Identification of autophagy receptors for the Crohn's disease-associated adherent-invasive Escherichia coli.

Introduction: Crohn's disease (CD) is a chronic inflammatory bowel disease, of which the etiology involves genetic, environmental and microbial factors. Adherent-invasive Escherichia coli (AIEC) and polymorphisms in autophagy-related genes have been implicated in CD etiology. Autophagy is a key process for the maintenance of cellular homeostasis, which allows the degradation of damaged cytoplasmic components and pathogens via lysosome. We have shown that a functional autophagy is necessary for AIEC clearance. Here, we aimed at identifying the autophagy receptor(s) responsible to target AIEC to autophagy for degradation. Methods: The levels of autophagy receptors p62, NDP52, NBR1, TAX1BP1 and Optineurin were knocked down in human intestinal epithelial cells T84 using siRNAs. The NDP52 knock-out (KO) and p62 KO HeLa cells, as well as NDP52 KO HeLa cells expressing the wild-type NDP52 or the mutated NDP52Val248Ala protein were used. Results and discussion: We showed that, among the tested autophagy receptors (p62, NDP52, NBR1, TAX1BP1 and Optineurin), diminished expression of p62 or NDP52 increased the number of the clinical AIEC LF82 strain inside epithelial cells. This was associated with increased pro-inflammatory cytokine production. Moreover, p62 or NDP52 directly colocalized with AIEC LF82 and LC3, an autophagy marker. As the NDP52Val248Ala polymorphism has been associated with increased CD susceptibility, we investigated its impact on AIEC control. However, in HeLa cell and under our experimental condition, no effect of this polymorphism neither on AIEC LF82 intracellular number nor on pro-inflammatory cytokine production was observed. Together, our results suggest that p62 and NDP52 act as autophagy receptors for AIEC recognition, controlling AIEC intracellular replication and inflammation.

Nucleoside-diphosphate kinase of uropathogenic Escherichia coli inhibits caspase-1-dependent pyroptosis facilitating urinary tract infection.

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infection (UTI). UPEC invades bladder epithelial cells (BECs) via fusiform vesicles, escapes into the cytosol, and establishes biofilm-like intracellular bacterial communities (IBCs). Nucleoside-diphosphate kinase (NDK) is secreted by pathogenic bacteria to enhance virulence. However, whether NDK is involved in UPEC pathogenesis remains unclear. Here, we find that the lack of ndk impairs the colonization of UPEC CFT073 in mouse bladders and kidneys owing to the impaired ability of UPEC to form IBCs. Furthermore, we demonstrate that NDK inhibits caspase-1-dependent pyroptosis by consuming extracellular ATP, preventing superficial BEC exfoliation, and promoting IBC formation. UPEC utilizes the reactive oxygen species (ROS) sensor OxyR to indirectly activate the regulator integration host factor, which then directly activates ndk expression in response to intracellular ROS. Here, we reveal a signaling transduction pathway that UPEC employs to inhibit superficial BEC exfoliation, thus facilitating acute UTI.

A bacterial toxin co-opts caspase-3 to disable active gasdermin D and limit macrophage pyroptosis.

During infections, host cells are exposed to pathogen-associated molecular patterns (PAMPs) and virulence factors that stimulate multiple signaling pathways that interact additively, synergistically, or antagonistically. The net effect of such higher-order interactions is a vital determinant of the outcome of host-pathogen interactions. Here, we demonstrate one such complex interplay between bacterial exotoxin- and PAMP-induced innate immune pathways. We show that two caspases activated during enterohemorrhagic Escherichia coli (EHEC) infection by lipopolysaccharide (LPS) and Shiga toxin (Stx) interact in a functionally antagonistic manner; cytosolic LPS-activated caspase-11 cleaves full-length gasdermin D (GSDMD), generating an active pore-forming N-terminal fragment (NT-GSDMD); subsequently, caspase-3 activated by EHEC Stx cleaves the caspase-11-generated NT-GSDMD to render it nonfunctional, thereby inhibiting pyroptosis and interleukin-1ß maturation. Bacteria typically subvert inflammasomes by targeting upstream components such as NLR sensors or full-length GSDMD but not active NT-GSDMD. Thus, our findings uncover a distinct immune evasion strategy where a bacterial toxin disables active NT-GSDMD by co-opting caspase-3.

