ABSTRACT
Cytochrome bd quinol oxidases, which have a greater affinity for oxygen than heme-copper cytochrome oxidases (HCOs), promote bacterial respiration and fitness in low-oxygen environments, such as host tissues. Here, we show that, in addition to the CydA and CydB subunits, the small protein CydX is required for the assembly and function of the cytochrome bd complex in the enteric pathogen Salmonella enterica serovar Typhimurium. Mutant S. Typhimurium lacking CydX showed a loss of proper heme arrangement and impaired oxidase activity comparable to that of a ΔcydABX mutant lacking all cytochrome bd subunits. Moreover, both the ΔcydX mutant and the ΔcydABX mutant showed increased sensitivity to β-mercaptoethanol and nitric oxide (NO). Cytochrome bd-mediated protection from β-mercaptoethanol was not a result of resistance to reducing damage but, rather, was due to cytochrome bd oxidase managing Salmonella respiration, while β-mercaptoethanol interacted with the copper ions necessary for the HCO activity of the cytochrome bo-type quinol oxidase. Interactions between NO and hemes in cytochrome bd and cytochrome bd-dependent respiration during nitrosative stress indicated a direct role for cytochrome bd in mediating Salmonella resistance to NO. Additionally, CydX was required for S. Typhimurium proliferation inside macrophages. Mutants deficient in cytochrome bd, however, showed a significant increase in resistance to antibiotics, including aminoglycosides, d-cycloserine, and ampicillin. The essential role of CydX in cytochrome bd assembly and function suggests that targeting this small protein could be a useful antimicrobial strategy, but potential drug tolerance responses should also be considered.
IMPORTANCE Cytochrome bd quinol oxidases, which are found only in bacteria, govern the fitness of many facultative anaerobic pathogens by promoting respiration in low-oxygen environments and by conferring resistance to antimicrobial radicals. Thus, cytochrome bd complex assembly and activity are considered potential therapeutic targets. Here we report that the small protein CydX is required for the assembly and function of the cytochrome bd complex in S. Typhimurium under stress conditions, including exposure to β-mercaptoethanol, nitric oxide, or the phagocytic intracellular environment, demonstrating its crucial function for Salmonella fitness. However, cytochrome bd inactivation also leads to increased resistance to some antibiotics, so considerable caution should be taken when developing therapeutic strategies targeting the CydX-dependent cytochrome bd.
INTRODUCTION
Bacterial oxidative respiration promotes energy generation and, consequently, increases in the growth rate in many bacteria. In the final step of the respiratory electron transport chain, molecular oxygen accepts electrons from terminal cytochrome oxidases. Similar to eukaryotic cells, which tend to live in environments with a plentiful oxygen supply, bacteria also contain heme-copper cytochrome oxidases (HCOs), including the cytochrome bo-type quinol oxidases, which have a low affinity for oxygen (1). However, owing to the different oxygen concentrations present in their various habitats, bacteria possess genes for another type of oxidase, the cytochrome bd-type quinol oxidases, which have a much higher affinity for oxygen than do HCOs. Therefore, the cytochrome bd proteins are found in many facultative anaerobic bacteria, where they promote the replication and, thus, the virulence of pathogenic bacteria that invade low-oxygen host tissues (2).
Cytochrome bd uses quinols as electron donors from the periplasmic side and protons from the cytoplasmic side to generate the proton motive force but does not directly pump protons, unlike the proton-translocating heme-copper cytochrome bo; thus, it produces less energy (3). It does not contain copper and carries three hemes: one low-spin heme (b558) and two high-spin hemes (b595 and d). In addition to its energy-producing role in bacterial respiration, cytochrome bd confers resistance to environmental stresses, including the reducing agent β-mercaptoethanol and reactive nitrogen and oxygen species (such as nitric oxide, hydrogen peroxide, superoxide, and peroxynitrite), thereby potentiating bacterial virulence (4–8). Therefore, this enzyme complex is considered a promising therapeutic target for controlling bacterial infections.
The cytochrome bd complex contains two subunits, CydA and CydB, that are encoded by the cydAB operon, which is conserved in many bacteria. However, recent genomic and phenotypic studies in some bacteria have revealed that the end of the cydAB operon also contains a third gene, cydX (also known as cydY or cydZ), that encodes the small protein CydX, which participates in cytochrome bd function (8–12). Moreover, the molecular structure of a cytochrome bd-type oxidase complexed with CydS (a potential functional homologue of CydX) in Geobacillus thermodenitrificans shows that this third subunit associates with CydA and may stabilize a triangular arrangement of three hemes located in the CydA subunit (13).
As a facultative anaerobe, Salmonella enterica serovar Typhimurium (S. Typhimurium) is predicted to use two types of quinol oxidases to respire under conditions of fluctuating oxygen levels. Based on biochemical studies of Escherichia coli cytochrome oxidases (4) and on the synteny between the E. coli and S. Typhimurium cytochrome quinol oxidase genes, it has been predicted that, in aerobic in vitro cultures, S. Typhimurium may mainly utilize cytochrome bo3. In contrast, in microaerobic environments, such as low-oxygen host tissues, S. Typhimurium replication largely depends on cytochrome bd, which has a higher oxygen affinity than cytochrome bo3 (6, 14, 15). The cydAB operon encoding cytochrome bd in S. Typhimurium appears to contain genes encoding the following three proteins: CydA, CydB, and CydX (encoded by ybgT [STM0742]).
