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

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.

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

“…Pectate/pectin lyases are characterized by cleavage of the α-1,4 bonds of polygalacturonic acid. Enzymes possessing such domain have been described for Pseudomonas phages Φ15 and AF (Cornelissen et al 2011, 2012), Klebsiella phages (NTUH-K2044-K1–1, KP36) (Lin et al 2014; Majkowska-Skrobek et al 2016), Vibrio phage JA1 (Linnerborg et al 2001), and Staphylococcus phage vB_SepiS-ΦIPLA7 (Gutierrez et al 2015) (Table 1). Alginate lyases (mannuronate or guluronate lyases) characteristic for Pseudomonas and Azobacter phages (PT6) are able to degrade the α-1,4 bond of alginate, a linear polysaccharide of β-D-mannuronate, and its C5 epimer α-L-guluronate common for mucoid strains infecting cystic fibrosis patients (Davidson et al 1977; Wong et al 2000; Glonti et al 2010) (Table 1).…”

“…The complex structure of highly interwoven β-sheets also determines the high stability of these proteins, since they were found to be resistant to high temperature (possessing relatively high melting point temperature), proteases, and detergents at room temperature. This high stability corresponds to the harsh external conditions these proteins have to withstand in different environments such as the presence of proteases and denaturing conditions (Yan et al 2014; Majkowska-Skrobek et al 2016).

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“…Spectrometric measurement of reducing sugars allows to determine the EPS/CPS digestion (Yurewicz et al 1971; Rieger et al 1975; Hsu et al 2013). Other methods include examination of periodate oxidation of saccharides by determining the consumption of sodium periodate and the amount of formic acid formed (Kwiatkowski et al 1983), evaluation of decrease of turbidity of insoluble EPS precipitated by cetylpyridinium chloride (CPC complexes) (Born et al 2014; Majkowska-Skrobek et al 2016), or uronic acid release (Glonti et al 2010). Also, the crude separation of released sugars from polymer was analyzed by TLC chromatography (Glonti et al 2010).…”

“…A couple of electrophoretic methods were also implemented to analyze depolymerase activity. The non-digested and digested polysaccharides can be compared on polyacrylamide gel electrophoresis (PAGE/SDS PAGE) stained by alcian blue/silver/methylene blue (Clarke et al 2000; Barbirz et al 2008; Majkowska-Skrobek et al 2016). Fluorophore-assisted carbohydrate electrophoresis (FACE) (Chua et al 1999) and capillary electrophoresis (Baker et al 2002) are other electrophoretic methods that have been used.…”

“…Although most of these proteins do not seem to possess bactericidal activity as described for VALs and endolysins, they should be considered in therapy for their antivirulent potential (Majkowska-Skrobek et al 2016). The loss or modification of bacterial surface structures that are used by many pathogens to promote virulence, host colonization, and biofilm formation makes bacteria less pathogenic and/or sensitizes them to either some antimicrobials or host defenses such as phagocytosis by macrophages and the bactericidal action of serum (Mushtaq et al 2004, 2005; Zelmer et al 2010; Bansal et al 2014; Pan et al 2015).…”