Stable mutants of quorum quenching lactonase and their use in treatment of pathogens

A lactonase, mutant-based technology for the management of fire blight and other plant diseases that addresses unmet issues of effective reagents and management, regulatory constraints, and public health restrictions on the long-term prospects for use of antibiotics and other reagents

Pending Publication Date: 2022-04-08
米加尔加利里研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, regulatory constraints, public health concerns, and the development of resistance severely limit the prospects for long-term use of antibiotics and other agents (5)
[0005] Thus, there is an unmet need for effective agents and management of fire blight and other debilitating plant diseases

Method used

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  • Stable mutants of quorum quenching lactonase and their use in treatment of pathogens
  • Stable mutants of quorum quenching lactonase and their use in treatment of pathogens
  • Stable mutants of quorum quenching lactonase and their use in treatment of pathogens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] Example 1. Recombinant expression, purification and biochemical characterization of enzymes utilizing c6-oxo-HSL

[0139] The genes encoding PPH and its evolutionary variants from Mycobacterium tuberculosis were cloned into the expression vector pMal-c4X and propagated in Escherichia coli-BL21(DE3) as a fusion protein with the high-binding mutant (A313V) maltose-binding protein. Expression (SEQ ID NO: 10). Cells were then lysed and proteins were purified using an amylose column (NEB). After purification, we tested the temperature optima of the enzymes, their thermostability and shelf life with a chromogenic substrate (TBBL, thiobutyrylbutyrolactone). Their association with C6-oxo-HSL (also known as N-hexanoyl-L-homoserine) as a lactone secreted by the plant pathogen Erwinia amylovora was tested using a pH indicator as previously described (7). Lactone, N-[(3S)-tetrahydro-2-oxo-3-furyl]hexanamide, HHL), see Figure 1B and 2A ; PPH exhibits high activity with C6-oxo-H...

Embodiment 2

[0140] Example 2. Construction of random gene libraries and isolation of improved variants with higher activity, thermostability and shelf life

[0141] The gene encoding PPH was used to construct a gene library using the Gene Morph Random Mutagenesis Kit (Agilent). After PCR amplification, the resulting PCR product was used as a template for nested PCR with outer primers, followed by digestion with EcoRI and PstI and ligation into pMAL-c2x, the library plasmid was electroporated into E. coli DH5α, and the estimated library size was 5000 , and isolate plasmid DNA. Single clones of unselected libraries were sequenced and an average of 2-3 point mutations per gene were identified. Use rounds of random mutations (3-4*10 5 library size of variants) and screening (600 variants per round) for increased thermostability, we isolated two variants with unique sequences, see Figure 2: variants with the following mutations: variant PPH_R2 : G58V [G59V; SEQ ID NO: 11] in P4-D5 and varia...

Embodiment 3

[0143] Example 3. Wild-type QQ PPH lactonase inhibits exopolysaccharide formation in culture

[0144] Levan production was observed spectroscopically at 400 nm after supplementation with 500 mM sucrose as a measure of bacterial virulence manifested in the formation of the exopolysaccharide (EPS) matrix according to a previously described protocol (Molina et al., 2005). .

[0145] When the purified wild-type PPH protein was applied to the cell culture of Erwinia amylovora, a 30% decrease in EPS production was observed ( image 3 ). The G59V and H172Y mutants are expected to be effective at least in reducing EPS production.

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Abstract

Provided are mutant phosphotriesterase-like lactonases or functional fragments thereof, as well as nucleic acid molecules and vectors encoding the same. Furthermore, in addition to cells expressing the mutant phosphotriesterase-like lactonase and methods of producing them, methods of treating or preventing bacterial infections in a host, such as a plant or a portion thereof, an organ or plant propagation material, are provided, the method comprises administering to it the mutant phosphotriesterase-like lactonase or wild type enzyme.

Description

technical field [0001] The present invention relates generally to the management of quorum sensing-dependent bacterial infections, and in particular to the management of fireblight and other plant diseases. Background technique [0002] Diseases caused by pathogens through quorum-sensing regulatory systems pose great challenges in clinical and agricultural settings. For example, effective management of fire blight, a contagious disease caused by Erwinia amylovora that affects apples, pears, and some other members of the family Rosaceae, is multifaceted and primarily preventive. Sexual, using sanitation, culturing practices, copper pesticides, products containing Streptomyces lydicus as an active ingredient, and antibiotics (eg, streptomycin or oxytetracycline) combination for preventive use (1). [0003] Other examples of common plant pathogens that can cause disease in various crops through quorum sensing regulatory systems are Pectobacterium carotovorum (2), Pseudomonass...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18A01N63/20A01N63/50C12N15/10
CPCC12N9/18A01N63/50C40B40/08C40B50/06C12N15/1058C12N15/70C12N9/16C12Y301/08001C12Y301/01081A01N59/20A01P1/00
Inventor L·阿弗里特-朱尔诺M·埃罗夫大卫·古列维奇M·D·叶林
Owner 米加尔加利里研究院有限公司
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