METTL3 enhances E. coli F18 resistance by targeting IKBKG/NF-κB signaling via an m6A-YTHDF1-dependent manner in IPEC-J2 cells.

Post-weaning diarrhea caused by enterotoxigenic E. coli F18 introduces enormous losses to the porcine industry. N6-methyladenosine (m6A) is a ubiquitous epitranscriptomic biomarker that modulates host cell resistance to pathogen infection, however, its significance in E. coli F18-treated IPEC-J2 cells remains unexplored. Herein, we revealed that m6A and associated modulators strongly controlled E. coli F18 susceptibility. The data indicated an enhancement of METTL3 contents in E. coli F18-treated IPEC-J2 cells. METTL3 is known to be a major modulator of E. coli F18 adhesion within IPEC-J2 cells. As expected, METTL3 deficiency was observed to reduce m6A content at the IKBKG 5'-UTR, leading to critical suppression of YTHDF1-dependent IKBKG translation. Therefore, the activation of the NF-κB axis was observed, which enhanced IPEC-J2 resistance to E. coli F18 infection. Taken together, these findings uncover a potential mechanism underlying the m6A-mediated control of E. coli F18 susceptibility. This information may contribute to the establishment of new approaches for combating bacteria-induced diarrhea in piglets.

Effect of estradiol after bacterial infection on the Wnt/ß-catenin pathway in bovine endometrium epithelial cells and organoids.

Endometritis is a disease caused by a postpartum bacterial infection with a poor prognosis that primarily affects dairy cows. Three-dimensional organoids have been used as a model for endometritis, because they exhibit a structure comparable to that of the endometrium, demonstrating both expansibility and hormone responsiveness. These characteristics render them an ideal platform for in vitro investigations of endometrial diseases. Estradiol (E2) is an endogenous steroid hormone with demonstrated anti-inflammatory properties, and the objective of this study was to determine the mechanism by which E2 modulates the inflammatory response and the Wnt signal transduction pathway in bovine endometrial epithelial cells and organoids following E. coli infection. We present the techniques for isolating and culturing primary bovine endometrial epithelial cells (BEECs), and producing endometrial organoids. For the experiments, the endometrial epithelial cells and organoids were infected with E. coli for 1 h, followed by incubation with E2 for 12 h. The mRNA and protein expressions of the inflammation-related genes, IL-1ß, IL-6, TLR4, and NF-κB, as well as the Wnt pathway-related genes, Wnt4, ß-catenin, c-Myc, and CyclinD1, were assessed using real-time quantitative-PCR and western blotting, respectively. The CCK8 viable cell counting assay was utilized to determine the optimal concentration of the Wnt inhibitor, IWR-1. The mRNA and protein expression of Wnt pathway-related genes was assessed following IWR-1 treatment, while the expression levels of proliferation-associated genes (Ki67, PCNA) and barrier repair genes (occludin, claudin, and Zo-1) in BEECs and organoids were evaluated after E2 treatment. The results of this study show that mRNA expression of the inflammatory genes, IL-1ß, TLR4, and NF-κB (P < 0.05) decreased in BEECs following E2 treatment compared to the E. coli group. The protein expression of the IL-1ß, IL-6, TLR4 and NF-κB genes was also inhibited (P < 0.05). Similar results were observed in tests on the organoids. Our findings demonstrate that E2 significantly upregulates the expression of Wnt-related genes, including ß-catenin and c-Myc, while concurrently downregulating the expression of GSK3ß (P < 0.05). Next, we treated E. coli-infected BEECs and organoids with the Wnt inhibitor, IWR-1. Compared with E. coli and E. coli + E2, the expression of mRNA and protein from Wnt 4, ß-catenin, and CyclinD1 in E. coli + E2 and E. coli + IWR-1 was down-regulated (P < 0.05). The expression of the proliferation genes, Ki67, PCNA, and the tight junction genes, occludin, claudin1, and Zo-1, in organoids was significantly higher than that in BEECs (P < 0.05). In summary, we found strong potential for E2 mitigation of the E. coli-induced inflammatory response in BEECs and organoids, through activation of the Wnt pathway. In addition, the proliferation and repair capacity of organoids was much higher than that of BEECs.

