Presentation Information
[P01-122]Development of phage-derived endolysins with bactericidal activity against Klebsiella pneumoniae
○Norina Hamada1, Hideto Tochikura2, Kazuki Takahashi2, Haruko Takeyama1,2,3, Masahito Hosokawa1,2,3 (1. Grad. Sch. Adv. Sci. Eng., Waseda Univ. (Japan), 2. Res. Org. Nano Life Innov., Waseda Univ. (Japan), 3. Inst. Adv. Res. Biosyst. Dyn., Waseda Res. Inst. Sci. Eng., Waseda Univ. (Japan))
Keywords:
bacteriophage-derived lytic enzyme,multidrug-resistant bacteria,gut microbiome,protein engineering
[Purpose]
The rapid emergence of multidrug-resistant (MDR) bacteria has become a serious global concern, particularly the ESKAPE pathogens, which pose major public health challenges. Klebsiella pneumoniae is one of the gram-negative bacteria in this group, and there is an urgent need for alternative antibacterial strategies beyond conventional antibiotics. Phage-derived cell wall hydrolases, termed endolysins, are attracting attention as potential antibacterial agents that degrade peptidoglycan. In this study, we aimed to develop endolysins active against the gram-negative bacterium K. pneumoniae.
[Method]
Endolysin candidate sequences were extracted from K. pneumoniae genomic data and screened based on domain architecture and amino acid sequence homology. Selected candidates were expressed in Escherichia coli, and their antibacterial activity against a specific K. pneumoniae strain was evaluated using growth inhibition assays. The antibacterial activity of these endolysins was also evaluated against bacterial species other than K. pneumoniae to assess their specificity. In addition, bactericidal activity was evaluated by counting colony-forming units following endolysin treatment. Furthermore, protein engineering approaches were applied to design and evaluate variants with potentially enhanced activity. The endolysins were engineered to reduce their molecular weight and improve their antibacterial activity.
[Results]
We identified 3 endolysins that inhibited the growth of this strain. When these endolysins were evaluated against other gram-negative bacteria, differences in antibacterial activity were observed among the tested species. In addition, the selected endolysins were confirmed to have a bactericidal activity of approximately 99.9%. Furthermore, protein engineering approaches were applied to design and evaluate variants with potentially enhanced activity. Several engineered variants exhibited comparable or enhanced antibacterial activity at lower concentrations than the original constructs.
[Conclusion]
In this study, endolysin candidates were identified from genomic data and screened for antibacterial activity, leading to the identification of three endolysins active against K. pneumoniae. These endolysins exhibited selective growth inhibition and bactericidal activity against K. pneumoniae. Furthermore, protein engineering approaches were applied to design and evaluate variants with potentially enhanced activity. Through these modifications, the roles of individual domains and their contributions to antibacterial function and mechanism of action were investigated. In addition, minimal constructs retaining functionally essential regions were designed with the aim of achieving higher antibacterial activity. These natural and engineered endolysins may serve as promising antibacterial agents with mechanisms of action distinct from those of conventional antibiotics, and may be promising against other multidrug-resistant Gram-negative bacteria.
The rapid emergence of multidrug-resistant (MDR) bacteria has become a serious global concern, particularly the ESKAPE pathogens, which pose major public health challenges. Klebsiella pneumoniae is one of the gram-negative bacteria in this group, and there is an urgent need for alternative antibacterial strategies beyond conventional antibiotics. Phage-derived cell wall hydrolases, termed endolysins, are attracting attention as potential antibacterial agents that degrade peptidoglycan. In this study, we aimed to develop endolysins active against the gram-negative bacterium K. pneumoniae.
[Method]
Endolysin candidate sequences were extracted from K. pneumoniae genomic data and screened based on domain architecture and amino acid sequence homology. Selected candidates were expressed in Escherichia coli, and their antibacterial activity against a specific K. pneumoniae strain was evaluated using growth inhibition assays. The antibacterial activity of these endolysins was also evaluated against bacterial species other than K. pneumoniae to assess their specificity. In addition, bactericidal activity was evaluated by counting colony-forming units following endolysin treatment. Furthermore, protein engineering approaches were applied to design and evaluate variants with potentially enhanced activity. The endolysins were engineered to reduce their molecular weight and improve their antibacterial activity.
[Results]
We identified 3 endolysins that inhibited the growth of this strain. When these endolysins were evaluated against other gram-negative bacteria, differences in antibacterial activity were observed among the tested species. In addition, the selected endolysins were confirmed to have a bactericidal activity of approximately 99.9%. Furthermore, protein engineering approaches were applied to design and evaluate variants with potentially enhanced activity. Several engineered variants exhibited comparable or enhanced antibacterial activity at lower concentrations than the original constructs.
[Conclusion]
In this study, endolysin candidates were identified from genomic data and screened for antibacterial activity, leading to the identification of three endolysins active against K. pneumoniae. These endolysins exhibited selective growth inhibition and bactericidal activity against K. pneumoniae. Furthermore, protein engineering approaches were applied to design and evaluate variants with potentially enhanced activity. Through these modifications, the roles of individual domains and their contributions to antibacterial function and mechanism of action were investigated. In addition, minimal constructs retaining functionally essential regions were designed with the aim of achieving higher antibacterial activity. These natural and engineered endolysins may serve as promising antibacterial agents with mechanisms of action distinct from those of conventional antibiotics, and may be promising against other multidrug-resistant Gram-negative bacteria.
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