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The bundle-forming pilus (BFP) system in enteropathogenic Escherichia coli (EPEC) produces type IVb pili that enable bacterial auto-aggregation, facilitating bacterial adhesion, colonization, and virulence. One of its components, lipoprotein BfpB, interacts with BfpG to form a secretin channel complex that enables pilus translocation across the outer membrane. Here, we report a high-resolution cryo-EM structure of the BfpB-BfpG complex, revealing a 17:17 stoichiometry with stable zigzag-like interactions between BfpG and BfpB near the N3 ring. Secretin BfpB consists of three β-barrels, including an additional N3 barrel that is crucial for BFP biogenesis. As a lipoprotein-type secretin, BfpB possesses an N-terminal LG domain that bridges the N0 domain and the outer membrane, ensuring its correct localization to the bacterial outer membrane. The C-terminal region of the LG domain mediates binding to BfpG, and disruption of these interactions impairs BFP biogenesis. Our results advance our understanding of the assembly mechanism of secretin complexes within the secretin superfamily. Enteropathogenic Escherichia coli produces bundle-forming pili that facilitate bacterial adhesion and colonization. Here, the authors report a high-resolution structure of the BfpB-BfpG secretin channel complex, which enables pilus translocation across the outer membrane, revealing an unusual 17:17 stoichiometry and providing insights into its assembly mechanism.

Abstract Context The hormone secretin (SCT) is released from intestinal S cells and acts via the SCT receptor (SCTR). Circulating SCT levels increase after Roux-en-Y gastric bypass surgery and have been associated with massive weight loss and high remission rates of type 2 diabetes (T2D) linked to these operations. Exogenous SCT was recently shown to reduce ad libitum food intake in healthy volunteers. Objective To understand SCT biology and its potential role in T2D pathophysiology, we examined the intestinal mucosal expression profile of SCT and SCTR and evaluated the density of S cells along the intestinal tract of individuals with T2D and healthy controls. Methods Using immunohistochemistry and messenger RNA (mRNA) sequencing, we analyzed intestinal mucosa biopsies sampled along the small intestine at 30-cm intervals and from 7 well-defined anatomical sites along the large intestine (during 2 sessions of double-balloon enteroscopy) in 12 individuals with T2D and 12 healthy controls. Results Both groups exhibited a progressive and similar decrease in SCT and SCTR mRNA expression and S-cell density along the small intestine, with reductions of 14, 100, and 50 times, respectively, in the ileum compared to the duodenum (used as reference). Negligible amounts of SCTR and SCT mRNA, as well as low S-cell density, were found in the large intestine. No significant differences were observed between the groups. Conclusion SCT and SCTR mRNA expression and S-cell density were abundant in the duodenum and decreased along the small intestine. Very low SCT and SCTR mRNA levels and S-cell numbers were observed in the large intestine, without aberrations in individuals with T2D compared to healthy controls.

Class B G protein-coupled receptors can form dimeric complexes important for high potency biological effects. Here, we apply pharmacological, biochemical, and biophysical techniques to cells and membranes expressing the prototypic secretin receptor (SecR) to gain insights into secretin binding to homo-dimeric and monomeric SecR. Spatial proximity between peptide and receptor residues, probed by disulfide bond formation, demonstrates that the secretin N-terminus moves from adjacent to extracellular loop 3 (ECL3) at wild type SecR toward ECL2 in non-dimerizing mutants. Analysis of fluorescent secretin analogs demonstrates stable engagement of the secretin C-terminal region within the receptor extracellular domain (ECD) for both dimeric and monomeric receptors, while the mid-region exhibits lower mobility while docked at the monomer. Moreover, decoupling of G protein interaction reduces mobility of the peptide mid-region at wild type receptor to levels similar to the mutant, whereas it has no further impact on the monomer. These data support a model of peptide engagement whereby the ability of SecR to dimerize promotes higher conformational dynamics of the peptide-bound receptor ECD and ECLs that likely facilitates more efficient G protein recruitment and activation, consistent with the higher observed functional potency of secretin at wild type SecR relative to the monomeric mutant receptor. GPCRs can form functionally important dimers. Here, authors study impact of dimerization of the secretin receptor on peptide ligand binding and show high receptor conformational dynamics that facilitate G protein recruitment and activation.

