Expression, Purification, and Cryo-EM Structural Analysis of an Outer Membrane Secretin Channel.

Secretin proteins form pores in the outer membranes of Gram-negative bacteria, and as such provide a means of transporting a wide variety of molecules out of or in to the cell. They are important components of several different bacterial secretion systems, surface filament assembly machineries, and virus assembly complexes. Despite accommodating a diverse assortment of molecules, including virulence factors, folded proteins, and whole viruses, the secretin family of proteins is highly conserved, particularly in their membrane-embedded ß-barrel domain. We describe here a protocol for the expression, purification and cryo-EM structural determination of the pIV secretin from the Ff family of filamentous bacteriophages.

Role of CFTR in diabetes-induced pancreatic ductal fluid and HCO3 - secretion.

Type 1 diabetes is a disease of the endocrine pancreas; however, it also affects exocrine function. Although most studies have examined the effects of diabetes on acinar cells, much less is known regarding ductal cells, despite their important protective function in the pancreas. Therefore, we investigated the effect of diabetes on ductal function. Diabetes was induced in wild-type and cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice following an i.p. administration of streptozotocin. Pancreatic ductal fluid and HCO3 - secretion were determined using fluid secretion measurements and fluorescence microscopy, respectively. The expression of ion transporters was measured by real-time PCR and immunohistochemistry. Transmission electron microscopy was used for the morphological characterization of the pancreas. Serum secretin and cholecystokinin levels were measured by an enzyme-linked immunosorbent assay. Ductal fluid and HCO3 - secretion, CFTR activity, and the expression of CFTR, Na+ /H+ exchanger-1, anoctamine-1 and aquaporin-1 were significantly elevated in diabetic mice. Acute or chronic glucose treatment did not affect HCO3 - secretion, but increased alkalizing transporter activity. Inhibition of CFTR significantly reduced HCO3 - secretion in both normal and diabetic mice. Serum levels of secretin and cholecystokinin were unchanged, but the expression of secretin receptors significantly increased in diabetic mice. Diabetes increases fluid and HCO3 - secretion in pancreatic ductal cells, which is associated with the increased function of ion and water transporters, particularly CFTR. KEY POINTS: There is a lively interaction between the exocrine and endocrine pancreas not only under physiological conditions, but also under pathophysiological conditions The most common disease affecting the endocrine part is type-1 diabetes mellitus (T1DM), which is often associated with pancreatic exocrine insufficiency Compared with acinar cells, there is considerably less information regarding the effect of diabetes on pancreatic ductal epithelial cells, despite the fact that the large amount of fluid and HCO3 - produced by ductal cells is essential for maintaining normal pancreatic functions Ductal fluid and HCO3 - secretion increase in T1DM, in which increased cystic fibrosis transmembrane conductance regulator activation plays a central role. We have identified a novel interaction between T1DM and ductal cells. Presumably, the increased ductal secretion represents a defence mechanism in the prevention of diabetes, but further studies are needed to clarify this issue.

Secretin-dependent signals in the ventromedial hypothalamus regulate energy metabolism and bone homeostasis in mice.

Secretin, though originally discovered as a gut-derived hormone, is recently found to be abundantly expressed in the ventromedial hypothalamus, from which the central neural system controls satiety, energy metabolism, and bone homeostasis. However, the functional significance of secretin in the ventromedial hypothalamus remains unclear. Here we show that the loss of ventromedial hypothalamus-derived secretin leads to osteopenia in male and female mice, which is primarily induced by diminished cAMP response element-binding protein phosphorylation and upregulation in peripheral sympathetic activity. Moreover, the ventromedial hypothalamus-secretin inhibition also contributes to hyperphagia, dysregulated lipogenesis, and impaired thermogenesis, resulting in obesity in male and female mice. Conversely, overexpression of secretin in the ventromedial hypothalamus promotes bone mass accrual in mice of both sexes. Collectively, our findings identify an unappreciated secretin signaling in the central neural system for the regulation of energy and bone metabolism, which may serve as a new target for the clinical management of obesity and osteoporosis.

Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system.

