The high-pathogenicity island (HPI) was initially identified in Yersinia and can be horizontally transferred to Escherichia coli to produce yersiniabactin (Ybt), which enhances the pathogenicity of E. coli by competing with the host for Fe³⁺. Pyroptosis is gasdermin-induced necrotic cell death. It involves the permeabilization of the cell membrane and is accompanied by an inflammatory response. It is still unclear whether Ybt HPI can cause intestinal epithelial cells to undergo pyroptosis and contribute to gut inflammation during E. coli infection. In this study, we infected intestinal epithelial cells of mice with E. coli ZB-1 and the Ybt-deficient strain ZB-1Δirp2. Our findings demonstrate that Ybt-producing E. coli is more toxic and exacerbates gut inflammation during systemic infection. Mechanistically, our results suggest the involvement of the NLRP3/caspase-1/GSDMD pathway in E. coli infection. Ybt promotes the assembly and activation of the NLRP3 inflammasome, leading to GSDMD cleavage into GSDMD-N and promoting the pyroptosis of intestinal epithelial cells, ultimately aggravating gut inflammation. Notably, NLRP3 knockdown alleviated these phenomena, and the binding of free Ybt to NLRP3 may be the trigger. Overall, our results show that Ybt HPI enhances the pathogenicity of E. coli and induces pyroptosis via the NLRP3 pathway, which is a new mechanism through which E. coli promotes gut inflammation. Furthermore, we screened drugs targeting NLRP3 from an existing drug library, providing a list of potential drug candidates for the treatment of gut injury caused by E. coli.

Hemolysin-coregulated protein 1 (Hcp1) is an effector released by the type VI secretion system (T6SS) in certain pathogenic strains of Escherichia coli (E. coli) that causes apoptosis and contributes to the development of meningitis. The exact toxic consequences of Hcp1 and whether it intensifies the inflammatory response by triggering pyroptosis are yet unknown. Here, utilizing the CRISPR/Cas9 genome editing method, we removed the gene expressing Hcp1 from wild-type E. coli W24 and examined the impact of Hcp1 on E. coli virulence in Kunming (KM) mice. It was found that Hcp1-sufficient E. coli was more lethal, exacerbating acute liver injury (ALI) and acute kidney injury (AKI) or even systemic infections, structural organ damage, and inflammatory factor infiltration. These symptoms were alleviated in mice infected with W24Δhcp1. Additionally, we investigated the molecular mechanism by which Hcp1 worsens AKI and found that pyroptosis is involved, manifested as DNA breaks in many renal tubular epithelial cells. Genes or proteins closely related to pyroptosis are abundantly expressed in the kidney. Most importantly, Hcp1 promotes the activation of the NLRP3 inflammasome and the expression of active caspase-1, thereby cleaving GSDMD-N and accelerating the release of active IL-1β and ultimately leading to pyroptosis. In conclusion, Hcp1 enhances the virulence of E. coli, aggravates ALI and AKI, and promotes the inflammatory response; moreover, Hcp1-induced pyroptosis is one of the molecular mechanisms of AKI.

Ellagic acid (EA) has improving function against oxidative damage and inflammatory reaction in many disorders. Hepatic ischemia–reperfusion injury (IRI) is a common pathophysiological phenomenon in the veterinary clinic. In the present study, the protective effects of EA pretreatment against hepatic IRI-induced injury and the underlying mechanisms were investigated. We found that pyroptosis is involved in hepatic IRI, which is manifested in increasing the expression of pyroptosis-related genes and promoting the expression of active caspase-1, thereby cleaving GSDMD-N to cause pyroptosis, and caspase-1^−/− mice were used to verify this conclusion. In addition, we found that EA protects against hepatic IRI by inhibiting pyroptosis, including reducing the activity of caspase-1 and its expression in the liver, inhibiting the lysis of GSDMD-N, and reducing the levels of IL-18 and IL-1β. The present results have demonstrated that prophylactic administration of EA ameliorated hepatic IRI by inhibiting pyroptosis induced in hepatic ischemia–reperfusion in vivo through the caspase-1-GSDMD axis, providing a potential therapeutic option prevent hepatic IRI in pets.

