AIEC-dependent pathogenic Th17 cell transdifferentiation in Crohn's disease is suppressed by rfaP and ybaT deletion.

Mucosal enrichment of the Adherent-Invasive E. coli (AIEC) pathotype and the expansion of pathogenic IFNγ-producing Th17 (pTh17) cells have been linked to Crohn's Disease (CD) pathogenesis. However, the molecular pathways underlying the AIEC-dependent pTh17 cell transdifferentiation in CD patients remain elusive. To this aim, we created and functionally screened a transposon AIEC mutant library of 10.058 mutants to identify the virulence determinants directly implicated in triggering IL-23 production and pTh17 cell generation. pTh17 cell transdifferentiation was assessed in functional assays by co-culturing AIEC-infected human dendritic cells (DCs) with autologous conventional Th17 (cTh17) cells isolated from blood of Healthy Donors (HD) or CD patients. AIEC triggered IL-23 hypersecretion and transdifferentiation of cTh17 into pTh17 cells selectively through the interaction with CD-derived DCs. Moreover, the chronic release of IL-23 by AIEC-colonized DCs required a continuous IL-23 neutralization to significantly reduce the AIEC-dependent pTh17 cell differentiation. The multi-step screenings of the AIEC mutant's library revealed that deletion of ybaT or rfaP efficiently hinder the IL-23 hypersecretion and hampered the AIEC-dependent skewing of protective cTh17 into pathogenic IFNγ-producing pTh17 cells. Overall, our findings indicate that ybaT (inner membrane transport protein) and rfaP (LPS-core heptose kinase) represent novel and attractive candidate targets to prevent chronic intestinal inflammation in CD.

Contribution of Toxin-Antitoxin Systems to Adherent-Invasive E. coli Pathogenesis.

Pathobionts have been implicated in various chronic diseases, including Crohn's disease (CD), a multifactorial chronic inflammatory condition that primarily affects the gastrointestinal tract, causing inflammation and damage to the digestive system. While the exact cause of CD remains unclear, adherent-invasive Escherichia coli (AIEC) strains have emerged as key contributors to its pathogenesis. AIEC are characterized by their ability to adhere to and invade intestinal epithelial cells and survive and replicate inside macrophages. However, the mechanisms underlying the virulence and persistence of AIEC within their host remain the subject of intensive research. Toxin-antitoxin systems (TAs) play a potential role in AIEC pathogenesis and may be therapeutic targets. These systems generally consist of two components: a toxin harmful to the cell and an antitoxin that neutralizes the toxin's effects. They contribute to bacterial survival in adverse conditions and regulate bacterial growth and behavior, affecting various cellular processes in bacterial pathogens. This review focuses on the current information available to determine the roles of TAs in the pathogenicity of AIEC. Their contribution to the AIEC stress response, biofilm formation, phage inhibition, the maintenance of mobile genetic elements, and host lifestyles is discussed.

Stratifying macrophages based on their infectious burden identifies novel host targets for intervention during Crohn's disease associated adherent-invasive Escherichia coli infection.

Bacterial infection is a dynamic process resulting in a heterogenous population of infected and uninfected cells. These cells respond differently based on their bacterial load and duration of infection. In the case of infection of macrophages with Crohn's disease (CD) associated adherent-invasive Escherichia coli (AIEC), understanding the drivers of pathogen success may allow targeting of cells where AIEC replicate to high levels. Here we show that stratifying immune cells based on their bacterial load identifies novel pathways and therapeutic targets not previously associated with AIEC when using a traditional homogeneous infected population approach. Using flow cytometry-based cell sorting we stratified cells into those with low or high intracellular pathogen loads, or those which were bystanders to infection. Immune cells transcriptomics revealed a diverse response to the varying levels of infection while pathway analysis identified novel intervention targets that were directly related to increasing intracellular AIEC numbers. Chemical inhibition of identified targets reduced AIEC intracellular replication or inhibited secretion of tumour necrosis factor alpha (TNFα), a key cytokine associated with AIEC infection. Our results have identified new avenues of intervention in AIEC infection that may also be applicable to CD through the repurposing of already available inhibitors. Additionally, they highlight the applicability of immune cell stratification post-infection as an effective approach for the study of microbial pathogens.

Two-component system RstAB promotes the pathogenicity of adherent-invasive Escherichia coli in response to acidic conditions within macrophages.

