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A novel depolymerase encoded by phage P5054 specifically degrades the K57-type capsular polysaccharide of Klebsiella pneumoniae

One Health Advances volume 3, Article number: 7 (2025) Cite this article

Abstract

Klebsiella pneumoniae is an important pathogen, especially hypervirulent and multidrug-resistant K. pneumoniae, which are increasingly becoming a serious threat to global public health. Bacteriophages and their depolymerases are promising therapeutic alternatives to antibiotics as they are effective against hypervirulent and multidrug-resistant K. pneumoniae infections. In this study, we identified the novel depolymerase K57-Dpo8 from K. pneumoniae phage P5054. K57-Dpo8 exhibited specific depolymerase activity against K57-type capsular polysaccharide, increasing the susceptibility of K57-type K. pneumoniae strains to serum killing, macrophage phagocytosis, and improving survival rates in a murine infection model. K57-Dpo8 could inhibit biofilm formation and degrade formed biofilms. Our results provide evidence that K57-Dpo8 is not only effective for capsular typing of K57-type K. pneumoniae but also represents a promising alternative therapeutic strategy for treating K57-type K. pneumoniae infections.

Introduction

Klebsiella pneumoniae is a Gram-negative, opportunistic pathogen that accounts for a large proportion of community-acquired infections, as well as up to 10% of nosocomial infections [1]. These vastly vary from urinary tract infections and bacteremia to pneumonia in immunocompromised populations and can include intracranial, epidermal, and surgically-complicated infections as well as colonization [2]. Hypervirulent K. pneumoniae (hvKP) have emerged as a major concern in hospital- and severe community-acquired infections [3]. Several virulence-associated genes, such as peg-344, rmpA, rmpA2, iroB, and iucA, are closely associated with hypervirulence [4]. Recent studies show that some hvKP strains also possess resistance mechanisms against multiple antibiotics, including extended-spectrum β-lactamases and carbapenemases, further complicating treatment strategies. The emergence and dissemination of hvKP strains have become a significant public health concern, particularly in hospitals [5, 6].

Recent studies, including our previous work [7,8,9], have highlighted the critical role of capsular polysaccharide (CPS) in the survival of K. pneumoniae against host immune defenses. The CPS forms a key component of the extracellular matrix, serving as a major structural defense and a pivotal virulence factor [9]. It plays a critical role in shielding strains from phagocytosis by macrophages and neutrophils, as well as in evasion from antimicrobial peptides and complement-mediated killing. Targeting CPS to enhance bacterial vulnerability represents a promising therapeutic strategy. Towards this end, bacteriophage-derived polymerases exhibit high specificity and efficacy against K. pneumoniae infections in vitro and in vivo [10,11,12].

CPS genes in K. pneumoniae strains are clustered in the cps genomic locus. To date, over 130 K. pneumoniae capsular types (K-types) have been described, and the clinical importance of different K-types has been investigated. Six K-types (K1, K2, K5, K20, K54, and K57) are thought to be closely related to hypervirulence and associated with comprehensive infections in animals and humans [13]. K57 K. pneumoniae has also been associated with a significant number of pyogenic liver abscesses, particularly in Asia [14]. Several capsule depolymerases targeting various CPS types have been reported, with some demonstrating antimicrobial activity [15, 16]. Depolymerases Dep_kpv79 and Dep_kpv767, encoded by the Klebsiella phages KpV79 and KpV767, are specific β-galactosidases that cleave K57-type CPS [17]. In this study, we isolated a novel phage targeting K57-type K. pneumoniae strains and identified its capsule-specific depolymerase. Our findings highlight the potential of this depolymerase in advancing capsular typing and as a therapeutic adjunct to combating K57-type strain infection.