In animal pathogens, assembly of the type III secretion system injectisome requires the presence of so-called pilotins, small lipoproteins that assist the formation of the secretin ring in the outer membrane. Using a combination of functional assays, interaction studies, proteomics, and live-cell microscopy, we determined the contribution of the pilotin to the assembly, function, and substrate selectivity of the T3SS and identified potential new downstream roles of pilotin proteins. In absence of its pilotin SctG, Yersinia enterocolitica forms few, largely polar injectisome sorting platforms and needles. Accordingly, most export apparatus subcomplexes are mobile in these strains, suggesting the absence of fully assembled injectisomes. Remarkably, while absence of the pilotin all but prevents export of early T3SS substrates, such as the needle subunits, it has little effect on secretion of late T3SS substrates, including the virulence effectors. We found that although pilotins interact with other injectisome components such as the secretin in the outer membrane, they mostly localize in transient mobile clusters in the bacterial membrane. Together, these findings provide a new view on the role of pilotins in the assembly and function of type III secretion injectisomes.

Secretin: a hormone for HCO3- homeostasis.

Secretin is a key hormone of the intestinal phase of digestion which activates pancreatic, bile duct and Brunner gland HCO3- secretion. Recently, the secretin receptor (SCTR) was also found in the basolateral membrane of the beta-intercalated cell (B-IC) of the collecting duct. Experimental addition of secretin triggers a pronounced activation of urinary HCO3- excretion, which is fully dependent on key functional proteins of the B-IC, namely apical pendrin and CFTR and the basolateral SCTR. Recent studies demonstrated that the SCTR knock-out mouse is unable to respond to an acute base load. Here, SCTR KO mice could not rapidly increase urine base excretion, developed prolonged metabolic alkalosis and exhibited marked compensatory hypoventilation. Here, we review the physiological effects of secretin with distinct focus on how secretin activates renal HCO3- excretion. We describe its new function as a hormone for HCO3- homeostasis.

Insights into dynamics and gating properties of T2SS secretins.

Secretins are outer membrane (OM) channels found in various bacterial nanomachines that secrete or assemble large extracellular structures. High-resolution 3D structures of type 2 secretion system (T2SS) secretins revealed bimodular channels with a C-module, holding a conserved central gate and an optional top gate, followed by an N-module for which multiple structural organizations have been proposed. Here, we perform a structure-driven in vivo study of the XcpD secretin, which validates one of the organizations of the N-module whose flexibility enables alternative conformations. We also show the existence of the central gate in vivo and its required flexibility, which is key for substrate passage and watertightness control. Last, functional, genomic, and phylogenetic analyses indicate that the optional top gate provides a gain of watertightness. Our data illustrate how the gating properties of T2SS secretins allow these large channels to overcome the duality between the necessity of preserving the OM impermeability while simultaneously promoting the secretion of large, folded effectors.

Expression of Secretin and its Receptor Along the Intestinal Tract in Type 2 Diabetes Patients and Healthy Controls.

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.

Prolonged administration of a secretin receptor antagonist inhibits biliary senescence and liver fibrosis in Mdr2 -/- mice.

BACKGROUND AND AIMS: Secretin (SCT) and secretin receptor (SR, only expressed on cholangiocytes within the liver) play key roles in modulating liver phenotypes. Forkhead box A2 (FoxA2) is required for normal bile duct homeostasis by preventing the excess of cholangiocyte proliferation. Short-term administration of the SR antagonist (SCT 5-27) decreased ductular reaction and liver fibrosis in bile duct ligated and Mdr2 -/- [primary sclerosing cholangitis (PSC), model] mice. We aimed to evaluate the effectiveness and risks of long-term SCT 5-27 treatment in Mdr2 -/- mice. APPROACH AND RESULTS: In vivo studies were performed in male wild-type and Mdr2 -/- mice treated with saline or SCT 5-27 for 3 months and human samples from late-stage PSC patients and healthy controls. Compared with controls, biliary SCT/SR expression and SCT serum levels increased in Mdr2 -/- mice and late-stage PSC patients. There was a significant increase in ductular reaction, biliary senescence, liver inflammation, angiogenesis, fibrosis, biliary expression of TGF-ß1/VEGF-A axis, and biliary phosphorylation of protein kinase A and ERK1/2 in Mdr2 -/- mice. The biliary expression of miR-125b and FoxA2 decreased in Mdr2 -/- compared with wild-type mice, which was reversed by long-term SCT 5-27 treatment. In vitro , SCT 5-27 treatment of a human biliary PSC cell line decreased proliferation and senescence and SR/TGF-ß1/VEGF-A axis but increased the expression of miR-125b and FoxA2. Downregulation of FoxA2 prevented SCT 5-27-induced reduction in biliary damage, whereas overexpression of FoxA2 reduced proliferation and senescence in the human PSC cell line. CONCLUSIONS: Modulating the SCT/SR axis may be critical for managing PSC.

