Low Abundance of Respiratory Nitrate Reductase Is Essential for Escherichia Coli in Resistance to Aminoglycoside and Cephalosporin

The increasing incidence of antibiotic-resistant bacteria has posed a serious threat to human health and aquaculture today and thus has become a scientific issue worldwide.  A line of evidence has indicated that four mechanisms contribute to the bacterial resistance, including modification or hydrolysis of enzymes, modification of drug targets, activation of efflux pump systems, and reduce of outer membrane permeability.  Despite these progresses over past sixty years, our understanding of the resistant mechanisms is still incomplete, which should be one of the major causes resulting in antibiotic-resistant bacteria out of control. Thus, further investigation of bacterial resistant strategies to antibiotics may be necessary for combating antibiotic-resistant bacteria.

The recently developed technologies are expected to facilitate the study of bacterial antibiotic-resistant mechanisms. For example, protein-protein interactions, being important for the majority of biological functions including antibiotic resistance, may be potentiated using highly throughput screening methods of 2-D native PAGE (N-PAGE) and 2-D blue native PAGE (BN-PAGE) based proteomics.  The two methods have been proven especially useful for investigation of native protein complexes in the context of proteomics. Comparatively, N-PAGE based on proteomics may offer more information on labile protein complexes containing metabolic enzymes than BN-PAGE based proteomics. Using the two approaches, researchers have investigated membrane and outer membrane protein complexes in Escherichia coli and identified a series of homogeneous and heterogeneous complexes. The identified membrane-related protein complexes may contain cytoplasmic and periplasmic proteins since approximately 30 - 40% E. coli proteins may function in the membrane of the cell envelope, in which there are soluble proteins tethered to the inner and outer membranes through hydrophobic patches, lipid moieties, or charge interactions or in membrane protein complexes. On the other hand, recent reports have indicated that metabolic enzymes are related to antibiotic resistance, and the regulation of metabolism elevates susceptibility of bacterial persisters to antibiotics. We have also shown that Na(+) NQR complex is essential for Vibrio alginolyticus in resistance to balofloxacin using N-PAGE based proteomics coupled with functional validation. We hypothesized that the comparative N-PAGE approach might provide metabolic-related protein complexes that contributed to antibiotic resistance for further functional investigation since many of metabolic enzymes are detected in membrane protein fraction. Here, N-PAGE based proteomics is used to identify a metabolic protein complex in membrane fraction of Escherichia coli K12 BW25113 and then the role of the complex in bacterial antibiotic resistance is further investigated.

Separation of membrane proteins and detection of Nar complex in E. coli K12 BW25113 membrane protein fraction using N-PAGE based proteomics. N-PAGE was used to separate the membrane proteins of E. coli K12 BW25113.  Approximately a hundred of clear protein spots were observed on each of the 2-DE gels stained with cCBB R-250, most of which were located in the range of 25-120 kDa with minimal streaks.  The streaks could be resulted from the diffusion of the same proteins during the first dimensional native-PAGE as reported previously.  These well visualized protein spots were excised from 2-DE gels and analyzed by MALDI/TOF-TOF analysis.  A hundred of proteins were successfully identified.  They represented ninety-four unique proteins since five proteins had more than one spot. We interestingly found that two components, NarG and NarH, of Nar complex were lined, which did not show in our previous N-PAGE to detection of E. coli DH5α membrane protein fraction. Nar complex includes four polypeptides NarG, NarH, NarJ and NarI of molecular weight 138.7, 57.7, 26.5 and 25.5 kDa, respectively.  NarJ and NarI did not appear in the gel probably due to the low molecular weight. NarG and NarH were further validated in 2-DE Western blotting.  The complex is capable of reducing nitrate using normal physiological substrates and is clearly the major respiratory Nar in E. coli since it accounts for 98% of the Nar activity when fully induced. The result provides a chance to investigate a role of the complex contributed into bacterial energy metabolism in bacterial antibiotic resistance. In addition, sixteen out of the 94 proteins were validated using Western blotting. We identified the subunits A and B of the BAM complex in two distinct protein complexes. The BAM complex consists of one outer membrane protein, BamA (formerly YaeT), and four lipoproteins, BamB, BamC, BamD and BamE (formerly YfgL, NlpB, YfiO and SmpA, respectively). The other three were absence, which might be related to protein abundance, molecular weight and binding force. Further pathway analysis was carried out and obtained 90 annotation out of the 119 proteins identified. Among the 90 annotated proteins, 76 were enriched in 16 pathways. They are mainly involved in carbon metabolism, nitrogen metabolism, oxidative phosphorylation. Furthermore, we investigated the relationships between protein complexes containing altered abundance of protein(s) and antibiotic resistance.