Allosteric inhibitor of SHP2 enhances macrophage endocytosis and bacteria elimination by increasing caveolae activation and protects against bacterial sepsis.

The uncontrolled bacterial infection-induced cytokine storm and sequential immunosuppression are commonly observed in septic patients, which indicates that the activation of phagocytic cells and the efficient and timely elimination of bacteria are crucial for combating bacterial infections. However, the role of dysregulated immune cells and their disrupted function in sepsis remains unclear. Here, we found that macrophages exhibited the impaired endocytosis capabilities in sepsis by Single-cell RNA sequencing and bulk RNA sequencing. Caveolae protein Caveolin-1 (Cav-1) of macrophages was inactivated by SHP2 rapidly during Escherichia coli (E.coli) infection. Allosteric inhibitor of SHP2 effectively maintains Cav-1 phosphorylation to enhance macrophage to endocytose and eliminate bacteria. Additionally, TLR4 endocytosis of macrophage was also enhanced upon E.coli infection by SHP099, inducing an increased and rapidly resolved inflammatory response. In vivo, pretreatment or posttreatment with inhibitor of SHP2 significantly reduced the bacterial burden in organs and mortality of mice subjected E.coli infection or CLP-induced sepsis. The cotreatment of inhibitor of SHP2 with an antibiotic conferred complete protection against mortality in mice. Our findings suggest that Cav-1-mediated endocytosis and bacterial elimination may play a critical role in the pathogenesis of sepsis, highlighting inhibitor of SHP2 as a potential therapeutic agent for sepsis.

Lcn2 deficiency accelerates the infection of Escherichia coli O157:H7 by disrupting the intestinal barrier function.

Bacterial infections result in intestinal inflammation and injury, which affects gut health and nutrient absorption. Lipocalin 2 (Lcn2) is a protein that reacts to microbial invasion, inflammatory responses, and tissue damage. However, it remains unclear whether Lcn2 has a protective effect against bacterial induced intestinal inflammation. Therefore, this study endeavors to investigate the involvement of Lcn2 in the intestinal inflammation of mice infected with Enterohemorrhagic Escherichia coli O157:H7 (E. coli O157:H7). Lcn2 knockout (Lcn2-/-) mice were used to evaluate the changes of inflammatory responses. Lcn2 deficiency significantly exacerbated clinical symptoms of E. coli O157:H7 infection by reducing body weight and encouraging bacterial colonization of. Compared to infected wild type mice, infected Lcn2-/- mice had significantly elevated levels of pro-inflammatory cytokines in serum and ileum, including interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α), as well as severe villi destruction in the jejunum. Furthermore, Lcn2 deficiency aggravated intestinal barrier degradation by significantly reducing the expression of tight junction proteins occludin and claudin 1, the content of myeloperoxidase (MPO) in the ileum, and the number of goblet cells in the colon. Our findings indicated that Lcn2 could alleviate inflammatory damage caused by E. coli O157:H7 infection in mice by enhancing intestinal barrier function.

PUFAs add fuel to Crohn's disease-associated AIEC-induced enteritis by exacerbating intestinal epithelial lipid peroxidation.