Results

Morphology and lytic spectrum of phage P5054

Phage P5054 was obtained from hospital sewage through co-culture with K. pneumoniae strain 8665 isolated from sputum samples of a patient in the intensive care unit [18]. The phage was added to a double-layer agar plate of K. pneumoniae strain 8665, resulting in the formation of clear plaques with faint halos. Over time, the size of these halos gradually increased (Fig. 1A), indicating the existence of a polysaccharide depolymerase. The lytic spectrum of phage P5054 was evaluated using a spot test against 16 K. pneumoniae strains representing seven different K-types. Only K57-type strains could be infected and produce plaques (Table 1). We also constructed a K57 capsular synthesis gene wbap knockout strain (8665Δwbap). Phage P5054 was unable to infect and form plaques in the 8665Δwbap mutant (Fig. 1B), indicating that it has high specificity for the K57-type encapsulated K. pneumoniae strains.

Fig. 1
figure 1

Characterization of Phage P5054. A Phage P5054 forms clear plaques surrounded by translucent halos on the double-layer agar plate, and the halos surrounding the plaques increase in size after 5 days. B Spot tests of Klebsiella pneumoniae strain 8665 and its uncapsulated mutant strain 8665Δwbap by phage P5054 and depolymerase. Phage P5054 and depolymerase K57-Dpo8 (0.75 mg/mL) were spotted on the bacterial lawns. K64-ORF41 (0.75 mg/mL), a depolymerase that targeted K64-type capsular polysaccharide [19], was used as a negative control

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Table 1 Host spectrum of phage P5054 and depolymerase K57-Dpo8
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Genomic analysis of phage P5054

The phage genome was assembled, and the obtained sequence was deposited in the NCBI Genbank database (accession: PQ133610). The phage genome was 39,590 bp with a GC content of 53.04%. According to the BLASTp, the sequence encodes 50 open reading frames (ORFs), including hypothetical proteins, phage-like proteins, tail proteins, and other proteins related to the phage (Fig. 2A). BLASTn analysis revealed the presence of two phages exhibiting high sequence homology to phage P5054: Klebsiella phage phi1_146049 (GenBank accession: PP889498.1, query cover = 97%, identity = 96.06%) and Klebsiella phage L2_1 (GenBank accession: PQ303613.1, query cover = 90%, identity = 94.37%). According to PhageScope [20] and the genome annotation, phage P5054 belongs to the Caudoviricetes.

Fig. 2
figure 2

Bioinformatic analysis of the phage P5054 genome and structural characterization of the putative depolymerase K57-Dpo8. A Genomic map of phage P5054, with open reading frame 8 (ORF8, highlighted in red) encoding the predicted polysaccharide depolymerase. B Bioinformatic analysis of the putative depolymerase K57-Dpo8. The tail fiber protein comprises 570 amino acids and contains a predicated domain, the pectate lyase domain (residues 27–436), as identified by BLASTp and HHpred analysis. C Structural modeling of depolymerase K57-Dpo8 using AlphaFold3. D Predicated active-site residues of K57-Dpo8. The 3D structural model of K57-Dpo8, visualized using PyMOL software, is shown in red, while the 3D model of rhamnogalacturonase A (RGase A) is shown in blue. The active-site residues Ala193 and Pro220 in K57-Dpo8 and Asp156 and Asp180 in RGase A are highlighted

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Phage depolymerases, typically located in the tail spike or tail fibers, degrade capsular polysaccharides into smaller oligosaccharides during infection [21]. Based on this, we hypothesized that ORF8 (named K57-Dpo8), a predicted tail fiber protein, may have polysaccharide depolymerase activity. Structural analysis using HHPred predicted that this protein possesses a β-helix fold, a hallmark of depolymerases [22], and that it exhibited a high degree of similarity to pectate lyase (Fig. 2B). AlphaFold3-bases structural modeling further confirmed that K57-Dpo8 adopts a β-helix conformation (Fig. 2C) [23]. Comparative amino acid sequence analysis revealed that K57-Dpo8 shares 79% coverage and 59.03% identity with Dep_kpv79, and 69% coverage and 71.26% identity with Dep_kpv767, previously characterized K57-type depolymerases [17]. However, no significant result was obtained when comparing nucleotide sequences using BLASTn because of the low sequence similarity.

