The Mutation in wbaP cps Gene Cluster Selected by Phage-Borne Depolymerase Abolishes Capsule Production and Diminishes the Virulence of Klebsiella pneumoniae.

Marta Kaszowska,Zuzanna Drulis-Kawa,Grazyna Majkowska-Skrobek,Anna Maciejewska,Cédric Lood,Wojciech Jachymek,Jolanta Lukasiewicz,Pawel Markwitz

doi:10.3390/ijms222111562

Abstract

Klebsiella pneumoniae is considered one of the most critical multidrug-resistant pathogens and urgently requires new therapeutic strategies. Capsular polysaccharides (CPS), lipopolysaccharides (LPS), and exopolysaccharides (EPS) are the major virulence factors protecting K. pneumoniae against the immune response and thus may be targeted by phage-based therapeutics such as polysaccharides-degrading enzymes. Since the emergence of resistance to antibacterials is generally considered undesirable, in this study, the genetic and phenotypic characteristics of resistance to the phage-borne CPS-degrading depolymerase and its effect on K. pneumoniae virulence were investigated. The K63 serotype targeting depolymerase (KP36gp50) derived from Klebsiella siphovirus KP36 was used as the selective agent during the treatment of K. pneumoniae 486 biofilm. Genome-driven examination combined with the surface polysaccharide structural analysis of resistant mutant showed the point mutation and frameshift in the wbaP gene located within the cps gene cluster, resulting in the loss of the capsule. The sharp decline in the yield of CPS was accompanied by the production of a larger amount of smooth LPS. The modification of the surface polysaccharide layers did not affect bacterial fitness nor the insensitivity to serum complement; however, it made bacteria more prone to phagocytosis combined with the higher adherence and internalization to human lung epithelial cells. In that context, it was showed that the emerging resistance to the antivirulence agent (phage-borne capsule depolymerase) results in beneficial consequences, i.e., the sensitization to the innate immune response.

Highlights

IntroductionKlebsiella pneumoniae belongs to the bacterial ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) and is placed on the top priority WHO list of “critical” pathogens and a target for the development of therapeutic strategies combating hypervirulence and multidrug-resistance problems [1,2,3]
While studying the antibiofilm potential of recombinant K63 capsule-degrading depolymerase (KP36gp50), phenotypically smaller K. pneumoniae K. pneumoniae strain 486 (Kp486) colonies were isolated, which turned out to be both resistant to KP36gp50 activity as well as to phages targeting K63 receptor (Figure 1A)
Based on comprehensive genome-wide examination combined with the surface polysaccharides structural analysis, it was showed that point mutation in the first nucleotides of the wbaP gene located within the cps cluster in the Kp7De strain impaired capsule formation without reduction in relative fitness in terms of growth rate and generation time compared to its parental strain (Kp486)

Summary

Introduction

Klebsiella pneumoniae belongs to the bacterial ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) and is placed on the top priority WHO list of “critical” pathogens and a target for the development of therapeutic strategies combating hypervirulence and multidrug-resistance problems [1,2,3]. The cell surface-associated polysaccharides and glycolipids, namely capsular polysaccharides (CPS known as K-antigen) and lipopolysaccharide (LPS; O-antigen), as well as the exopolysaccharides (EPS) are the major virulence factors of K. pneumoniae [4,5]. Klebsiella strains can effectively be discriminated against based on the O- and K-typing.

Methods

Results

Conclusion