Polyunsaturated fatty acids (PUFAs) have been shown to exacerbate Crohn's disease (CD) by promoting lipid peroxidation (LPO) of intestinal epithelial cells (IECs). Dysbiosis of the gut microbiota may play a crucial role in this process. CD patients often exhibit an increased abundance of Escherichia coli (E. coli) in the gut, and the colonization of adherent-invasive E. coli (AIEC) is implicated in the initiation of intestinal inflammation in CD. However, the impact of AIEC on LPO remains unclear. In this study, we observed that AIEC colonization in the terminal ileum of CD patients was associated with decreased levels of glutathione peroxidase 4 (GPX4) and ferritin heavy chain (FTH) in the intestinal epithelium, along with elevated levels of 4-Hydroxynonenal (4-HNE). In vitro experiments demonstrated that AIEC infection reduced the levels of GPX4 and FTH, increased LPO, and induced ferroptosis in IECs. Furthermore, arachidonic acid (AA) and docosahexaenoic acid (DHA) supplementation in AIEC-infected IECs significantly aggravated LPO and ferroptosis. However, overexpression of GPX4 rescued AIEC-induced LPO and ferroptosis in IECs. Our results further confirmed that AIEC with AA supplementation, associated with excessive LPO and cell death in IECs, worsened colitis in the DSS mouse model and induced enteritis in the antibiotic cocktail pre-treatment mouse model in vivo. Moreover, treatment with ferrostatin-1, a ferroptosis inhibitor, alleviated AIEC with AA supplementation-induced enteritis in mice, accompanied by reduced LPO and cell death in IECs. Our findings suggest that AIEC, in combination with PUFA supplementation, can induce and exacerbate intestinal inflammation, primarily through increased LPO and ferroptosis in IECs.

Response mechanisms to acid stress promote LF82 replication in macrophages.

Background: Adherent-invasive E. coli (AIEC) LF82 is capable of adhering to and invading intestinal epithelial cells, as well as replicating within macrophages without inducing host cell death. Methods: We compared the transcriptomics of LF82 at pH=7.5 and pH=5.8 by RNA-sequencing, and qRT-PCR verified differentially expressed genes (DEGs). The deletion mutants of DEGs in the treatment group (pH=5.8) compared to the control group (pH=7.5) were constructed by λ recombinant. The replication differences between the mutants and WT infected Raw 264.7 at 24 h.p.i were analyzed by combining LB solid plate count and confocal observation. NH4Cl and chloroquine diphosphate (CQ) were used for acid neutralization to study the effect of pH on the replication of LF82 in macrophages. Na2NO3 was added to RPMI 1640 to study the effect of nitrate on the replication of LF82 in macrophages. 0.3% solid LB was used for flagellar motility assay and Hela was used to study flagellar gene deletion mutants and WT adhesion and invasion ability. Results: In this study, we found that infection with LF82 results in acidification of macrophages. Subsequent experiments demonstrated that an intracellular acidic environment is necessary for LF82 replication. Transcriptome and phenotypic analysis showed that high expression of acid shock genes and acid fitness genes promotes LF82 replication in macrophages. Further, we found that the replication of LF82 in macrophages was increased under nitrate treatment, and nitrogen metabolism genes of LF82 were upregulated in acid treatment. The replication in macrophages of ΔnarK, ΔnarXL, ΔnarP, and Δhmp were decreased. In addition, we found that the expression of flagellar genes was downregulated in acidic pH and after LF82 invading macrophages. Motility assay shows that the movement of LF82 on an acidic semisolid agar plate was limited. Further results showed that ΔfliC and ΔfliD decreased in motility, adhesion ability, and invasion of host cells, but no significant effect on replication in macrophages was observed. Conclusion: In this study, we simulated the acidic environment in macrophages, combined with transcriptome technology, and explained from the genetic level that LF82 promotes replication by activating its acid shock and fitness system, enhancing nitrate utilization, and inhibiting flagellar function.

Shiga toxin targets the podocyte causing hemolytic uremic syndrome through endothelial complement activation.

BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS) is the leading cause of acute kidney injury in children, with an associated mortality of up to 5%. The mechanisms underlying STEC-HUS and why the glomerular microvasculature is so susceptible to injury following systemic Stx infection are unclear. METHODS: Transgenic mice were engineered to express the Stx receptor (Gb3) exclusively in their kidney podocytes (Pod-Gb3) and challenged with systemic Stx. Human glomerular cell models and kidney biopsies from patients with STEC-HUS were also studied. FINDINGS: Stx-challenged Pod-Gb3 mice developed STEC-HUS. This was mediated by a reduction in podocyte vascular endothelial growth factor A (VEGF-A), which led to loss of glomerular endothelial cell (GEnC) glycocalyx, a reduction in GEnC inhibitory complement factor H binding, and local activation of the complement pathway. Early therapeutic inhibition of the terminal complement pathway with a C5 inhibitor rescued this podocyte-driven, Stx-induced HUS phenotype. CONCLUSIONS: This study potentially explains why systemic Stx exposure targets the glomerulus and supports the early use of terminal complement pathway inhibition in this devastating disease. FUNDING: This work was supported by the UK Medical Research Council (MRC) (grant nos. G0901987 and MR/K010492/1) and Kidney Research UK (grant nos. TF_007_20151127, RP42/2012, and SP/FSGS1/2013). The Mary Lyon Center is part of the MRC Harwell Institute and is funded by the MRC (A410).

GILZ Modulates the Recruitment of Monocytes/Macrophages Endowed with a Resolving Phenotype and Favors Resolution of Escherichia coli Infection.

Macrophages are important effectors of inflammation resolution that contribute to the elimination of pathogens and apoptotic cells and restoration of homeostasis. Pre-clinical studies have evidenced the anti-inflammatory and pro-resolving actions of GILZ (glucocorticoid-induced leucine zipper). Here, we evaluated the role of GILZ on the migration of mononuclear cells under nonphlogistic conditions and Escherichia coli-evoked peritonitis. TAT-GILZ (a cell-permeable GILZ-fusion protein) injection into the pleural cavity of mice induced monocyte/macrophage influx alongside increased CCL2, IL-10 and TGF-ß levels. TAT-GILZ-recruited macrophages showed a regulatory phenotype, exhibiting increased expression of CD206 and YM1. During the resolving phase of E. coli-induced peritonitis, marked by an increased recruitment of mononuclear cells, lower numbers of these cells and CCL2 levels were found in the peritoneal cavity of GILZ-deficient mice (GILZ-/-) when compared to WT. In addition, GILZ-/- showed higher bacterial loads, lower apoptosis/efferocytosis counts and a lower number of macrophages with pro-resolving phenotypes. TAT-GILZ accelerated resolution of E. coli-evoked neutrophilic inflammation, which was associated with increased peritoneal numbers of monocytes/macrophages, enhanced apoptosis/efferocytosis counts and bacterial clearance through phagocytosis. Taken together, we provided evidence that GILZ modulates macrophage migration with a regulatory phenotype, inducing bacterial clearance and accelerating the resolution of peritonitis induced by E. coli.

Yersiniabactin-Producing E. coli Induces the Pyroptosis of Intestinal Epithelial Cells via the NLRP3 Pathway and Promotes Gut Inflammation.

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.

C-X-C Motif Chemokine 3 Promotes the Inflammatory Response of Microglia after Escherichia coli-Induced Meningitis.

