Hemolysin Co-Regulatory Protein 1 Enhances the Virulence of Clinically Isolated Escherichia coli in KM Mice by Increasing Inflammation and Inducing Pyroptosis

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.


Introduction
It has been revealed that certain Gram-negative bacteria have a complex secretion mechanism called the type VI secretion system (T6SS) [1][2][3]. Its effectors are cytotoxic, as shown in previous studies [4][5][6]. Hemolysin-coregulated protein 1 (Hcp1) is a member of the Hcp protein family found in Escherichia coli (E. coli), which is unique to T6SS and makes up the bulk of its extracellular structure [7,8]. Hcp1 contributes to the virulence of Edwardsiella ictaluri in catfish [9]. In Burkholderia pseudomallei, Hcp1 stimulates strong IFN-γ-secreting T-cell responses in patients with acute melioidosis and correlates with patient survival [10]. Additionally, in E. coli RS218, Hcp1 is injected into the host cell HBMEC as a bacterial toxin via the T6SS to promote cysteinyl aspartate specific protease-8 (caspase-8) expression and induce apoptosis [11]. Similarly, Zhao et al. found that Hcp1 can promote apoptosis and cause E. coli meningitis [12].
This work aims for a better understanding of how Hcp1 contributes to the virulence of E. coli. We initially removed Hcp1 from wild-type E. coli W24 (WT W24) with CRISPR/Cas9, then evaluated the virulence and histopathological damage posed by WT W24 and Hcp1 knockout E. coli (W24∆hcp1) strains and studied the molecular mechanism of how Hcp1 induces pyroptosis and promotes the occurrence of acute kidney injury (AKI).

Deletion of E. coli Toxin Gene Hcp1
To investigate the role of Hcp1 in E. coli pathogenesis, we deleted the gene expressing Hcp1 in WT W24. The gene structure of Hcp1 is shown in Figure 1A. We designed two sgRNAs to target the Hcp1 gene. Gene editing is shown in Figure 1B. Finally, W24∆hcp1 was obtained, which was confirmed by PCR and gene sequencing ( Figure 1C,D).

Hcp1 Enhances E. coli Virulence against KM Mice
First, we established a mouse model of acute infection caused by E. coli (Figure 2A). Dissection revealed that Hcp1-sufficient WT W24 induced severe organ damage, a consequence similar to that caused by direct injection of LPS. In contrast, in mice infected with the W24∆hcp1 strain, the damage was not present ( Figure 2B). Acutely infected mice were clinically scored, with those infected with WT W24 showing severe damage, those injected with LPS showing moderate damage, and those infected with W24∆hcp1 showing mild damage. Moreover, we measured the body temperature of infected mice, which peaked 24 or 36 h after infection ( Figure 2C,D). Mice infected with WT W24 developed a more serious systemic infection than those infected with W24∆hcp1, with significantly increased bacterial loads in the blood, spleen, liver, and kidney. As expected, mice injected with LPS also showed serious systemic disease ( Figure 2E-I).
Moreover, we found a large number of inflammatory factors, including IL-18, IL-1β, TNF-α, and IFN-γ in the serum of infected mice, and the level of inflammatory factors in the serum of mice infected with the W24∆hcp1 strain was reduced in the serum of mice infected with WT W24. ( Figure 2J). Additionally, acute infection with the WT W24 strain caused tissue damage to the lung, spleen, and small intestine, but in the mice infected with the W24∆hcp1 strain, these symptoms were not present ( Figure 2K).    . All data are shown as the mean ± SD. **** p < 0.0001, *** p < 0.001, and * p < 0.05.

