ABSTRACT
Bacteriophage can be used as an alternative or complementary therapy to antibiotics for treating multidrug-resistant bacterial infections. However, the rapid emergence of resistant host variants during phage treatment has limited its therapeutic applications. In this study, a potential phage-resistant mechanism of Klebsiella pneumoniae was revealed. After phage GH-K3 treatment, a smooth-type colony, named K7RB, was obtained from the K. pneumoniae K7 culture. Treatment with IO4− and/or proteinase K indicated that polysaccharides of K7 played an important role in phage recruitment, and protein receptors on K7 were essential for effective infection by GH-K3. Differences in protein expression between K7 and K7RB were quantitatively analyzed by liquid chromatography-tandem mass spectrometry. Among differentially expressed proteins, OmpC, OmpN, KPN_02430, and OmpF were downregulated significantly in K7RB. trans-Complementation of OmpC in K7RB conferred rapid adsorption and sensitivity to GH-K3. In contrast, a single-base deletion mutation of ompC in K7, which resulted in OmpC silencing, led to lower adsorption efficiency and resistance to GH-K3. These assays proved that OmpC is the key receptor-binding protein for GH-K3. In addition, the native K. pneumoniae strains KPP14, KPP27, and KPP36 showed low or no sensitivity to GH-K3. However, these strains became more sensitive to GH-K3 after their native receptors were replaced by OmpC of K7, suggesting that OmpCK7 was the most suitable receptor for GH-K3. This study revealed that K7RB became resistant to GH-K3 due to gene mutation of ompC and that OmpC of K7 is essential for effective infection by GH-K3.
IMPORTANCE With increased incidence of multidrug-resistant (MDR) bacterial strains, phages have regained attention as promising potential antibacterial agents. However, the rapid emergence of resistant variants during phage treatment has limited the therapeutic applications of phage. According to our trans-complementation, ompC mutation, and phage adsorption efficiency assays, we identified OmpC as the key receptor-binding protein (RBP) for phage GH-K3, which is essential for effective infection. This study revealed that the phage secondary receptor of K. pneumoniae, OmpC, is the essential RBP not only for phage infecting Gram-negative bacteria, such as Escherichia coli and Salmonella, but also for K. pneumoniae.
INTRODUCTION
The discovery and application of bacteriophages as antibacterial agents has been going on for a century (1, 2). Due to failure in some treatment cases, phage therapies gradually declined after the advent of antibiotics (3). In recent years, with the rising incidence of MDR strains, therapeutic and preventive strategies using antibiotics have been restricted. Because the infection ability of bacteriophages is unrelated to the antibiotic resistance of bacteria, phages have regained attention as promising potential therapies (4). However, phage-resistant or -insensitive strains emerge by natural mutations. Thus, the rapid appearance of phage-resistant mutant strains in the process of treatment limits the clinical applications of phage (5). Phage-resistant mutant bacteria largely arise during the processes of phage adsorption (6–9), DNA injection (10, 11), and replication (12–14). Overall, bacteriophage resistance mechanisms are numerous and sophisticated, with some strains able to defeat phage invasion in several different ways. Bacteria prevent phage adsorption by changing receptors, which is the most common form of bacteriophage resistance. Some outer membrane receptors from Gram-negative strains have been identified. Outer membrane protein C (OmpC) was proved to be the receptor of T4-like Salmonella bacteriophage vB_SenM-S16, and the phage adsorption could be transferred to Escherichia coli by replacement of OmpC with the Salmonella homologue (15). After hydrolyzing lipopolysaccharide (LPS), phage Sf6 infects Shigella flexneri through OmpA or OmpC, with OmpA being the preferred receptor (16, 17).
Klebsiella pneumoniae is an important nosocomial and community-acquired opportunistic pathogen that mainly triggers pneumonia and urinary tract and blood infections. In recent years, MDR K. pneumoniae has appeared frequently alongside the overuse and abuse of antibiotics. In particular, strains producing extended-spectrum β-lactamases and carbapenemases (including KPC, MBL, and OXA-48) have become leading pathogens associated with nosocomial infection (18, 19).