Meningitis is a major clinical manifestation of Escherichia coli (E. coli) infection characterized by inflammation of the meninges and subarachnoid space. Many chemokines are secreted during meningitic E. coli infection, of which C-X-C motif chemokine 3 (CXCL3) is the most highly expressed. However, it is unclear how CXCL3 plays a role in meningitic E. coli infection. Therefore, this study used in vitro and in vivo assays to clarify these contributions and to identify novel therapeutic targets for central nervous system inflammation. We found a significantly upregulated expression of CXCL3 in human brain microvascular endothelial cells and U251 cells after meningitic E. coli infection, and the CXCL3 receptor, C-X-C motif chemokine receptor 2 (CXCR2), was expressed in microglia. Furthermore, CXCL3 induced M1 microglia by selectively activating mitogen-activated protein kinases signaling and significantly upregulating tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, nitric oxide synthase 2 (NOS2), and cluster of differentiation 86 (CD86) expression levels, promoting an inflammatory response. Our findings clarify the role of CXCL3 in meningitic E. coli-induced neuroinflammation and demonstrate that CXCL3 may be a potential therapeutic target for future investigation and prevention of E. coli-induced neuroinflammation.

Enteroaggregative Escherichia coli induced activation of epidermal growth factor receptor contributes to IL-8 secretion by cultured human intestinal epithelial cells.

Enteroaggregative Escherichia coli (EAEC) has been identified as a new enteropathogen that causes acute and chronic diarrhea in children and travelers. One defining aspect of EAEC-pathogenesis is the induction of an inflammatory response in intestinal epithelium. In this study, we have found that EAEC-induced EGFR activation in human small intestinal and colonic epithelial was attenuated in the presence of a specific inhibitor of EGFR (Tyrphostin AG1478). Further, the aggregative stacked-brick type of adherence of this organism to both the cell lines and this pathogen-induced cytoskeletal rearrangement of these cells was also reduced in the presence of Tyrphostin AG1478. Moreover, EAEC-induced activation of downstream effectors (ERK-1/2, PI3K and Akt) of EGFR mediated cell signaling pathways were found to be suppressed in the presence of EGFR inhibitor. A decrease in IL-8 response in EAEC infected both the cell types were also noted in the presence of specific inhibitors of these downstream effectors, transcription factors and Tyrphostin AG1478. We propose that EAEC-induced activation of EGFR is quintessential for stacked-brick adherence of EAEC to human intestinal epithelial cells, their cytoskeletal rearrangements and stimulation of ERK-1/2 and PI3K/Akt mediated signal transduction pathways, resulting in the activation of NF-κB, AP-1, STAT-3 and finally IL-8 secretion by these cells.

Z. morio Hemolymph Relieves E. coli-Induced Mastitis by Inhibiting Inflammatory Response and Repairing the Blood-Milk Barrier.

Escherichia coli (E. coli) is a major environmental pathogen causing coliform mastitis, characterized by cell death and mammary tissue damage. Our previous study has shown the antimicrobial effect of Zophobas morio (Z. morio) hemolymph against mastitis pathogens. In this study, we established E. coli-induced cellular and animal models for mastitis, aiming to evaluate the protective effect of Z. morio hemolymph against E. coli-induced mastitis in vivo and in vitro. In mice with E. coli, Z. morio hemolymph attenuated bacterial burden and histopathological impairment, reduced the production of interleukin (IL)-1ß, IL-18, tumor necrosis factor-α (TNF-α) and the ratio of CD4+ T/CD8+ T, and increased the production of IL-2 triggered by E. coli. Z. morio hemolymph also enhanced the integrity of the blood-milk barrier in E. coli-induced mastitis. In E. coli-stimulated porcine mammary epithelial cells, Z. morio hemolymph inhibited E. coli-induced inflammatory responses and upregulated tight junction proteins (ZO-1, Claudin-3 and Occludin). Moreover, we found that the anti-inflammatory effect of Z. morio hemolymph was mediated by inhibiting E. coli-induced NLRP3 inflammasome assembly, Caspase-1 activation, and reversing the inhibitory effect of E. coli on autophagy. Besides, Z. morio hemolymph augmented ATG5/ATG16L1-mediated autophagy activation, negatively regulated NLRP3 inflammasome activation. Our results reveal that Z. morio hemolymph alleviates E. coli-induced mastitis via lessening the inflammatory response by regulating the NLRP3 and ATG5/ATG16L1 signaling pathway, as well as repairing the blood-milk barrier.