In this study, we investigated whether the S. Typhimurium CydX homologue is required for cytochrome bd complex assembly and activity and cytochrome bd-related metabolism, by examining the phenotypes of mutant S. Typhimurium strains lacking CydX or all of the cytochrome bd subunits. This phenotypic study identifies CydX to be an essential component of cytochrome bd in S. Typhimurium. However, both ΔcydX and ΔcydABX mutants showed a significant increase in resistance to antibiotics, including aminoglycosides, d-cycloserine, and ampicillin. Therefore, our findings suggest that although targeting the CydX-containing cytochrome bd complex could be a useful antimicrobial strategy, inhibiting this enzyme activity may also promote drug resistance.
RESULTS
CydX is required for cytochrome bd stability and activity in S. Typhimurium.The role of CydX in cytochrome bd function and stable assembly of the active site (the diheme center) has been shown in E. coli and Brucella abortus (10, 12). However, in Shewanella oneidensis, CydX is required only for the function, and not the assembly, of cytochrome bd (11), which raises the question of whether this small protein is required for cytochrome bd assembly, function, or both in Salmonella. To test whether CydX is required for the assembly of this enzyme complex in S. Typhimurium, we employed UV-visible (UV-Vis) electronic absorption spectroscopy, which can superimpose the individual absorption spectra of all hemes in the cytochrome bd complex (16). Usually, the absorption spectra of all b and d hemes overlap in the Soret (blue wavelength) region, whereas in the visible region, the α-bands of the hemes are distinguishable (2, 17, 18). The analysis was performed using cytoplasmic membranes purified from wild-type (WT) S. Typhimurium producing both bo- and bd-type cytochrome quinol oxidases (bo+ bd+), a ΔcyoA mutant lacking the cyoA gene for the cytochrome bo subunit CyoA (bo− bd+), a ΔcydABX mutant lacking the entire cydABX operon encoding all of the cytochrome bd subunits (bo+ bd−), a ΔcydX mutant deficient in the cydX gene only, and a complemented ΔcydX mutant harboring a plasmid containing cydX (Fig. 1A). The spectra of air-oxidized membranes from the WT, complemented ΔcydX mutant, and ΔcyoA mutant strains presented a Soret peak maximum at 410 nm and a distinct shoulder peak near 645 nm, and when reduced with sodium dithionite, the spectra displayed peaks at 427, 530, 560, and 630 nm. In the ΔcydABX and ΔcydX mutants, the spectra of the oxidized membranes also showed a Soret peak maximum at 410 nm but did not show the shoulder peak near 645 nm; and in reduced membranes, peaks were observed at 427, 530, and 560 nm, but there was no peak at 630 nm. For the E. coli cytochrome bd, the peaks observed at 530 and 560 nm in reduced membranes correspond to heme b, and the peak observed at 630 nm for the reduced membrane and the peak observed at 645 nm for oxidized membranes are specific for heme d (19). Thus, the loss of peaks at 645 nm and 630 nm for the oxidized and reduced membranes, respectively, strongly indicates the loss of heme d in both the ΔcydABX and ΔcydX mutants. The reduced-minus-oxidized difference spectra confirmed that the spectra from the WT, complemented ΔcydX mutant, and ΔcyoA mutant strains were characteristic of the complete cytochrome bd complex, as they displayed typical changes in the heme d spectra in response to oxidation and reduction, while both the ΔcydABX mutant and the ΔcydX mutant lacked these heme d spectral changes (Fig. 1A). This shows that CydX is required for stable heme assembly of the cytochrome bd complex in S. Typhimurium.
CydX is required for cytochrome bd stability and function in S. Typhimurium. (A) UV-Vis spectrophotometry of cytoplasmic membranes from S. Typhimurium. (Top) Absorption spectra of oxidized (black line) and reduced (red line) cytoplasmic membranes from S. Typhimurium strains. The insets show magnified (×5) spectra from the visible region (500 nm to 700 nm). (Bottom) The reduced-minus-oxidized difference spectra. (B, C) O2 consumption rate of S. Typhimurium. The O2 concentration of the bacterial culture in the absence (B) and presence (C) of 0.2% l-arabinose was recorded and graphed as the O2 concentration as percentage of that at t0, as described in Materials and Methods. The data shown are representative of those from three independent experiments. (D) Colony morphologies of S. Typhimurium strains grown overnight on LB agar plates in the absence (−Ara) and presence (+Ara) of 0.2% l-arabinose. To photograph the colonies, slowly growing ΔcydABX, ΔcydX, and ΔcydX/vector strains were incubated 4 h longer (18 h) than the other strains (14 h). A representative image is shown. pcydX, plasmid harboring cydX.
Next, we examined the respiratory activity of S. Typhimurium by measuring bacterial oxygen consumption rates. The oxygen consumption rate of the ΔcydX mutant was severely decreased to a level comparable to that of the ΔcydABX mutant, while the WT and ΔcyoA mutant strains consumed much more oxygen than the ΔcydX and ΔcydABX mutants (Fig. 1B). Notably, the ΔcyoA mutant consumed oxygen at the same rate as the WT strain, suggesting that enough active cytochrome bd may have been present to compensate for the absence of the cytochrome bo under our culture conditions, which contained approximately 8% O2. When grown on Luria-Bertani (LB) agar plates, both the ΔcydABX mutant and the ΔcydX mutant exhibited mixed colony sizes, with some small-colony variants (Fig. 1D), as has been observed previously for analogous E. coli mutants (8). Complementation of the cydX mutation with a cydX-encoding plasmid restored the oxygen consumption rate and colony size to levels comparable to those of the WT strain (Fig. 1B and C). Collectively, these results indicate that the third gene, cydX, contained in the S. Typhimurium cydABX operon is required for the complete assembly and function of cytochrome bd.
