Serine metabolism is crucial for cGAS-STING signaling and viral defense control in the gut.

Inflammatory bowel diseases are characterized by the chronic relapsing inflammation of the gastrointestinal tract. While the molecular causality between endoplasmic reticulum (ER) stress and intestinal inflammation is widely accepted, the metabolic consequences of chronic ER stress on the pathophysiology of IBD remain unclear. By using in vitro, in vivo models, and patient datasets, we identified a distinct polarization of the mitochondrial one-carbon metabolism and a fine-tuning of the amino acid uptake in intestinal epithelial cells tailored to support GSH and NADPH metabolism upon ER stress. This metabolic phenotype strongly correlates with IBD severity and therapy response. Mechanistically, we uncover that both chronic ER stress and serine limitation disrupt cGAS-STING signaling, impairing the epithelial response against viral and bacterial infection and fueling experimental enteritis. Consequently, the antioxidant treatment restores STING function and virus control. Collectively, our data highlight the importance of serine metabolism to allow proper cGAS-STING signaling and innate immune responses upon gut inflammation.

Intelectin-1 binds and alters the localization of the mucus barrier-modifying bacterium Akkermansia muciniphila.

Intelectin-1 (ITLN1) is a lectin secreted by intestinal epithelial cells (IECs) and upregulated in human ulcerative colitis (UC). We investigated how ITLN1 production is regulated in IECs and the biological effects of ITLN1 at the host-microbiota interface using mouse models. Our data show that ITLN1 upregulation in IECs from UC patients is a consequence of activating the unfolded protein response. Analysis of microbes coated by ITLN1 in vivo revealed a restricted subset of microorganisms, including the mucolytic bacterium Akkermansia muciniphila. Mice overexpressing intestinal ITLN1 exhibited decreased inner colonic mucus layer thickness and closer apposition of A. muciniphila to the epithelial cell surface, similar to alterations reported in UC. The changes in the inner mucus layer were microbiota and A. muciniphila dependent and associated with enhanced sensitivity to chemically induced and T cell-mediated colitis. We conclude that by determining the localization of a select group of bacteria to the mucus layer, ITLN1 modifies this critical barrier. Together, these findings may explain the impact of ITLN1 dysregulation on UC pathogenesis.

DNA methyltransferase 3A controls intestinal epithelial barrier function and regeneration in the colon.

Genetic variants in the DNA methyltransferase 3 A (DNMT3A) locus have been associated with inflammatory bowel disease (IBD). DNMT3A is part of the epigenetic machinery physiologically involved in DNA methylation. We show that DNMT3A plays a critical role in maintaining intestinal homeostasis and gut barrier function. DNMT3A expression is downregulated in intestinal epithelial cells from IBD patients and upon tumor necrosis factor treatment in murine intestinal organoids. Ablation of DNMT3A in Caco-2 cells results in global DNA hypomethylation, which is linked to impaired regenerative capacity, transepithelial resistance and intercellular junction formation. Genetic deletion of Dnmt3a in intestinal epithelial cells (Dnmt3aΔIEC) in mice confirms the phenotype of an altered epithelial ultrastructure with shortened apical-junctional complexes, reduced Goblet cell numbers and increased intestinal permeability in the colon in vivo. Dnmt3aΔIEC mice suffer from increased susceptibility to experimental colitis, characterized by reduced epithelial regeneration. These data demonstrate a critical role for DNMT3A in orchestrating intestinal epithelial homeostasis and response to tissue damage and suggest an involvement of impaired epithelial DNMT3A function in the etiology of IBD.

Activating Transcription Factor 6 Mediates Inflammatory Signals in Intestinal Epithelial Cells Upon Endoplasmic Reticulum Stress.