Secretin (Sct) is released into the circulation postprandially from the duodenal S-cells. The major functions of Sct originated from the gastrointestinal system are to delay gastric emptying, stimulate fluid secretion from pancreas and liver, and hence optimize the digestion process. In recent years, Sct and its receptor (Sctr) have been identified in discrete nuclei of the hypothalamus, including the paraventricular nucleus (PVN) and the arcuate nucleus (Arc). These nuclei are the primary brain sites that are engaged in regulating body energy homeostasis, thus providing anatomical evidence to support a functional role of Sct in appetite control. In this study, the effect of Sct on feeding behavior was investigated using wild-type (wt), Sct −/− , and secretin receptor-deficient ( Sctr −/− ) mice. We found that both central and peripheral administration of Sct could induce Fos expression in the PVN and Arc, suggesting the activation of hypothalamic feeding centers by this peptide. Consistent with this notion, Sct was found to increase thyrotropin-releasing hormone and melanocortin-4 receptor (Mc4r) transcripts in the PVN, and augment proopiomelanocortin, but reduces agouti-related protein mRNA expression in the Arc. Injection of Sct was able to suppress food intake in wt mice, but not in Sctr −/− mice, and that this effect was abolished upon pretreatment with SHU9119, an antagonist for Mc4r. In summary, our data suggest for the first time that Sct is an anorectic peptide, and that this function is mediated by the melanocortin system.

The traditional nomenclature of enteroendocrine cells (EECs), established in 1977, applied the “one cell - one hormone” dogma, which distinguishes subpopulations based on the secretion of a specific hormone. These hormone-specific subpopulations included S cells for secretin (SCT), K cells for glucose-dependent insulinotropic polypeptide (GIP), N cells producing neurotensin (NTS), I cells producing cholecystokinin (CCK), D cells producing somatostatin (SST), and others. In the past 15 years, reinvestigations into murine and human organoid-derived EECs, however, strongly questioned this dogma and established that certain EECs coexpress multiple hormones. Using the Gut Cell Atlas, the largest available single-cell transcriptome dataset of human intestinal cells, this study consolidates that the original dogma is outdated not only for murine and human organoid-derived EECs, but also for primary human EECs, showing that the expression of certain hormones is not restricted to their designated cell type. Moreover, specific analyses into SCT-expressing cells reject the presence of any cell population that exhibits significantly elevated secretin expression compared to other cell populations, previously referred to as S cells. Instead, this investigation indicates that secretin production is realized jointly by other enteroendocrine subpopulations, validating corresponding observations in murine EECs also for human EECs. Furthermore, our findings corroborate that SCT expression peaks in mature EECs, in contrast, progenitor EECs exhibit markedly lower expression levels, supporting the hypothesis that SCT expression is a hallmark of EEC maturation.

