Construction method and application of pseudomonas aeruginosa engineering bacteria duec

By knocking out the retS, tssB2, and tssB3 genes in Pseudomonas aeruginosa PAO1 and mutating the catalytic site of the H1-T6SS secretory effector protein, a DUEC engineered strain was constructed. This solved the problem of Pseudomonas aeruginosa specifically responding to T6SS+ cells in mixed communities and achieved specific delivery of Cre recombinase and regulation of downstream pathways.

CN115992162BActive Publication Date: 2026-03-03SHANGHAI JIAOTONG UNIV
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Patent Information

Application Number
CN202211049053.3
Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Filing Date
2022-08-30
Publication Date
2026-03-03
Estimated Expiration
2042-08-30

AI Technical Summary

Technical Problem

When Pseudomonas aeruginosa responds to T6SS attacks from heterologous or sister cells, its effector proteins do not kill sister cells in the presence of homologous immune proteins, and the mechanism is unclear. This limits its application as a signal sensing module, and it lacks tools that specifically respond to physical contact signals, making it difficult to regulate microorganisms in mixed communities.

Method used

By knocking out the retS and tssB2 and tssB3 genes in Pseudomonas aeruginosa PAO1 and mutating the catalytic sites of the H1-T6SS secretory effector proteins Tse1-Tse4 and Tse6-Tse8, a DUEC engineered strain was constructed, enabling it to respond to physical contact with T6SS and specifically deliver cargo proteins such as Cre recombinase to T6SS+ cells.

Benefits of technology

The DUEC engineered strain can specifically distinguish between T6SS+ and T6SS- strains in complex microbial communities, deliver Cre recombinase for site-specific recombination, and regulate downstream pathways, thus having broad application potential.

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Abstract

The present application relates to a kind of pseudomonas aeruginosa engineering bacteria DUEC construction method and application.The DUEC engineering strain construction method of the present application is: response to kinase coding gene retS and sheath protein coding gene tssB2, tssB3 Knockout, H1-T6SS secretory effector protein Tse1-Tse4, Tse6-Tse8 catalytic site mutation, Tse5 Coding gene knockout.DUEC engineering strain can not only constitutively express and assemble H1-T6SS, but also can respond to adjacent sister cell source T6SS attack, assemble H1-T6SS at the position of being attacked.The present application constructs a series of effector protein combination inactivation mutant by gene knockout or catalytic site mutation method.Microscope observation and statistical analysis results show that no single effector protein is necessary for T6SS attack and assembly.By bacterial competition experiment, it is verified that all effector protein inactivation mutant 8eff c Lose T6SS + Vibrio cholerae and T6SS + Aeromonas dhaka's bactericidal activity, but 8eff c Can respond to T6SS physical attack, Tse6 N -Cre is specifically delivered to T6SS + In Vibrio cholerae.
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