Capsule-Targeting Depolymerase, Derived from Klebsiella KP36 Phage, as a Tool for the Development of Anti-Virulent Strategy

Abstract

The rise of antibiotic-resistant Klebsiella pneumoniae, a leading nosocomial pathogen, prompts the need for alternative therapies. We have identified and characterized a novel depolymerase enzyme encoded by Klebsiella phage KP36 (depoKP36), from the Siphoviridae family. To gain insights into the catalytic and structural features of depoKP36, we have recombinantly produced this protein of 93.4 kDa and showed that it is able to hydrolyze a crude exopolysaccharide of a K. pneumoniae host. Using in vitro and in vivo assays, we found that depoKP36 was also effective against a native capsule of clinical K. pneumoniae strains, representing the K63 type, and significantly inhibited Klebsiella-induced mortality of Galleria mellonella larvae in a time-dependent manner. DepoKP36 did not affect the antibiotic susceptibility of Klebsiella strains. The activity of this enzyme was retained in a broad range of pH values (4.0-7.0) and temperatures (up to 45 °C). Consistently, the circular dichroism (CD) spectroscopy revealed a highly stability with melting transition temperature (Tm) = 65 °C. In contrast to other phage tailspike proteins, this enzyme was susceptible to sodium dodecyl sulfate (SDS) denaturation and proteolytic cleavage. The structural studies in solution showed a trimeric arrangement with a high β-sheet content. Our findings identify depoKP36 as a suitable candidate for the development of new treatments for K. pneumoniae infections.

Keywords: Klebsiella sp.; bacteriophage; capsule; polysaccharide depolymerase.

Figure 1

Bioinformatic analysis of Klebsiella phage KP36. (a) BlastP analysis in non-redundant sequence database; (b) HHPred analysis in Protein Data Bank (PDB).

Figure 2

Expression and purification of the depolymerase enzyme encoded by Klebsiella phage KP36 (depoKP36); (a) Ni-NTA His∙Bind® Resins affinity column purification. Protein samples run on a reducing 12% gel. Lane MW: molecular weight (MW) markers; lane 1: lysate of induced E. coli BL21 pEXP-5-CT/TOPO^®-depoKP36 cells; lane 2: column wash with buffer containing 10 mM imidazole; lane 3: eluted proteins; (b) Gel filtration chromatogram; (c) Eluted protein after gel filtration chromatography. Protein samples were separated on 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Lane MW: MW markers; lane 1: aggregate protein; lanes 2, 3, 4, 5: non-aggregate protein fractions. Samples were stained using Coomassie brilliant blue R-250. The arrow indicates protein of interest, depoKP36; SEC: size-exclusion chromatography.

Figure 3

The activity of depoKP36 for its natural substrate. (a) Spot test with serial dilutions of depoKP36 on host strain. Phosphate-buffered saline (PBS) was used as a control; (b) Zymography. Protein samples run on a 12% standard Laemmli SDS-PAGE gel containing crude exopolysaccharide (EPS) as a substrate. Zymograms were stained with methylene blue. Lane MW: MW markers; lane 1: recombinant depoKP36 (2.5 μg/lane); lane 2: phage KP36 (10⁹ PFU/lane).

Figure 4

Inhibition of K. pneumoniae–induced mortality by depoKP36 using G. mellonella model. Larvae (n = 30) were injected with either bacteria (10⁷ CFUs/per larvae), enzyme (final concentration, 280 μg/mL) administered within 5 min after untreated bacteria inoculation, or depoKP36-treated bacteria, and monitored for mortality. The experiments were controlled by observation of uninfected larvae, PBS-injected larvae and larvae receiving the enzyme only. Survival for each control group was 100%, so for simplicity, these groups were not included in the figure. Survival curves were plotted using the Kaplan-Meier method, and differences in survival were calculated by using the log-rank test (GraphPad); * p < 0.003 (considered to be statistically significant); Results are the means of at least three independent experiments.

Figure 5

EPS-degrading activity of depoKP36. (a) Effect of pH on the activity of depoKP36. The optimal pH was determined at 37 °C in 50 mM CH₃COONa-HCl buffer (pH 3.0–5.0), 50 mM Na₂HPO₄ buffer (pH 6.0–7.0), and 50 mM Tris-HCl buffer (pH 8.0–9.0); (b) Influence of various temperatures on the activity of depoKP36 at pH 5.0; (c) Time evolution of depoKP36 activity at 37 °C, 45 °C, 56 °C and 65 °C. Enzyme was pre-incubated in the absence substrate for 10, 20, 30, 40, 50, and 60 min at desired temperature before measuring its activity. Relative enzyme activity was calculated and is expressed as a percent reduction of turbidity compared with control without depoKP36. Each experiment was performed in quadruplicate and repeated at least twice. The data represent means ± standard deviation (SD).

Figure 6

(a) Circular dichroism (CD) spectrum of depoKP36 (0.2 mg/mL) in sodium acetate 20 mM, pH 6.0; (b) Thermal denaturation curve measured at 214 nm. The midpoint of the curve was used to calculate the melting transition temperatures (Tm) for protein.

Figure 7

(a) Analytical SEC, coupled with multi-angle static light scattering (MALS) of depoKP36. The black curve represents the Rayleigh ratio (left scale) against the retention time. Molecular mass (right scale) values correspond to a trimeric state; (b) Native PAGE electrophoresis of depoKP36. Lane MM: molecular weight markers, lane 1: recombinant depoKP36.

Figure 8

Susceptibility of depoKP36 to denaturation in the presence of 1% SDS and proteolysis. Lane MW: molecular weight markers; lanes: (1) depoKP36 boiled, (2) depoKP36 non-boiled, (3) depoKP36 + trypsin, boiled, (4) depoKP36 + trypsin, non-boiled, (5) BSA boiled, (6) BSA + trypsin, boiled, (7) trypsin boiled, (8) BSA non-boiled, (9) BSA + trypsin, non-boiled.