To further characterize its structural features, a three-dimensional model of K57-Dpo8 was generated using the SWISS-MODEL online tool [24], followed by visualization and comparative analysis with PyMOL. Structural homology analysis identified rhamnogalacturonase A (RGase A, PDB: 1rmg) from Aspergillus aculeatus as the closest structural match, with a sequence identity of 19.92% [25]. Using RGase A as a template, a 3D structural model of K57-Dpo8 was constructed. Based on previous studies, the putative active center of RGase A was located at Asp156 and Asp180. Structural alignment of K57-Dpo8 with RGase A revealed that Ala193 and Pro220 in K57-Dpo8 corresponded to these catalytic residues, suggesting that they may constitute the active site (Fig. 2D). These findings suggested that K57-Dpo8 is a novel phage-derived polysaccharide depolymerase, with a putative catalytic center at Ala193 and Pro220.

K57-Dpo8 exhibits depolymerase activity specific to the K57 capsular polysaccharide of K. pneumoniae

The ORF8 gene from phage P5054 was cloned into an expression plasmid and expressed in Escherichia coli BL21 cells. The recombinant protein was purified using Ni–NTA affinity chromatography, yielding a single distinct band of approximately 63 kDa, as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 3A). To assess depolymerase activity, a spot assay was performed on a lawn of K. pneumoniae strain 8665 using serial dilutions of the recombinant protein (0.75 mg/mL to 0.075 μg/mL). The formation of halo zones, indicative of capsular degradation, was observed, with a minimum effective concentration of 7.5 μg/mL required to produce a detectable effect (Fig. 3B).

Fig. 3
figure 3

Expression and depolymerase activity of recombinant K57-Dpo8. A Purified K57-Dpo8 was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue. B Polysaccharide-degrading activity of K57-Dpo8 was assessed using a modified single-spot assay on a lawn of K. pneumoniae strain 8665, with K57-Dpo8 dilutions ranging from 0.75 mg/mL to 75 ng/mL; K64-ORF41 (0.75 mg/mL) served as a negative control. C Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of CPS samples treated with K57-Dpo8. The red line represents purified, untreated K57 CPS; the green line indicates K57-Dpo8 alone; the blue line shows K57 CPS incubated with 0.75 mg/mL K57-Dpo8 at 37 °C for 30 min; the gray line represents K57 CPS treated with 0.75 mg/mL K64-ORF41 at 37 °C for 30 min

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The depolymerase activity of K57-Dpo8 was further validated using purified K57 CPS. Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of the untreated CPS solution revealed a prominent peak with a retention time of 12–14 min. Following a 30-min incubation with K57-Dpo8 (75 μg/mL) at 37 °C, the CPS peak was no longer detectable within this retention time range, indicating complete degradation of K57 polysaccharide. In contrast, the control protein K64-ORF41 [19], a depolymerase that specifically targets K64-type K. pneumoniae CPS, exhibited no detectable CPS-degrading activity (Fig. 3C).

To further assess the substrate specificity of K57-Dpo8, a spot assay was performed on double-layer lysogeny broth (LB) agar plates containing sixteen different K. pneumoniae strains. Translucent halo formation was observed exclusively in K57-type encapsulated K. pneumoniae strains, while no halo was detected in K57-type nonencapsulated strain 8665Δwbap (Fig. 1B) or strains of other K-types (Table 1). These findings demonstrate that K57-Dpo8 specifically degrades K57 K. pneumoniae CPS.

Serum susceptibility of K. pneumoniae following K57-Dpo8 treatment

CPS is a key virulence factor of K. pneumoniae, conferring resistance to host immune defenses, particularly bactericidal effects of serum. To assess the capacity of K57-Dpo8 to enhance serum susceptibility, a serum killing assay was performed on K. pneumoniae strain 8665. Bacterial cells were preincubated with K57-Dpo8 or control depolymerase K64-ORF41, followed by exposure to 75% baby rabbit serum. The result showed a significant reduction in the viable bacterial count following treatment with depolymerase K57-Dpo8 compared with K64-ORF41 treated controls. No significant difference in bacterial viability was observed when K. pneumoniae strain 8665 was treated with heat-inactivated rabbit serum (Fig. 4A). These findings indicate that K57-Dpo8 effectively enhances serum bactericidal activity against K57-type encapsulated K. pneumoniae strains.