According to previous data, bacteriophage therapy can effectively control infection caused by K. pneumoniae (20, 21). However, during the infection of K. pneumoniae by phages, antiphage mutagenesis among K. pneumoniae strains occurs with high frequency (5). Based on our previous study, K. pneumoniae K7 can form large, smooth colonies. However, most of the phage-resistant strains form small, rough colonies (22). In addition, the phage adsorption efficiencies of rough colonies are greatly reduced due to the loss of capsular polysaccharide (CPS). In the 1970s, the capsular polysaccharides of K. pneumoniae were first reported as the primary receptor for phage adsorption. Klebsiella bacteriophage KP11 adsorbs to the host by identifying and hydrolyzing β-d-glucosyl-(1-3)-β-d-glucuronic acid linkages (23). O-antigen-specific LPS was also verified as a cell surface receptor for K. pneumoniae phage FC3-2, FC3-3, and FC3-6 (24). In the present study, a rare smooth colony was isolated from a GH-K3-resistant mutant of K7, named K7RB. Contrary to rough phage-resistant variants, phage adsorption efficiency of K7RB approaches that of K7. To our knowledge, the phage resistance mechanisms of smooth-type mutant K. pneumoniae strains have not been reported. For elucidating the novel mechanism by which K. pneumoniae establishes resistance to phage treatments, K7RB was selected and studied. By quantitative analysis with liquid chromatography-tandem mass spectrometry (LC-MS-MS), we determined that the abundance of several outer membrane porins was dramatically altered in K7RB compared with that of the native K7. The potential phage secondary receptor then was identified among the porins by trans-complementation, gene mutation, and phage adsorption efficiency assays. Finally, OmpC from K. pneumoniae was confirmed to be the major receptor-binding protein (RBP) of GH-K3.
RESULTS
Protein receptors on K7 are essential for effective adsorption of GH-K3.Compared with the growth curve of K. pneumoniae K7, phage GH-K3 efficiently inhibited the growth of K7 within 1.5 h (Fig. 1A). However, the OD600 of the mixture of GH-K3 and K7 started to rise after 5 h, indicating that phage-resistant mutations appeared and were enriched. At 10 h, the phage-resistant variants were separated. Most mutant strains formed small, rough colonies (K7RR), and very few colonies were of the smooth type. A smooth colony was picked up and named K7RB. The colony morphology of K7RB looked just like that of K7 (Fig. 1B). In addition, there was no significant divergence in the adsorption efficiency of GH-K3 on K7 and on K7RB (Fig. 1C).
Characteristics of K. pneumoniae K7, K7RB, and K7RR. (A) Growth curves of K. pneumoniae K7 with or without GH-K3 treatment. K7 culture (2 × 107 CFU/ml) was infected with GH-K3 for 10 h at an MOI of 0.01. The same culture without phage treatment served as the control. (B) Colony morphologies of K7, K7RB, and K7RR. Colonies of K7, K7RB, and K7RR were cultured on LB plates at 37°C for 12 h after streak plating. Both K7 and K7RB can form smooth-type colonies, but K7RR can form rough-type colonies. (C) Adsorption efficiencies of GH-K3 binding to K. pneumoniae K7, K7RB, and K7RR with different treatments. Untreated strains, proteinase K-treated strains, periodate-treated strains, and double-treated strains were tested for adsorption as indicated on the x axis. *, **, and *** indicate significant differences at P values of <0.05, <0.01, and <0.001, respectively. Data represent the means ± SEM from triplicate experiments.
For identifying the receptor types, K7 was treated with periodate and/or proteinase K. GH-K3 was almost unable to bind the IO4−-treated K7 and IO4−-treated K7RB in 10 min. Interestingly, the adsorption efficiency of GH-K3 on the IO4−-treated K7 was substantially increased within 1 h (up to 93.9%), but the adsorption efficiency of GH-K3 on the IO4−-treated K7RB remained unchanged within the same time period (9.5%) (Fig. 1C).
The adsorption efficiency of GH-K3 on proteinase K-treated K7 seemed not to be affected compared with that of nontreated K7, with the same observed for K7RB. However, the adsorption efficiencies of GH-K3 on the double-treated (IO4− and proteinase K) K7 and K7RB were significantly lower than those of strains treated with only IO4− (3.2% and 2.0%, respectively). Thus, as the primary receptors, polysaccharides on K. pneumoniae K7 were related to GH-K3 recruitment, but they were not essential for adsorption of this phage. In contrast, proteins (secondary receptors) on K7 were essential for effective adsorption of GH-K3.