BACKGROUND & AIMS: Excess and unresolved endoplasmic reticulum (ER) stress in intestinal epithelial cells (IECs) promotes intestinal inflammation. Activating transcription factor 6 (ATF6) is one of the signaling mediators of ER stress. We studied the pathways that regulate ATF6 and its role for inflammation in IECs. METHODS: We performed an RNA interference screen, using 23,349 unique small interfering RNAs targeting 7783 genes and a luciferase reporter controlled by an ATF6-dependent ERSE (ER stress-response element) promoter, to identify proteins that activate or inhibit the ATF6 signaling pathway in HEK293 cells. To validate the screening results, intestinal epithelial cell lines (Caco-2 cells) were transfected with small interfering RNAs or with a plasmid overexpressing a constitutively active form of ATF6. Caco-2 cells with a CRISPR-mediated disruption of autophagy related 16 like 1 gene (ATG16L1) were used to study the effect of ATF6 on ER stress in autophagy-deficient cells. We also studied intestinal organoids derived from mice that overexpress constitutively active ATF6, from mice with deletion of the autophagy related 16 like 1 or X-Box binding protein 1 gene in IECs (Atg16l1ΔIEC or Xbp1ΔIEC, which both develop spontaneous ileitis), from patients with Crohn's disease (CD) and healthy individuals (controls). Cells and organoids were incubated with tunicamycin to induce ER stress and/or chemical inhibitors of newly identified activator proteins of ATF6 signaling, and analyzed by real-time polymerase chain reaction and immunoblots. Atg16l1ΔIEC and control (Atg16l1fl/fl) mice were given intraperitoneal injections of tunicamycin and were treated with chemical inhibitors of ATF6 activating proteins. RESULTS: We identified and validated 15 suppressors and 7 activators of the ATF6 signaling pathway; activators included the regulatory subunit of casein kinase 2 (CSNK2B) and acyl-CoA synthetase long chain family member 1 (ACSL1). Knockdown or chemical inhibition of CSNK2B and ACSL1 in Caco-2 cells reduced activity of the ATF6-dependent ERSE reporter gene, diminished transcription of the ATF6 target genes HSP90B1 and HSPA5 and reduced NF-κB reporter gene activation on tunicamycin stimulation. Atg16l1ΔIEC and or Xbp1ΔIEC organoids showed increased expression of ATF6 and its target genes. Inhibitors of ACSL1 or CSNK2B prevented activation of ATF6 and reduced CXCL1 and tumor necrosis factor (TNF) expression in these organoids on induction of ER stress with tunicamycin. Injection of mice with inhibitors of ACSL1 or CSNK2B significantly reduced tunicamycin-mediated intestinal inflammation and IEC death and expression of CXCL1 and TNF in Atg16l1ΔIEC mice. Purified ileal IECs from patients with CD had higher levels of ATF6, CSNK2B, and HSPA5 messenger RNAs than controls; early-passage organoids from patients with active CD show increased levels of activated ATF6 protein, incubation of these organoids with inhibitors of ACSL1 or CSNK2B reduced transcription of ATF6 target genes, including TNF. CONCLUSIONS: Ileal IECs from patients with CD have higher levels of activated ATF6, which is regulated by CSNK2B and HSPA5. ATF6 increases expression of TNF and other inflammatory cytokines in response to ER stress in these cells and in organoids from Atg16l1ΔIEC and Xbp1ΔIEC mice. Strategies to inhibit the ATF6 signaling pathway might be developed for treatment of inflammatory bowel diseases.

NOD2 Influences Trajectories of Intestinal Microbiota Recovery After Antibiotic Perturbation.

