphate- or Tris-buffered M9 minimal medium containing 18.7 mM NH4Cl, 0.1 mM CaCl2, 0.1 mM MgSO4, 40 mg liter−1 each of methionine, threonine, histidine, and leucine, and 2 mg liter−1 thiamine, with 0.2% (wt/vol) glucose as a carbon source at pH 7.2. In addition, phosphate-M9 contained 7 g liter−1 Na2HPO4, 3 g liter−1 KH2PO4, 0.5 g liter−1 NaCl; Tris-M9 contained 20 mM Tris-HCl, pH 7.2, 1.36 g liter−1 KH2PO4, and 2.3 g liter−1 NaCl (51). It should be noted that 18.7 mM NH4Cl is a limiting ammonium concentration for growth of E. coli (29), whereas the amount of phosphate (10 mM KH2PO4) in Tris-buffered medium is not limiting for V. cholerae (52). Growth was monitored with a diode array spectrophotometer (HP 8452A) at 600 nm or with a Tecan Infinite F200 Pro plate reader at 595 nm using 96-well plates (Nunclon 96 flat-bottom, transparent polystyrene; Thermo Fisher Scientific).

Cell growth in the presence of CuSO4.To compare the copper sensitivities of the V. cholerae reference and Δnqr strains, cells were grown either in LB or in Tris-based minimal medium. Tris buffer was used instead of phosphate buffer to avoid precipitation of M9 medium components upon addition of CuSO4. To obtain inocula for growth experiments performed in LB or in M9 with glucose, cells were grown overnight in LB at 37°C, washed, and resuspended in the corresponding medium to a normalized optical density at 600 nm (OD600) of 0.1. To start growth, an aliquot of 0.1 ml was added to 0.1 ml growth medium with appropriate amounts of CuSO4 added from a stock solution prepared with the corresponding medium. The final volume was 0.2 ml per well, with CuSO4 concentrations ranging from 0 to 3 mM in LB or from 0 to 120 μM in M9 with glucose. Bacteria were grown at 37°C, and growth was measured every 13 min at 595 nm. Microtiter plates were shaken repeatedly between measurements. Four wells per condition were analyzed with three reads per well. Mean values and standard deviations of these 12 data points are presented. Student's t test was performed to test the difference in growth upon CuSO4 treatments. The P value significance threshold was set to 0.05.

Cell growth for comparative proteome analysis.Experiments were performed in triplicates. Single colonies of the V. cholerae reference or nqr deletion strain were used to inoculate 5 ml LB medium. The next day, cell pellets obtained by centrifugation were resuspended in M9 medium to obtain a normalized OD600 of 2. For each strain, three Erlenmeyer flasks with 15 ml phosphate-buffered M9 with glucose were inoculated to give a starting OD of 0.05, and cells were grown for 5 to 6 h until an OD600 of 0.2 to 0.3 was reached. From each culture, 13 ml was retrieved and harvested by centrifugation for 15 min (7,000 rpm, 4°C).

Protein extraction for comparative proteome analysis.Cell pellets were resuspended in cell lysis buffer containing 2% SDS (sodium dodecyl sulfate), 20 mM dithiothreitol (DTT) and 150 mM Tris-HCl, pH 8.5, and incubated for 10 min at 95°C. After centrifugation at 4°C for 30 min at 13,700 rpm, the supernatant was removed and proteins were precipitated using chloroform-methanol (53). Protein pellets were resuspended in 6 M urea in 50 mM Tris-HCl, pH 8.5, and protein concentrations were determined by the Bradford assay (54).

In-solution digestion of proteins and peptide purification with C18 stage tips.To 25 μg protein in 60 μl 50 mM Tris-HCl (pH 8.5) with 6 M urea, DTT was added to a final concentration of 10 mM for the reduction of cysteines. Samples were incubated for 30 min at 56°C under shaking at 1,000 rpm. Alkylation of cysteines was performed by adding 30 mM iodoacetamide and incubating for 45 min at room temperature in the dark. Alkylation was stopped by adding 50 mM DTT, and samples were incubated for another 10 min at room temperature. LysC protease (500 ng; Roche) in 50 mM Tris-HCl, pH 8.5, was added and samples were digested overnight at 30°C. The urea in the reaction mixture next was diluted to 2 M by adding the appropriate amount of 50 mM Tris-HCl, pH 8.5. Trypsin (1 μg; Roche) in 50 mM Tris-HCl, pH 8.5, was added and digestion was continued for 4 h at 37°C. The digestion was stopped by adding 3 μl 10% trifluoroacetic acid (TFA). Peptide mixtures next were concentrated and desalted on C18 stage tips (55) and dried under vacuum. Samples were dissolved in 30 μl 0.1% TFA. Aliquots of 2 μl were subjected to nano-LC-MS/MS analysis.