The CydX-dependent cytochrome bd is required for S. Typhimurium resistance to β-mercaptoethanol independently of the disulfide bond formation system.Susceptibility to the reducing agent β-mercaptoethanol is a characteristic phenotype of mutant E. coli strains lacking cytochrome bd (8). We therefore examined the resistance of the S. Typhimurium ΔcydX mutant to β-mercaptoethanol, as shown in Fig. 2A. The growth of the ΔcydX mutant in β-mercaptoethanol-containing medium was severely impaired compared with that of the WT strain to an extent similar to that for the ΔcydAB and ΔcydABX mutants. This growth impairment was completely rescued by the presence of a plasmid harboring cydX or cydABX, confirming the essential role of CydX in cytochrome bd-mediated resistance to β-mercaptoethanol in S. Typhimurium (Fig. 2B).
CydX-dependent cytochrome bd is required for S. Typhimurium resistance to β-mercaptoethanol (BME). The growth of S. Typhimurium in medium containing β-mercaptoethanol (10 mM) (A to C) or DTT (5 mM) (C) was monitored by measuring the OD600. The OD600 values for the bacterial cultures are shown as the mean ± SD from three independent experiments. Control cultures without the reducing agent are also shown (control). (B) The growth of bacteria, including the complemented strains, is shown as the time to the half-maximal OD600 based on the growth curve for each strain. ****, P < 0.0001, as determined by two-way analysis of variance; ns, not significant.
The β-mercaptoethanol sensitivity of E. coli cydAB mutants is presumed to result from the impaired formation of disulfide bonds on periplasmic proteins, a process that is catalyzed by the DsbA and DsbB disulfide oxidoreductases and supported by cytochrome bd (8, 20). To confirm the role of cytochrome bd in disulfide formation in S. Typhimurium, the ΔcydABX mutant was treated with another reducing agent, dithiothreitol (DTT). Surprisingly, treatment with DTT did not impair the growth of the ΔcydABX mutant to a greater extent than the WT strain, whereas, as expected, a mutant Salmonella strain lacking both dsbA and dsbB was susceptible to DTT (Fig. 2C). In contrast, the ΔdsbA ΔdsbB mutant was not sensitive to β-mercaptoethanol under conditions that impaired the growth of the ΔcydABX mutant. This suggests that the sensitivity of cytochrome bd-deficient mutants to β-mercaptoethanol may be related to β-mercaptoethanol activities other than the reduction of disulfide bonds on periplasmic proteins.
β-Mercaptoethanol inhibits respiration in cytochrome bd-deficient S. Typhimurium mutants.As β-mercaptoethanol-mediated inhibition of the growth of cytochrome bd-deficient S. Typhimurium mutants is independent of disulfide bond formation, we hypothesized that cytochrome bd protects S. Typhimurium from β-mercaptoethanol toxicity via an unknown activity or that β-mercaptoethanol inhibits factors other than cytochrome bd that are involved in energy production in S. Typhimurium. To test this, we first compared the growth of the ΔcydABX mutant in minimal medium containing the fermentable sugar glucose to its growth in minimal medium containing the less fermentable glycerol, which requires redox-balanced quinone pools to enter glycolysis and promotes oxidative respiration over fermentation (21, 22). The growth rate of the ΔcydABX mutant was comparable to that of the WT strain in minimal medium containing glucose, irrespective of β-mercaptoethanol treatment, but was largely impaired by β-mercaptoethanol in glycerol-containing minimal medium, which was similar to the growth rate observed in LB medium cultures (Fig. 3A and B). This suggests that β-mercaptoethanol may target other bacterial components that play compensatory roles in energy production in S. Typhimurium in the absence of cytochrome bd. It has been reported that copper can be removed from HCOs by β-mercaptoethanol (23) and that copper ions can interact chemically with mercaptans, including β-mercaptoethanol (24). Thus, we hypothesized that the function of the heme-copper-containing cytochrome bo might be inhibited by β-mercaptoethanol. Indeed, supplementation with copper ions significantly alleviated the sensitivity of the ΔcydABX mutant to β-mercaptoethanol (Fig. 3B). Additionally, treatment with β-mercaptoethanol did not affect the growth of the ΔcyoA mutants compared with that of the WT strain, suggesting that it has little effect on cytochrome bd-mediated respiration. Next, the effect of β-mercaptoethanol on Salmonella respiration was examined by monitoring the bacterial oxygen consumption rate. Treatment with β-mercaptoethanol significantly slowed the oxygen consumption rate of the ΔcydABX and ΔcydX mutants but had no effect on the oxygen consumption rate of the WT and ΔcyoA mutant strains, which express functional cytochrome bd (Fig. 3C). UV-Vis spectrophotometry of membranes from the ΔcydABX (bo+ bd−) mutant showed that there was a remarkably decreased absorption intensity at the peak at 427 nm when the membranes were treated with β-mercaptoethanol (Fig. 4). This was also observed in nitric oxide (NO)-treated membranes, consistent with the characteristic change caused by the interaction of NO with copper in the binuclear center of the E. coli cytochrome bo3 (25). Thus, a similar decrease in absorption intensity in the Soret band of the β-mercaptoethanol-treated membranes suggests that β-mercaptoethanol interacts with cytochrome bo. In the ΔcyoA (bo− bd+) mutant, however, there was little change in the spectra for the β-mercaptoethanol-treated membranes, whereas NO caused characteristic changes in the peaks for heme d (from 630 nm to 645 nm) for the reduced cytochrome bd, as previously observed (26). This spectral analysis demonstrates that β-mercaptoethanol may alter the stability of cytochrome bo but not that of cytochrome bd. Taken together, these results suggest that the β-mercaptoethanol-sensitive phenotype of mutants deficient in cytochrome bd may be related to the inactivation of cytochrome bo by β-mercaptoethanol, which causes a synergistic reduction in the respiratory activity and, consequently, replication of S. Typhimurium.
