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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.
Highlights
Klebsiella pneumoniae ranks among the eight most common etiological factors of nosocomial infections, including pneumonia and urinary tract infections, accounting for up to 8% of all cases [1].it is the second cause of both hospital- and community-acquired gram-negative bacteremia [1]
We proved that the K. pneumoniae phage KP36 expresses an enzyme able to degrade the bacterial capsule and that this activity is conditioned by a tailspike protein with depolymerase activity
In a single dose of enzyme enzyme given within 5 min after bacteria injection as well as inoculation of bacteria pretreated with depoKP36 for 2 h, significantly inhibited K. pneumoniae-induced death in a time-dependent manner (p < 0.003)
Summary
Klebsiella pneumoniae ranks among the eight most common etiological factors of nosocomial infections, including pneumonia and urinary tract infections, accounting for up to 8% of all cases [1]. Multiple applications of depolymerases have been proposed, including determination of Klebsiella capsular types for clinical strains [8,17], production of oligosaccharides from polysaccharides [18,19], or as alternative therapeutic agents to treat Klebsiella infections [17,20]. The latter application is of particular importance, as this opportunistic pathogen is a growing concern for public health. We recombinantly produced this protein and characterized its biochemical properties, structural features in solution, as well as its activity in vitro and in vivo on Galleria mellonella model
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