Identification of a Depolymerase Specific for K64-Serotype Klebsiella pneumoniae: Potential Applications in Capsular Typing and Treatment

Abstract

Carbapenem-resistant Klebsiella pneumoniae (CRKP), one of the major nosocomial pathogens, is increasingly becoming a serious threat to global public health. There is an urgent need to develop effective therapeutic and preventive approaches to combat the pathogen. Here, we identified and characterized a novel capsule depolymerase (K64-ORF41) derived from Klebsiella phage SH-KP152410, which showed specific activities for K. pneumoniae K64-serotype. We showed that this depolymerase could be used in the identification of K64 serotypes based on the capsular typing, and the results agreed well with those from the conventional serotyping method using antisera. From this study, we also identified K64 mutant strains, which showed typing discrepancy between wzi-sequencing based genotyping and depolymerase-based or antiserum-based typing methods. Further investigation indicated that the mutant strain has an insertion sequence (IS) in wcaJ, which led to the alteration of the capsular serotype structure. We further demonstrated that K64-ORF41 depolymerase could sensitize the bacteria to serum or neutrophil killing by degrading the capsular polysaccharide. In summary, the identified K64 depolymerase proves to be an accurate and reliable tool for capsular typing, which will facilitate the preventive intervention such as vaccine development. In addition, the polymerase may represent a potential and promising therapeutic biologics against CRKP-K64 infections.

Characterization and Functional Studies of a Novel Depolymerase Against K19-Type Klebsiella pneumoniae

“…The phage was spread over K. pneumoniae strain 6570 to form a double-layer agar plate and cleared plaques formations and faint halos were observed. The size of the faint halos increased over time (Figure 1A), which we suspected indicated the presence of a phage-derived depolymerase based on our previous study (Li J. et al, 2021). To understand the relationship between SH-KP156570 and capsular types, we performed a host spectrum analysis of SH-KP156570 on all 29 strains using spot tests.…”

“…Klebsiella pneumoniae strain 6570 was cultured in fresh TSB medium at 37 • C for 5 days. The purification of CPS was performed according to the method from previous studies with some modifications (Gorodnichev et al, 2021;Li J. et al, 2021). Firstly, 60 µL of formaldehyde solution (36.5%) was added to 10 mL of bacterial culture and incubated 100 rpm at room temperature for 1 h. Subsequently, 1M NaOH was added to the system, with agitation at room temperature for 3 h. Cell suspensions were centrifuged at 16,800 g for 1 h at 4 The activity of K19-Dpo41 was assessed by spot test (Khan Mirzaei and Nilsson, 2015).…”

“…It allows the bacterium to survive inside the host by overcoming the protective mechanisms of the immune system. The capsule hinders the bactericidal action of antimicrobial peptides and blocks complement components, preventing complement-mediated killing ( Paczosa and Mecsas, 2016 ; Li J. et al, 2021 ). It further impairs phagocytosis and opsonophagocytosis of K. pneumoniae by immune cells ( Opoku-Temeng et al, 2019 ; Assoni et al, 2021 ).…”

Characterization of phage resistance and phages capable of intestinal decolonization of carbapenem-resistant Klebsiella pneumoniae in mice

“…P24 encodes a tail fiber protein (Supplementary Table 3 ), which had 97.54% identity and 100% coverage with the depolymerase (a tail fiber protein, called ORF42, accession no. YP_009797016.1) of Klebsiella phage SH-KP152410 11 . The tail fiber protein of P39 (Supplementary Table 6 ) had 83.61% identity and 80% coverage with the depolymerase (a tail fiber protein, called Gp50, accession no.…”

In Vitro and In Vivo Assessments of Two Newly Isolated Bacteriophages against an ST13 Urinary Tract Infection Klebsiella pneumoniae

“…Klebsiella bacteriophage specificity for the capsular type is well-known [ 25 , 51 ]. This specificity could be linked to a specific interaction of the bacteriophage’s depolymerase, highlighted in silico for both bacteriophages in this study, with the bacterial capsule [ 52 , 53 , 54 ], even if a broad host range of K. pneumoniae bacteriophages have already been described [ 24 , 55 ]. In this study, vB_KpnP_K3-ULINTkp1 seemed more specific than vB_KpnP_K3-ULINTkp2 and could replicate within two of the tested strains (QAMH 130326/0185 and SB5904) that both belonged to K-type 3.…”