Mutated Lactonase for Quorum Sensing Disruption
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Solution Overview
Problem
Current management strategies for quorum sensing-dependent bacterial infections, such as fire blight and other plant diseases, are limited by regulatory restrictions and the development of antibiotic resistance, necessitating the need for effective and sustainable control methods.
Innovation Solution
A mutated phosphotriesterase-like lactonase with specific amino acid substitutions is developed, maintaining the TIM-barrel fold and catalytic residues, which is used to treat or prevent infections by hydrolyzing quorum sensing lactones, thereby reducing bacterial virulence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If antibiotics are used to treat bacterial infections, then infection control effectiveness is improved, but antibiotic resistance development occurs
Solution Approach 1:
The patent converts the harmful quorum sensing signaling molecules (lactones) produced by bacteria into a beneficial target for enzymatic degradation. By using lactonase to specifically hydrolyze these signaling molecules, the system disables bacterial virulence without killing the bacteria, thus avoiding the selection pressure that drives antibiotic resistance while still achieving effective infection control.
Solution Approach 2:
The patent introduces lactonase as an intermediary substance that mediates between the host and pathogenic bacteria. Instead of direct antibiotic-bacteria interaction that causes resistance, the lactonase enzyme acts as a middleman that degrades bacterial quorum sensing signals, indirectly disrupting virulence expression without direct antibacterial pressure.
2Reliability
If copper pesticide and Streptomyces lydicus are used for fire blight management, then disease control is improved, but regulatory restrictions and application limitations increase
Solution Approach 1:
The patent creates a universal solution by developing lactonase that can degrade multiple types of quorum sensing lactones (C4-HSL, C6-HSL, C8-HSL, C10-HSL) produced by various plant pathogens including Erwinia amylovora, Pectobacterium carotovorum, and Pseudomonas species. This single enzyme provides broad-spectrum activity against different bacterial diseases, replacing the need for pathogen-specific treatments.
Solution Approach 2:
The patent optimizes the lactonase enzyme's operational parameters through mutation to enhance its thermal stability and activity at plant-relevant temperatures. The mutated lactonase maintains catalytic function while showing improved stability, allowing effective application in diverse environmental conditions without the regulatory and application constraints of copper-based pesticides.
3Reliability
If wild-type phosphotriesterase-like lactonase is used, then quorum quenching activity is achieved, but thermal stability and shelf life are insufficient
Solution Approach 1:
The patent systematically changes the physical-chemical parameters of the lactonase enzyme through directed evolution and site-directed mutagenesis. Specific amino acid substitutions are introduced to enhance thermal stability and structural rigidity while preserving the catalytic triad residues essential for quorum quenching activity, thereby extending the enzyme's shelf life and operational duration.
Solution Approach 2:
The patent performs preliminary stabilization of the lactonase enzyme through mutation before application. The mutated lactonase is pre-optimized for enhanced thermal stability and storage longevity, ensuring the enzyme remains active and effective throughout the intended application period without degradation, thus extending its functional duration.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The mutated lactonase effectively inhibits the production of virulence factors in bacteria, demonstrating enhanced thermal stability and shelf life, and shows similar efficacy to existing antibiotics in reducing fire blight symptoms, offering a potential solution to antibiotic resistance.
Implementation Method 1
the mutated phosphotriesterase-like lactonase has substantially identical TIM-barrel fold to the wild-type phosphotriesterase-like lactonase and preserved catalytic residues in its active site
Implementation Method 2
A mutated phosphotriesterase-like lactonase with specific amino acid substitutions is developed, maintaining the TIM-barrel fold and catalytic residues, which is used to treat or prevent infections by hydrolyzing quorum sensing lactones
Data Source
AI summary
Mutated phosphotriesterase-like lactonases or functional fragments can be used in methods for treating or preventing infection of a bacterium in a host, such as a plant or a part, organ or a plant propagation material. The methods include applying the mutated phosphotriesterase-like lactonases or the wild-type enzyme to the host Cells expressing the mutated phosphotriesterase-like lactonases can also be produced using nucleic acid molecules and vectors encoding the mutated phosphotriesterase-like lactonases or functional fragments.