Bacterial Sphingolipids Exacerbate Colitis by Inhibiting ILC3-derived IL-22 Production.

Background & Aims: Gut bacterial sphingolipids, primarily produced by Bacteroidetes, have dual roles as bacterial virulence factors and regulators of the host mucosal immune system, including regulatory T cells and invariant natural killer T cells. Patients with inflammatory bowel disease display altered sphingolipids profiles in fecal samples. However, how bacterial sphingolipids modulate mucosal homeostasis and regulate intestinal inflammation remains unclear.

IRE1α recognizes a structural motif in cholera toxin to activate an unfolded protein response.

IRE1α is an endoplasmic reticulum (ER) sensor that recognizes misfolded proteins to induce the unfolded protein response (UPR). We studied cholera toxin (CTx), which invades the ER and activates IRE1α in host cells, to understand how unfolded proteins are recognized. Proximity labeling colocalized the enzymatic and metastable A1 segment of CTx (CTxA1) with IRE1α in live cells, where we also found that CTx-induced IRE1α activation enhanced toxicity.

Protocol for measuring transcytosis and recycling of IgG in intestinal epithelial Caco-2 cells and primary human intestinal organoids.

Transcytosis is the primary mechanism by which macro-molecules transverse epithelial cell barriers. Here, we present an assay for measuring transcytosis and recycling of IgG in intestinal epithelial Caco-2 cells and primary human intestinal organoids. We describe steps for establishing human enteroids or Caco-2 cells and plating monolayers.

Structural basis for acyl chain control over glycosphingolipid sorting and vesicular trafficking.

The complex sphingolipids exhibit a diversity of ceramide acyl chain structures that influence their trafficking and intracellular distributions, but it remains unclear how the cell discerns among the different ceramides to affect such sorting. To address the mechanism, we synthesize a library of GM1 glycosphingolipids with naturally varied acyl chains and quantitatively assess their sorting among different endocytic pathways. We find that a stretch of at least 14 saturated carbons extending from C1 at the water-bilayer interface dictate lysosomal sorting by exclusion from endosome sorting tubules.

The epithelial-specific ER stress sensor ERN2/IRE1β enables host-microbiota crosstalk to affect colon goblet cell development.

Epithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express ERN2/IRE1β, a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor, ERN1/IRE1α. How ERN2 functions at the host-environment interface and why a second paralogue evolved remain incompletely understood. Using conventionally raised and germ-free Ern2-/- mice, we found that ERN2 was required for microbiota-induced goblet cell maturation and mucus barrier assembly in the colon.

Depletion of the apical endosome in response to viruses and bacterial toxins provides cell-autonomous host defense at mucosal surfaces.

Polarized epithelial cells form an essential barrier against infection at mucosal surfaces. Many pathogens breach this barrier to cause disease, often by co-opting cellular endocytosis mechanisms to enter the cell through the lumenal (apical) cell surface. We recently discovered that the loss of the cell polarity gene PARD6B selectively diminishes apical endosome function.

Intestinal goblet cells sample and deliver lumenal antigens by regulated endocytic uptake and transcytosis.

Intestinal goblet cells maintain the protective epithelial barrier through mucus secretion and yet sample lumenal substances for immune processing through formation of goblet cell associated antigen passages (GAPs). The cellular biology of GAPs and how these divergent processes are balanced and regulated by goblet cells remains unknown. Using high-resolution light and electron microscopy, we found that in mice, GAPs were formed by an acetylcholine (ACh)-dependent endocytic event remarkable for delivery of fluid-phase cargo retrograde into the trans-golgi network and across the cell by transcytosis - in addition to the expected transport of fluid-phase cargo by endosomes to multi-vesicular bodies and lysosomes.

Cholera Toxin as a Probe for Membrane Biology.

Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly.

Small-molecule modulators of INAVA cytosolic condensate and cell-cell junction assemblies.