Adherent-invasive Escherichia coli (AIEC) strain LF82, isolated from patients with Crohn's disease, invades gut epithelial cells, and replicates in macrophages contributing to chronic inflammation. In this study, we found that RstAB contributing to the colonization of LF82 in a mouse model of chronic colitis by promoting bacterial replication in macrophages. By comparing the transcriptomes of rstAB mutant- and wild-type when infected macrophages, 83 significant differentially expressed genes in LF82 were identified. And we identified two possible RstA target genes (csgD and asr) among the differentially expressed genes. The electrophoretic mobility shift assay and quantitative real-time PCR confirmed that RstA binds to the promoters of csgD and asr and activates their expression. csgD deletion attenuated LF82 intracellular biofilm formation, and asr deletion reduced acid tolerance compared with the wild-type. Acidic pH was shown by quantitative real-time PCR to be the signal sensed by RstAB to activate the expression of csgD and asr. We uncovered a signal transduction pathway whereby LF82, in response to the acidic environment within macrophages, activates transcription of the csgD to promote biofilm formation, and activates transcription of the asr to promote acid tolerance, promoting its replication within macrophages and colonization of the intestine. This finding deepens our understanding of the LF82 replication regulation mechanism in macrophages and offers new perspectives for further studies on AIEC virulence mechanisms.

The CpxRA two-component system of adherent and invasive Escherichia coli contributes to epithelial cell invasion and early-stage intestinal fitness in a dysbiotic mouse model mediated by type 1 fimbriae expression.

Adherent and invasive Escherichia coli (AIEC) is a pathobiont that is involved in the onset and exacerbation of Crohn's disease. Although the inducible expression of virulence traits is a critical step for AIEC colonization in the host, the mechanism underlying AIEC colonization remains largely unclear. We here showed that the two-component signal transduction system CpxRA contributes to AIEC gut competitive colonization by activating type 1 fimbriae expression. CpxRA from AIEC strain LF82 functioned as a transcriptional regulator, as evidenced by our finding that an isogenic cpxRA mutant exhibits reduced expression of cpxP, a known regulon gene. Transcription levels of cpxP in LF82 increased in response to envelope stress, such as exposure to antimicrobials compromising the bacterial membrane, whereas the cpxRA mutant did not exhibit this response. Furthermore, we found that the cpxRA mutant exhibits less invasiveness into host cells than LF82, primarily due to reduced expression of the type 1 fimbriae. Finally, we found that the cpxRA mutant is impaired in gut competitive colonization in a mouse model. The colonization defects were reversed by the introduction of a plasmid encoding the cpxRA gene or expressing the type 1 fimbriae. Our findings indicate that modulating CpxRA activity could be a promising approach to regulating AIEC-involved Crohn's disease.

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.

Metabolic network of the gut microbiota in inflammatory bowel disease.

Gut dysbiosis is closely linked to the pathogenesis of inflammatory bowel disease (IBD). Emerging studies highlight the relationship between host metabolism and the modulation of gut microbiota composition through regulating the luminal microenvironment. In IBD, various disease-associated factors contribute to the significant perturbation of host metabolism. Such disturbance catalyzes the selective proliferation of specific microbial populations, particularly pathobionts such as adherent invasive Escherichia coli and oral-derived bacteria. Pathobionts employ various strategies to adapt better to the disease-associated luminal environments. In addition to the host-microbe interaction, recent studies demonstrate that the metabolic network between commensal symbionts and pathobionts facilitates the expansion of pathobionts in the inflamed gut. Understanding the metabolic network among the host, commensal symbionts, and pathobionts provides new insights into the pathogenesis of IBD and novel avenues for treating IBD.

The Efficacy of Selected Probiotic Strains and Their Combination to Inhibit the Interaction of Adherent-Invasive Escherichia coli (AIEC) with a Co-Culture of Caco-2:HT29-MTX Cells.