Mass spectrometry analysis.Nano-LC-ESI-MS/MS experiments were performed on an EASY-nLC 1000 system (Thermo Fisher Scientific) coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) using an EASY-Spray nanoelectrospray ion source (Thermo Fisher Scientific). Tryptic peptides were directly injected into an EASY-Spray analytical column (2 μm; 100-Å PepMapRSLC C18; 25 cm by 75 μm; Thermo Fisher Scientific) operated at a constant temperature of 35°C. Peptides were separated at a flow rate of 250 nl/min using a 235-min gradient with the following profile: 2% to 10% solvent B in 100 min, 10% to 22% solvent B in 80 min, 22% to 45% solvent B in 55 min, 45% to 90% solvent B in 5 min, and 15 min isocratic at 90% solvent B, followed by 90% to 2% solvent B in 1 min and reequilibration at 2% solvent B for 40 min. Solvents used were 0.5% acetic acid (solvent A) and 0.5% acetic acid in acetonitrile-H2O (80/20, vol/vol; solvent B).

The Q-Exactive Plus was operated under the control of XCalibur 3.0.63 software. MS spectra (m/z = 300 to 1,600) were detected in the Orbitrap at a resolution of 70,000 (m/z = 200) using a maximum injection time (MIT) of 50 ms and an automatic gain control (AGC) value of 1 × 106. Internal calibration of the Orbitrap analyzer was performed using lock-mass ions from ambient air as described by Olsen et al. (64). Data-dependent MS/MS spectra were generated for the 10 most abundant peptide precursors in the Orbitrap using high-energy collision dissociation (HCD) fragmentation at a resolution of 17,500, a normalized collision energy of 25, and an intensity threshold of 1 × 105. Only ions with charge states from +2 to +5 were selected for fragmentation using an isolation width of 1.6 Da. For each MS/MS scan, the AGC was set at 5 × 105 and the MIT was 50 ms. Fragmented precursor ions were dynamically excluded for 30 s within a 5-ppm mass window to avoid repeated fragmentation.

Protein quantification and data analysis.Raw files were imported into MaxQuant (56), version 1.5.3.8, for protein identification and label-free quantification (LFQ) of proteins. For protein identification in MaxQuant, MS spectra and MS/MS spectra were searched against a V. cholerae protein sequence database downloaded from UniProt (57) using the database search engine Andromeda (58). Reversed sequences as a decoy database and common contaminant sequences were added automatically by MaxQuant. Mass tolerances of 4.5 ppm for MS spectra and 20 ppm for MS/MS spectra were used. Trypsin was specified as an enzyme, and two missed cleavages were allowed. Carbamidomethylation of cysteines was set as a fixed modification, and protein N-terminal acetylation and oxidation were allowed as variable modifications. The “match between runs” feature of MaxQuant was enabled with a match time window of 1 min and an alignment time window of 20 min. Peptide false discovery rate (FDR) and protein FDR thresholds were set to 0.01.

Multiple-sample analysis of variance (ANOVA) test, principal-component analysis (PCA), hierarchical clustering, and Volcano plots were performed using Perseus, version 1.5.2.4 (59). Matches to contaminant (e.g., keratins and trypsin) and reverse databases identified by MaxQuant were excluded from further analysis. Proteins were considered for LFQ if they were identified by at least two peptides. First, normalized LFQ values from MaxQuant were log2 transformed. For analyzing the V. cholerae reference (referred to as the wild type) versus Δnqr strains, missing values were replaced from normal distribution using a width of 0.3 and a downshift of 1.8. For multiple-sample ANOVA, a permutation-based FDR with a cutoff of 0.05 was used. Volcano plots were used for pairwise comparison of sample groups. A P value of <0.05 and a regulation factor of >2 were considered significant changes in protein abundance. Information about protein function and pathway assignments were received from UniProt and KEGG (Kyoto Encyclopedia of Genes and Genomes [60–62]). If the functional assignment of a protein was unknown or weak, NCBI-based BLAST search (24) was used to define functionality by detection of homologous proteins or protein domains. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE (63) partner repository with the data set identifier PXD009379.

ACKNOWLEDGMENTS

This work was supported by Deutsche Forschungsgemeinschaft grant FR 1321/5-1 (to J.S.).

We thank K. Hantke (University of Tübingen) for discussions and B. Würtz and L. Proksch for excellent technical assistance.

FOOTNOTES

    • Received 31 December 2017.
    • Accepted 1 May 2018.
    • Accepted manuscript posted online 7 May 2018.
  • Address correspondence to Julia Steuber, julia.steuber{at}uni-hohenheim.de.

  • Citation Toulouse C, Metesch K, Pfannstiel J, Steuber J. 2018. Metabolic reprogramming of Vibrio cholerae impaired in respiratory NADH oxidation is accompanied by increased copper sensitivity. J Bacteriol 200:e00761-17. https://doi.org/10.1128/JB.00761-17.

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KEYWORDS

Vibrio cholerae
Na+-translocating NADH:quinone oxidoreductase
NQR
sodium bioenergetics
copper tolerance
comparative proteomics
nontraditional drug target
sodium-translocating NADH:quinone oxidoreductase

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