Hcp1 Promotes E. coli-Induced Acute Liver Injury in Mice
WT W24 infection caused more severe liver damage than W24∆hcp1 infection, as revealed by higher serum ALT and AST levels in WT W24-infected mice ( Figure 3A,B). The macroscopic image of the liver is presented in Figure 3C. It can be seen that the infection with Hcp1-sufficient WT W24 significantly promotes hepatomegaly. Transcript levels of IL-18 and IL-1β were significantly increased in liver tissues after WT W24 infection, and this consequence was similar to that caused by LPS injection. However, the inflammatory factor levels were significantly lower in mice with W24∆hcp1 infection than in those with WT W24 infection ( Figure 3D), which was confirmed by liver ELISA and liver IHC ( Figure 3E-G).  coli Hcp1 damage to hepatocytes (typical injuries are depicted by the red arrows). All data are shown as the mean ± SD. **** p < 0.0001, *** p < 0.001, and ** p < 0.01.
Given the apparent detrimental effects of Hcp1 on liver function, a histopathological examination of morphological changes in the liver was performed. Histopathology revealed that WT W24 infection caused severe congestion, hemorrhage, and structural damage in liver tissues ( Figure 3F). Similarly, TEM showed that WT W24 infection resulted in chromatin alteration and liver cell death, which were alleviated in livers infected with W24∆hcp1 ( Figure 3G). These results suggest that Hcp1 of E. coli promotes acute liver injury (ALI).

Hcp1 Promotes E. coli-Induced Acute Kidney Injury in Mice
We addressed the toxicological effects of Hcp1 on the kidney after finding that it can cause ALI. Kidneys were significantly enlarged after WT W24 infection, while the kidney index was relatively lower after W24∆hcp1 infection ( Figure 4A). Based on serum renal function (BUN and SCr) indicators, it was found that W24 infection-induced AKI was similar to that caused by direct LPS injection in mice. In addition, WT W24 infection caused more severe renal injury than W24∆hcp1 infection ( Figure 4B,C). IHC showed that in WT W24-infected kidneys, a large number of IL-18 and IL-1β were expressed, mainly in renal tubular epithelial cells, while their expression was reduced in W24∆hcp1-infected kidneys ( Figure 4D). Similarly, ELISA of kidney tissue showed that in WT W24-infected kidneys, a large number of IL-18, IL-1β, and TNF-α, were expressed, but their expression was significantly reduced in W24∆hcp1-infected kidneys ( Figure 4E).
Moreover, renal histopathology demonstrated that WT W24 was more virulent, manifested as the loss of more nuclei and invasion of more inflammatory cells. In extravasated blood, the glomerulus was enlarged, the renal tubular epithelial cells were necrotic, and the lumen was filled with serous fluid, fibrin, and neutrophils. No visible lesions were found in the control group. Similarly, TEM observation showed that cell damage was severe after WT W24 infection in mice. For example, nucleus pycnosis of most renal tubular epithelial cells and dissolution of small nucleoli occurred; euchromatin edge set part of the cell nucleolus side together. The microvilli on the cell surface were sparsely arranged, the base was swollen, and the pleura disappeared. Mitochondria increased and swelled. The damage was alleviated in W24∆hcp1-infected livers ( Figure 4D,F). These results suggest that the Hcp1 promotes AKI. After finding that Hcp1 can promote ALI, we further explored the toxicological effects of Hcp1 on the kidney.