The gastrointestinal tract's microbiota plays a crucial role in human health, with dysbiosis linked to the development of diseases such as inflammatory bowel disease (IBD). Whilst the pathogenic mechanisms underlying IBD remain poorly characterised, adherent-invasive Escherichia coli (AIEC) has been implicated as a microbiological factor in disease pathogenesis. These strains show an enhanced ability to diffusely adhere to and invade intestinal epithelial cells, along with the ability to survive and replicate within macrophages. Probiotics, such as Lactobacillus strains, have been identified as potential treatment options due to their abilities to compete with pathogens for binding sites and regulate the host immune response. In this study, we used four well-characterised Lactobacillus strains and their combination to test their ability to inhibit the adhesion, invasion, and translocation of a well-characterized AIEC strain, F44A-1, in a co-culture of Caco-2 and HT29-MTX cell lines representing the gut epithelium. The results demonstrated that the pre-inoculation of the probiotic candidates 90 min prior to the introduction of the AIEC was more effective in inhibiting AIEC interaction than the co-inoculation of the strains. While the individual probiotic strains greatly reduced AIEC colonisation and invasion of the co-cultured cells, their combination was only more effective in reducing the translocation of the AIEC. These results suggest that probiotics are more effective when used prophylactically against pathogens and that the combination of strains may enhance their efficacy against AIEC translocation once used as a prophylactic measure.

Categorization of the effects of E. coli LF82 and mutants lacking the chuT and shuU genes on survival, the transcriptome, and metabolome in germ-free honeybee.

The precise etiology of inflammatory bowel diseases (IBDs) remains elusive. The Escherichia coli strain LF82 (LF82) is known to be associated with IBD, and we hypothesized that this association may be related to the chuT and shuU genes. Here we constructed a germ-free (GF) honeybee model to investigate the effects of LF82 chuT and shuU genes on the honeybee intestine and their mechanisms. The chuT and shuU gene deletion strains LF82∆chuT and LF82∆shuU were generated by CRISPR-Cas9. These strains, together with nonpathogenic E. coli MG1655 (MG1655) and wildtype LF82, were allowed to colonize the guts of GF honeybees to establish single bacterial colonization models. Intestinal permeability was assessed following the administration of a sterile Brilliant Blue (FCF) solution. Comprehensive transcriptomic and metabolomic analyses of intestinal samples indicated that MG1655 had few disadvantageous effects on honeybees. Conversely, colonization with LF82 and its gene-deletion mutants provoked pronounced activation of genes associated with innate immune pathways, stimulated defensive responses, and induced expression of genes associated with inflammation, oxidative stress, and glycosaminoglycan degradation. Crucially, the LF82∆chuT and LF82∆shuU strains perturbed host heme and iron regulation, as well as tryptophan metabolism. These findings suggest that the deletion of chuT and shuU genes in E. coli LF82 may alleviate intestinal inflammation by partially modulating tryptophan catabolism. Our study proposes that targeting iron uptake mechanisms could be a potential strategy to mitigate the virulence of IBD-associated bacteria.

Cyclic-di-AMP confers an invasive phenotype on Escherichia coli through elongation of flagellin filaments.

BACKGROUND: Adherent-invasive Escherichia coli (AIEC) is isolated from patients with Crohn's disease (CD). AIEC can invade the intestinal epithelium, suggesting that it is involved in the development and pathogenesis of CD. However, the mechanism by which AIEC acquired the invasive phenotype remains unknown. RESULTS: This study was designed to examine the mechanisms of AIEC invasiveness. We found that the flagellin (fliC) expression in AIEC was two-fold higher than that in non-AIEC strains, and this overexpression induced the formation of long-filament flagellin. Deletion of fliC in the AIEC LF82 strain resulted in the disappearance of flagellar filaments and attenuated the motility and invasive ability of the bacterium, suggesting that the formation of long filament flagellin induced by increased fliC expression is required by AIEC to invade the intestinal epithelium. In AIEC and non-AIEC K12 strains cultured in the presence of cyclic-di-AMP (c-di-AMP), the expression of fliC was enhanced, and flagellar filaments were elongated. Stimulation with c-di-AMP enhanced the bacterial motility and ability to invade epithelial cells, even in the non-AIEC K12 strain. CONCLUSIONS: Our findings show that c-di-AMP confers an AIEC-like phenotype on non-AIEC strains by enhancing the expression of fliC. The results should be useful for understanding the pathogenesis of CD.

A novel avian intestinal epithelial cell line: its characterization and exploration as an in vitro infection culture model for Eimeria species.

