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Application of nitrosation modification of transcriptional regulatory factors WalR and MgrA in treatment of staphylococcus aureus infection

A staphylococcus infection, nitrosylation technology, applied in the field of genetic engineering

Pending Publication Date: 2022-02-25
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report about the modification of bacterial endogenous NO to its own proteome. Therefore, we used the method of protein modification omics to identify protein targets in Staphylococcus aureus that can be nitrosylated by its endogenous NO. It can provide new targets and strategies for the treatment of Staphylococcus aureus VISA infection and other bacterial pathogens

Method used

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  • Application of nitrosation modification of transcriptional regulatory factors WalR and MgrA in treatment of staphylococcus aureus infection
  • Application of nitrosation modification of transcriptional regulatory factors WalR and MgrA in treatment of staphylococcus aureus infection
  • Application of nitrosation modification of transcriptional regulatory factors WalR and MgrA in treatment of staphylococcus aureus infection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Construction of embodiment 1.nos knockout strain:

[0095] The nos gene is used to encode the NOS protein (nitric oxide synthase), which produces NO, and then mediates the nitrosylation modification of the cysteine ​​site of the protein.

[0096] In this example, the target gene traceless knockout technology is adopted, and the homologous recombination plasmid is constructed with the temperature-sensitive plasmid pBTs as the carrier. The method is as follows: use HS DNA Polymerase (TaKaRa, DR010A) amplifies the upstream and downstream homology arm fragments of the nnos gene with bNOS-up-F and bNOS-up-R, bNOS-down-F and bNOS-down-R from the XN108 genome, respectively, by PCR was performed to overlap the upstream and downstream to obtain combined fragments, and the corresponding fast restriction endonucleases KpnI (Thermo, FD0524) and SacI (Thermo, FD1133) were used to treat the vector pBTs and fragments respectively, and after recovery and purification, T4 DNA ligase (T...

Embodiment 2

[0108] Example 2. Identification of protein targets that can be modified by bacterial endogenous NO nitrosylation:

[0109] In this example, iodoTMT (irreversible isobaric iodoacetyl Cys-reactivetandem mass tag) is used to carry out isotope labeling and enrichment of proteins modified by SNO nitrosylation, and then combined with mass spectrometry-based proteomics analysis, and nos knockout strains were combined with Wild-type strains were compared to identify protein targets in S. aureus that could be modified by nitrosylation by endogenous bacterial NO. iodoTMT markers (thermo, 90100, 90101, 90102, 90103) and proteomics were completed by Jingjie Biological Company.

Embodiment 3

[0110] Example 3. Construction of amino acid point mutation strains:

[0111] The walR (C67S) and mgrA (C12S) single-point mutant strains in the strain XN108 were constructed using the target gene seamless knockout technology, and the temperature-sensitive plasmid pBTs was used as a carrier to construct a homologous recombination plasmid. The method is as follows:

[0112] use HS DNA Polymerase (TaKaRa, DR010A) amplifies the walR gene from the XN108 genome with walR-C67S-up-F and walR-C67S-up-R, walR-C67S-down-F and walR-C67S-down-R, respectively The 67th cysteine ​​(C67) upstream and downstream homology arm fragment; use mgrA-C12S-up-F and mgrA-C12S-up-R, mgrA-C12S-down-F and mgrA-C12S-down-R respectively Amplify the homology arm fragments upstream and downstream of the 12th cysteine ​​(C12) of the mgrA gene. The combined fragments were obtained by overlapping the upstream and downstream by PCR respectively. The upstream and downstream combined fragments of walR (C67S) we...

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Abstract

Proteomics is combined with mass spectrum identification to find that nitric oxide generated by staphylococcus aureus endogenously can cause nitrosation modification of protein of the staphylococcus aureus. Experiments prove that nitrosation modification of important transcriptional regulatory factors WalR and MgrA has the potential to become targets of vancomycin drug resistance treatment.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the application of nitrosylation modification of transcriptional regulators WalR and MgrA in the treatment of Staphylococcus aureus infection, which can provide a new target for clinical treatment of Staphylococcus aureus drug-resistant bacteria . Background technique [0002] Staphylococcus aureus (Staphylococcus aureus) is the main opportunistic bacterial pathogen of human beings. It exists widely in nature. Its infection may cause many serious diseases such as pneumonia, endocarditis, osteomyelitis, and even sepsis. The treatment of Staphylococcus aureus infection mainly relies on antibiotics. However, the use and abuse of antibiotics have spawned and enriched drug-resistant strains, especially methicillin-resistant Staphylococcus aureus (MRSA) in hospitals and communities. Widespread infection and spread has become the most difficult infectious problem...

Claims

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Application Information

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IPC IPC(8): G01N33/68A61K38/14A61K45/06A61P31/04
CPCG01N33/6806A61K45/06A61K38/14A61P31/04A61K2300/00
Inventor 孙宝林舒雪琴黄奕
Owner UNIV OF SCI & TECH OF CHINA
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