Effect of β-mercaptoethanol on S. Typhimurium growth and respiration. (A and B) The effect of β-mercaptoethanol on bacterial growth was examined by measuring the OD600 of cultures grown in minimal medium (glucose and glycerol [A] or glucose only [B]; 0.2% each) with or without β-mercaptoethanol (10 mM) or CuCl2 (1 mM) and is shown as the time to the half-maximal OD600 based on the growth curve for each culture. ****, P < 0.0001, as determined by two-way analysis of variance. (C) The effect of β-mercaptoethanol on the bacterial O2 consumption rate was examined by recording the concentration of O2 that remained in each culture. Bacterial cells were grown to early log phase (OD600 = 0.4) in LB medium with (red line) or without (black line) β-mercaptoethanol (10 mM) treatment for 1 min before recording, and the results are expressed as the O2 concentration as a percentage of that at t0. The data shown are representative of those from three independent experiments.
UV-Vis spectrophotometry of cytoplasmic membranes from S. Typhimurium ΔcydABX and ΔcyoA mutants. The data shown are the absorption spectra for membranes reduced by sodium dithionate without any treatment (R) or after treatment with β-mercaptoethanol (5 mM; BME-R) or spermine NONOate (1 mM; NO-R).
The CydX-dependent cytochrome bd is required for S. Typhimurium resistance to nitric oxide.In E. coli, cytochrome bd promotes a much higher rate of NO dissociation than the cytochrome bo, thereby promoting bacterial respiration and, consequently, resistance to cytotoxic levels of NO (27, 28). A mutant strain of S. Typhimurium lacking cydA and cydB is also susceptible to nitrosative stress and shows synergistic susceptibility when hmp (encoding the NO-metabolizing flavohemoglobin Hmp) is also deleted (6, 29). To determine whether CydX is also required for the cytochrome bd-dependent S. Typhimurium resistance to nitrosative stress, we compared the growth rate of a Δhmp mutant with that of Δhmp mutants also lacking cydX, cyoA, or cydABX. In NO-producing cultures, the growth rates of the Δhmp ΔcydX and Δhmp ΔcydABX mutant strains were impaired to similar extents, demonstrating the synergistic effect of mutations in genes encoding both Hmp and cytochrome bd complex components compared with the effect of the Δhmp or Δhmp ΔcyoA mutation (Fig. 5A). S. Typhimurium respiration also required CydX under nitrosative stress (Fig. 5B). The oxygen consumption of the ΔcydABX and ΔcydX mutant strains was almost completely abolished when NO was injected into the culture, indicating the CydX dependence of cytochrome bd in S. Typhimurium respiration under nitrosative stress. Notably, the respiratory activity of the WT strain was significantly inhibited for 3 min and then mostly recovered, whereas the ΔcyoA mutant showed a gradual decrease in respiration over time. This suggests that NO inhibited cytochrome bo-mediated respiration, after which cytochrome bd accounted for most of the respiration in S. Typhimurium. Cumulatively, these data indicate that S. Typhimurium resistance to NO relies on cytochrome bd-dependent respiration, in which CydX plays an essential role.
Effect of NO on S. Typhimurium growth and respiration. (A) Identical amounts of overnight cultures were inoculated into minimal medium containing glucose (0.2%) with or without the NO congener S-nitrosoglutathione (GSNO; 100 μM). The OD600 values for the bacterial cultures are shown as the mean ± SD from three independent experiments. (B) The effect of NO on the bacterial oxygen consumption rate was examined as described in the legend to Fig. 3C. Black line, not treated with NO; red line, treated with NO. Spermine NONOate (50 μM) was used as an NO congener.
CydX is required for S. Typhimurium replication in a macrophage cell line.It has been shown that cytochrome bd-type quinol oxidases, including those present in S. Typhimurium, are required for the replication of bacterial pathogens in macrophages (2). To test the role of CydX in S. Typhimurium proliferation in macrophages, we compared the intracellular replication rate of the ΔcydX, ΔcydAB, ΔcydABX, and ΔcyoA S. Typhimurium mutant strains in RAW264.7 macrophages. As expected, based on the phenotypic results described above, the intramacrophage replication rate of the ΔcydX mutant was substantially reduced to a level comparable to that of the ΔcydAB and ΔcydABX mutants, and the ΔcyoA mutant and WT strains replicated at similar rates (Fig. 6). This demonstrates that CydX and an intact cytochrome bd complex are required for S. Typhimurium replication in macrophages.