BACKGROUND: The gastrointestinal epithelium plays an important role in directing recognition by the immune system, and epithelial cells provide the host's front line of defense against microorganisms. However, it is difficult to cultivate avian intestinal epithelial cells in vitro for lengthy periods, and the lack of available cell lines limits the research on avian intestinal diseases and nutritional regulation. Chicken coccidiosis is a serious intestinal disease that causes significant economic losses in the poultry industry. In vitro, some cell line models are beneficial for the development of Eimeria species; however, only partial reproduction can be achieved. Therefore, we sought to develop a new model with both the natural host and epithelial cell phenotypes. METHODS: In this study, we use the SV40 large T antigen (SV40T) gene to generate an immortalized cell line. Single-cell screening technology was used to sort positive cell clusters with epithelial characteristics for passage. Polymerase chain reaction (PCR) identification, immunofluorescence detection, and bulk RNA sequencing analysis and validation were used to check the expression of epithelial cell markers and characterize the avian intestinal epithelial cell line (AIEC). AIECs were infected with sporozoites, and their ability to support the in vitro endogenous development of Eimeria tenella was assessed. RESULTS: This novel AIEC consistently expressed intestinal epithelial markers. Transcriptome assays revealed the upregulation of genes associated with proliferation and downregulation of genes associated with apoptosis. We sought to compare E. tenella infection between an existing fibroblast cell line (DF-1) and several passages of AIEC and found that the invasion efficiency was significantly increased relative to that of chicken fibroblast cell lines. CONCLUSIONS: An AIEC will serve as a better in vitro research model, especially in the study of Eimeria species development and the mechanisms of parasite-host interactions. Using AIEC helps us understand the involvement of intestinal epithelial cells in the digestive tract and the immune defense of the chickens, which will contribute to the epithelial innate defense against microbial infection in the gastrointestinal tract.

The IbeA protein from adherent invasive Escherichia coli is a flavoprotein sharing structural homology with FAD-dependent oxidoreductases.

Invasion of brain endothelium protein A (IbeA) is a virulence factor specific to pathogenic Escherichia coli. Originally identified in the K1 strain causing neonatal meningitis, it was more recently found in avian pathogenic Escherichia coli (APEC) and adherent invasive Escherichia coli (AIEC). In these bacteria, IbeA facilitates host cell invasion and intracellular survival, in particular, under harsh conditions like oxidative stress. Furthermore, IbeA from AIEC contributes to intramacrophage survival and replication, thus enhancing the inflammatory response within the intestine. Therefore, this factor is a promising drug target for anti-AIEC strategies in the context of Crohn's disease. Despite such an important role, the biological function of IbeA remains largely unknown. In particular, its exact nature and cellular localization, i.e., membrane-bound invasin versus cytosolic factor, are still of debate. Here, we developed an efficient protocol for recombinant expression of IbeA under native conditions and demonstrated that IbeA from AIEC is a soluble, homodimeric flavoprotein. Using mass spectrometry and tryptophan fluorescence measurements, we further showed that IbeA preferentially binds flavin adenine dinucleotide (FAD), with an affinity in the one-hundred nanomolar range and optimal binding under reducing conditions. 3D-modeling with AlphaFold revealed that IbeA shares strong structural homology with FAD-dependent oxidoreductases. Finally, we used ligand docking, mutational analyses, and molecular dynamics simulations to identify the FAD binding pocket within IbeA and characterize possible conformational changes occurring upon ligand binding. Overall, we suggest that the role of IbeA in the survival of AIEC within host cells, notably macrophages, is linked to modulation of redox processes.

A consortia of clinical E. coli strains with distinct in vitro adherent/invasive properties establish their own co-colonization niche and shape the intestinal microbiota in inflammation-susceptible mice.