Fig. 4
figure 4

K57-Dpo8 enhances serum susceptibility, promotes phagocytosis, and confers therapeutic protection of K. pneumoniae infection. A Serum-killing assay of K. pneumoniae strain 8665 following K57-Dpo8 treatment. Bacteria were preincubated with K57-Dpo8 or K64-ORF41 at 37 °C for 1 h, followed by exposure to 75% rabbit serum or heat-inactivated rabbit serum for an additional 3 h at 37 °C. Surviving bacteria were counted by a dilution plating method. B Phagocytosis assay evaluating macrophage-mediated uptake of K. pneumoniae. Strain 8665 was pretreated with K57-Dpo8, K64-ORF41, or 0.01 M phosphate-buffered saline (pH 7.4) at 37 °C for 1 h before co-incubation with RAW264.7 macrophages for 2 h. Data are presented as the mean ± SEM from three independent experiments. Statistical significance was determined using a two-tailed unpaired Student’s t-test. C Therapeutic efficacy of K57-Dpo8 in a murine infection model. C57BL/6 J mice were intraperitoneally infected with 2 × 108 colony-forming units (CFU) of K. pneumoniae strain 8665. After 30 min, mice were administered either PBS or 50 μg K57-Dpo8 intraperitoneally. Each group (n = 5) was monitored over 8 days, and survival rates were analyzed using the Kaplan–Meier method with a log-rank test. * p < 0.05; **** p < 0.0001

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Phagocytosis assay of K. pneumoniae following K57-Dpo8 treatment

To assess the effect of K56-Dpo8 on macrophage-mediated phagocytosis. K. pneumoniae strain 8665 was pretreated with K57-Dpo8, K64-ORF41 or phosphate-buffered saline (PBS), followed by incubation with RAW264.7 macrophage for 2 h at a multiplicity of infection of 20. Phagocytosis efficiency was significantly (p < 0.0001) enhanced in K57-Dpo8-treated bacteria, with an approximately two-fold increase compared with K64-ORF41- or PBS-treated controls (Fig. 4B). These findings indicate that K57-Dpo8 facilitates the phagocytosis of K57 K. pneumoniae by macrophages.

K57-Dpo8 exhibits therapeutic effects in a murine infection model

The therapeutic potential of K57-Dpo8 was assessed in a murine infection model. Mice were infected intraperitoneally with K. pneumoniae strain 8665 at a dose of 2 × 108 colony-forming units (CFU). After 30 min, mice in the control group received an intraperitoneal injection of 0.01 M PBS (pH 7.4) while those in the treatment group were administered 50 μg K57-Dpo8 intraperitoneally. All PBS-treated mice succumbed to infection within 48 h. In contrast, K57-Dpo8 treatment significantly improved survival, with 60% of mice remaining alive at the end of the observation period (Fig. 4C). These findings indicate that K57-Dpo8 exerts a protective therapeutic effect against K57 K. pneumoniae infection in vivo.

Antibiofilm activity of K57-Dpo8

The inhibitory effect impact of K57-Dpo8 on biofilm formation by K. pneumoniae strain 8665 was evaluated using a 96-well plates assay. K. pneumoniae strain 8665 was incubated with K57-Dpo8 or control depolymerase K64-ORF41 at 10 μg/mL for 48 h. Quantification of biofilm biomass, determined by absorbance measurements, revealed a significant reduction in the K57-Dpo8 treated group compared with the K64-ORF41 treated group (Fig. 5A). These results demonstrate that K57-Dpo8 effectively inhibits biofilm formation by K57 K. pneumoniae strains.

