A Smooth-Type, Phage-Resistant Klebsiella pneumoniae Mutant Strain Reveals that OmpC Is Indispensable for Infection by Phage GH-K3

Isolation of a phage-resistant K. pneumoniae mutant strain.K. pneumoniae strain K7 and phage GH-K3 (Siphoviridae) were isolated from a clinical specimen and water samples collected from a sewage treatment plant, respectively, in a previous study (22). K7 culture was initiated from a single colony. The culture then was grown to the exponential growth phase and transferred to fresh Luria-Bertani (LB) broth at a final bacterial count of 2 × 10⁷ CFU/ml. Phage GH-K3 (multiplicity of infection [MOI] of 0.01) was then added and incubated for 10 h. Growth curves were constructed by measuring optical density at 600 nm (OD₆₀₀) by using a BioPhotometer plus (Eppendorf, Hamburg, Germany). Phage-resistant variants in the final culture were isolated by the streak-plating method. In addition to the vast majority of small, rough-type colonies, a K7-like smooth colony was picked up and named K7R^B.

Phage adsorption assay.Adsorption assays were performed as previously described, with modest modifications (38). Briefly, 1 ml late-exponential-phase culture of each K. pneumoniae strain (2 × 10⁹ CFU/ml) was mixed with 10 μl diluted phage (1 × 10⁷ PFU), and the mixture was incubated for 10 min at 37°C with shaking. The mixture was then centrifuged at 4°C and 13,000 ×g for 5 min. Supernatants were filtered through 0.22-μm filters (Millipore, Billerica, MA, USA). The number of phage particles remaining in the filtrates was determined by plaque assay in triplicate. The phage adsorption efficiency was calculated using the equation [(initial titer − residual titer in the supernatant)/initial titer] × 100%.

Identification of the phage receptor type.The receptor properties of GH-K3 were measured as described previously (39). Briefly, K. pneumoniae strains were treated (protected from light) with 100 mM IO₄⁻ in sodium acetate (50 mM, pH 5.2) to destroy surface carbohydrates at 25°C for 2 h. Alternatively, K. pneumoniae strains were treated with proteinase K (0.2 mg/ml) in sodium acetate (50 mM, pH 5.2) to destroy surface proteins at 37°C for 3 h. K. pneumoniae strains treated with IO₄⁻ or proteinase K were then used to detect phage adsorption and to perform double-layer agar plate assays to detect sensitivity to phage GH-K3.

Proteomic and metabolomic sample preparation.K7 and K7R^B cultures were harvested, pelleted, and washed three times with phosphate-buffered saline (PBS). After that, the suspensions were mixed with 4× protein SDS-PAGE loading buffer (TaKaRa, Dalian, China) and then boiled for 10 min. These samples were prefractionated by 10% SDS-PAGE. Subsequently, each sample was processed into eight gel bands and subjected to in-gel trypsin digestion (40). The resulting tryptic peptides were extracted twice from the gels by equilibrating the samples with 50% acetonitrile and 5% formic acid for 20 min at 37°C. The final peptide samples were vacuum dried and reconstituted in high-performance liquid chromatography (HPLC)-grade water (Thermo Fisher Scientific, Waltham, MA, USA) prior to LC-MS-MS analyses. Four biological replicates of K7 and K7R^B were analyzed. We carried out 136 experiments (no technical runs) with eight gel fractions per bacterial sample.

LC-MS-MS analyses.LC-MS-MS for proteomic analyses was performed using a linear ion trap mass spectrometer (LTQ Velos Pro; Thermo Scientific, San Jose, CA, USA) equipped with nanoflow reverse-phase liquid chromatography (EASY-nLC 1000; Thermo Scientific, San Jose, CA, USA) at Peking University. The capillary column (75 μm by 150 mm) with a laser-pulled electrospray tip (model P-2000; Sutter Instruments) was packed with 4-μm, 100-Å Magic C18AQ silica-based particles (Michrom BioResources Inc., Auburn, CA, USA). The mobile phase included solvent A (97% H₂O, 3% acetonitrile, and 0.1% formic acid) and solvent B (100% acetonitrile and 0.1% formic acid). The gradient of LC separation started with 7% solvent B for 3 min and then increased to 35% solvent B in 40 min; subsequently, solvent B was rapidly raised to 90% in 2 min and maintained for 10 min before 100% solvent A was used for column equilibration. The mass spectrometer was operated in a data-dependent mode with one full MS scan (m/z 350 to 1,500) followed by MS-MS analyses of the 10 most intense ions. Dynamic exclusion was set with repeat duration of 12 s and exclusion duration of 6 s.

Proteomic data analyses.Raw MS files were searched with Mascot (version 2.3.02; Matrix Science Inc.) against annotated protein sequences of K. pneumoniae K7 and K7R^B (GenBank accession numbers NKQH00000000 and PKKQ00000000, respectively). Average mass with a peptide tolerance of 1.5 ppm and a fragment mass tolerance of 0.8 Da was chosen. The maximum missed cleavage was set to 2. Oxidation (M) was set as a variable modification. The screening procedure was designed to achieve a false discovery rate (FDR) of <1% at both the protein and peptide levels. Spectral counts were used to assess the relative abundances of proteins from different samples. This index represents the total number of repeated identifications of peptides for a given protein throughout the analysis, which provides a semiquantitative measurement of protein abundance. Raw spectral counts were normalized against the total counts of all identified proteins in a given sample. For comparing the data differences visually, heat map analysis was generated by Heml 1.0 (41). With the purpose of analyzing the relevance of differentially expressed proteins, COG analyses were performed by STRING (42).