Integrative structural analysis of the type III secretion system needle complex from Shigella flexneri.

The type III secretion system (T3SS) is a large, transmembrane protein machinery used by various pathogenic gram-negative bacteria to transport virulence factors into the host cell during infection. Understanding the structure of T3SSs is crucial for future developments of therapeutics that could target this system. However, much of the knowledge about the structure of T3SS is available only for Salmonella, and it is unclear how this large assembly is conserved across species. Here, we combined cryo-electron microscopy, cross-linking mass spectrometry, and integrative modeling to determine the structure of the T3SS needle complex from Shigella flexneri. We show that the Shigella T3SS exhibits unique features distinguishing it from other structurally characterized T3SSs. The secretin pore complex adopts a new fold of its C-terminal S domain and the pilotin MxiM[SctG] locates around the outer surface of the pore. The export apparatus structure exhibits a conserved pseudohelical arrangement but includes the N-terminal domain of the SpaS[SctU] subunit, which was not present in any of the previously published virulence-related T3SS structures. Similar to other T3SSs, however, the apparatus is anchored within the needle complex by a network of flexible linkers that either adjust conformation to connect to equivalent patches on the secretin oligomer or bind distinct surface patches at the same height of the export apparatus. The conserved and unique features delineated by our analysis highlight the necessity to analyze T3SS in a species-specific manner, in order to fully understand the underlying molecular mechanisms of these systems. The structure of the type III secretion system from Shigella flexneri delineates conserved and unique features, which could be used for the development of broad-range therapeutics.

Secretin modulates appetite via brown adipose tissue-brain axis.

PURPOSE: Secretin 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 RESULTS: In 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. CONCLUSION: These 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 REGISTRATION: EudraCT no. 2016-002373-35, registered 2 June 2016; Clinical Trials no. NCT03290846, registered 25 September 2017.

Secretin alleviates biliary and liver injury during late-stage primary biliary cholangitis via restoration of secretory processes.

BACKGROUND & AIMS: Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples. METHODS: At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late-stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models. RESULTS: Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA-sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late-stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment. CONCLUSION: Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC. IMPACT AND IMPLICATIONS: Secretin (Sct) regulates biliary proliferation and bicarbonate secretion in cholangiocytes via its receptor, SR, and in mouse models and human samples of late-stage primary biliary cholangitis (PBC), the Sct/SR axis is blunted along with loss of the protective 'bicarbonate umbrella'. We found that both short- and long-term Sct treatment ameliorated ductular reaction, immune cell influx, and liver fibrosis in late-stage PBC mouse models. Importantly, Sct treatment promoted bicarbonate and mucin secretion and hepatic bile acid efflux, thus reducing cholestatic and toxic bile acid-associated injury in late-stage PBC mouse models. Our work perpetuates the hypothesis that PBC pathogenesis hinges on secretory defects, and restoration of secretory processes that promote the 'bicarbonate umbrella' may be important for amelioration of PBC-associated damage.

Structural lessons on bacterial secretins.

To exchange and communicate with their surroundings, bacteria have evolved multiple active and passive mechanisms for trans-envelope transport. Among the pore-forming complexes found in the outer membrane of Gram-negative bacteria, secretins are distinctive homo-oligomeric channels dedicated to the active translocation of voluminous structures such as folded proteins, assembled fibers, virus particles or DNA. Members of the bacterial secretin family share a common cylinder-shaped structure with a gated pore-forming part inserted in the outer membrane, and a periplasmic channel connected to the inner membrane components of the corresponding nanomachine. In this mini-review, we will present what recently determined 3D structures have told us about the mechanisms of translocation through secretins of large substrates to the bacterial surface or in the extracellular milieu.

Isolation and functional identification of secretin family G-protein coupled receptor from Y-organ of the mud crab, Scylla olivacea.

