The CRISPR/Cas9-Mediated Knockout of <i>VgrG2</i> in Wild Pathogenic <i>E. coli</i> to Alleviate the Effects on Cell Damage and Autophagy
CRISPR/Cas9, as a well-established gene editing technology, has been applied in numerous model organisms, but its application in wild-type <i>E. coli</i> remains limited. Pathogenic wild-type <i>E. coli</i>, a major cause of foodborne illnesses and intestinal inflammation in...
Saved in:
| Main Authors: | , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-03-01
|
| Series: | Veterinary Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2306-7381/12/3/249 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850207700767473664 |
|---|---|
| author | Tian-Ling Pan Jin-Long Cha Hao Wang Jing-Song Zhang Jin-Long Xiao Jue Shen Meng Zhou Yue Li Jin-Zhi Ma Kai-Yuan Zhao Yong-Kang Zhang Peng Xiao Hong Gao |
| author_facet | Tian-Ling Pan Jin-Long Cha Hao Wang Jing-Song Zhang Jin-Long Xiao Jue Shen Meng Zhou Yue Li Jin-Zhi Ma Kai-Yuan Zhao Yong-Kang Zhang Peng Xiao Hong Gao |
| author_sort | Tian-Ling Pan |
| collection | DOAJ |
| description | CRISPR/Cas9, as a well-established gene editing technology, has been applied in numerous model organisms, but its application in wild-type <i>E. coli</i> remains limited. Pathogenic wild-type <i>E. coli</i>, a major cause of foodborne illnesses and intestinal inflammation in humans and animals, poses a significant global public health threat. The valine-glycine repeat protein G (VgrG) is a key virulence factor that enhances <i>E. coli</i> pathogenicity. In this study, PCR was used to identify 50 strains carrying the virulence gene <i>VgrG2</i> out of 83 wild pathogenic <i>E. coli</i> strains, with only one strain sensitive to kanamycin and spectinomycin. A homologous repair template for <i>VgrG2</i> was constructed using overlap PCR. A dual-plasmid CRISPR/Cas9 system, combining pTarget (spectinomycin resistance) and pCas (kanamycin resistance) with Red homologous recombination, was then used to induce genomic cleavage and knock out <i>VgrG2</i>. PCR and sequencing confirmed the deletion of a 1708 bp fragment of the <i>VgrG2</i> gene in wild-type <i>E. coli</i>. IPEC-J2 cells were infected with <i>E. coli</i>-WT and <i>E. coli</i> ∆<i>VgrG2</i>, and treated with the mTOR inhibitor rapamycin to study the effects of <i>VgrG2</i> on the mTOR signaling pathway. The qPCR results showed that <i>VgrG2</i> activated the mTOR pathway, suppressed <i>mTOR</i> and <i>p62</i> mRNA levels, and upregulated the autophagy-related genes and LC3-II protein expression. In conclusion, we utilized CRISPR/Cas9 technology to achieve large-fragment deletions in wild-type <i>E. coli</i>, revealing that <i>VgrG2</i> activates the mTOR signaling pathway and upregulates autophagy markers. These findings offer new insights into <i>E. coli</i> genome editing and clarifies the pathogenic mechanisms through which <i>VgrG2</i> induces cellular damage. |
| format | Article |
| id | doaj-art-6ea02f0b297445a7a3a7f5d5b5c4469b |
| institution | OA Journals |
| issn | 2306-7381 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Veterinary Sciences |
| spelling | doaj-art-6ea02f0b297445a7a3a7f5d5b5c4469b2025-08-20T02:10:25ZengMDPI AGVeterinary Sciences2306-73812025-03-0112324910.3390/vetsci12030249The CRISPR/Cas9-Mediated Knockout of <i>VgrG2</i> in Wild Pathogenic <i>E. coli</i> to Alleviate the Effects on Cell Damage and AutophagyTian-Ling Pan0Jin-Long Cha1Hao Wang2Jing-Song Zhang3Jin-Long Xiao4Jue Shen5Meng Zhou6Yue Li7Jin-Zhi Ma8Kai-Yuan Zhao9Yong-Kang Zhang10Peng Xiao11Hong Gao12College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, ChinaCollege of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, ChinaCollege of Food Science and Technology, Yunnan Agricultural University, Kunming 650201, ChinaCollege of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, ChinaCollege of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, ChinaCollege of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, ChinaCollege of Food Science and Technology, Yunnan Agricultural University, Kunming 650201, ChinaCollege of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, ChinaCollege of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, ChinaCollege of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, ChinaCollege of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, ChinaCollege of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, ChinaCollege of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, ChinaCRISPR/Cas9, as a well-established gene editing technology, has been applied in numerous model organisms, but its application in wild-type <i>E. coli</i> remains limited. Pathogenic wild-type <i>E. coli</i>, a major cause of foodborne illnesses and intestinal inflammation in humans and animals, poses a significant global public health threat. The valine-glycine repeat protein G (VgrG) is a key virulence factor that enhances <i>E. coli</i> pathogenicity. In this study, PCR was used to identify 50 strains carrying the virulence gene <i>VgrG2</i> out of 83 wild pathogenic <i>E. coli</i> strains, with only one strain sensitive to kanamycin and spectinomycin. A homologous repair template for <i>VgrG2</i> was constructed using overlap PCR. A dual-plasmid CRISPR/Cas9 system, combining pTarget (spectinomycin resistance) and pCas (kanamycin resistance) with Red homologous recombination, was then used to induce genomic cleavage and knock out <i>VgrG2</i>. PCR and sequencing confirmed the deletion of a 1708 bp fragment of the <i>VgrG2</i> gene in wild-type <i>E. coli</i>. IPEC-J2 cells were infected with <i>E. coli</i>-WT and <i>E. coli</i> ∆<i>VgrG2</i>, and treated with the mTOR inhibitor rapamycin to study the effects of <i>VgrG2</i> on the mTOR signaling pathway. The qPCR results showed that <i>VgrG2</i> activated the mTOR pathway, suppressed <i>mTOR</i> and <i>p62</i> mRNA levels, and upregulated the autophagy-related genes and LC3-II protein expression. In conclusion, we utilized CRISPR/Cas9 technology to achieve large-fragment deletions in wild-type <i>E. coli</i>, revealing that <i>VgrG2</i> activates the mTOR signaling pathway and upregulates autophagy markers. These findings offer new insights into <i>E. coli</i> genome editing and clarifies the pathogenic mechanisms through which <i>VgrG2</i> induces cellular damage.https://www.mdpi.com/2306-7381/12/3/249wild-type <i>E. coli</i>CRISPR/Cas9<i>VgrG2</i> genelarge fragment deletionmTOR signaling pathway |
| spellingShingle | Tian-Ling Pan Jin-Long Cha Hao Wang Jing-Song Zhang Jin-Long Xiao Jue Shen Meng Zhou Yue Li Jin-Zhi Ma Kai-Yuan Zhao Yong-Kang Zhang Peng Xiao Hong Gao The CRISPR/Cas9-Mediated Knockout of <i>VgrG2</i> in Wild Pathogenic <i>E. coli</i> to Alleviate the Effects on Cell Damage and Autophagy Veterinary Sciences wild-type <i>E. coli</i> CRISPR/Cas9 <i>VgrG2</i> gene large fragment deletion mTOR signaling pathway |
| title | The CRISPR/Cas9-Mediated Knockout of <i>VgrG2</i> in Wild Pathogenic <i>E. coli</i> to Alleviate the Effects on Cell Damage and Autophagy |
| title_full | The CRISPR/Cas9-Mediated Knockout of <i>VgrG2</i> in Wild Pathogenic <i>E. coli</i> to Alleviate the Effects on Cell Damage and Autophagy |
| title_fullStr | The CRISPR/Cas9-Mediated Knockout of <i>VgrG2</i> in Wild Pathogenic <i>E. coli</i> to Alleviate the Effects on Cell Damage and Autophagy |
| title_full_unstemmed | The CRISPR/Cas9-Mediated Knockout of <i>VgrG2</i> in Wild Pathogenic <i>E. coli</i> to Alleviate the Effects on Cell Damage and Autophagy |
| title_short | The CRISPR/Cas9-Mediated Knockout of <i>VgrG2</i> in Wild Pathogenic <i>E. coli</i> to Alleviate the Effects on Cell Damage and Autophagy |
| title_sort | crispr cas9 mediated knockout of i vgrg2 i in wild pathogenic i e coli i to alleviate the effects on cell damage and autophagy |
| topic | wild-type <i>E. coli</i> CRISPR/Cas9 <i>VgrG2</i> gene large fragment deletion mTOR signaling pathway |
| url | https://www.mdpi.com/2306-7381/12/3/249 |
| work_keys_str_mv | AT tianlingpan thecrisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT jinlongcha thecrisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT haowang thecrisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT jingsongzhang thecrisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT jinlongxiao thecrisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT jueshen thecrisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT mengzhou thecrisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT yueli thecrisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT jinzhima thecrisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT kaiyuanzhao thecrisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT yongkangzhang thecrisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT pengxiao thecrisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT honggao thecrisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT tianlingpan crisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT jinlongcha crisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT haowang crisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT jingsongzhang crisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT jinlongxiao crisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT jueshen crisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT mengzhou crisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT yueli crisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT jinzhima crisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT kaiyuanzhao crisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT yongkangzhang crisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT pengxiao crisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy AT honggao crisprcas9mediatedknockoutofivgrg2iinwildpathogeniciecoliitoalleviatetheeffectsoncelldamageandautophagy |