Differential protein expression between K7 and K7RB by LC-MS-MS.We identified and compared ∼3,100 proteins between K7 and K7RB by LC-MS-MS. The abundance of 60 proteins in K7RB was significantly altered compared with that of K7, with 13 of the proteins induced and 47 proteins repressed (Fig. 2). The differentially expressed proteins were distributed among several functional categories, including outer membrane proteins, sugar and amino acid metabolism, metal ion transport systems, and respiratory chains for energy generation.
Overview of differentially expressed proteins between K. pneumoniae K7 and K7RB. Heat-map analysis of differentially expressed proteins between K. pneumoniae K7 and K7RB generated by Heml 1.0. Lanes 1 to 3 represent three biological replicates at each sampling time. Protein functional classification was performed by STRING, version 10.5.
By SDS-PAGE analysis, the differential protein band (35 to 40 kDa) between K7 and K7RB was clearly visible (see Fig. S1A in the supplemental material). Similarly, LC-MS-MS indicated some β-barrel outer membrane porins in K7RB were robustly downregulated in abundance, including OmpC (∼9.7-fold), OmpN (∼133.2-fold), KPN02430 (∼23.7-fold), and OmpF (∼18.3-fold) (Fig. 2 and 3A and Fig. S1B). Western blot analyses demonstrated that the changes in OmpC, OmpN, KPN02430, and OmpF expression levels were generally all in accordance with the mass spectrometry data. The expression levels of the four porins were drastically downregulated in K7RB (Fig. 3B). Interestingly, genome sequence BLAST analysis indicated that a single-base (G712) deletion occurred in the ompC gene in K7RB and resulted in the generation of incorrect translation products (OmpC-1 and OmpC-2) (Fig. S2).
Differences in expression of outer membrane porins between K7 and K7RB. (A) Extracted ion chromatograms of protonated peptides from OmpC, OmpN, KPN_02430, and OmpF. Arrows indicate peaks of OmpF (top, K7; bottom, K7RB). DnaK was used as a loading control. The following peptides were used: DnaK,S453LGQFNLDGINPAPR467 (m/z 800.4); OmpC-1, I38DGLHYFSDDKSVDGDQTYMR58 (m/z 812.9); OmpC-2, Y316VDVGATYYFNK327 (m/z 720.8); OmpN, L97AFAGIK103 (m/z 360.5); KPN_02430, N28GNKLDLYGK37 (m/z 561.6); OmpF, F331NQLDDNDYTK341 (m/z 687.2). (B) Western blot analyses of OmpC-1, OmpC-2, OmpN, KPN_02430, and OmpF. Expression levels of OmpC-1, OmpC-2, OmpN, KPN_02430, and OmpF in K. pneumoniae K7 and K7RB were detected. Expression of DnaK was detected as a control.
OmpC is the outer membrane receptor for GH-K3.When K7RB cells were trans-complemented with ompC, ompN, KPN_02430, or ompF, the expression levels of these four porins were dramatically upregulated (Fig. 4A). Despite the plaques not being as clear as those in K7, K7RB harboring pUC18K-ompCK7 became sensitive to GH-K3. However, K7RBC-ompNK7, K7RBC-KPN_02430K7, and K7RBC-ompFK7 were still resistant to GH-K3 (Fig. 4B). In addition, when K. pneumoniae K7 was preincubated with anti-Omp sera (1:10) before infection by GH-K3, only anti-OmpC serum affected the infection efficiency of GH-K3 on K7 (27.2% decreased) (Fig. S3). Further, single-base deletion at ompC712 in K7 was performed with reference to the sequence of K7RB. The OmpC expression level of K7(ΔompC712) was similar to that in K7RB (Fig. 4C). The infection assay indicated that K7(ΔompC712) lost its sensitivity to GH-K3 (Fig. 4D).
