[Expression, purification and functional validation of phage depolymerase from hypervirulent Klebsiella pneumoniae serotype K1].

Abstract

Objective: To express and purify the phage depolymerase from hypervirulent Klebsiella pneumoniae (hvKp) serotype K1 and validate its function. Methods: Phage that infected serotype K1-type hvKp was isolated from hospital sewage. The biology and morphology of the phage were determined by plaque assay and transmission electron microscopy. The whole genome of the phage was sequenced by the Illumina HiSeq 2500 platform. The presence of depolymerase was determined by observing the plaque halo. Bioinformatic analysis and prokaryotic protein expression system were further used to predict and identify phage depolymerase. The depolymerase gene fragment was obtained by PCR and cloned into the pET28a expression vector, and the expression and purification of the depolymerase were completed in strain BL21. The depolymerase activities on the capsular polysaccharide of serotype K1-type hvKp clinical isolates were detected by plaque assay and low-speed centrifugation assay. Results: A lytic phage (phiA2) that infected serotype K1-type hvKp clinical isolate was isolated from hospital sewage. It was typical of the Caudovirales order and Autographiviridae family, and its whole genome was 43 526 bp in length and contained 51 coding domain sequences. The phage phiA2-derived depolymerase phiA2-dep was predicted, expressed and purified. The plaque assay and low-speed centrifugation assay indicated that the depolymerase phiA2-dep had good lytic activity on the capsular polysaccharide of serotype K1-type hvKp clinical isolates. Conclusion: Depolymerase phiA2-dep can specifically degrade the capsular polysaccharide of serotype K1-type hvKp, which has potential application value in treating bacterial infection.