Hcp1 Induces Pyroptosis in the Kidney
One of the characteristics of pyroptosis is the large production of IL-18 and IL-1β [23,24]. According to the above results, it is speculated that pyroptosis occurs in the kidneys of mice infected with E. coli W24. IHC results showed that each factor in the pyroptosis classical pathway NLRP3-caspase-1-GSDMD was mainly expressed in renal tubular epithelial cells. Furthermore, WT W24 infection more significantly promotes the expression of these factors than W24∆hcp1 infection ( Figure 5B-E). Additionally, mRNA levels of pyroptosis-related genes (NLRP3, ASC, caspase-1, GSDMD, IL-18, and IL-1β) in the kidneys with W24∆hcp1 infection are considerably lower than in those with WT W24 infection ( Figure 5A).   . The livers' mean density of NLRP3, caspase-1, and GSDMD was determined (n = 5). All data are shown as the mean ± SD. **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05.
Genomic DNA in pyroptosis nuclei is disrupted, exposing 3′-OH and making the cells TUNEL positive [16,25]. WT W24 infection can promote DNA fragmentation in renal tubular epithelial cells, increasing the TUNEL-positive ratio; in W24Δhcp1-infected kidneys, TUNEL-positive cells are significantly fewer ( Figure 6A,B). Furthermore, WB results showed that WT W24 infection activated the NLRP3 inflammasome, while the degree of inflammasome activation was significantly reduced in W24Δhcp1-infected kidneys, which was consistent with the IHC results. In addition, we also found that caspase-1 was activated in infected kidneys, which was facilitated by Hcp1-replete E. coli ( Figure 6C,D). We observed the cleavage of GSDMD in WT W24-infected kidneys and found abundant GSDMD-N, an executor of pyroptosis [26,27]. Furthermore, active forms of IL-1β were All data are shown as the mean ± SD. **** p < 0.0001, *** p < 0.001, ** p < 0.01, and * p < 0.05.
Genomic DNA in pyroptosis nuclei is disrupted, exposing 3 -OH and making the cells TUNEL positive [16,25]. WT W24 infection can promote DNA fragmentation in renal tubular epithelial cells, increasing the TUNEL-positive ratio; in W24∆hcp1-infected kidneys, TUNEL-positive cells are significantly fewer ( Figure 6A,B). Furthermore, WB results showed that WT W24 infection activated the NLRP3 inflammasome, while the degree of inflammasome activation was significantly reduced in W24∆hcp1-infected kidneys, which was consistent with the IHC results. In addition, we also found that caspase-1 was activated in infected kidneys, which was facilitated by Hcp1-replete E. coli ( Figure 6C,D). We observed the cleavage of GSDMD in WT W24-infected kidneys and found abundant GSDMD-N, an executor of pyroptosis [26,27]. Furthermore, active forms of IL-1β were found in infected kidneys, and they were significantly reduced in W24∆hcp1-infected kidneys ( Figure 6C,D). These results validate our hypothesis that pyroptosis occurs in W24 E. coli infection-induced ALI and that Hcp1-sufficient W24 promotes pyroptosis and then exacerbates ALI.
found in infected kidneys, and they were significantly reduced in W24Δhcp1-infected kidneys ( Figure 6C,D). These results validate our hypothesis that pyroptosis occurs in W24 E. coli infection-induced ALI and that Hcp1-sufficient W24 promotes pyroptosis and then exacerbates ALI.  D). Western blotting was used to detect the expression levels of NLRP3, ASC, caspase-1, GSDMD, IL-1β, and IL-18 in the kidneys of mice in different treatment groups and compared with β-actin (n = 5). All data are shown as the mean ± SD. **** p < 0.0001, *** p < 0.001, and * p < 0.05.

Hcp1 Binds to NLRP3 Protein
Section 2.5 show that Hcp1 can promote NLRP3 expression. It is hypothesized that Hcp1 will be detected by NLRP3 after entering cells and might adhere to it, promoting the expression of NLRP3. HDOCK SERVER was used for protein docking, and then SWISS-MODEL was used to generate the three-dimensional structure of Hcp1. This structure is  D). Western blotting was used to detect the expression levels of NLRP3, ASC, caspase-1, GSDMD, IL-1β, and IL-18 in the kidneys of mice in different treatment groups and compared with β-actin (n = 5). All data are shown as the mean ± SD. **** p < 0.0001, *** p < 0.001, and * p < 0.05.

Hcp1 Binds to NLRP3 Protein
Section 2.5 show that Hcp1 can promote NLRP3 expression. It is hypothesized that Hcp1 will be detected by NLRP3 after entering cells and might adhere to it, promoting the expression of NLRP3. HDOCK SERVER was used for protein docking, and then SWISS-MODEL was used to generate the three-dimensional structure of Hcp1. This structure is displayed in Figure 7A (PDB: 4W64). Then, AlphaFold was used to obtain the 3D NLRP3 result ( Figure 7B).  Figure 7A (PDB: 4W64). Then, AlphaFold was used to obtain the 3D NLRP3 result ( Figure 7B). HDOCK SERVER predicted 10 good combination models (docking scores and confidence scores are shown in Supplementary Table S4). The optimal results we selected (Figure 7C) show that Hcp1 can combine well with NLRP3, with an interconnection score of −266.70 and a confidence score of 0.9117 (the docking score is around −200 and when the confidence score is above 0.7, it is better, and the two molecules are most likely to bind), which supports our hypothesis. HDOCK SERVER predicted 10 good combination models (docking scores and confidence scores are shown in Supplementary Table S4). The optimal results we selected ( Figure 7C) show that Hcp1 can combine well with NLRP3, with an interconnection score of −266.70 and a confidence score of 0.9117 (the docking score is around −200 and when the confidence score is above 0.7, it is better, and the two molecules are most likely to bind), which supports our hypothesis.