Intramacrophage replication of S. Typhimurium. The intracellular replication of WT and quinol oxidase-deficient S. Typhimurium mutants in RAW 264.7 cells was determined as described in Materials and Methods. The numbers of bacterial CFU were determined after lysis of infected RAW 264.7 cells at 1 h and 18 h postinfection, and then the fold replication of each strain was calculated as the ratio of the number of CFU at 18 h postinfection/number of CFU at 1 h postinfection. For each experiment, the intracellular fold replication of the mutants is expressed as a percentage of the fold replication of the WT strain. Data are shown as the mean ± SD from two independent experiments, with each condition being assessed in triplicate. P values were determined by one-way analysis of variance. *, P < 0.05; **, P < 0.01; ns, not significant.
Inactivation of cytochrome bd causes S. Typhimurium resistance to aminoglycosides, d-cycloserine, and ampicillin.Reports have shown that bacterial respiration is related to antibiotic efficacy in vitro, suggesting that host factors affecting bacterial respiration may modulate pathogen resistance to antibiotics (30). For example, inhibiting bacterial respiration blocks the energy-dependent stages of aminoglycoside uptake in S. Typhimurium and decreases the efficacy of other bactericidal antibiotics in E. coli (31, 32). Therefore, we asked whether quinol oxidase deficiency affects antibiotic resistance in S. Typhimurium. The MICs of aminoglycosides (kanamycin, neomycin, and streptomycin) and antibiotics that inhibit peptidoglycan synthesis (d-cycloserine and ampicillin) were higher for the ΔcydABX mutant than for WT S. Typhimurium (see Table S1 in the supplemental material). However, the MICs for two other bactericidal antibiotics, norfloxacin and nalidixic acid, and the bacteriostatic antibiotic chloramphenicol were not affected by a deficiency in either cytochrome bo or cytochrome bd. We next examined the role of CydX in the cytochrome bd-mediated modulation of antibiotic susceptibility. When log-phase cultures were treated with kanamycin, neomycin, streptomycin, d-cycloserine, or ampicillin at concentrations close to their respective MICs, the survival of the ΔcydX and ΔcydABX mutant strains was higher than that of the WT strain (Fig. 7). The percent survival of the WT strain decreased drastically 2 h after treatment with kanamycin (1,000-fold), streptomycin (100-fold), or neomycin (25-fold), whereas the impact on the survival of the ΔcydX and ΔcydABX strains was considerably lower. The ΔcydX and ΔcydABX mutant strains also exhibited significantly higher resistance to d-cycloserine and ampicillin than the WT strain, with the ΔcydX mutant showing slightly more resistance than the ΔcydABX mutant. Notably, the ΔcyoA mutant was just as sensitive to aminoglycosides (except neomycin), d-cycloserine, and ampicillin as the WT strain, suggesting that the two cytochromes play different roles in antibiotic resistance. These results indicate that CydX deficiency promotes antibiotic resistance in the same way as cytochrome bd deficiency and that the inactivation of cytochrome bd can increase S. Typhimurium resistance to aminoglycosides, d-cycloserine, and ampicillin, thus revealing a strong relationship between cytochrome bd-mediated respiration and antibiotic resistance in S. Typhimurium.
Antibiotic susceptibility of WT and quinol oxidase-deficient S. Typhimurium mutants. Salmonella cells cultured to early log phase (OD600 = 0.4) were challenged with antibiotics (512 μg ml−1 of streptomycin, 32 μg ml−1 of kanamycin, 128 μg ml−1 of neomycin, 512 μg ml−1 of d-cycloserine, or 16 μg ml−1 of ampicillin), and before and at 2 h after antibiotic treatment the cultures were serially diluted in PBS before plating onto LB agar. Percent survival was calculated by dividing the number of CFU that grew from samples taken 2 h after antibiotic treatment by the number of CFU that grew from samples taken before treatment. Data are shown as the mean ± SD from three independent cultures. *, P < 0.05, as determined by two-way analysis of variance.
DISCUSSION
Since their discovery, the role of CydX proteins (which are mostly 37 amino acids in length) in the formation and activation of the cytochrome bd complex has remained poorly understood. In this study, we found that CydX plays an essential role in cytochrome bd assembly and function in S. Typhimurium. We characterized the full spectral properties of cytoplasmic membranes containing cytochrome bd and/or cytochrome bo in S. Typhimurium. The spectra of membranes from mutant strains lacking cytochrome bo or cytochrome bd indicated the involvement of hemes in redox reactions in both cytochromes (Fig. 1A). The heme d peaks (at 645 nm for oxidized membranes and 630 nm for reduced membranes) for the ΔcyoA mutant (bo− bd+) may be especially useful for determining the oxidoreduction status and stability of cytochrome bd, as observed previously in a study of cytochrome bd purified from E. coli (18, 33). It was speculated that the loss of CydX might downregulate cydAB expression or induce aberrant conformational changes in the CydA and CydB subunits, resulting in cytochrome bd-deficient phenotypes, including the loss of the heme d spectra. However, cydA transcription did not decrease but, rather, slightly increased in the ΔcydX mutant strain (see Fig. S1 in the supplemental material), and the heme d spectra and other phenotypes were stably restored by the sole expression of CydX from a cydX-harboring plasmid in this strain to an extent that they were comparable to those observed in the WT strain (Fig. 1 and 2B). Therefore, it is unlikely that cydX mutation causes aberrant expression of CydA and CydB subunits.