BACKGROUND & AIMS: Loss-of-function variants in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) impair the recognition of the bacterial cell wall component muramyl-dipeptide and are associated with an increased risk for developing Crohn's disease. Likewise, exposure to antibiotics increases the individual risk for developing inflammatory bowel disease. Here, we studied the long-term impact of NOD2 on the ability of the gut bacterial and fungal microbiota to recover after antibiotic treatment. METHODS: Two cohorts of 20-week-old and 52-week-old wild-type (WT) C57BL/6J and NOD2 knockout (Nod2-KO) mice were treated with broad-spectrum antibiotics and fecal samples were collected to investigate temporal dynamics of the intestinal microbiota (bacteria and fungi) using 16S ribosomal RNA and internal transcribed spacer 1 sequencing. In addition, 2 sets of germ-free WT mice were colonized with either WT or Nod2-KO after antibiotic donor microbiota and the severity of intestinal inflammation was monitored in the colonized mice. RESULTS: Antibiotic exposure caused long-term shifts in the bacterial and fungal community composition. Genetic ablation of NOD2 was associated with delayed body weight gain after antibiotic treatment and an impaired recovery of the bacterial gut microbiota. Transfer of the postantibiotic fecal microbiota of Nod2-KO mice induced an intestinal inflammatory response in the colons of germ-free recipient mice compared with respective microbiota from WT controls based on histopathology and gene expression analyses. CONCLUSIONS: Our data show that the bacterial sensor NOD2 contributes to intestinal microbial community composition after antibiotic treatment and may add to the explanation of how defects in the NOD2 signaling pathway are involved in the etiology of Crohn's disease.

A Phage Protein Aids Bacterial Symbionts in Eukaryote Immune Evasion.

Phages are increasingly recognized as important members of host-associated microbiomes, with a vast genomic diversity. The new frontier is to understand how phages may affect higher order processes, such as in the context of host-microbe interactions. Here, we use marine sponges as a model to investigate the interplay between phages, bacterial symbionts, and eukaryotic hosts. Using viral metagenomics, we find that sponges, although massively filtering seawater, harbor species-specific and even individually unique viral signatures that are taxonomically distinct from other environments. We further discover a symbiont phage-encoded ankyrin-domain-containing protein, which is widely spread in phages of many host-associated contexts including human. We confirm in macrophage infection assays that the ankyrin protein (ANKp) modulates the eukaryotic host immune response against bacteria. We predict that the role of ANKp in nature is to facilitate coexistence in the tripartite interplay between phages, symbionts, and sponges and possibly many other host-microbe associations.

Missense variants in NOX1 and p22phox in a case of very-early-onset inflammatory bowel disease are functionally linked to NOD2.

Whole-genome and whole-exome sequencing of individual patients allow the study of rare and potentially causative genetic variation. In this study, we sequenced DNA of a trio comprising a boy with very-early-onset inflammatory bowel disease (veoIBD) and his unaffected parents. We identified a rare, X-linked missense variant in the NAPDH oxidase NOX1 gene (c.C721T, p.R241C) in heterozygous state in the mother and in hemizygous state in the patient. We discovered that, in addition, the patient was homozygous for a common missense variant in the CYBA gene (c.T214C, p.Y72H). CYBA encodes the p22phox protein, a cofactor for NOX1. Functional assays revealed reduced cellular ROS generation and antibacterial capacity of NOX1 and p22phox variants in intestinal epithelial cells. Moreover, the identified NADPH oxidase complex variants affected NOD2-mediated immune responses, and p22phox was identified as a novel NOD2 interactor. In conclusion, we detected missense variants in a veoIBD patient that disrupt the host response to bacterial challenges and reduce protective innate immune signaling via NOD2. We assume that the patient's individual genetic makeup favored disturbed intestinal mucosal barrier function.

ATG16L1 orchestrates interleukin-22 signaling in the intestinal epithelium via cGAS-STING.

