Metabolite-based inter-kingdom communication controls intestinal tissue recovery following chemotherapeutic injury.

Cytotoxic chemotherapies have devastating side effects, particularly within the gastrointestinal tract. Gastrointestinal toxicity includes the death and damage of the epithelium and an imbalance in the intestinal microbiota, otherwise known as dysbiosis. Whether dysbiosis is a direct contributor to tissue toxicity is a key area of focus. Here, from both mammalian and bacterial perspectives, we uncover an intestinal epithelial cell death-Enterobacteriaceae signaling axis that fuels dysbiosis. Specifically, our data demonstrate that chemotherapy-induced epithelial cell apoptosis and the purine-containing metabolites released from dying cells drive the inter-kingdom transcriptional re-wiring of the Enterobacteriaceae, including fundamental shifts in bacterial respiration and promotion of purine utilization-dependent expansion, which in turn delays the recovery of the intestinal tract. Inhibition of epithelial cell death or restriction of the Enterobacteriaceae to homeostatic levels reverses dysbiosis and improves intestinal recovery. These findings suggest that supportive therapies that maintain homeostatic levels of Enterobacteriaceae may be useful in resolving intestinal disease.

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. In vitro, CTxA1 bound the IRE1α lumenal domain (IRE1αLD), but global unfolding was not required. Rather, the IRE1αLD recognized a seven-residue motif within an edge ß-strand of CTxA1 that must locally unfold for binding. Binding mapped to a pocket on IRE1αLD normally occupied by a segment of the IRE1α C-terminal flexible loop implicated in IRE1α oligomerization. Mutation of the CTxA1 recognition motif blocked CTx-induced IRE1α activation in live cells, thus linking the binding event with IRE1α signal transduction and induction of the UPR.

Evolution and function of the epithelial cell-specific ER stress sensor IRE1ß.

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. Several dedicated molecules with a unique tissue-specific expression in mucosal epithelia govern mucosal homeostasis. Here, we review the biology of Inositol-requiring enzyme 1ß (IRE1ß), an ER-resident endonuclease and paralogue of the most evolutionarily conserved ER stress sensor IRE1α. IRE1ß arose through gene duplication in early vertebrates and adopted functions unique from IRE1α which appear to underlie the basic development and physiology of mucosal tissues.

IRE1ß negatively regulates IRE1α signaling in response to endoplasmic reticulum stress.

IRE1ß is an ER stress sensor uniquely expressed in epithelial cells lining mucosal surfaces. Here, we show that intestinal epithelial cells expressing IRE1ß have an attenuated unfolded protein response to ER stress. When modeled in HEK293 cells and with purified protein, IRE1ß diminishes expression and inhibits signaling by the closely related stress sensor IRE1α. IRE1ß can assemble with and inhibit IRE1α to suppress stress-induced XBP1 splicing, a key mediator of the unfolded protein response. In comparison to IRE1α, IRE1ß has relatively weak XBP1 splicing activity, largely explained by a nonconserved amino acid in the kinase domain active site that impairs its phosphorylation and restricts oligomerization. This enables IRE1ß to act as a dominant-negative suppressor of IRE1α and affect how barrier epithelial cells manage the response to stress at the host-environment interface.

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. Here, we describe a novel quantitative and effectively real-time single-cell flow cytometry assay to directly measure retrograde membrane transport. The assay takes advantage of the well-known retrograde trafficking of cholera toxin engineered with split-fluorescent proteins to generate novel tools for immediate monitoring of intracellular trafficking. This approach will greatly extend the ability to study the underlying biology of intracellular membrane trafficking, and how trafficking systems can adapt to the physiologic needs of different cell types and cell states.