Like E. coli, S. Typhimurium appears to produce an additional cytochrome bd, bd-II, which has a terminal oxidase function under hypoxic conditions with very limited oxygen tensions (∼0.8% O2) (15, 34). Similar to cytochrome bd (bd-I), the cytochrome bd-II complex is encoded by the cyxAB operon, which also contains an additional gene (STM1794) downstream of cyxB that encodes a CydX paralog. In E. coli, this CydX paralog, AppX, is produced by the appCBX operon, which encodes cytochrome bd-II, and its expression is induced under low-oxygen conditions that also induce CydX expression (9). Furthermore, purified AppX can interact with CydA and CydB to replace CydX in vitro, suggesting that there is functional redundancy between AppX and CydX with regard to cytochrome bd function (8). However, the S. Typhimurium ΔcydX mutant showed a complete failure to maintain cytochrome bd stability and function, suggesting that the AppX homologue in S. Typhimurium does not interact with the CydA and CydB subunits in vivo. Furthermore, a bd-II-deficient mutant (the ΔcyxA mutant) had no effect on cytochrome bd function in S. Typhimurium resistance to β-mercaptoethanol (Fig. S2). The oxygen levels (∼8% O2) in our broth cultures, at which S. Typhimurium primarily depends on cytochrome bd-I for respiration (Fig. 1B), may have been too high to induce enough AppX expression to compensate for the loss of CydX. The potential functional overlap between CydX and AppX in Salmonella or other bacteria should be explored in future studies conducted under oxygen-limited conditions.
The sensitivity of cytochrome bd-deficient mutants to the reducing agent β-mercaptoethanol has been presumed to be due to impairment of the disulfide bond (Dsb) formation system (8), based on an in vitro study using purified enzymes (20). However, our results suggest that this sensitivity arises from a Dsb-independent mechanism, as β-mercaptoethanol and another reducing agent (DTT) did not impair the growth of the ΔcydABX and ΔdsbA ΔdsbB mutant strains in the same way (Fig. 2). Moreover, the β-mercaptoethanol sensitivity of the ΔcydABX mutant strain was alleviated by supplementing the cultures with copper ions (Fig. 3), and treatment with β-mercaptoethanol induced changes in cytochrome bo hemes but not in cytochrome bd hemes (Fig. 4), suggesting that the β-mercaptoethanol sensitivity of the ΔcydABX S. Typhimurium mutant strain may be due to β-mercaptoethanol-mediated damage to cytochrome bo that causes a synergistic decrease in respiration. This should be confirmed by further physiological investigation of the role of quinol oxidases in maintaining redox homeostasis within the Dsb pathway.
Like other heme-containing proteins, NO can bind to cytochrome bo and cytochrome bd, thereby inhibiting bacterial respiration. However, cytochrome bd metabolizes NO and the NO-mediated reactive species peroxynitrite, which can inactivate cytochrome bo, and has a much higher NO dissociation rate than cytochrome bo, which confers bacterial resistance to NO and leads to the rapid recovery of bacterial respiration, as confirmed in this study (27, 28, 35). These abilities depend on the reduction of heme d and b595 in cytochrome bd, while NO binds to the reduced hemes (27). Consistent with the spectral changes observed for NO-mediated heme d reduction (Fig. 4) and the dependence of heme d assembly on CydX (Fig. 1A), growth and respiration were sensitive to NO in the S. Typhimurium ΔcydX mutant (Fig. 5). In addition to resistance to NO, cytochrome bd also promotes bacterial tolerance to oxidative stress, due to its quinol peroxidase and catalase activities (36, 37). This cytochrome bd-mediated tolerance to nitrosative and oxidative stresses confers bacterial virulence in animal hosts (2), as phagocytic cells, such as macrophages, mainly produce cytotoxic levels of NO, as well as reactive oxygen species. We also showed that cytochrome bd significantly promotes S. Typhimurium proliferation inside macrophages and that CydX plays an essential role in this process (Fig. 6).
Because cytochrome bd-type quinol oxidases are found only in bacteria, they are considered a drug target for selectively controlling bacterial infections. Drugs targeting cytochrome bd are of particular interest for combatting infections caused by multidrug-resistant Mycobacterium tuberculosis strains. The presence of cytochrome bd promotes mycobacterial persistence and the establishment of chronic infections (38), and deletion of cytochrome bd synergistically potentiates the effect of antituberculosis drugs that act on other factors involved in mycobacterial respiration and ATP synthesis. In M. smegmatis, treatment with the NADH-dehydrogenase inhibitor clofazimine or the F1Fo ATP synthase inhibitor bedaquiline is bacteriostatic to WT bacteria but kills mutants that lack cytochrome bd (39). Additionally, inactivating cytochrome bd completely abolishes the residual maintenance of respiration and ATP synthesis in M. tuberculosis treated with the cytochrome bc1-aa3 inhibitor Q203 (40). These observations illustrate the need for new drugs targeting the cytochrome bd to help control bacterial infections. However, our study also shows that cytochrome bd-deficient S. Typhimurium strains have increased resistance to aminoglycosides, d-cycloserine, and ampicillin (Fig. 7).
