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Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi
Published on: September 28, 2022
Stepwise phage resistance and collateral phage susceptibility in Klebsiella pneumoniae
Xin Yin1,2,3, Yu Feng1,2,3, Huan Luo4
1Center of Infectious Diseases, West China Hospital, Sichuan University, Chengdu, People's Republic of China.
Abstract:
Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a difficult-to-treat pathogen. Phages recovered by a "stepwise" approach may constitute a cocktail able to prolong retardation of bacterial regrowth. We stepwise recovered three lytic phages (namely P04, P40, and P49) from different families against ST11KL64 CRKP and created a cocktail that restrained CRKP growth for 15 h. Phage P04 recognized bacterial capsular polysaccharide (CPS). P04/P40-resistant mutants lost the large fragments containing CPS gene due to homologous recombination between two insertion sequences. Through gene cloning and complementation experiments, CPS and lipopolysaccharide (LPS) quantification, and untargeted lipid metabolism assays, deletion of the ugd/wbgU genes alters lipid A modification and outer membrane environment. This change affects the accessibility of an unidentified membrane protein or lipid A itself, which serves as the receptor for P40. P49 recognized bacterial transmembrane protein involved in vitamin B12 transportation. Synergistic antibacterial activity was observed because the three phages recognize different receptors. Notably, P49 also lysed Salmonella enterica, Escherichia coli, Enterobacter ludwigii, and Kluyvera tianfuensis, suggesting new receptors would be exposed when the synthesis of CPS was inhibited, thereby allowing efficient attack by phages originally targeting other species (the "close-one-door-but-open-another" phenomenon). The conserved structure of BtuB provides a molecular basis for the rare cross-genus activity of P49. Insertion sequences provide a generalized anti-phage defence in encapsulated bacteria. Our findings provide critical insights into the versatile mechanisms underpinning bacteria-phages interactions. Specifically, the collateral susceptibility to phages targeting other bacterial species may provide a novel and highly promising approach for creating clinically viable phage cocktails.
Insights
A novel phage cocktail effectively restrains carbapenem-resistant Klebsiella pneumoniae (CRKP) growth. This strategy leverages phages targeting diverse bacterial receptors, offering a promising approach for combating difficult-to-treat infections.
Area of Science:
- Microbiology and Virology
- Bacteriophage Therapy
- Antimicrobial Resistance
Background:
- Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a significant global health threat due to limited treatment options.
- Bacteriophage therapy presents a potential alternative or adjunct to antibiotics for combating resistant bacterial infections.
- Understanding phage-bacterial interactions is crucial for developing effective phage cocktails.
Purpose of the Study:
- To develop a phage cocktail capable of prolonging the inhibition of CRKP regrowth.
- To elucidate the specific bacterial receptors targeted by individual phages within the cocktail.
- To investigate the mechanisms of phage resistance and cross-genus activity.
Main Methods:
- Stepwise isolation of lytic phages (P04, P40, P49) against ST11-KL64 CRKP.
- Construction and evaluation of a three-phage cocktail for CRKP growth inhibition.
- Analysis of phage-resistant mutants, including genomic analysis (insertion sequences, gene deletions), lipid metabolism assays, and receptor identification (CPS, LPS, transmembrane proteins, BtuB).
Main Results:
- The developed phage cocktail (P04, P40, P49) restrained CRKP growth for 15 hours.
- Phage P04 targets capsular polysaccharide (CPS); P04/P40 resistance involved homologous recombination and deletion of ugd/wbgU genes, altering lipid A modification and outer membrane.
- Phage P40 targets an unidentified membrane protein or lipid A; Phage P49 targets a vitamin B12 transporter (BtuB), exhibiting cross-genus activity against Salmonella, E. coli, E. ludwigii, and K. tianfuensis.
Conclusions:
- The synergistic activity of phages targeting distinct receptors (CPS, outer membrane components, BtuB) is key to cocktail efficacy.
- The 'close-one-door-but-open-another' phenomenon, where inhibiting CPS synthesis exposes new receptors, enhances phage effectiveness.
- Insertion sequences act as a generalized anti-phage defense, and BtuB's conserved structure explains P49's cross-genus activity, offering insights for novel phage cocktail development.
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