Production and characterization of a phage-encoded capsular polysaccharide endoglycosidase targeting Sequence Type 307 Klebsiella pneumoniae strains

Bacteriophages are viruses that infect and lyse bacterial cells by using several types of specialized proteins. Among these, endoglycosidases (EGs) target and degrade capsular polysaccharides (CPS), key virulence factors protecting bacterial cells. Here we characterized a phage-derived endoglycosidase (EG-GP1) able to specifically target and degrade the CPS expressed by an emerging high-risk clone of multidrug-resistant (MDR) Klebsiella pneumoniae. Bioinformatics analysis of the genome of the vB_KpS_GP-1 phage, able to lyse Sequence Type (ST) 307 K. pneumoniae strains, revealed the presence of two putative endoglycosidase-encoding genes (orf57 and orf58). The latter, encoding a protein showing 77.4% identity with a previously characterized endoglycosidase, was selected for heterologous expression in Escherichia coli by using a N-terminal His-Tag cloning strategy. The EG-GP1 recombinant protein was produced in soluble form and its activity was proved by spot-test on the vB_KpS_GP-1 indicator strain. SDS-PAGE and western-blot revealed a band of ≈135 kDa, in line with theoretical calculations. EG-GP1 tested positive by spot-test for 11/14 (79 %) ST307 strains, showing results in agreement with vB_KpS_GP-1 host-range, i.e. being inactive on capsule-deficient ST307 strains. In addition, EG-GP1 tested negative also on strains belonging to other STs (n=7). Negative-stain microscopy showed a statistically significant reduction of extracellular area of EG-GP1–treated cells of the indicator strain when compared to untreated, while no significant changes were observed by using K. pneumoniae ATCC 13883. To summarize, EG-GP1 may represent a possible additional therapeutic tool to be combined with standard or novel antibiotics against infections by MDR ST307 K. pneumoniae strains.