The CRISPR/Cas9-Mediated Knockout of VgrG2 in Wild Pathogenic E. coli 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 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.