Aminoglycoside resistance might be caused by respiration arrest, which blocks the energy flow required for drug uptake (31, 41). Also, attenuation of bacterial respiration has been suggested to decrease the efficacy of bactericidal antibiotics, including gentamicin (another aminoglycoside) and ampicillin, by an as-yet-unidentified mechanism potentially involving the generation of reactive oxygen species in response to the bactericidal antibiotic-triggered acceleration of bacterial respiration (32, 42, 43). The mechanism underlying d-cycloserine resistance in cytochrome bd-deficient bacteria has never been explored. However, two recent studies of mycobacterial l-alanine dehydrogenase (Ald) suggest a possible interaction between bacterial respiration and d-cycloserine efficacy. Ald catalyzes a reversible reaction converting pyruvate to l-alanine (l-Ala) in an oxygen-dependent manner. l-Ala is the precursor of d-Ala, which is incorporated into peptidoglycans during their synthesis, and d-cycloserine competes with d-Ala in serial enzyme reactions leading to d-Ala production. Thus, increased d-Ala levels in bacteria can reduce the toxicity of d-cycloserine. A genomic study showed that a mutation in ald that interrupts the conversion of l-Ala to pyruvate is responsible for the resistance of many clinical M. tuberculosis isolates to d-cycloserine (44). Another study showed that interrupting mycobacterial respiration by targeting bcc1-aa3 cytochrome oxidases induces a concomitant increase in NADH levels and Ald expression. Ald catalyzes the conversion of pyruvate to L-Ala by oxidizing NADH (45). Interestingly, we found that the ΔcydX mutant strain had higher resistance to d-cycloserine and ampicillin than did the ΔcydABX mutant strain. This may suggest that other unknown factors are involved in interactions between cytochrome bd subunits and the metabolic changes that antibiotics induce in cell wall biosynthesis; however, further study is required to understand the underlying mechanism.
In conclusion, our results show that the small protein CydX is required for cytochrome bd assembly and function in S. Typhimurium. The role of the CydX-dependent cytochrome bd was further evaluated under stress conditions induced by treatment with β-mercaptoethanol, treatment with NO, or exposure to the phagocytic intracellular environment, demonstrating its crucial role in maintaining S. Typhimurium fitness. Thus, assembly of the cytochrome bd complex subunits, including CydX, could be a potential target for antimicrobial therapy to control infections with drug-resistant bacteria. However, the increased tolerance or resistance to antibiotics induced by cytochrome bd inactivation suggests that considerable caution should be taken when designing drugs and treatment strategies that target cytochrome bd.
MATERIALS AND METHODS
Bacterial strains and culture media.All Salmonella strains used in this study are isogenic strains of the WT S. Typhimurium 14028s strain and are listed in Table S2 in the supplemental material. Strains were grown in Luria-Bertani (LB; Difco, USA) complex medium or minimal E medium (46) supplemented with glucose (0.2%) or glycerol (0.2%) at 37°C with shaking. For mutant construction and cloning, the following antibiotics were added to LB medium when necessary: ampicillin (100 μg ml−1), kanamycin (50 μg ml−1), or chloramphenicol (20 μg ml−1). The S. Typhimurium mutant strains were constructed via the bacteriophage λ Red-mediated recombination method, as described previously (47–49), using the DNA oligonucleotides listed in Table S3. All mutant constructs were confirmed by PCR analysis using the gene-flanking primers listed in Table S3 and then transduced via P22 phage into the WT strain. Complementation of the cydABX, cydX, and cydAB deletion mutations was achieved by cloning the respective genes amplified from S. Typhimurium chromosomal DNA using the oligonucleotides listed in Table S3 into pBAD33 or pBAD18 plasmids (50). The sequences of all of the clones were verified by DNA sequence analysis (Macrogen Inc., Republic of Korea). l-Arabinose was added to the medium to induce cydX transcription from an l-arabinose-inducible promoter in the vector plasmid. All reagents, including medium supplements and antibiotics, were purchased from Sigma (Republic of Korea), unless otherwise noted.
Isolation of cytoplasmic membrane and UV-Vis spectroscopy.S. Typhimurium strains were grown overnight and subcultured in LB broth until the cultures reached mid-log phase (optical density at 600 nm [OD600], ∼1.0). Cytoplasmic membranes were prepared as described by Miller and Gennis (51), with minor modifications. Briefly, bacterial pellets collected from 1-liter cultures were washed with and resuspended in buffer containing 10 mM EDTA and 100 mM Tris-HCl, pH 8.5. After sonication (15 20-s pulses at 35% power with 15 s of rest on ice between each pulse), the cell debris was obtained by centrifugation for 30 min at 27,000 × g using a Sorvall SL-50T rotor (Sorvall Products, USA) and discarded. The supernatant was centrifuged at 200,000 × g using a Beckman type MLA-130 rotor (Beckman Instruments, USA) for 1 h, and then the pellet was solubilized in buffer containing 75 mM potassium phosphate, 150 mM KCl, 5 mM EDTA, and 60 mM N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate, pH 6.4. The solution was centrifuged once more at 200,000 × g for 1 h. The supernatants, containing the inner membranes, were collected, and the protein concentrations were determined using a Bradford protein assay kit (Bio-Rad, USA). The protein concentration was adjusted to 1.5 mg ml−1 using the same buffer. UV-visible (UV-Vis) absorbance spectroscopy was performed using a Hitachi U-1900 spectrophotometer at room temperature, and the spectra were analyzed using UV Solutions software (Hitachi, Japan). Selected groups of membranes were treated with β-mercaptoethanol (5 mM) or spermine NONOate (1 mM) for 10 min before measuring the absorption spectra. Sodium dithionite was treated to reduce all cytochromes in membrane samples.