Sensitivity of Omp overexpression strains and K7(ΔompC712) to GH-K3. (A) Expression levels of OmpC (K7RBC-ompCK7), OmpN (K7RBC-ompNK7), KPN_02430 (K7RBC-KPN_02430K7), and OmpF (K7RBC-ompFK7) were detected by Western blotting. K7RB was used as a control. DnaK served as a loading control. (B) Plaque assays. Infection ability of GH-K3 on K7RBC-ompCK7, K7RBC-ompNK7, K7RBC-KPN_02430K7, and K7RBC-ompFK7 was determined. K7 and K7RB were used as controls. (C) Expression levels of OmpC in K7 and K7(ΔompC712). DnaK served as a loading control. (D) Sensitivity of K7(ΔompC712) to GH-K3. K7 was used as a control. (E) Adsorption efficiency of GH-K3 binding to K7RBC-ompCK7 and K7(ΔompC712) with different treatments. Untreated strains, proteinase K-treated strains, periodate-treated strains, and double-treated strains were tested for adsorption as indicated on the x axis. *, **, and *** indicate significant differences at P values of <0.05, <0.01, and <0.001, respectively. Data represent the means ± SEM from triplicate experiments.
In order to identify the function of OmpC, K7RBC-ompCK7 and K7(ΔompC712) were treated with periodate and/or proteinase K. The adsorption efficiency of GH-K3 on the IO4−-treated K7(ΔompC712) was markedly reduced (42.2% left). Interestingly, unlike K7, the majority of GH-K3 could bind to K7RBC-ompCK7 in 10 min after IO4− treatment (87.7%). However, the adsorption capacity of IO4−-treated K7RBC-ompCK7 and K7(ΔompC712) did not change significantly with the passing of time (up to 1 h). In addition, the adsorption efficiency of GH-K3 on the double-treated (IO4− and proteinase K) K7RBC-ompCK7 (57.4%) was drastically lower than that of proteinase K or IO4− treatment but was still higher than that of K7 (ΔompC712) (34.6%) (Fig. 4E). These results indicate that OmpC plays a critical role in the adsorption of GH-K3.
Phage sensitivity of other K. pneumoniae strains can be improved by receptor transplantation.Besides K7, clinically isolated K. pneumoniae KPP14, KPP27, and KPP36 were all identified as K2 capsular type strains according to the description in the previous study (25). GH-K3 can form small and blurred plaques on a lawn of K. pneumoniae KPP27. This phage can also form spots on lawns of K. pneumoniae KPP14 and KPP36 but cannot form plaques. The amino acid sequences of OmpC in K7, KPP14, KPP27, and KPP36 were compared by BLAST analysis. Compared with the OmpC amino acid sequence of K7, Ser255 was replaced by Thr in KPP27; there were four residue replacements in the OmpC of both KPP14 and KPP36, including Thr255 (Ser255 in OmpC of K7), Glu342 (Asp342 in OmpC of K7), Asp344 (Ser344 in OmpC of K7), and Lys348 (Asn348 in OmpC of K7) (Fig. 5A). There were no obvious differences in the expression levels of OmpC among K7 and the three other K. pneumoniae strains (Fig. 5B and Fig. S4).
Sensitivity of different K. pneumoniae strains to GH-K3. (A) Amino acid sequence alignment of OmpC among K7, KPP14, KPP27, and KPP36. The picture was generated by CLC Main Workbench, version 7.7.3 (CLC Bio-Qiagen, Aarhus, Denmark). (B) Expression levels of OmpC in K7, KPP14, KPP27, and KPP36. DnaK was used as a loading control. (C) Sensitivity of KPP14, KPP27, and KPP36 to GH-K3.
To determine the specificity of OmpC recognition by GH-K3, phage secondary receptors (the native OmpC) in KPP14, KPP27, and KPP36 were replaced by OmpCK7. Higher OmpC expression levels were detected in three receptor replacement ompCK7-overexpressing strains (Fig. 6A). Compared with the native strains, the ompCK7-overexpressing strains were more easily absorbed by GH-K3 before or after treatment with IO4− (Fig. 6B). In addition, the infection efficiency of GH-K3 on KPP27(ΔompC)C-ompCK7 was considerably enhanced, with more and clearer plaques formed than those formed on the native KPP27. Additionally, GH-K3 could also form small and blurred plaques on a lawn of either KPP14(ΔompC)C-ompCK7 or KPP36(ΔompC)C-ompCK7 (Fig. 6C).