Expression and purification of outer membrane proteins.Protein expression, solubilization of inclusion bodies (IBs), and refolding were performed by referring to a previous study, with modest modifications (43). The open reading frames (ORFs) of ompC, ompN, KPN_02430, and ompF were amplified by PCR using the primers shown in Table S1 in the supplemental material. The PCR products were cloned into pET28a(+) vector (Novagen, Madison, WI, USA). The constructed plasmids were then transformed into competent E. coli BL21(DE3) cells (TransGen Biotech, Beijing, China). Exponentially growing cultures were induced with 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) (Sigma-Aldrich, St. Louis, MO, USA) and incubated for 5 h at 25°C for expression. Cell pellets were obtained by centrifugation at 8,000 × g for 10 min at 4°C and washed twice with Tris buffer (20 mM Tris, pH 8.0, 0.2 M NaCl). After disruption by sonication, crude IBs were obtained by centrifuging at 13,000 × g for 15 min at 4°C. IBs were further washed twice with TTN buffer (50 mM Tris, pH 8.0, 0.1 M NaCl, and 2% Triton X-100). After each washing, the suspensions were centrifuged at 13,000 × g for 15 min at 4°C. IBs were dissolved in Tris-urea buffer (20 mM Tris, pH 8.0, 0.2 M NaCl, and 8 M urea) for 4 h with gentle shaking at 4°C. For obtaining unfolded proteins, the suspensions were centrifuged at 13,000 × g for 20 min at 4°C. Aggregates and particulate matter were removed by 0.22-μm filters (Millipore, Billerica, MA, USA). Unfolded proteins were rapidly added to refolding buffer (20 mM Tris, pH 8.0, 0.2 M NaCl, 10% [vol/vol] glycerol, and 0.2% [vol/vol] polyoxyethylene-9-lauryl ether [Sigma-Aldrich, St. Louis, MO, USA]) at a ratio of 1:5 (protein solution to buffer) and kept at 4°C with stirring overnight. The solutions were then centrifuged at 35,000 × g for 90 min at 4°C to remove small aggregates and particulate matter from refolded proteins.

Neutralizing antibody production.The refolded proteins were emulsified with equal amounts of Freund's complete adjuvant or Freund's incomplete adjuvant (Sigma-Aldrich, St. Louis, MO, USA). New Zealand White rabbits (male, 16 weeks) were immunized with different refolded proteins three times at 2-week intervals (44). At 10 days after the last immunization, the rabbits were euthanized by sodium pentobarbital (100 mg/kg of body weight, intravenously). Sera were collected and stored at −80°C.

All of the animal experiments were conducted in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals, approved by the State Council of the People's Republic of China (11 January 1988), and were approved by the Animal Welfare and Research Ethics Committee at Jilin University.

Construction of ompC-deficient strains.Suicide plasmid pCVD442 was supplied by Addgene (plasmid 11074; Cambridge, MA, USA) (45). In this study, this plasmid was reconstructed by kanamycin resistance gene or gentamicin resistance gene replacement. The ompC-deficient strains were created as previously described (46). Briefly, the replacement sequences were individually cloned in pCVD442-derived plasmids before electrotransformation into E. coli SM10 λpir. The recombinant strains were then used as donors in conjugation with recipient strains. K7(ΔompC₇₁₂) transconjugants were selected on LB plates with 30 μg/ml kanamycin and 100 μg/ml ampicillin, while KPP14(ΔompC), KPP27(ΔompC), and KPP36(ΔompC) transconjugants were selected on LB plates containing 20 μg/ml gentamicin and 100 μg/ml ampicillin. All primers used for amplification and sequencing are listed in Table S1. The strains and plasmids are listed in Table S2.

Construction of overexpression strains.The genes ompC, ompN, KPN_02430, and ompF of K. pneumoniae K7 were individually cloned into pUC18K (47, 48). In accordance with a standard transformation protocol by using electrotransformation, the recombinant plasmids were transformed into K7R^B and other ompC-deficient K. pneumoniae strains, including KPP14(ΔompC), KPP27(ΔompC), and KPP36(ΔompC). Recombinant strains were then selected on LB plates containing kanamycin (30 μg/ml). All primers used for amplification and sequencing are listed in Table S1. The strains and plasmids are listed in Table S2.

Western blot analyses.All primary antibodies against the peptide antigens of different Omp proteins were made by PolyExpress at GenScript Biological Technology Co., Ltd. (Nanjing, China). The sequences of peptide antigens are listed in Table S3. The following antibodies were used in this study: anti-OmpC-1 (1:1,000), anti-OmpC-2 (1:1,000), anti-OmpN (1:2,000), anti-KPN_02430 (1:2,000), anti-OmpF (1:1,000), and anti-DnaK (1:2,000). These antibodies were diluted with TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% [vol/vol] Tween 20). Equal amounts of each protein sample were separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Immunoblot analyses were performed using the mentioned primary antibodies and horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (ab205718; 1:5,000; Abcam, MA, USA). Immunoblots were developed with Immobilon Western chemiluminescent HRP substrate (Millipore, Billerica, MA, USA) and measured using a chemiluminescence imaging system (Tanon 5200; Shanghai, China). Protein expression analyses were performed by Image J (https://imagej.nih.gov/ij/index.html).

Statistical analysis.Experimental data were analyzed by Student's t tests or one-way analysis of variance (ANOVA) tests through GraphPad Prism 6 (GraphPad Software, Inc., CA, USA). P values of <0.05 were considered to represent statistically significant differences. Error bars represent the standard errors of the means (SEM).

Accession number(s).Whole-genome sequences of K. pneumoniae K7 and K7R^B have been deposited in NCBI GenBank (accession numbers NKQH00000000 and PKKQ00000000, respectively).