Fig. 5
figure 5

Antibiofilm activity of K57-Dpo8 against K. pneumoniae strain 8665. A Inhibition of biofilm formation by K57-Dpo8. K. pneumoniae strain 8665 was incubated with K57-Dpo8 or control depolymerase K64-ORF41 at 10 μg/mL in 96-well plates for 48 h. Residual biofilm biomass was assessed by crystal violet staining, with absorbance measured at 595 nm. B Disruption of mature biofilm by K57-Dpo8. Biofilms formed by K. pneumoniae strain 8665 in 96-well plates over 48 h were treated with K57-Dpo8 or control depolymerase K64-ORF41 at 10 μg/mL for an additional 3 h. Residual biofilm biomass was evaluated by crystal violet staining with absorbance measured at 595 nm. Data are presented as the mean ± SEM and represent three separate experiments. Two-tailed unpaired Student’s t-test was used for statistical analysis. * p < 0.05; ** p < 0.01

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We also evaluated the ability of K57-Dpo8 to degrade mature biofilms. The enzyme was applied at 10 μg/mL to 48-h-old K. pneumoniae strain 8665 biofilms. Post-treatment quantification of residual biofilm biomass revealed a significant reduction in the K57-Dpo8-treated group compared with the K64-ORF41-treated control group (Fig. 5B). These findings indicate that K57-Dpo8 both inhibits biofilm formation and effectively degrades pre-existing biofilms.

Discussion

K. pneumoniae is a clinically significant pathogen that poses a substantial threat to public health. The K57 K-type is associated with hypervirulence and increased antibiotic resistance, leading to severe infections that are often challenging to treat [26, 27]. The emergence of carbapenem-resistant K57 strains has further exacerbated this issue, complicating treatment and increasing public health risks [28, 29]. There is an urgent need to develop novel and effective therapeutic strategies to combat K57-type K. pneumoniae infections.

Depolymerase-mediated treatments have shown significant therapeutic potential in addressing K. pneumoniae infections [30, 31]. These enzymes degrade the CPS, enhancing susceptibility to host immune responses such as complement-mediated killing. Depolymerase treatment can successfully rescue K. pneumoniae-infected animal models, including both mice and Galleria mellonella larvae, highlighting its potential as a novel intervention strategy [32,33,34]. To date, two K57-type capsular depolymerases have been identified that have demonstrated significant therapeutic efficacy in murine models of K. pneumoniae K57-type infection [17].

In this study, we identified a novel K57-type depolymerase, K57-Dpo8, which demonstrated significant therapeutic efficacy against K57-type K. pneumoniae. K57-Dpo8 effectively compromised bacterial defenses, enhancing both the phagocytic ability of immune cells and serum-mediated bactericidal effects. These findings are consistent with previous research highlighting the therapeutic potential of depolymerases in bacterial infections [1, 12, 15]. In vivo K57-Dpo8 significantly improved survival outcomes in animal models, further supporting its potential as a novel therapeutic strategy. Our in vitro biofilm assays also demonstrated that K57-Dpo8 effectively inhibits biofilm formation and disrupts established biofilms. These findings underscore the potential of K57-Dpo8 as a promising therapeutic strategy for biofilm-associated K. pneumoniae infections, which are typically resistant to conventional antibiotic therapies.

Capsular depolymerases have been shown to provide a more accurate method of capsule typing by directly detecting polysaccharide composition and structure, surpassing the accuracy of wzi genotyping [12, 35]. Unlike wzi gene-based typing, which may fail to identify disruptions in the capsular synthesis region, the K57-Dpo8 capsule typing approach directly targets the intact capsule, providing a more reliable tool for specifically identifying the K57 K-type. Our results indicate that K57-Dpo8 is a valuable tool for capsule typing, particularly in the clinical and epidemiological surveillance of capsule-deficient mutants of K57-type K. pneumoniae.

Conclusion

In this study, we isolated the novel phage P5054, from which we identified and characterized K57-Dpo8 depolymerase, which exhibited high degradation activity against K57-type K. pneumoniae CPS. Our findings demonstrate that K57-Dpo8 enhances serum susceptibility and phagocytosis of K57-type K. pneumoniae, increases survival rates in K57 K. pneumoniae-infected mice, and degrades biofilms formed by these strains. Additionally, K57-Dpo8 offers a more accurate and efficient method for capsular typing of K57 K. pneumoniae. These results suggest that K57-Dpo8 holds significant potential for the identification and control of K57 K. pneumoniae infections.