Gram-negative bacteria have two cell membranes sandwiching a peptidoglycan net that together form a robust protective cell envelope. To translocate effector proteins across this multilayer envelope, bacteria have evolved several specialized secretion systems. The phytopathogenic proteobacterium Dickeya dadantii secretes an array of plant cell wall-degrading enzymes and other virulence factors via the type 2 secretion system (T2SS). T2SSs are widespread among important plant, animal, and human bacterial pathogens. This multiprotein complex spans the double membrane cell envelope and secretes fully folded proteins through a large outer membrane pore formed by 15 subunits of the secretin GspD. Secretins are also found in the type 3 secretion system and the type 4 pili. Usually, specialized lipoproteins termed pilotins assist the targeting and assembly of secretins into the outer membrane. Here, we show that in D. dadantii , the pilotin acts in concert with the scaffolding protein GspB. Deletion of gspB profoundly impacts secretin assembly, pectinase secretion, and virulence. Structural studies reveal that GspB possesses a conserved periplasmic homology region domain that interacts directly with the N-terminal secretin domain. Site-specific photo-cross-linking unravels molecular details of the GspB-GspD complex in vivo . We show that GspB facilitates outer membrane targeting and assembly of the secretin pores and anchors them to the inner membrane while the C-terminal extension of GspB provides a scaffold for the secretin channel in the peptidoglycan cell wall. Phylogenetic analysis shows that in other bacteria, GspB homologs vary in length and domain composition and act in concert with either a cognate ATPase GspA or the pilotin GspS. IMPORTANCE Gram-negative bacteria have two cell membranes sandwiching a peptidoglycan net that together form a robust protective cell envelope. To translocate effector proteins across this multilayer envelope, bacteria have evolved several specialized secretion systems. In the type 2 secretion system and some other bacterial machineries, secretins form large multimeric pores that allow transport of effector proteins or filaments across the outer membrane. The secretins are essential for nutrient acquisition and pathogenicity and constitute a target for development of new antibacterials. Targeting of secretin subunits into the outer membrane is often facilitated by a special class of lipoproteins called pilotins. Here, we show that in D. dadantii and some other bacteria, the scaffolding protein GspB acts in concert with pilotin, facilitating the assembly of the secretin pore and its anchoring to both the inner membrane and the bacterial cell wall. GspB homologs of varied domain composition are present in many other T2SSs.

Secretin-induced duodenal aspiration (SIDA) of pancreatic duct fluid has been proposed for pancreatic neoplasm screening in very high-risk patients. We sought to determine the clinical yield and safety of commercially-analyzed SIDA samples in patients at moderately elevated risk. A prospectively maintained institutional database of pancreatic fluid DNA profiles was retrospectively reviewed. Fifty-seven patients underwent SIDA testing, most commonly for intraductal papillary mucinous neoplasms (n=43) and not otherwise specified solitary cysts (n=9). SIDA mutation yield was low compared to 37 concomitant endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) samples of pancreatic fluid: KRAS (2.5% vs. 40.0%), GNAS (2.6% vs. 11.1%) and allelic loss of heterozygosity (3.1% vs. 0%). Patients undergoing SIDA alone experienced no complications while 3 patients with concomitant EUS-FNA had post-procedural pancreatitis. The genetic yield of commercially-analyzed SIDA samples was relatively low in a moderately elevated risk cohort. SIDA testing may have a better safety profile than EUS-FNA.

PurposeSecretin activates brown adipose tissue (BAT) and induces satiation in both mice and humans. However, the exact brain mechanism of this satiety inducing, secretin-mediated gut-BAT-brain axis is largely unknown.Methods and resultsIn this placebo-controlled, single-blinded neuroimaging study, firstly using [18F]-fluorodeoxyglucose (FDG) PET measures (n = 15), we established that secretin modulated brain glucose consumption through the BAT-brain axis. Predominantly, we found that BAT and caudate glucose uptake levels were negatively correlated (r = -0.54, p = 0.037) during secretin but not placebo condition. Then, using functional magnetic resonance imaging (fMRI; n = 14), we found that secretin improved inhibitory control and downregulated the brain response to appetizing food images. Finally, in a PET-fMRI fusion analysis (n = 10), we disclosed the patterned correspondence between caudate glucose uptake and neuroactivity to reward and inhibition, showing that the secretin-induced neurometabolic coupling patterns promoted satiation.ConclusionThese findings suggest that secretin may modulate the BAT-brain metabolic crosstalk and subsequently the neurometabolic coupling to induce satiation. The study advances our understanding of the secretin signaling in motivated eating behavior and highlights the potential role of secretin in treating eating disorders and obesity.Trial registrationEudraCT no. 2016-002373-35, registered 2 June 2016; Clinical Trials no. NCT03290846, registered 25 September 2017.