Gastrointestinal infections often induce epithelial damage that must be repaired for optimal gut function. While intestinal stem cells are critical for this regeneration process [R. C. van der Wath, B. S. Gardiner, A. W. Burgess, D. W. Smith,PLoS One8, e73204 (2013); S. Kozaret al.,Cell Stem Cell13, 626–633 (2013)], how they are impacted by enteric infections remains poorly defined. Here, we investigate infection-mediated damage to the colonic stem cell compartment and how this affects epithelial repair and recovery from infection. Using the pathogenClostridioides difficile,we show that infection disrupts murine intestinal cellular organization and integrity deep into the epithelium, to expose the otherwise protected stem cell compartment, in a TcdB-mediated process. Exposure and susceptibility of colonic stem cells to intoxication compromises their function during infection, which diminishes their ability to repair the injured epithelium, shown by altered stem cell signaling and a reduction in the growth of colonic organoids from stem cells isolated from infected mice. We also show, using both mouse and human colonic organoids, that TcdB from epidemic ribotype 027 strains does not require Frizzled 1/2/7 binding to elicit this dysfunctional stem cell state. This stem cell dysfunction induces a significant delay in recovery and repair of the intestinal epithelium of up to 2 wk post the infection peak. Our results uncover a mechanism by which an enteric pathogen subverts repair processes by targeting stem cells during infection and preventing epithelial regeneration, which prolongs epithelial barrier impairment and creates an environment in which disease recurrence is likely.

Clostridioides (formerly Clostridium) difficile is a gram-positive, spore-forming anaerobe and a leading cause of hospital-acquired infection and gastroenteritis-associated death in U.S. hospitals 1. The disease state is most often preceded by disruption of the host microbiome in response to antibiotic treatment and is characterized by mild to severe diarrhea. C. difficile infection (CDI) is dependent on the secretion of one or more AB-type toxins: toxin A (TcdA), toxin B (TcdB), and the C. difficile transferase toxin (CDT) 2. While TcdA and TcdB are considered the primary virulence factors, recent studies suggest that CDT increases the severity of CDI in some of the most problematic clinical strains 3. To better understand how CDT functions, we used cryo-electron microscopy (cryo-EM) to define the structure of the cell-binding component of CDT (CDTb). We obtained structures of several oligomeric forms that highlight the conformational changes that permit conversion from a prepore to a β-barrel pore. The structural analysis also reveals a glycan binding domain and residues involved in binding the host cell-receptor, lipolysis-stimulated lipoprotein receptor (LSR). Together, these results provide a framework to understand how CDT functions at the host cell interface. CDT belongs to the Iota family of binary toxins and consists of two proteins: an ADPribosyltransferase (CDTa) and a cell binding, pore-forming, and CDTa translocating protein (CDTb) 4. CDTb engages host cells through LSR 5 and undergoes proteolytic cleavage and oligomerization to facilitate CDTa binding. The complex is internalized by endocytosis, and endosome acidification leads to CDTb pore formation and CDTa translocation. Inside the cell, CDTa catalyzes ADP-ribosylation of G-actin which leads to disruption of the cytoskeleton, formation of microtubule protrusions, and increased C. difficile adherence Reprints and permissions information is available at www.nature.com/reprintsUsers may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://

Type III protein secretion systems (T3SS) deliver effector proteins from the Gram-negative bacterial cytoplasm into a eukaryotic host cell through a syringe-like, multi-protein nanomachine. Cytosolic components of T3SS include a portion of the export apparatus, which traverses the inner membrane and features the opening of the secretion channel, and the sorting complex for substrate recognition and for providing the energetics required for protein secretion. Two components critical for efficient effector export are the export gate protein and the ATPase, which are proposed to be linked by the central stalk protein of the ATPase. We present the structure of the soluble export gate homo-nonamer, CdsV, in complex with the central stalk protein, CdsO, of its cognate ATPase, both derived from Chlamydia pneumoniae . This structure defines the interface between these essential T3S proteins and reveals that CdsO engages the periphery of the export gate that may allow the ATPase to catalyze an opening between export gate subunits to allow cargo to enter the export apparatus. We also demonstrate through structure-based mutagenesis of the homologous export gate in Pseudomonas aeruginosa that mutation of this interface disrupts effector secretion. These results provide novel insights into the molecular mechanisms governing active substrate recognition and translocation through a T3SS.

S100B is a damage-associated molecular pattern protein that, when released into the extracellular milieu, triggers initiation of the inflammatory response through the receptor for advanced glycation end products (RAGE). Recognition of S100B is accomplishedviathe amino-terminal variable immunoglobulin domain (V-domain) of RAGE. To gain insights into this interaction, a complex between S100B and a 15-amino-acid peptide derived from residues 54–68 of the V-domain was crystallized. The X-ray crystal structure was solved to 2.55 Å resolution. There are two dimers of S100B and one peptide in the asymmetric unit. The binding interface of this peptide is compared with that found in the complex between S100B and the 12-amino-acid CapZ-derived peptide TRTK-12. This comparison reveals that although the peptides adopt completely different backbone structures, the residues buried at the interface interact with S100B in similar regions to form stable complexes. The binding affinities of S100B for the intact wild-type V-domain and a W61A V-domain mutant were determined to be 2.7 ± 0.5 and 1.3 ± 0.7 µM, respectively, using fluorescence titration experiments. These observations lead to a model whereby conformational flexibility in the RAGE receptor allows the adoption of a binding conformation for interaction with the stable hydrophobic groove on the surface of S100B.

Characterization of MAbs against SARS-CoV-2, elicited through vaccination or natural infection, has provided vital immunotherapeutic options for curbing the COVID-19 pandemic and has supplied critical insights into SARS-CoV-2 escape, transmissibility, and mechanisms of viral inactivation. Neutralizing MAbs that target the RBD but do not block ACE2 binding are of particular interest because the epitopes are well conserved within sarbecoviruses and MAbs targeting this area demonstrate cross-reactivity.