Materials and methods

Bacterial strain, phage isolation, and purification

The K. pneumoniae strains used in this study are listed in Table 1. All bacterial strains were stored at − 80 °C in 50% (v/v) glycerol and routinely cultured in LB at 37 °C. The control depolymerase protein, K64-ORF41, was previously purified and stored in our laboratory [19].

Phage P5054 was isolated from sewage samples collected at Ruijin Hospital, affiliated with Shanghai Jiaotong University (Shanghai, China). To isolate the phage, K. pneumoniae strain 8665 was co-cultured with filtered hospital sewage in LB medium at 37 °C overnight. The culture was then centrifuged at 6000 × g for 10 min, after which the supernatant containing phages was filtered and plated onto a double-layer LB agar overlaid with K. pneumoniae strain 8665. Following Pires’s method [36], a single phage plaque was selected and re-inoculated several times until a uniform plaque morphology was observed. This purification process was repeated three times. The purified phages were then stored at − 80 °C in sodium magnesium buffer (8 mM MgCl2, 100 mM NaCl, 50 mM Tris–HCl, pH 7.5) containing 50% (v/v) glycerol.

Capsule knockout strain and complementary plasmid construction

The mutant K. pneumoniae strain 8665Δwbap strain was constructed using the λ-red-dependent recombination and FLP/FRT systems, according to established methods [37]. Briefly, the thermosensitive plasmid pKOBEG, encoding apramycin resistance and the λ-red phage operon, was introduced into strain 8665 through electroporation (ECM 630 electroporator, BTX, MA, USA). Donor DNA fragments were constructed using Gibson assembly with 500 bp flanking regions of wbap, the hygromycin B phosphotransferase gene, and pSUMO. Mutant strains were selected on LB agar supplemented with apramycin and hygromycin and confirmed by PCR and Sanger sequencing. The pKOBEG plasmid was removed by incubating the culture at 42 °C with shaking. Primers used for the construction of the deletion clone and complementation of the engineered mutants are listed in Table 2.

Table 2 List of primers
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Phage host spectrum analysis

The host range of phage P5054 was assessed using a spot test with the strains listed in Table 1. Briefly, 400 µL of log-phase bacterial culture was mixed with 4 mL of 0.5% agar LB and spread onto a 1.5% agar LB plate. When the overlay agar had solidified, 5 µL of purified phage was spotted onto the plate and incubated overnight at 37 °C for 12 h.

Genomic DNA sequencing and annotation

Extraction of genomic DNA from phages followed a previously established protocol [38]. Briefly, the purified phage solution was initially incubated with 1 μg/mL DNase I and RNase A at 37 °C for 1 h, after which the enzyme activity was halted by heating at 80 °C for 15 min. Proteinase K was then added at 50 μg/mL, and the solution was incubated at 56 °C for 1 h. DNA was isolated from the lysate using phenol–chloroform extraction and was precipitated by adding an equal volume of isopropanol, followed by incubation at − 20 °C for at least 2 h. The DNA was then pelleted by centrifugation at 12,000 × g for 10 min at 4 °C, rinsed twice with 75% ethanol, and dissolved in nuclease-free water for storage at − 20 °C. Phage genome sequencing was performed using Illumina HiSeq 3000 (Illumina, San Diego, CA, USA). The resulting raw sequence reads were trimmed and quality filtered using FastQC v0.12.1 [39], assembled using SPAdes v3.14 [39], and genome circularity was verified using Bandage (https://rrwick.github.io/Bandage/) [40]. The PHASTER platform (http://phaster.ca) was used to annotate ORFs in the assembled genome [41].

