Klebsiella phages representing a novel clade of viruses with an unknown DNA modification and biotechnologically interesting enzymes

Abstract

Lytic bacteriophages and phage-encoded endolysins (peptidoglycan hydrolases) provide a source for the development of novel antimicrobial strategies. In the present study, we focus on the closely related (96 % DNA sequence identity) environmental myoviruses vB_KpnM_KP15 (KP15) and vB_KpnM_KP27 (KP27) infecting multidrug-resistant Klebsiella pneumoniae and Klebsiella oxytoca strains. Their genome organisation and evolutionary relationship are compared to Enterobacter phage phiEap-3 and Klebsiella phages Matisse and Miro. Due to the shared and distinct evolutionary history of these phages, we propose to create a new phage genus “Kp15virus” within the Tevenvirinae subfamily. In silico genome analysis reveals two unique putative homing endonucleases of KP27 phage, probably involved in unrevealed mechanism of DNA modification and resistance to restriction digestion, resulting in a broader host spectrum. Additionally, we identified in KP15 and KP27 a complete set of lysis genes, containing holin, antiholin, spanin and endolysin. By turbidimetric assays on permeabilized Gram-negative strains, we verified the ability of the KP27 endolysin to destroy the bacterial peptidoglycan. We confirmed high stability, absence of toxicity on a human epithelial cell line and the enzymatic specificity of endolysin, which was found to possess endopeptidase activity, cleaving the peptide stem between l-alanine and d-glutamic acid.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7928-3) contains supplementary material, which is available to authorized users.

Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process

“…The cleavage specificity of phages lysins is only rarely experimentally examined and is often based on in silico homology and therefore biased by historic misannotations. However, in those few experimentally validated cases, liquid chromatography–mass spectrometry is the most common method (Caldentey and Bamford 1992; Sudiarta et al 2010; Stockdale et al 2013; Rodríguez-Rubio et al 2016; Maciejewska et al 2017). …”

Gram-Negative Bacterial Lysins

“…Typhimurium (Table 1). Lysin KP27, isolated from environmental phage vB_KpnM_KP27, has bactericidal activity against multidrug-resistant K. pneumonia [26,52]. AP3gp15, a muralytic enzyme originally encoded on the Burkholderia AP3 phage, has broad in vitro antibacterial activity against B. cenocepacia, as well as E. coli, K. pneumoniae, P. aeruginosa, and S. enterica ser.…”

“…AP3gp15, a muralytic enzyme originally encoded on the Burkholderia AP3 phage, has broad in vitro antibacterial activity against B. cenocepacia, as well as E. coli, K. pneumoniae, P. aeruginosa, and S. enterica ser. Typhimurium [26,52].…”

Untitled

“…However, recent research has produced some promising results regarding the use of endolysins against Gram-negative bacteria also. Maciejewska et al (2017) produced a recombinant endolysin from the K. pneumoniae phage KP27 and analysed its peptidoglycan-degrading activity on a range of Gram-negative bacteria, including strains of K. pneumoniae, P. aeruginosa, Salmonella enterica and E. coli, by co-incubation of bacteria and endolysin. The recombinant enzyme successfully lysed all strains of bacteria that were tested.…”

“…However, the outer membrane of bacteria was permeabilised prior to endolysin treatment. This suggests that any potential endolysin-based infection control agents require mixing with outer-membrane-permeabilising agents to be effective against K. pneumoniae (Maciejewska et al, 2017).…”