Discussion
Our results demonstrate that Hcp1 can increase the virulence of E. coli in KM mice, leading to a more serious systemic infection and promoting both ALI and AKI. We found that pyroptosis is involved in AKI and is highly correlated with Hcp1, and Hcp1 promotes the expression of pyroptosis-related genes or proteins in the kidney. Furthermore, pyroptosis promotes the activation of the NLRP3 inflammasome and promotes the expression of active caspase-1, which cleaves GSDMD-N and accelerates the release of active IL-1β and ultimately leads to cell death. These studies reveal that Hcp1 is an essential bacterial toxin in pathogenic E. coli and promotes AKI by inducing pyroptosis.
In this study, we identified and isolated WT W24 ( Figure S2) from the feces of diarrheal piglets from a pig farm in Yunnan, China, and performed a sequencing analysis, which has not been published yet. Hcp1, a bacterial-derived toxin protein of the T6SS, can also be secreted into host cells to cause damage [28,29]. When mice were infected with WT W24, we found high levels of TNF-α, IFN-γ, IL-1β, and IL-18 in their blood, livers, and kidneys. These are typical inflammatory factors, and their massive release means a robust inflammatory response e [30]. In WT W24, Hcp1 increases bacterial load across tissues and promotes systemic infection; E. coli PCN003 infection achieves similar results [7]. In E. coli RS218, Hcp1 causes the release of massive IL-6 and IL-8. The induction of HBMC apoptosis by promoting the expression of active caspase-8 is a mechanism for inducing bacterial meningitis [11]. These results suggested that Hcp1 could promote inflammation and enhance the pathogenicity of different strains. Although Hcp1 has been shown to promote injury as a one-minute toxin protein, no systematic studies on its pathogenicity have been conducted. Our study found that Hcp1-expressing E. coli W24 was more lethal, promoting systemic infection like strain PCN003. We also observed pathological damage to internal organs, including the spleen, lung, and small intestine, but the damage was alleviated in W24∆hcp1-infected mice. These findings imply that Hcp1 increases the pathogenicity of E. coli in KM mice.
We also found that the damage to the liver caused by E. coli W24 is typical ALI [31], which is similar to that induced by LPS. WT W24 damages liver function and structure and promotes the expression and release of many inflammatory factors. Furthermore, it can also be observed by TEM that liver cells are in a state of death edge, and all of these phenomena are highly correlated with Hcp1. Our data also show that E. coli W24 promotes AKI, which is consistent with the findings in previous studies [32,33]. The attenuation of kidney and liver function, the infiltration of inflammatory cells, the expression and release of inflammatory factors, and the accumulation of a large number of mitochondria in renal tubular epithelial cells were observed by TEM. The reason may be that the host needs a lot of energy and ROS to clear bacteria after infection, and progress has been made in studies on this topic. In contrast, these problems were alleviated in W24∆hcp1-infected mice, suggesting that Hcp1 can act as a toxin protein to enhance the virulence of E. coli, causing severe damage in mice.
Pyroptosis is a gene-regulated programmed death whose canonical pathway is mediated by the NLRP3 inflammasome [34,35]. Recent studies have found that Citrobacter freundii can activate the NLRP3 inflammasome through the T6SS and cause pyroptosis in BMMs. Furthermore, one of the hallmarks of pyroptosis is the massive release of IL-18 and IL-1β [23,24]. Our results show thatin E. coli-induced AKI, Hcp1 can promote the massive expression and release of IL-18 and IL-1β in the kidney. It is speculated that pyroptosis is involved in E. coli-induced AKI, and Hcp1 can promote pyroptosis. The research findings are in line with the expectations. Hcp1 promotes the activation of the NLRP3 inflammasome and the production of active caspase-1. Active caspase-1 cleaves GS-DMD, and pro-IL-1β generates GSDMD-N to induce pyroptosis, this is a typical pyroptosis pathway [36]. After WT W24 strain infection, NLRP3, caspase-1, GSDMD-N, and other factors in the classical pyroptosis pathway were found in the kidney, mainly in renal tubular epithelial cells. Moreover, TUNEL staining shows that many renal tubular epithelial cells have DNA breaks, which is another sign of pyroptosis [16,25]. DNA breaks were localized in renal tubular epithelial cells, suggesting that pyroptosis may occur in there, but further studies are needed. Although our findings indicate that pyroptosis is weakened following Hcp1 infection, it can still be found in the Hcp1 infection group. The results suggest that other virulence factors in E. coli may promote pyroptosis, which needs further confirmation. Additionally, using protein docking, we found that Hcp1 and NLRP3 would bind together in idealized situations. The result requires additional experimental confirmation using techniques such as CO-IP. Evidence suggests that pyroptosis is one cause of AKI [37]. Hcp1 can promote the occurrence of AKI in mice and the pyroptosis of renal tubular epithelial cells. It is suggested that Hcp1-induced pyroptosis is one of the mechanisms by which E. coli causes AKI.
This study has certain limitations. Although it is confirmed that pyroptosis is involved in AKI and pyroptosis-related proteins are mainly expressed in renal tubular epithelial cells, cell experiments still need to be conducted for further research. In addition, our research is limited to the classical signaling pathway of pyroptosis. Whether the non-classical pathway is involved in Hcp1-promoted AKI requires further investigation.
In conclusion, Hcp1 can increase the pathogenicity of WT W24, promotes ALI and AKI in KM mice, and accelerates inflammatory responses. Hcp1-induced pyroptosis is one of the molecular mechanisms of AKI.These results provide new insights into the pathogenic mechanism of pathogenic E. coli, and revealed the virulence contribution of Hcp1 as an essential bacterial toxin in pathogenic E. coli.