Cloning, expression, and purification of depolymerase

The gene encoding the predicted depolymerase K57-Dpo8 was amplified from phage DNA by PCR using the primers listed in Table 2. The PCR product was then inserted into an N-terminal 6 × His-tagged pET24a expression vector using NdeI and XhoI restriction sites. After verification by DNA sequencing, the recombinant plasmid was transformed into E. coli BL21 (DE3) for protein expression. Transformed cells were cultured in 1 L of LB medium containing 50 μg/mL kanamycin at 37 °C until the optical density at 600 nm (OD600) reached 0.6. Induction was performed by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside and cultures were incubated at 16 °C for 12 h. Cells were harvested and lysed using a high-pressure homogenizer (JNBIO, Guangzhou, China), and cell debris was removed by centrifugation. The depolymerase was purified using a Ni–NTA column, and purity was confirmed by SDS-PAGE.

Purification of CPS

CPS was purified following a previously described protocol [42]. Briefly, 10 mL of an overnight K. pneumoniae strain 8665 culture grown at 37 °C was treated with 2% formaldehyde and incubated at 100 rpm for 8 h. The cell suspensions were then collected by centrifugation at 16,800 × g for 1 h at 4 °C, and the supernatant was filtered through a 0.45 μm filter. The resulting supernatant was dialyzed overnight against a buffer (25 mM NaH2PO4, pH 7.5) using a 100 KD membrane. To precipitate the polysaccharide, 0.5% (w/v) cetyltrimethylammonium bromide was added, followed by dissociation with graded concentrations of CaCl2 and centrifugation of the supernatant. Ethanol was then used for selective precipitation, and the precipitate was washed with NaCl solution. After ultrafiltration, the sample was subjected to Capto adhesive chromatography. The dialyzed CPS was filtered, dried, and weighed.

Assessment of depolymerase activity

K57-Dpo8 activity was evaluated on K. pneumoniae strain 8665 using a spot assay with enzyme concentrations ranging from 0.75 mg/mL to 0.075 μg/mL, while K64-ORF41 at 0.75 mg/mL served as a negative control. The presence of translucent halos on the agar plate indicated depolymerase activity.

To further confirm the depolymerase activity against the CPS, SEC-HPLC was performed. Purified CPS was dissolved in 200 μL of 50 mM Na2HPO4 buffer to a final concentration of 0.5 mg/mL and incubated with either 10 μL of capsular-specific depolymerase (0.75 mg/mL), 10 μL of deionized distilled water (ddH2O), or 10 μL of K64-ORF41 (0.75 mg/mL) at 37 °C for 30 min [41]. The reaction mixtures were inactivated by heating to 100 °C for 10 min, followed by analysis on Waters HPLC equipment (Waters, Milford, MA, USA).

Serum resistance assay

The serum resistance assay was performed as previously described with minor modifications [30]. K. pneumoniae strain 8665 (approximately 107 CFU) was incubated with K57-Dpo8 (final concentration 7.5 μg/mL) or K64-ORF41 (final concentration 7.5 μg/mL) for 1 h at 37 °C. Active or inactivated (heated at 56 °C for 30 min) 75% baby rabbit serum (Cat# RCM-20200320; Shanghai Reinovax Biologics Co., Ltd., Shanghai, China) was added to depolymerase-pretreated bacteria at 37 °C for 3 h. The mixture was serially diluted and plated to quantify the number of bacteria. This experiment was independently repeated three times.

Phagocytosis assay

The murine macrophage cell line RAW264.7 was used to evaluate the effect of K57-Dpo8 on the phagocytic activity of immune cells against K. pneumoniae strain 8665. Briefly, K. pneumoniae strain 8665 (3 × 108 CFU/mL) was resuspended in dulbecco's modified eagle medium (DMEM) following treatment with PBS, K57-Dpo8 (7.5 μg/mL) or K64-ORF41 (7.5 μg/mL) at 37 °C for 1 h. RAW264.7 cells (3 × 105) were cultured in DMEM to form a monolayer in a 24-well plate. After washing the cells three times with PBS, 300 μL of PBS, K57-Dpo8 or K64-ORF41 treated K. pneumoniae strain 8665 was added at a multiplicity of infection of 20:1 and incubated at 37 °C for 2 h. The cells were then washed and incubated in a DMEM medium containing 100 μg/mL gentamicin at 37 °C for 2 h. After a final wash, the cells were lysed by resuspension in 1 mL of 1% Triton X-100, with repeated pipetting to ensure complete lysis. The bacterial count was determined to assess anti-phagocytic activity.