Epithelial cells lining mucosal surfaces distinctively express the inflammatory bowel disease risk gene INAVA. We previously found that INAVA has dual and competing functions: one at lateral membranes where it affects mucosal barrier function and the other in the cytosol where INAVA enhances IL-1β signal transduction and protein ubiquitination and forms puncta. We now find that IL-1β-induced INAVA puncta are biomolecular condensates that rapidly assemble and physiologically resolve.

Evolution and function of the epithelial cell-specific ER stress sensor IRE1β.

Barrier epithelial cells lining the mucosal surfaces of the gastrointestinal and respiratory tracts interface directly with the environment. As such, these tissues are continuously challenged to maintain a healthy equilibrium between immunity and tolerance against environmental toxins, food components, and microbes. An extracellular mucus barrier, produced and secreted by the underlying epithelium plays a central role in this host defense response.

A Quantitative Single-cell Flow Cytometry Assay for Retrograde MembraneTrafficking Using Engineered Cholera Toxin.

The organization and distribution of proteins, lipids, and nucleic acids in eukaryotic cells is an essential process for cell function. Retrograde trafficking from the plasma membrane to the Golgi and endoplasmic reticulum can greatly modify cell membrane composition and intracellular protein dynamics, and thus typifies a key sorting step. However, methods to efficiently quantify the extent or kinetics of these events are currently limited.

Extracellular cyclic dinucleotides induce polarized responses in barrier epithelial cells by adenosine signaling.

Cyclic dinucleotides (CDNs) are secondary messengers used by prokaryotic and eukaryotic cells. In mammalian cells, cytosolic CDNs bind STING (stimulator of IFN gene), resulting in the production of type I IFN. Extracellular CDNs can enter the cytosol through several pathways but how CDNs work from outside eukaryotic cells remains poorly understood.

Everything Illuminated-Clostridium perfringens β-toxin.

In this issue of Cell Host & Microbe, Bruggisser et al. show that Clostridium perfringens β-toxin (CPB) binds platelet endothelial cell adhesion molecule-1 (PECAM-1) (also known as CD31) to induce membrane pores. The discovery explains the cell type specificity for CPB and, in large part, the basic pathophysiology of disease.

Structured clustering of the glycosphingolipid GM1 is required for membrane curvature induced by cholera toxin.

AB bacterial toxins and polyomaviruses induce membrane curvature as a mechanism to facilitate their entry into host cells. How membrane bending is accomplished is not yet fully understood but has been linked to the simultaneous binding of the pentameric B subunit to multiple copies of glycosphingolipid receptors. Here, we probe the toxin membrane binding and internalization mechanisms by using a combination of superresolution and polarized localization microscopy.

Conjugation of peptides to short-acyl-chain ceramides for delivery across mucosal cell barriers.

Robust transport of therapeutic peptides and other medicinal molecules across tight epithelial barriers would overcome the major obstacle to oral delivery. We have already demonstrated that peptides conjugated to gangliosides (GM1 and GM3) having non-native short N-acyl groups hijack the endogenous process of intracellular lipid sorting resulting in transcytosis and delivery across epithelial barriers in vitro and in vivo. Here, we report synthetic methodologies to covalently conjugate peptides directly to short-acyl-chain C-ceramides.

IRE1β negatively regulates IRE1α signaling in response to endoplasmic reticulum stress.

IRE1β is an ER stress sensor uniquely expressed in epithelial cells lining mucosal surfaces. Here, we show that intestinal epithelial cells expressing IRE1β have an attenuated unfolded protein response to ER stress. When modeled in HEK293 cells and with purified protein, IRE1β diminishes expression and inhibits signaling by the closely related stress sensor IRE1α.

A quantitative single-cell assay for retrograde membrane traffic enables rapid detection of defects in cellular organization.

Retrograde membrane trafficking from plasma membrane to Golgi and endoplasmic reticulum typifies one of the key sorting steps emerging from the early endosome that affects cell surface and intracellular protein dynamics underlying cell function. While some cell surface proteins and lipids are known to sort retrograde, there are few effective methods to quantitatively measure the extent or kinetics of these events. Here we took advantage of the well-known retrograde trafficking of cholera toxin and newly defined split fluorescent protein technology to develop a quantitative, sensitive, and effectively real-time single-cell flow cytometry assay for retrograde membrane transport.