Sensitivity of OmpCK7-expressed K. pneumoniae strains to GH-K3. (A) Expression levels of OmpC in different K. pneumoniae. Lanes: 1, KPP14; 2, KPP14(ΔompC); 3, KPP14(ΔompC)C-ompCK7; 4, KPP27; 5, KPP27(ΔompC), 6, KPP27(ΔompC)C-ompCK7; 7, KPP36; 8, KPP36(ΔompC); 9, KPP36(ΔompC)C-ompCK7. DnaK was used as a loading control. (B) Adsorption efficiency of GH-K3 binding to different K. pneumoniae strains before or after periodate treatment. *, **, and *** indicate significant differences at P values of <0.05, <0.01, and <0.001, respectively. Data represent the means ± SEM from triplicate experiments. (C) Sensitivity of different K. pneumoniae strains to GH-K3. 1, KPP14(ΔompC); 2, KPP14(ΔompC)C-ompCK7; 3, KPP27(ΔompC); 4, KPP27(ΔompC)C-ompCK7; 5, KPP36(ΔompC); 6, KPP36(ΔompC)C-ompCK7.
DISCUSSION
To date, several studies have detailed the phage adsorption receptors of K. pneumoniae (23, 24). CPS on bacterial surfaces often serves as the primary receptor (26). Phage depolymerases specifically recognize the sugar chains, leading the phage to move closer to the cell surface by hydrolyzing the polysaccharides and then bind to outer membrane receptors (secondary receptors) (16, 27). In our previous study, the occurrence of phage-resistant bacterial variants of K. pneumoniae was found (22). The majority of K. pneumoniae K7-derived phage-resistant variants were small, rough colonies, suggesting that the modification of the primary receptor (CPS) occurred. However, to our knowledge, there was no previous study on secondary receptors and bacteriophage resistance mechanisms of K. pneumoniae. In this study, K7RB, a smooth-type phage-resistant mutant strain was picked up, and the phage resistance mechanism of K7RB was studied.
Our adsorption assays of IO4−-treated K7 and K7RB indicated that CPS played an important role in the recruitment of phages. However, IO4−-treated K7 could be adsorbed by the majority of virions within 1 h, demonstrating that CPS is not the only factor during phage adsorption. Compared with IO4−-treated strains, the adsorption efficiencies of GH-K3 on the double-treated (IO4− and proteinase K) K7 and K7RB were further decreased. These data suggest that protein receptors on K. pneumoniae K7 play an irreplaceable role during GH-K3 infection. Thus, the phage resistance of K7RB may be related to protein receptor alteration.
The data obtained by LC-MS-MS indicated OmpC, OmpN, KPN_02430, and OmpF in K7RB were drastically downregulated in abundance (Fig. 2 and 3). Porins on the outer membrane usually act as receptors for a variety of phages (26). The resistance of K7RB to GH-K3 may be associated with the downregulation of these proteins, while for K7RR the abundances for these four porins were almost not altered (data not shown). In order to verify the MS data, ompC, ompN, KPN_02430, and ompF were transformed in K7RB. However, only K7RBC-ompCK7 showed sensitivity to phage GH-K3, suggesting OmpC plays a critical role for GH-K3 infection (Fig. 4B). Additionally, OmpC overexpression did not result in the upregulation of the other porins, suggesting that OmpC is the single factor that determines the effective infection of GH-K3 (Fig. 4A). OmpC has been identified as the phage receptor in several Gram-negative bacteria, such as E. coli and Salmonella, and its gene mutation results in phage resistance (15, 16). In this study, K7(ΔompC712) showed resistance to GH-K3, which further supported our hypothesis (Fig. 4D). The functions performed by OmpC during phage infection were also detected. Compared with K7 and K7RB (Fig. 1C), K7RBC-ompCK7 could still be adsorbed by GH-K3 after IO4− treatment (within 10 min), while proteinase K- and/or IO4−-treated K7(ΔompC712) showed much lower phage adsorption efficiency than K7RBC-ompCK7 (Fig. 4E), suggesting that OmpC serves as the secondary phage receptor for GH-K3 adsorption after CPS degradation.
