Capsule-Targeting Depolymerase, Derived from Klebsiella KP36 Phage, as a Tool for the Development of Anti-Virulent Strategy

Abstract

The rise of antibiotic-resistant <i>Klebsiella pneumoniae</i>, a leading nosocomial pathogen, prompts the need for alternative therapies. We have identified and characterized a novel depolymerase enzyme encoded by <i>Klebsiella</i> phage KP36 (depoKP36), from the <i>Siphoviridae</i> 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 <i>K. pneumoniae</i> host. Using in vitro and in vivo assays, we found that depoKP36 was also effective against a native capsule of clinical <i>K. pneumoniae</i> strains, representing the K63 type, and significantly inhibited <i>Klebsiella</i>-induced mortality of <i>Galleria mellonella</i> larvae in a time-dependent manner. DepoKP36 did not affect the antibiotic susceptibility of <i>Klebsiella</i> 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 (T<i>m</i>) = 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 <i>K. pneumoniae</i> infections.