Strains and Plasmids
All E. coli W24 and E. coli DH5α strains were grown at 37 • C or 30 • C in Luria-Bertani (LB) broth with appropriate antibiotics and shaking. The bacterial strains and plasmids used in this study are given in Supplementary Table S1.

Deletion of E. coli Hcp1
In brief, for the convenience of gene editing, we used the primers in Supplementary  Table S2 to construct the pUC-spacer-Donor plasmid according to the process in Supplementary Figure S1.
W24 competent cells harboring pCas were prepared as described previously [38]. In accordance with the procedure, arabinose (30 mM final concentration) was given to the culture for λ-Red induction. The transformation was done by electroporation using the GenePulser Xcell Electroporation System (Bio-Rad, Hercules, CA, USA). For electroporation, 80 µL of cells were mixed with 200 ng of pUC-spacer-Donor series DNA; electroporation was done in a 2 mm Gene Pulser cuvette (Bio-Rad) at 1.8 kV, and the product was suspended immediately in 1 mL of ice-cold SOC medium. Cells were recovered at 30 • C for 2 h before being spread onto LB agar containing kanamycin (50 mg/L) and Ampicillin (100 mg/L) and incubated overnight at 30 • C. Transformants were identified by colony PCR, and DNA sequencing was prepared as described previously [39,40].

KM Mice Infection Model
The Experimental Animal Center at Kunming Medical University sold us Kunming mice (20-25 g). (Kunming, China). Mice were randomly allocated to experimental animal groups and kept in a pathogen-free cage, in a room with a photoperiod of 12 h of light and 12 h of darkness, at a temperature of 25 ± 2 • C and ambient humidity levels of 40-60%. The Yunnan Agriculture University's Animal Ethics Committee (Kunming, China) approved the experimental program.
The mice intraperitoneally injected with LPS were set as positive controls since it has been demonstrated that LPS may cause inflammation to accelerate pyroptosis [20,36].
Sixty Kunming mice were randomized into four groups: the control group, the WT W24 group, the W24∆hcp1 group, and the LPS group. The LPS group was only intraperitoneal (i.p.) injected with LPS (4 mg/kg, mice), the control group was i.p. injected with LB, the WT W24 group and the W24∆hcp1 group were challenged with 2 mL of E. coli strains (1 × 10 7 CFU/mL) by an i.p. injection to evaluate the pathogenicity of the different E. coli strains. The survival rate and body temperature were recorded.