TcpC Inhibits M1 but Promotes M2 Macrophage Polarization via Regulation of the MAPK/NF-κB and Akt/STAT6 Pathways in Urinary Tract Infection.

TcpC is a multifunctional virulence factor of Uropathogenic Escherichia coli (UPEC). Macrophages can differentiate into two different subsets M1 and M2 that play distinct roles in anti-infection immunity. Here, we investigate the influence of TcpC on M1/M2 polarization and the potential mechanisms. Our data showed that M1 markers CD86 and iNOS were significantly inhibited, while the M2 markers CD163, CD206 and Arg-1 were enhanced in macrophages in kidneys from the TcpC-secreting wild-type CFT073 (CFT073wt)-infected pyelonephritis mouse model, compared with those in macrophages in kidneys from TcpC knockout CFT073 mutant (CFT073Δtcpc)-infected mice. CFT073wt or recombinant TcpC (rTcpC) treatment inhibits LPS + IFN-γ-induced CD80, CD86, TNF-α and iNOS expression, but promotes IL-4-induced CD163, CD206, Arg-1 and IL-10 expression in both human and mouse macrophage cell lines THP-1 and J774A.1. Moreover, rTcpC significantly attenuated LPS + IFN-γ-induced phosphorylation of p38, ERK, p50 and p65 but enhanced IL-4-induced phosphorylation of Akt and STAT6. These data suggest that TcpC inhibits M1 but promotes M2 macrophage polarization by down-regulation of p38, ERK/NF-κB and up-regulation of the Akt/STAT6 signaling pathway, respectively. Our findings not only illuminate the regulatory effects of TcpC on macrophage M1/M2 polarization and its related signaling pathways, but also provide a novel mechanism underlying TcpC-mediated immune evasion of macrophage-mediated innate immunity.

An adherent-invasive Escherichia coli-colonized mouse model to evaluate microbiota-targeting strategies in Crohn's disease.

Adherent-invasive Escherichia coli (AIEC) were investigated for their involvement in the induction/chronicity of intestinal inflammation in Crohn's disease (CD). AIEC gut establishment is favoured by overexpression of the glycoprotein CEACAM6 in the ileal epithelium. We generated a transgenic mouse model, named 'Vill-hCC6', in which the human CEACAM6 gene was under the control of the villin promoter, conditioning expression in the small intestine. We demonstrated that CEACAM6 is strongly expressed in the small intestine mucosa and is correlated with numerous glycosylations displayed at the brush border of enterocytes. Ex vivo, the AIEC-enterocyte interaction was enhanced by CEACAM6 expression and necessitated the presence of the bacterial adhesive factor FimH. Finally, AIEC bacteria preferentially persisted in a FimH-dependent manner in the ileal mucosa of Vill-hCC6 mice compared to wild-type mice. This preclinical model opens new perspectives in the mechanistic study of the AIEC pathobiont and represents a valuable tool to evaluate the efficacy of new strategies to eliminate AIEC implanted in the ileal mucosa, such as phages, inhibitory and/or anti-virulence molecules, or CRISPR-based strategies targeting virulence or fitness factors of AIEC bacteria.

Lactobacillus reuteri 1 Enhances Intestinal Epithelial Barrier Function and Alleviates the Inflammatory Response Induced by Enterotoxigenic Escherichia coli K88 via Suppressing the MLCK Signaling Pathway in IPEC-J2 Cells.