A modified cholera toxin B subunit containing an ER retention motif enhances colon epithelial repair an unfolded protein response.

Cholera toxin B subunit (CTB) exhibits broad-spectrum biologic activity upon mucosal administration. Here, we found that a recombinant CTB containing an endoplasmic reticulum (ER) retention motif (CTB-KDEL) induces colon epithelial wound healing in colitis the activation of an unfolded protein response (UPR) in colon epithelial cells. In a Caco2 cell wound healing model, CTB-KDEL, but not CTB or CTB-KDE, facilitated cell migration interaction with the KDEL receptor, localization in the ER, UPR activation, and subsequent TGF-β signaling.

Transcytosis Assay for Transport of Glycosphingolipids across MDCK-II Cells.

Absorption and secretion of peptide and protein cargoes across single-cell thick mucosal and endothelial barriers occurs by active endocytic and vesicular trafficking that connects one side of the epithelial or endothelial cell (the lumen) with the other (the serosa or blood). Assays that assess this pathway must robustly control for non-specific and passive solute flux through weak or damaged intercellular junctions that seal the epithelial or endothelial cells together. Here we describe an cell culture Transwell assay for transcytosis of therapeutic peptides linked covalently to various species of the glycosphingolipid GM1.

INAVA-ARNO complexes bridge mucosal barrier function with inflammatory signaling.

Homeostasis at mucosal surfaces requires cross-talk between the environment and barrier epithelial cells. Disruption of barrier function typifies mucosal disease. Here we elucidate a bifunctional role in coordinating this cross-talk for the inflammatory bowel disease risk-gene .

CRISPR Screen Reveals that EHEC's T3SS and Shiga Toxin Rely on Shared Host Factors for Infection.

Enterohemorrhagic (EHEC) has two critical virulence factors-a type III secretion system (T3SS) and Shiga toxins (Stxs)-that are required for the pathogen to colonize the intestine and cause diarrheal disease. Here, we carried out a genome-wide CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats with Cas9) loss-of-function screen to identify host loci that facilitate EHEC infection of intestinal epithelial cells. Many of the guide RNAs identified targeted loci known to be associated with sphingolipid biosynthesis, particularly for production of globotriaosylceramide (Gb3), the Stx receptor.

Mucosal absorption of therapeutic peptides by harnessing the endogenous sorting of glycosphingolipids.

Transport of biologically active molecules across tight epithelial barriers is a major challenge preventing therapeutic peptides from oral drug delivery. Here, we identify a set of synthetic glycosphingolipids that harness the endogenous process of intracellular lipid-sorting to enable mucosal absorption of the incretin hormone GLP-1. Peptide cargoes covalently fused to glycosphingolipids with ceramide domains containing C6:0 or smaller fatty acids were transported with 20-100-fold greater efficiency across epithelial barriers in vitro and in vivo.

Advances in Evaluation of Chronic Diarrhea in Infants.

Diarrhea is common in infants (children less than 2 years of age), usually acute, and, if chronic, commonly caused by allergies and occasionally by infectious agents. Congenital diarrheas and enteropathies (CODEs) are rare causes of devastating chronic diarrhea in infants. Evaluation of CODEs is a lengthy process and infrequently leads to a clear diagnosis.

Hepatic FcRn regulates albumin homeostasis and susceptibility to liver injury.

The neonatal crystallizable fragment receptor (FcRn) is responsible for maintaining the long half-life and high levels of the two most abundant circulating proteins, albumin and IgG. In the latter case, the protective mechanism derives from FcRn binding to IgG in the weakly acidic environment contained within endosomes of hematopoietic and parenchymal cells, whereupon IgG is diverted from degradation in lysosomes and is recycled. The cellular location and mechanism by which FcRn protects albumin are partially understood.