Recently, genes in the superfamily of GPCR are gaining more interest in crustaceans as more evidence shows that they are involved in molting. This study identified four forms of the secretin family of G-protein coupled receptor (GPCR) from the Y-organ of mud crab, Scylla olivacea (ScoGPCR). A full-length sequence of ScoGPCR-B2 was isolated and identified as a lipoprotein receptor while three forms of GPCR in Methuselah-like (Mthl) or B3 subfamilies were reported as ScoGPCR-B3a, -B3b, and -B3c. These four forms exhibit common features of the 7-trans membrane (7TM) domain and distinct aspects in the extracellular region (ECR) at the N-terminus. At the ECR, disulfide bridges are predicted to generate structural stability in all four forms while the putative ScoGPCR-B3 proteins retain conserved Tyr, Trp, Pro, and Phe residues, possibly to form the aromatic-proline interactions and function as key residues for receptor recognition. Expression levels of ScoGPCR-B2 and -B3 in eyestalk, thoracic ganglion, and hindgut between intermolt and premolt stages are similar. Only ScoGPCR-B2 and ScoGPCR-B3a in Y-organ (YO) seem to be premolt-specific responses. An upregulation of ScoGPCR-B2 in YO at the premolt stage is correlated with the demand for cholesterol used in ecdysteroid synthesis, resulting in increased ecdysteroid titers. The effects of ecdysone on YO were pursued by in vitro incubation and revealed that ScoGPCR-B3a and -B3b expressions were induced in a different time frame: early in ScoGPCR-B3b and late in ScoGPCR-B3a. The early response of ScoGPCR-B3b was followed through immunohistology and showed that the newly synthesized protein was located primarily in the cytosol.

Secretin receptor deletion in the subfornical organ attenuates the activation of excitatory neurons under dehydration.

In mammals, thirst is strongly influenced by the subfornical organ (SFO), a forebrain structure that integrates circulating signals including osmotic pressure and sodium contents. Secretin (SCT), a classical gastrointestinal hormone, has been implicated as a humoral factor regulating body-fluid homeostasis. However, the neural mechanism of secretin in the central nervous system in managing thirst remains unclear. In this study, we report that the local ablation of SCT receptor (SCTR) in the SFO reduces water but not salt intake in dehydrated mice and this effect could not be rescued by exogenous SCT administration. Electrophysiology with single-cell RT-PCR indicates that SCT elicits inward currents in the SFO neuronal nitric oxide synthase (SFOnNOS) neurons via SCTR in the presence of glutamate receptor antagonists. We further show that the SCTR in the SFO permits the activation of SFOnNOS neurons under distinct thirst types. Projection-specific gene deletion of SCTR in SFO to the median preoptic nucleus (MnPO) pathway also reduces water intake in dehydrated animals. SCT signaling thus plays an indispensable role in driving thirst. These data not only expand the functional boundaries of SCTR but also provide insights into the central mechanisms of homeostatic regulation.

Differential effects of glucose-dependent insulinotropic polypeptide receptor/glucagon-like peptide-1 receptor heteromerization on cell signaling when expressed in HEK-293 cells.

The incretin hormones: glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are important regulators of many aspects of metabolism including insulin secretion. Their receptors (GIPR and GLP-1R) are closely related members of the secretin class of G-protein-coupled receptors. As both receptors are expressed on pancreatic ß-cells there is at least the hypothetical possibility that they may form heteromers. In the present study, we investigated GIPR/GLP-1R heteromerization and the impact of GIPR on GLP-1R-mediated signaling and vice versa in HEK-293 cells. Real-time fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) saturation experiments confirm that GLP-1R and GIPR form heteromers. Stimulation with 1 µM GLP-1 caused an increase in both FRET and BRET ratio, whereas stimulation with 1 µM GIP caused a decrease. The only other ligand tested to cause a significant change in BRET signal was the GLP-1 metabolite, GLP-1 (9-36). GIPR expression had no significant effect on mini-Gs recruitment to GLP-1R but significantly inhibited GLP-1 stimulated mini-Gq and arrestin recruitment. In contrast, the presence of GLP-1R improved GIP stimulated mini-Gs and mini-Gq recruitment to GIPR. These data support the hypothesis that GIPR and GLP-1R form heteromers with differential consequences on cell signaling.

Discovery of small-molecule modulators of the secretin receptor: Purmorphamine as novel anti-hypertensive agent.

The Secretin/Secretin receptor (SCTR) axis is well-known for its important role in water/salt homeostasis and blood pressure control. Recent studies revealed that absence of Secretin could lead to hypertension in animals and the administration of external Secretin leads to a sharp drop in blood pressure. Therefore, Secretin receptor has emerged as a crucial drug target of interest. In this report, using structure based drug design strategy, we have identified a small compound-based Secretin receptor modulator (i.e. purmorphamine or KSD179019). The virtual docking of KSD179019 with SCTR crystal structure and homology models revealed similar binding interactions. Based on active pharmacophores of KSD179019, several derivatives were designed and sythesized. SAR studies revealed that KSD179019 is the most effective SCTR modulator and chosen for further biological evaluation, including drug like properties and anti-hypertensive effect. KSD179019 not only has a similar blood pressure lowering effect as SCT peptide, but more importantly, it has a much longer half-life (∼8 h) and can be taken orally. Preliminary preclinical studies revealed extended bioavailability and low toxicity of this compound.