 

Validation of Nar complex in E. coli K12 BW25113 membrane proteins. Then, the Nar complex was validated using two assays.  First, far-Western blotting was used to investigate the interaction of NarH with NarG. Our results indicated that the bait protein was NarH when NarG was used as the prey protein.  Then, the complex was further validated by N-PAGE analysis of narH and narG deletion mutants. This was based on the hypothesis that when one protein within a protein complex is absent, the others may disappear from N-PAGE gel.  Our results showed that loss of narG caused disappearance of NarH, while deletion of narH resulted in loss of NarG in the same line, meaning that NarG-NarH complex existed in the line. These results indicate that Nar complex detected by the N-PAGE based proteomics is reliable, and thus may be used for investigation of antibiotic resistance.

Characterization of Nar complex in five antibiotic-resistant E. coli strains. In order to investigate whether abundance of Nar complex components was altered to resist antibiotics, the N-PAGE based proteomics approach was used to detect NarG and NarH levels in SM-R, GEN-R, CAZ-R, TET-R and NA-R.  Significant changes were detected, showing that NarG and NarH disappeared in the SM-R, CAZ-R, TET-R and decreased in GEN-R and NA-R.  These results not only indicate that Nar complex may play an important role in antibiotic resistance, but also show that the role is related the classes of antibiotics.

Functional characterization of narG and narH deletion mutants in response to five antibiotics. We next performed functional characterization of the Nar complex proteins using narG or narH deletion mutants. The performance was carried out using two antimicrobial susceptibility assays: MIC assay and survival capability assay.  Deletion of narG or narH resulted in distinct elevation of MICs in medium with SM or GEN, and two-fold decrease of MICs in medium with TET, and no significant changes in medium with CAZ or NA. The survival capability assay was also carried out to further investigate functional characterization of the mutants due the assay is more sensitive than MIC assay.  These bacteria including ΔnarI and ΔnarJ were cultured in tubes with series of two-fold dilution of SM, GEN, CAZ, TET and NA concentrations from 0.625 to 5 μg/mL, 0.1953-1.1719 μg/mL, 0.0781-0.3125 μg/mL, 0.78125-3.25 μg/mL, 1.25-7.5 μg/mL, respectively. The resulting cultures were assayed by measurement of optical density at 600 nm and the survival capability was determined by division of OD values of the cultures with an antibiotic over those without the drug. The survival rates of the mutants cultured in medium with SM, GEN or CAZ were significantly increased (P<0.01) in a dose-dependent manner. However, lower survival capabilities were detected in the mutants cultured in medium with TET and NA. Generally, the results obtained from survival capability assay were in consistence with those obtained from MIC assay.  In summary, the results obtained from functional characterization of ΔnarG and ΔnarH were consistent with the changes of NarG and NarH at the protein abundance which was detected by 2-DE analysis of these antibiotic-resistant bacterial strains. 

In summary, N-PAGE based proteomics approach was used to investigate E. coli K12 BW25113 membrane protein complexes and resulted in the detection of NarG and NarH, two main components of Nar complex.  The decreased abundance of the two proteins was characterized in SM-R, GEN-R, CAZ-R, TET-R and NA-R.  Functional tests using narG- and narH-deleted mutants demonstrated distinctly elevated resistance to aminoglycosides and CAZ. It is documented that low abundance of Nar complex is required for E. coli resistance to aminoglycosides and CAZ, which is the first report on that E. coli Nar metabolism alters bacterial resistance to antibiotics. This finding can serve to propose novel strategies for treating infections caused by aminoglycoside- and CAZ-resistant E. coli through activation of Nar activity.

 

 

ACKNOWLODGEMENTS

This work was sponsored by grants from NSFC projects (30972279, 40976080), Guangdong Provincial Science and technology projects (2012A031100004). Doctoral Fund of Ministry of Education of China (100171110029)