Mouse infection model

To evaluate the therapeutic efficacy of K57-Dpo8 in a murine model of bacteremia, we adapted a previously described protocol with modifications [34]. Two groups of female C57BL/6 J mice (6–8 weeks old; n = 5 per group) were intraperitoneally inoculated with K. pneumoniae strain 8665 at 2 × 108 CFU. After 30 min, mice received a single intraperitoneal injection of either K57-Dpo8 (50 μg) or PBS as a control. Mortality was monitored for 8 days, and survival rates were analyzed using the Kaplan–Meier method with a log-rank test.

Antibiofilm activity of depolymerase

Biofilm formation was performed in a 96-well plate as described previously with some modifications [43]. In brief, K. pneumoniae strain 8665 was grown to OD600 = 0.6 and then diluted with fresh TSB medium to OD600 = 0.1. Samples of 100 μL were transferred to each well of a 96-well plate. K57-Dpo8 or K64-ORF41 (7.5 μg/mL) were added to different wells and incubated at 37 °C for 48 h. Residual biofilm was quantified using crystal violet staining with absorbance measured at 595 nm.

To investigate the biofilm-disrupting capability of K57-Dpo8 on K. pneumoniae strain 8665, bacteria were cultured in a 96-well plate for 48 h. After removal of the supernatant, the adherent bacteria were treated with K57-Dpo8 or K64-ORF41 (7.5 μg/mL) for 3 h. Biofilm degradation was assessed by crystal violet staining with absorbance and measured at 595 nm.

Statistical analyses

Experimental data are presented as means ± SEM and statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). A two-tailed, unpaired Student’s t-test was used for statistical analysis in the serum resistance, phagocytosis, and biofilm formation assays. Survival rates were analyzed using the Kaplan–Meier method with a log-rank test. p-value < 0.05 was considered statistically significant.

Data availability

The complete genome sequence of bacteriophage P5054 has been deposited in NCBI database (GenBank accession: PQ133610). Data are available from the corresponding author on request.

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Acknowledgements

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Funding

The study was funded by National Natural Science Foundation of China (81971896).

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Author notes

  1. Heyuan Lun and Juanjuan Wang contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology and Microbiology, School of Medicine, Shanghai Jiao Tong University, Huangpu District, Shanghai, 200025, China

    Heyuan Lun, Ruijing Ma, Tingting Qu, Aixi Wang, Kai He, Jingran Yu & Ping He

  2. Shanghai Reinovax Biologics Co., Ltd, Pudong New District, Shanghai, 201210, China

    Juanjuan Wang, Huagen Chen & Ruijing Ma

  3. Key Laboratory of Alkene-Carbon Fibers-Based Technology & Application for Detection of Major Infectious Diseases, MOE Key Laboratory of Geriatric Diseases and Immunology, Pasteurien College, Suzhou Medical College, Soochow University, Gusu District, Suzhou, 215123, China

    Heng Li

  4. Zhejiang Key Laboratory of Integrative Chinese and Western Medicine for Diagnosis and Treatment of Circulatory Diseases, Zhejiang Hospital, Zhejiang University, West Lake District, Hangzhou, 310007, China

    Yuqing Pan

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Contributions

P.H. conceived and supervised the study; P.H. and H.L. designed the experiments and wrote the manuscript; H.L. and J.W. performed the experiments and analyzed the data; R.M., Y.P. and T.Q. helped with experiments; A.W. and K.H. helped with data analysis; J.Y., H.C., and H.L. took part in the editing of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Ping He.

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Lun, H., Wang, J., Chen, H. et al. A novel depolymerase encoded by phage P5054 specifically degrades the K57-type capsular polysaccharide of Klebsiella pneumoniae. One Health Adv. 3, 7 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s44280-025-00073-3

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One Health Advances

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