Clinical Signs and Assigned Clinical Scores
Following Figure 3A, all experimental KM mice were scored and assigned clinical scores as follows [41]: 0 indicates a normal reaction to stimuli, 1 a ruffled coat and a delayed response, 2 a response to repeated stimuli only, 3 an inability to respond or circling in place, and 4 a dead. Mice were mercifully put to death if they had significant lethargy or neurological signs (score = 3).

Biochemical Analysis and ELISA Assay
A tail-vein bleed collected blood from mice 24 h after infection. An automated biochemical analyzer model 7020 was used to test the levels of AST, ALT, BUN, and SCr (Hitachi, Chiyoda-ku, Japan).
The levels of IL-1β, IL-18, TNF-α and IFN-γ (Jiancheng Bioengineering, Nanjing, China) in serum or tissue samples were determined by ELISA kit, according to the manufacturer's protocols.

Liver and Kidney Organ Index
36 h after infection, the mice in each group were weighed (free access to water), the liver and kidneys were removed in a clean environment, the surface fat and mesangium were removed, and an electronic balance was used to weigh and record the results. The organ index was calculated using the formula: organ index = organ weight (mg)/body weight (g).

Histopathology
Tissues were fixed with 4% paraformaldehyde overnight at 4 • C, followed by dehydration through an alcohol-xylene series, and finally embedded in paraffin. Leica 2235 (Wetzlar, Germany)was used to cut the kidney sections (5 m thick), which were then dried at 37 • C, deparaffinized, rehydrated utilizing a succession of xylene-alcohol solutions, washed with deionized water, and lastly stained with H&E (Servicebio, Wuhan, China).

Transmission Electron Microscopy
Two percent glutaraldehyde was used to fix renal tissue samples. Then, they were washed in PBS (0.01 M) and postfixed with 1% osmium tetroxide. After gradient dehydration in acetone, the tissue samples were embedded in Araldite M (Sigma Aldrich, St. Louis, MO, USA). Ultrathin sections were cut using an ultramicrotome (Leica, Wetzlar, Germany) and stained with uranyl acetate and lead citrate. The sections were examined with a transmission electron microscope (H-7700, Hitachi, Japan).

RNA Extraction and Real-Time PCR
Following the manufacturer's instructions, total RNA was extracted from mouse tissues using a Takara-made RNAiso Plus kit (Dalian, China). The PrimeScript RT Master Mix kit was used to reverse-transcribe one microgram of total RNA into cDNA (Takara, Dalian, China). SYBR Premix Ex Taq II (Takara, Dalian, China) was used for real-time PCR experiments, and CFX96TM real-time equipment was used for analysis (Bio-Rad, Hercules, CA, USA). The housekeeping gene β-actin was the reference to which relative changes in mRNA were computed using the ∆∆Ct method. Supplementary Table S3 provides the primer sequences that were employed, and Supplementary Table S5 provides the PCR conditions and reaction mixtures.

TUNEL Staining
The previously stated procedures were utilized to make kidney slices, and a Colorimetric TUNEL Apoptosis Assay Kit (Beyotime, Haimen, China) was used to look for cellular DNA damage. The processes were carried out as directed by the manufacturer.

Statistical Analysis
The findings are given as mean ± SD and were statistically analyzed using Graph-Pad Prism 9.0 software (GraphPad Software, San Diego, CA, USA). Analysis of variance (ANOVA) or a t-test was used to analyze the differences between groups. A p-value of <0.05 was regarded as significant.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/toxins15030171/s1, Figure S1: sgRNA expression cassette and plasmid construction process; Figure S2: Clinical isolation of E. coli; Table S1: Strains and plasmids used in this study; Table S2: Primers used for the PCR amplification; Table S3: Primers used for qPCR analysis; Table S4: Docking scores and confidence scores; Table S5: Reaction system and procedure for qPCR analysis.