CRISPR/Cas9, as a well-established gene editing technology, has been applied in numerous model organisms, but its application in wild-type E. coli remains limited. Pathogenic wild-type E. coli, 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 E. coli pathogenicity. In this study, PCR was used to identify 50 strains carrying the virulence gene VgrG2 out of 83 wild pathogenic E. coli strains, with only one strain sensitive to kanamycin and spectinomycin. A homologous repair template for VgrG2 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 VgrG2. PCR and sequencing confirmed the deletion of a 1708 bp fragment of the VgrG2 gene in wild-type E. coli. IPEC-J2 cells were infected with E. coli-WT and E. coli ∆VgrG2, and treated with the mTOR inhibitor rapamycin to study the effects of VgrG2 on the mTOR signaling pathway. The qPCR results showed that VgrG2 activated the mTOR pathway, suppressed mTOR and p62 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 E. coli, revealing that VgrG2 activates the mTOR signaling pathway and upregulates autophagy markers. These findings offer new insights into E. coli genome editing and clarifies the pathogenic mechanisms through which VgrG2 induces cellular damage.

The high-pathogenicity island (HPI) is a major virulence determinant in pathogenic Escherichia coli (E. coli), contributing to severe inflammation and tissue damage. Autophagy plays a critical role in clearing intracellular pathogens and modulating inflammation, but whether HPI manipulates this process remains unknown. Here, using a swine-pathogenic E. coli strain and its HPI-deficient mutant (Δirp2) generated by CRISPR/Cas9, we investigated the interplay between HPI and autophagy in RAW264.7 macrophages and a mouse intestinal infection model. We found that HPI⁺ infection induced autophagic activation, as evidenced by increased LC3 puncta (immunofluorescence), upregulated Beclin-1 and autophagy-related gene mRNA levels (qPCR), and downregulated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) expression at both mRNA (qPCR) and protein (immunohistochemistry) levels. In a mouse model, HPI⁺ infection upregulated intestinal Microfold (M) cell markers and secretory Immunoglobulin A (IgA), triggered robust production of pro-inflammatory cytokines, and induced more severe tissue pathology than the HPI-deficient mutant. Pharmacological activation of autophagy with rapamycin alleviated HPI-induced inflammation and injury, whereas inhibition of autophagy by 3-methyladenine (3-MA) or Beclin-1 silencing exacerbated damage. These findings suggest that HPI induces autophagy, but the endogenous autophagic response is insufficient to counteract HPI-induced pathology; pharmacological enhancement of autophagy partially alleviated this insufficiency and reduced tissue damage. Notably, Beclin-1 knockdown blunted HPI-induced upregulation of PI3K and autophagy-related genes, suggesting a role for Beclin-1 in the transcriptional regulation of these responses. In conclusion, HPI simultaneously exerts direct pro-inflammatory effects and induces Beclin-1-dependent autophagy. Enhancing this autophagic response pharmacologically, rather than relying on the endogenous level triggered by HPI alone, limits excessive tissue damage. Thus, boosting autophagy may represent a promising therapeutic strategy against HPI-bearing pathogenic E. coli infections.

Pathogenic bacteria frequently manipulate host cell death pathways to facilitate infection, though the precise mechanisms remain elusive. Here, we demonstrate that pathogenic Escherichia coli disrupts copper homeostasis through upregulation of copper transporter CTR2, thereby triggering cuproptosis to drive infectious pathology. During infection, circulating lipopolysaccharide activates the morphological hallmarks of cuproptosis via the gut-LPS-liver axis. Furthermore, mitochondrial malate dehydrogenase 2 (MDH2) functions as a transcriptional regulator, triggering CTR2 expression and metabolic reprogramming via acetylation-dependent nuclear translocation in response to infection. Notably, inhibiting cuproptosis mitigates liver damage caused by infection, highlighting its critical role in pathogen-host interactions. These findings identify a mechanism underlying E. coli pathogenesis and support therapeutic approaches based on targeted modulation of metal-dependent cell death.