Measurement of bacterial oxygen consumption.S. Typhimurium strains grown overnight in LB broth were diluted 1:200 in LB broth and grown at 37°C with shaking until the OD600 value reached 0.4. The cultures were then transferred into a multiport measurement chamber (model NOCHM-4; WPI Inc., USA) equipped with an Iso-Oxy-2 O2 probe connected to a free-radical analyzer (model TBR4100; WPI Inc.) at 37°C. The data were collected using the Labchart program (WPI Inc.). The remaining O2 concentrations in the cultures were recorded over time, and, when necessary, the cultures were treated for 1 min with β-mercaptoethanol (10 mM) or spermine NONOate (50 μM) before measurement. The data were calculated as molar concentrations of O2 and graphed as the O2 concentration as a percentage of the O2 concentration recorded when the measurement started (time zero [t0]).
Measurement of bacterial growth.The effects of β-mercaptoethanol, dithiothreitol (DTT), NO, and various antibiotics on the growth of WT and quinol oxidase-deficient mutant S. Typhimurium strains were examined by spectrophotometrically measuring the optical density of bacterial cultures using a Bioscreen C microbiology microplate reader (Labsystems, Helsinki, Finland). Salmonella cultures grown overnight were diluted to the same amount with phosphate-buffered saline (PBS) and were inoculated into microplate wells containing LB broth or minimal E medium (0.2% glucose). The optical density of the bacterial cultures was recorded at 1-h intervals.
Measurement of Salmonella replication in RAW 264.7 cells.To measure the intracellular proliferation of S. Typhimurium strains, a murine macrophage-like cell line, RAW 264.7 (ATCC TIB-7), was purchased from the American Type Culture Collection (ATCC; USA). RAW 264.7 cells were grown in RPMI 1640 cell culture medium (HyClone, USA) containing 10% heat-inactivated fetal bovine serum (HyClone, USA) at 37°C with 5% CO2. Infection of RAW 264.7 cells with S. Typhimurium was performed as described previously (52). Briefly, S. Typhimurium bacteria grown to the early stationary phase in LB were collected, washed with PBS, and used to infect RAW 264.7 cells at a multiplicity of infection of 10. Extracellular bacteria were killed by washing with RPMI medium containing gentamicin (10 μg ml−1). To determine the replication rate of S. Typhimurium in RAW 264.7 cells, the cells were washed with PBS and lysed at 1 h and 18 h postinfection in lysis buffer (0.1% Triton X-100 in PBS). Serial dilutions of lysates containing S. Typhimurium were plated on LB agar plates and incubated overnight at 37°C, and the CFU were enumerated.
Antibiotic susceptibility tests.To determine the MICs of various antibiotics for the S. Typhimurium strains, equal amounts of bacteria cultured overnight in Mueller-Hinton broth were added to fresh medium (to an OD600 of 0.02) containing antibiotics in serial 2-fold dilutions, and the newly inoculated cultures were then incubated at 37°C with shaking for 24 h. Antibiotic susceptibility was also examined by measuring bacterial survival at 37°C 2 h after adding antibiotics into early-log-phase (OD600 = 0.4) LB broth cultures. Bacterial cultures before and after antibiotic treatment were serially diluted and plated on LB agar. After overnight incubation, the CFU were enumerated and used to calculate percent survival.
Measurement of cydA gene transcription by real-time RT-PCR.To compare the amount of cydA mRNA expressed by the ΔcydX mutant and WT S. Typhimurium strains, overnight cultures were used to inoculate fresh LB broth, and the cultures were then incubated until they reached log phase (OD600 = 0.4). Bacterial transcription was stopped by adding 1/5 volume of an ice-cold phenol-ethanol (5% phenol in 95% ethanol) solution before harvesting the cells. Total bacterial RNA was extracted and purified with an RNAiso Plus kit (TaKaRa) according to the manufacturer’s instructions. Real-time reverse transcription (RT)-PCR was performed using a QuantiTect SYBR green RT-PCR kit (Qiagen, Hilden, Germany), as described previously (53). The sequences of the primer pairs used in this study are listed in Table S3.
Statistical analysis.Statistical analyses were performed using the program Prism (version 6; GraphPad Software, USA).
ACKNOWLEDGMENTS
This study was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education and the Ministry of Science and ICT (grants NRF-2018R1D1A1B07044085 and NRF-2016R1A2B1015928).
K.M.D., B.G.K., and I.S.B. designed the research; K.M.D., B.G.K., C.L., H.J.P., and Y.M.P. performed experiments; K.M.D., B.G.K., C.L., H.J.P., Y.M.P., Y.H.J., and I.S.B. made major contributions to the acquisition, analysis, and interpretation of the data; K.M.D., B.G.K., Y.H.J., and I.S.B. wrote the manuscript.
FOOTNOTES
- Received 20 May 2019.
- Accepted 21 October 2019.
- Accepted manuscript posted online 28 October 2019.
Supplemental material is available online only.
- Copyright © 2020 American Society for Microbiology.