Computational characterization of homologous ligands binding to a deep hydrophobic pocket in Shigella flexneri pilot protein MxiM.

The type III secretion system (T3SS) is an important molecular machinery in gram-negative bacteria Shigella flexneri as it provides ways for translocating virulence factors from the bacteria into host cells, eventually leading to severe disease symptoms such as bacillary dysentery. Due to the rising concerns of antibiotics resistance in bactericidal strategy, the anti-virulence strategy that primarily targets the T3SS components becomes an attractive alternative. MxiM, the secretin pilot protein of Shigella flexneri, binds the secretin MxiD and facilitates the formation of the secretin ring in outer membrane in T3SS assembly. MxiM harbors a large hydrophobic pocket that has been shown to be important in MxiM-MxiD interaction. In this work, I examined the ligand binding property of MxiM by performing molecular dynamics (MD) simulations of the association between MxiM and a series of hydrophobic ligands, with simulation time amounted to 30 µs. MD simulations successfully captured spontaneous ligand binding events in 153 of the 300 trajectories. The ligand binding can be categorized into two types: a fast type, in which the ligand binds quickly into the hydrophobic pocket and a slow type, in which the ligand forms an encounter complex with the protein before binding into the hydrophobic pocket. Using the MxiM-ligand binding poses captured in MD simulations, I additionally performed umbrella-sampling MD simulations with total simulation time amounted to 63 µs to obtain protein-ligand binding free energies. The relationship between the ligand binding free energy and ligand size appears to be nonlinear and exhibits an exponential decay pattern. In summary, I performed computational characterization of MxiM-hydrophobic ligand binding capabilities and properties, which may provide valuable insights into designing anti-bacterial medicine against antibiotics resistance in Shigella flexneri.

The M13 Phage Assembly Machine Has a Membrane-Spanning Oligomeric Ring Structure.

Bacteriophage M13 assembles its progeny particles in the inner membrane of the host. The major component of the assembly machine is G1p and together with G11p it generates an oligomeric structure with a pore-like inner cavity and an ATP hydrolysing domain. This allows the formation of the phage filament, which assembles multiple copies of the membrane-inserted major coat protein G8p around the extruding single-stranded circular DNA. The phage filament then passes through the G4p secretin that is localized in the outer membrane. Presumably, the inner membrane G1p/G11p and the outer G4p form a common complex. To unravel the structural details of the M13 assembly machine, we purified G1p from infected E. coli cells. The protein was overproduced together with G11p and solubilized from the membrane as a multimeric complex with a size of about 320 kDa. The complex revealed a pore-like structure with an outer diameter of about 12 nm, matching the dimensions of the outer membrane G4p secretin. The function of the M13 assembly machine for phage generation and secretion is discussed.

Maternal secretin ameliorates obesity by promoting white adipose tissue browning in offspring.

Our knowledge of the coordination of intergenerational inheritance and offspring metabolic reprogramming by gastrointestinal endocrine factors is largely unknown. Here, we showed that secretin (SCT), a brain-gut peptide, is downregulated by overnutrition in pregnant mice and women. More importantly, genetic loss of SCT in the maternal gut results in undesirable phenotypes developed in offspring including enhanced high-fat diet (HFD)-induced obesity and attenuated browning of inguinal white adipose tissue (iWAT). Mechanistically, loss of maternal SCT represses iWAT browning in offspring by a global change in genome methylation pattern through upregulation of DNMT1. SCT functions to facilitate ubiquitination and degradation of DNMT1 by activating AMPKα, which contributes to the observed alteration of DNMT1 in progeny. Lastly, we showed that SCT treatment during pregnancy can reduce the development of obesity and improve glucose tolerance and insulin resistance in offspring of HFD-fed females, suggesting that SCT may serve as a novel biomarker or a strategy for preventing metabolic diseases.

Scaffolding Protein GspB/OutB Facilitates Assembly of the Dickeya dadantii Type 2 Secretion System by Anchoring the Outer Membrane Secretin Pore to the Inner Membrane and to the Peptidoglycan Cell Wall.

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.