BACKGROUND: Inflammatory bowel disease (IBD) patients experience recurrent episodes of intestinal inflammation and often follow an unpredictable disease course. Mucosal colonization with adherent-invasive Escherichia coli (AIEC) are believed to perpetuate intestinal inflammation. However, it remains unclear if the 24-year-old AIEC in vitro definition fully predicts mucosal colonization in vivo. To fill this gap, we have developed a novel molecular barcoding approach to distinguish strain variants in the gut and have integrated this approach to explore mucosal colonization of distinct patient-derived E. coli isolates in gnotobiotic mouse models of colitis. RESULTS: Germ-free inflammation-susceptible interleukin-10-deficient (Il10-/-) and inflammation-resistant WT mice were colonized with a consortium of AIEC and non-AIEC strains, then given a murine fecal transplant to provide niche competition. E. coli strains isolated from human intestinal tissue were each marked with a unique molecular barcode that permits identification and quantification by barcode-targeted sequencing. 16S rRNA sequencing was used to evaluate the microbiome response to E. coli colonization. Our data reveal that specific AIEC and non-AIEC strains reproducibly colonize the intestinal mucosa of WT and Il10-/- mice. These E. coli expand in Il10-/- mice during inflammation and induce compositional dysbiosis to the microbiome in an inflammation-dependent manner. In turn, specific microbes co-evolve in inflamed mice, potentially diversifying E. coli colonization patterns. We observed no selectivity in E. coli colonization patterns in the fecal contents, indicating minimal selective pressure in this niche from host-microbe and interbacterial interactions. Because select AIEC and non-AIEC strains colonize the mucosa, this suggests the in vitro AIEC definition may not fully predict in vivo colonization potential. Further comparison of seven E. coli genomes pinpointed unique genomic features contained only in highly colonizing strains (two AIEC and two non-AIEC). Those colonization-associated features may convey metabolic advantages (e.g., iron acquisition and carbohydrate consumption) to promote efficient mucosal colonization. CONCLUSIONS: Our findings establish the in vivo mucosal colonizer, not necessarily AIEC, as a principal dysbiosis driver through crosstalk with host and associated microbes. Furthermore, we highlight the utility of high-throughput screens to decode the in vivo colonization dynamics of patient-derived bacteria in murine models. Video Abstract.

Colonization of larval zebrafish (Danio rerio) with adherent-invasive Escherichia coli prevents recovery of the intestinal mucosa from drug-induced enterocolitis.

IMPORTANCE: Although inflammatory bowel diseases are on the rise, what factors influence IBD risk and severity, and the underlying mechanisms remain to be fully understood. Although host genetics, microbiome, and environmental factors have all been shown to correlate with the development of IBD, cause and effect are difficult to disentangle in this context. For example, AIEC is a known pathobiont found in IBD patients, but it remains unclear if gut inflammation during IBD facilitates colonization with AIEC, or if AIEC colonization makes the host more susceptible to pro-inflammatory stimuli. It is critical to understand the mechanisms that contribute to AIEC infections in a susceptible host in order to develop successful therapeutics. Here, we show that the larval zebrafish model recapitulates key features of AIEC infections in other animal models and can be utilized to address these gaps in knowledge.

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.

Corrigendum: Motility increase of Adherent Invasive Escherichia coli (AIEC) induced by a sub-inhibitory concentration of recombinant endolysin LysPA90.

[This corrects the article DOI: 10.3389/fmicb.2022.1093670.].

PF-431396 hydrate inhibition of kinase phosphorylation during adherent-invasive Escherichia coli infection inhibits intra-macrophage replication and inflammatory cytokine release.

Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohn's disease (CD). They are characterized by an ability to adhere to and invade intestinal epithelial cells, and to replicate intracellularly in macrophages resulting in inflammation. Proline-rich tyrosine kinase 2 (PYK2) has previously been identified as a risk locus for inflammatory bowel disease and a regulator of intestinal inflammation. It is overexpressed in patients with colorectal cancer, a major long-term complication of CD. Here we show that Pyk2 levels are significantly increased during AIEC infection of murine macrophages while the inhibitor PF-431396 hydrate, which blocks Pyk2 activation, significantly decreased intramacrophage AIEC numbers. Imaging flow cytometry indicated that Pyk2 inhibition blocked intramacrophage replication of AIEC with no change in the overall number of infected cells, but a significant reduction in bacterial burden per cell. This reduction in intracellular bacteria resulted in a 20-fold decrease in tumour necrosis factor α secretion by cells post-AIEC infection. These data demonstrate a key role for Pyk2 in modulating AIEC intracellular replication and associated inflammation and may provide a new avenue for future therapeutic intervention in CD.

A consortia of clinical E. coli strains with distinct in-vitro adherent/invasive properties establish their own co-colonization niche and shape the intestinal microbiota in inflammation-susceptible mice.

