Two T7-like Bacteriophages, K5-2 and K5-4, Each Encodes Two Capsule Depolymerases: Isolation and Functional Characterization

Abstract

Two Klebsiella bacteriophages K5-2 and K5-4, which are able to infect and grow on either capsular types K30/K69 and K5 or K8 and K5 of Klebsiella strains, were isolated and characterized. Each phage contained two open reading frames (ORFs), which encoded two putative capsule depolymerases, respectively. The first ORF encoded tail fiber proteins, which have K30/K69 depolymerase and K8 depolymerase activities. The second ORF encoded hypothetical proteins, which are almost identical in amino acid sequences, and have K5 depolymerase activity. Alcian blue staining of enzyme-treated capsular polysaccharides (CPS) showed that purified depolymerases can cleave purified Klebsiella CPS in vitro and liberate monosaccharaides. Capsule K5 deletion mutants were not lysed by either phage, suggesting that the capsule was essential for phage infection. Bacterial killing was observed when incubated Klebsiella strains with phages but not with purified depolymerases. Treatment with the K5-4 phage significantly increased the survival of mice infected with a K. pneumoniae K5 strain. In conclusion, two dual host-specific Klebsiella phages and their tailspikes exhibit capsule depolymerase activity were characterized. Each phage and phage-encoded depolymerase has specificity for capsular type K30/K69, K8 or K5, and could be used for the typing and treatment of K. pneumoniae infection.

Identification of three podoviruses infecting Klebsiella encoding capsule depolymerases that digest specific capsular types

“…), with 53% G+C content and 67 probable protein‐coding genes (> 300 bp in length). Nucleotide BLAST analysis revealed that the three phages exhibited high DNA similarity with Klebsiella phage K5‐2 (Hsieh et al ., ) (Accession No. , 83–85% query coverage and 92–95% identity).…”

“…The genes located in this region have capsule depolymerase activities. These results were consistent with a previous study, which showed that this analogous variable region (between the genes encoding internal virion protein D and gp17.5) harbours genes encoding capsule depolymerases in different T7‐like phages (Hsieh et al ., ). Thus, PCR amplification of this region from other phages belonging to this group will be useful for the identification of capsule depolymerases (Hsieh et al ., ).…”

“…To our knowledge, 19 capsule depolymerases have been identified (capsule‐degrading activity has been demonstrated and the sequence is available) for 17 Klebsiella capsular types (Table S4), including three K1 enzymes (Lin et al ., ; Pan et al ., ; Solovieva et al ., ), one K2/K13 enzyme (Solovieva et al ., ), one K3 enzyme (Majkowska‐Skrobek et al ., ), one K5 enzyme (Hsieh et al ., ), one K8 enzyme (Hsieh et al ., ), two K30/K69 enzymes (Hsieh et al ., ; Pan et al ., ), one K11 enzyme (Pan et al ., ), two K21 enzymes (Pan et al ., ) (Majkowska‐Skrobek et al ., ), one K25 enzyme (Pan et al ., ), one K35 enzyme (Pan et al ., ), one K63 enzyme (Majkowska‐Skrobek et al ., ), one K64 enzyme (Pan et al ., ), one KN2 enzyme (Hsu et al ., ), one KN4 enzyme (Pan et al ., ) and one KN5 enzyme (Pan et al ., ). We analysed the sequence similarity of enzymes that depolymerize the same capsular type.…”

“…Therefore, the use of phages to determine the K types of Klebsiella has been described (Rakieten et al, 1940;Adams and Park, 1956;Thurow et al, 1974;Rieger-Hug and Stirm, 1981;Gaston et al, 1987;Pieroni et al, 1994). Moreover, several phages have been reported to carry capsule depolymerases that contribute to the recognition and digestion of capsule; some capsule depolymerases were demonstrated to be critical for phage infection Lin et al, 2014;Pan et al, 2015bPan et al, , 2017Hsieh et al, 2017). The proteins displaying capsule depolymerase activities are often tail fibres or tail spikes, components of the viral surface that interact with bacterial cells.…”

“…A K64 depolymerase was demonstrated to enhance serum and neutrophil killing in vitro and increase survival rates for K64 K. pneumoniae-inoculated mice (Pan et al, 2015b). Treatment with a K5 phage increased the survival of mice infected with a K. pneumoniae K5 strain (Hsieh et al, 2017). A K63 depolymerase can inhibit Klebsiella-induced mortality of Galleria mellonella larvae in a time-dependent manner (Majkowska-Skrobek et al, 2016).…”