A coding variant of the inflammatory bowel disease (IBD) risk gene ATG16L1 has been associated with defective autophagy and deregulation of endoplasmic reticulum (ER) function. IL-22 is a barrier protective cytokine by inducing regeneration and antimicrobial responses in the intestinal mucosa. We show that ATG16L1 critically orchestrates IL-22 signaling in the intestinal epithelium. IL-22 stimulation physiologically leads to transient ER stress and subsequent activation of STING-dependent type I interferon (IFN-I) signaling, which is augmented in Atg16l1 ΔIEC intestinal organoids. IFN-I signals amplify epithelial TNF production downstream of IL-22 and contribute to necroptotic cell death. In vivo, IL-22 treatment in Atg16l1 ΔIEC and Atg16l1 ΔIEC/Xbp1 ΔIEC mice potentiates endogenous ileal inflammation and causes widespread necroptotic epithelial cell death. Therapeutic blockade of IFN-I signaling ameliorates IL-22-induced ileal inflammation in Atg16l1 ΔIEC mice. Our data demonstrate an unexpected role of ATG16L1 in coordinating the outcome of IL-22 signaling in the intestinal epithelium.

Membrane Microdomain Disassembly Inhibits MRSA Antibiotic Resistance.

A number of bacterial cell processes are confined functional membrane microdomains (FMMs), structurally and functionally similar to lipid rafts of eukaryotic cells. How bacteria organize these intricate platforms and what their biological significance is remain important questions. Using the pathogen methicillin-resistant Staphylococcus aureus (MRSA), we show here that membrane-carotenoid interaction with the scaffold protein flotillin leads to FMM formation, which can be visualized using super-resolution array tomography. These membrane platforms accumulate multimeric protein complexes, for which flotillin facilitates efficient oligomerization. One of these proteins is PBP2a, responsible for penicillin resistance in MRSA. Flotillin mutants are defective in PBP2a oligomerization. Perturbation of FMM assembly using available drugs interferes with PBP2a oligomerization and disables MRSA penicillin resistance in vitro and in vivo, resulting in MRSA infections that are susceptible to penicillin treatment. Our study demonstrates that bacteria possess sophisticated cell organization programs and defines alternative therapies to fight multidrug-resistant pathogens using conventional antibiotics.

Attenuating Staphylococcus aureus Virulence by Targeting Flotillin Protein Scaffold Activity.

Scaffold proteins are ubiquitous chaperones that bind proteins and facilitate physical interaction of multi-enzyme complexes. Here we used a biochemical approach to dissect the scaffold activity of the flotillin-homolog protein FloA of the multi-drug-resistant human pathogen Staphylococcus aureus. We show that FloA promotes oligomerization of membrane protein complexes, such as the membrane-associated RNase Rny, which forms part of the RNA-degradation machinery called the degradosome. Cells lacking FloA had reduced Rny function and a consequent increase in the targeted sRNA transcripts that negatively regulate S. aureus toxin expression. Small molecules that altered FloA oligomerization also reduced Rny function and decreased the virulence potential of S. aureus in vitro, as well as in vivo, using invertebrate and murine infection models. Our results suggest that flotillin assists in the assembly of protein complexes involved in S. aureus virulence, and could thus be an attractive target for the development of new antimicrobial therapies.

Evolution of resistance to a last-resort antibiotic in Staphylococcus aureus via bacterial competition.

Antibiotic resistance is a key medical concern, with antibiotic use likely being an important cause. However, here we describe an alternative route to clinically relevant antibiotic resistance that occurs solely due to competitive interactions among bacterial cells. We consistently observe that isolates of Methicillin-resistant Staphylococcus aureus diversify spontaneously into two distinct, sequentially arising strains. The first evolved strain outgrows the parent strain via secretion of surfactants and a toxic bacteriocin. The second is resistant to the bacteriocin. Importantly, this second strain is also resistant to intermediate levels of vancomycin. This so-called VISA (vancomycin-intermediate S. aureus) phenotype is seen in many hard-to-treat clinical isolates. This strain diversification also occurs during in vivo infection in a mouse model, which is consistent with the fact that both coevolved phenotypes resemble strains commonly found in clinic. Our study shows how competition between coevolving bacterial strains can generate antibiotic resistance and recapitulate key clinical phenotypes.