Background: Inflammatory bowel disease (IBD) patients experience recurrent episodes of intestinal inflammation and often follow an unpredictable disease course. Mucosal colonization with adherent-invasive Escherichia coli (AIEC) are believed to perpetuate intestinal inflammation. However, it remains unclear if the 24-year-old AIEC in-vitro definition fully predicts mucosal colonization in-vivo. To fill this gap, we have developed a novel molecular barcoding approach to distinguish strain variants in the gut and have integrated this approach to explore mucosal colonization of distinct patient-derived E. coli isolates in gnotobiotic mouse models of colitis. Results: Germ-free inflammation-susceptible interleukin-10-deficient (Il10-/-) and inflammation-resistant WT mice were colonized with a consortia of AIEC and non-AIEC strains, then given a murine fecal transplant to provide niche competition. E. coli strains isolated from human intestinal tissue were each marked with a unique molecular barcode that permits identification and quantification by barcode-targeted sequencing. 16S rRNA sequencing was used to evaluate the microbiome response to E. coli colonization. Our data reveal that specific AIEC and non-AIEC strains reproducibly colonize the intestinal mucosa of WT and Il10-/- mice. These E. coli expand in Il10-/- mice during inflammation and induce compositional dysbiosis to the microbiome in an inflammation-dependent manner. In turn, specific microbes co-evolve in inflamed mice, potentially diversifying E. coli colonization patterns. We observed no selectivity in E. coli colonization patterns in the fecal contents, indicating minimal selective pressure in this niche from host-microbe and interbacterial interactions. Because select AIEC and non-AIEC strains colonize the mucosa, this suggests the in vitro AIEC definition may not fully predict in vivo colonization potential. Further comparison of seven E. coli genomes pinpointed unique genomic features contained only in highly colonizing strains (two AIEC and two non-AIEC). Those colonization-associated features may convey metabolic advantages (e.g., iron acquisition and carbohydrate consumption) to promote efficient mucosal colonization. Conclusions: Our findings establish the in-vivo mucosal colonizer, not necessarily AIEC, as a principal dysbiosis driver through crosstalk with host and associated microbes. Furthermore, we highlight the utility of high-throughput screens to decode the in-vivo colonization dynamics of patient-derived bacteria in murine models.

Adherent-invasive Escherichia coli associated with granulomatous colitis and extraintestinal dissemination in a Sphynx cat.

This case report describes a case of granulomatous colitis (GC) associated with adherent-invasive Escherichia coli (AIEC) with extension to cecum and ileum and dissemination to multiple lymph nodes, the spleen, and brain in a 10-year-old, male Sphynx cat. The cat had an episode of diarrhea 4 months prior to consultation due to sudden blindness. Signs rapidly progressed to ataxia, seizures, and death. Gross and histologic findings were consistent with granulomatous inflammation in all affected organs. In situ hybridization confirmed the presence of intracellular E. coli within enterocytes and infiltrating macrophages, and whole genome sequencing identified virulence traits commonly linked to AIEC strain. This is the first characterization of GC in a cat associated to AIEC resembling the metastatic form of Crohn's disease in humans and GC of dogs. Extraintestinal involvement might provide evidence of the ability of AIEC to promote granulomatous inflammation beyond the gut.

An IBD-associated pathobiont synergises with NSAID to promote colitis which is blocked by NLRP3 inflammasome and Caspase-8 inhibitors.

Conflicting evidence exists on the association between consumption of non-steroidal anti-inflammatory drugs (NSAIDs) and symptomatic worsening of inflammatory bowel disease (IBD). We hypothesized that the heterogeneous prevalence of pathobionts [e.g., adherent-invasive Escherichia coli (AIEC)], might explain this inconsistent NSAIDs/IBD correlation. Using IL10-/- mice, we found that NSAID aggravated colitis in AIEC-colonized animals. This was accompanied by activation of the NLRP3 inflammasome, Caspase-8, apoptosis, and pyroptosis, features not seen in mice exposed to AIEC or NSAID alone, revealing an AIEC/NSAID synergistic effect. Inhibition of NLRP3 or Caspase-8 activity ameliorated colitis, with reduction in NLRP3 inflammasome activation, cell death markers, activated T-cells and macrophages, improved histology, and increased abundance of Clostridium cluster XIVa species. Our findings provide new insights into how NSAIDs and an opportunistic gut-pathobiont can synergize to worsen IBD symptoms. Targeting the NLRP3 inflammasome or Caspase-8 could be a potential therapeutic strategy in IBD patients with gut inflammation, which is worsened by NSAIDs.