Intestinal epithelial barrier injury disrupts immune homeostasis and leads to many intestinal disorders. Lactobacillus reuteri (L. reuteri) strains can influence immune system development and intestinal function. However, the underlying mechanisms of L. reuteri LR1 that regulate inflammatory response and intestinal integrity are still unknown. The present study aimed to determine the effects of LR1 on the ETEC K88-induced intestinal epithelial injury on the inflammatory response, intestinal epithelial barrier function, and the MLCK signal pathway and its underlying mechanism. Here, we showed that the 1 × 109 cfu/ml LR1 treatment for 4 h dramatically decreased interleukin-8 (IL-8) and IL-6 expression. Then, the data indicated that the 1 × 108 cfu/ml ETEC K88 treatment for 4 h dramatically enhanced IL-8, IL-6, and tumor necrosis factor-α (TNF-α) expression. Furthermore, scanning electron microscope (SEM) data indicated that pretreatment with LR1 inhibited the ETEC K88 that adhered on IPEC-J2 and alleviated the scratch injury of IPEC J2 cells. Moreover, LR1 pretreatment significantly reversed the declined transepithelial electrical resistance (TER) and tight junction protein level, and enhanced the induction by ETEC K88 treatment. Additionally, LR1 pretreatment dramatically declined IL-8, IL-17A, IL-6, and TNF-α levels compared with the ETEC K88 group. Then, ETEC K88-treated IPEC-J2 cells had a higher level of myosin light-chain kinase (MLCK), higher MLC levels, and a lower Rho-associated kinase (ROCK) level than the control group, while LR1 pretreatment significantly declined the MLCK and MLC expression and enhanced ROCK level in the ETEC K88-challenged IPEC-J2 cells. Mechanistically, depletion of MLCK significantly declined MLC expression in IPEC-J2 challenged with ETEC K88 compared to the si NC+ETEC K88 group. On the other hand, the TER of the si MLCK+ETEC K88 group was higher and the FD4 flux in the si MLCK+ETEC K88 group was lower compared with the si NC+ETEC K88 group. In addition, depletion of MLCK significantly enhanced Claudin-1 level and declined IL-8 and TNF-α levels in IPEC-J2 pretreated with LR1 followed by challenging with ETEC K88. In conclusion, our work indicated that L. reuteri LR1 can decline inflammatory response and improve intestinal epithelial barrier function through suppressing the MLCK signal pathway in the ETEC K88-challenged IPEC-J2.

Targeting of microvillus protein Eps8 by the NleH effector kinases from enteropathogenic E. coli.

Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical "proline-rich" motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal "DY" segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection.

Persistent Escherichia coli infection in renal tubular cells enhances calcium oxalate crystal-cell adhesion by inducing ezrin translocation to apical membranes via Rho/ROCK pathway.

Recent evidence has suggested that recurrent urinary tract infection (UTI) can cause not only infection stones but also metabolic stones (e.g., those containing calcium oxalate monohydrate or COM). However, precise mechanisms underlying UTI-induced metabolic stones remained unknown. In this study, Escherichia coli, the most common bacterium found in recurrent UTI was used to establish the in vitro model for persistent infection of renal epithelial cells. The promoting effects of persistent E. coli infection on kidney stone formation were validated by COM crystal-cell adhesion assay, followed by immunofluorescence study for changes in surface expression of the known COM crystal receptors. Among the five receptors examined, only ezrin had significantly increased level on the surface of persistently infected cells without change in its total level. Such translocation of ezrin to apical membranes was confirmed by Western blotting of apical membrane and cytosolic fractions and confocal microscopic examination. Additionally, persistent infection increased phosphorylation (Thr567) of ezrin. However, all of these changes induced by persistent E. coli infection were significantly inhibited by small-interfering RNA (siRNA) specific for ezrin or a Rho-associated kinase (ROCK)-specific inhibitor (Y-27632). In summary, this study provides a piece of evidence demonstrating that persistent infection by E. coli, one of the non-urease-producing bacteria, may contribute to COM metabolic stone formation by translocation of ezrin to apical membranes, thereby promoting COM crystal-cell adhesion. Such ezrin translocation was mediated via Rho/ROCK signaling pathway. These findings may, at least in part, explain the pathogenic mechanisms underlying recurrent UTI-induced metabolic kidney stone disease.