OmpC is also named OmpK36 in K. pneumoniae and has high abundance on the cell surface (28). Due to a single-base deletion mutation in K7RB, the open reading frame (ORF) of ompC is divided into two parts (ompC1–732 and ompC837–1097). The abundances of defective translation products, OmpC-1 and OmpC-2, were sharply reduced (Fig. 3B) compared with that of OmpC in K7. OmpC with a defective three-dimensional conformation may stimulate cellular stress responses. Subsequently, the mutant porin is degraded by proteases such as DegP, DegS, RseP, etc. (29, 30). Indeed, this might also be caused by misfolding during the translation process. In Gram-negative bacteria, some chaperones, like Skp in E. coli, prevent the aggregation of outer membrane proteins during transport across the periplasm (31, 32). The defective translation products may have been severely hampered in the processes of folding or transportation to the outer membrane.
In general, masking phage receptors with antisera most likely prevents adsorption of the particles, resulting in neutralization of the virions (33). However, our data indicated that the infection efficiency was decreased by only 27.2% (Fig. S3). After purification under denaturing conditions, OmpC might not be fully folded into the correct natural conformation during the refolding process, causing the neutralization effect of anti-OmpC sera to be limited.
Compared with that of K7, KPP14, KPP27, and KPP36 had almost equivalent abundance of OmpC and high sequence similarity, except for a few residues (Fig. 5A and B and Fig. S4). KPP27 had much weaker sensitivity to GH-K3 than did K7, while neither KPP14 nor KPP36 could be infected by GH-K3 (Fig. 5C). These findings imply that the OmpC342–348 domain plays an important role in GH-K3 infection. Residues Asp342, Ser344, and Asn348 are located in the extracellular helix domain away from the center of the trimer and may serve as key binding sites for GH-K3 (Fig. S5).
Besides K7RB, pUC18K-ompCK7 was also transformed into K. pneumoniae KPP14(ΔompC), KPP27(ΔompC), and KPP36(ΔompC). These OmpCK7-overexpressing strains showed higher phage adsorption efficiency and plaque formation capacity (Fig. 6B and C). This demonstrates that OmpCK7 may be the most efficient secondary receptor for GH-K3.
OmpC has also been identified as a transferrin-binding protein that brings transferrin complexes close to the cell surface. Mutagenesis of ompC could affect transferrin binding and iron uptake efficiency (34). Some of the iron uptake proteins were altered to various degrees in K7RB, which might be associated with the inactivation of OmpC. Besides that of iron, alterations in the uptake of other metal ions (zinc, copper, etc.), energy generation, and other metabolism-related proteins were also detected in K7RB (Fig. 2).
GH-K3 infection is determined by OmpC, but the mechanism of interaction between the phage and the porin remains unclear. However, the interaction of E. coli bacteriophage T4 gp37 with OmpC has been elucidated (35). T4 gp37 has a region with a large proportion of histidine, which is responsible for binding with OmpC. In recent years, the mechanism has been further resolved by structural biology methods. The needle domain of the long tail fiber binds with the host outer membrane in either head-on or transverse orientation, but the head domain is consistently located inside the extracellular cavity of the porin (36). Moreover, the binding mode of podovirus with the host outer membrane has also been characterized by cryoelectron microscopy (cryo-EM). SP6 infection of Salmonella enterica serovars Typhimurium and Newport leads to the rotation of the tail spikes, providing direct proof of dual host adsorption specificity of the phage (37). Nevertheless, GH-K3 belongs to the family Siphoviridae, and knowledge gained from T4 or SP6 may not be relevant. Therefore, the interaction mechanisms of our phage, GH-K3, and host OmpC are still worth exploring further.
MATERIALS AND METHODS
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 K7RB.
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 K7RB 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 K7RB 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 K7RB (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 K7RB 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 K7RB have been deposited in NCBI GenBank (accession numbers NKQH00000000 and PKKQ00000000, respectively).
ACKNOWLEDGMENTS
We thank Yu Zheng (Tianjin University of Science & Technology, China) for generously providing the overexpression vector pUC18K. We also thank Addgene (Cambridge, MA, USA) for providing the suicide plasmid pCVD442.
This work was financially supported through grants from the National Natural Science Foundation of China (no. 31502103 and 31572553) and the National Key Research and Development Program of China (no. 2017YFD0501000).
We have no conflicts of interest to declare.
FOOTNOTES
- Received 27 June 2018.
- Accepted 22 August 2018.
- Accepted manuscript posted online 31 August 2018.
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.01585-18.
REFERENCES
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