Gastrobodies are engineered antibody mimetics resilient to pepsin and hydrochloric acid

Protein-based targeting reagents, such as antibodies and non-antibody scaffold proteins, are rapidly inactivated in the upper gastrointestinal (GI) tract. Hydrochloric acid in gastric juice denatures proteins and activates pepsin, concentrations of which reach 1 mg/mL in the mammalian stomach. Two stable scaffold proteins (nanobody and nanofitin), previously developed to be protease-resistant, were completely digested in less than 10 min at 100-fold lower concentration of pepsin than found in the stomach. Here we present gastrobodies, a protein scaffold derived from Kunitz soybean trypsin inhibitor (SBTI). SBTI is highly resistant to the challenges of the upper GI tract, including digestive proteases, pH 2 and bile acids. Computational prediction of SBTI’s evolvability identified two nearby loops for randomization, to create a potential recognition surface which was experimentally validated by alanine scanning. We established display of SBTI on full-length pIII of M13 phage. Phage selection of gastrobody libraries against the glucosyltransferase domain of Clostridium difficile toxin B (GTD) identified hits with nanomolar affinity and enzyme inhibitory activity. Anti-GTD binders retained high stability to acid, digestive proteases and heat. Gastrobodies show resilience to exceptionally harsh conditions, which should provide a foundation for targeting and modulating function within the GI tract.

Ontogenetic Pattern Changes of Nucleobindin-2/Nesfatin-1 in the Brain and Intestinal Bulb of the Short Lived African Turquoise Killifish

Nesfatin-1 (Nesf-1) was identified as an anorexigenic and well conserved molecule in rodents and fish. While tissue distribution of NUCB2 (Nucleobindin 2)/Nesf-1 is discretely known in vertebrates, reports on ontogenetic expression are scarce. Here, we examine the age-related central and peripheral expression of NUCB2/Nesf-1 in the teleost African turquoise killifish Nothobranchius furzeri, a consolidated model organism for aging research. We focused our analysis on brain areas responsible for the regulation of food intake and the rostral intestinal bulb, which is analogous of the mammalian stomach. We hypothesize that in our model, the stomach equivalent structure is the main source of NUCB2 mRNA, displaying higher expression levels than those observed in the brain, mainly during aging. Remarkably, its expression significantly increased in the rostral intestinal bulb compared to the brain, which is likely due to the typical anorexia of aging. When analyzing the pattern of expression, we confirmed the distribution in diencephalic areas involved in food intake regulation at all age stages. Interestingly, in the rostral bulb, NUCB2 mRNA was localized in the lining epithelium of young and old animals, while Nesf-1 immunoreactive cells were distributed in the submucosae. Taken together, our results represent a useful basis for gaining deeper knowledge regarding the mechanisms that regulate food intake during vertebrate aging.

Helicobacter pylori Senses Bleach as a Chemoattractant Using a Cytosolic Chemoreceptor

SUMMARYThe gastric pathogenHelicobacter pylorirequires a non-canonical cytosolic chemoreceptor transducer-like protein D (TlpD) for efficient colonization of the mammalian stomach. Here we reconstituted a complete chemotransduction signaling complexin vitrowith TlpD and the chemotaxis proteins CheW and CheA, enabling quantitative assays for potential chemotaxis ligands. We found that TlpD is selectively sensitive at micromolar concentrations to bleach (hypochlorous acid, HOCl), a potent antimicrobial produced by neutrophil myeloperoxidase during inflammation. Counterintuitively, HOCl acts as a chemoattractant by reversibly oxidizing a conserved cysteine within a 3His/1Cys Zn-binding motif in TlpD that inactivates the chemotransduction signaling complex. We found thatH. pyloriis resistant to killing by millimolar concentrations of HOCl and responds to bleach in the micromolar range by increasing its smooth swimming behavior, leading to chemoattraction to HOCl sources. We found that related protein domains fromSalmonella entericaandEscherichia colishowed a similar reactivity toward bleach. We propose that this family of proteins enables host-associated bacteria to sense sites of tissue inflammation, a strategy thatH. pyloriuses to aid in colonizing and persisting in inflamed gastric tissue.

Intracellular Accumulation of High Levels of γ-Aminobutyrate by Listeria monocytogenes 10403S in Response to Low pH: Uncoupling of γ-Aminobutyrate Synthesis from Efflux in a Chemically Defined Medium

ABSTRACT It is well established that the glutamate decarboxylase (GAD) system is central to the survival of Listeria monocytogenes at low pH, both in acidic foods and within the mammalian stomach. The accepted model proposes that under acidic conditions extracellular glutamate is transported into the cell in exchange for an intracellular γ-aminobutyrate (GABAi). The glutamate is then decarboxylated to GABAi, a reaction that consumes a proton, thereby helping to prevent acidification of the cytoplasm. In this study, we show that glutamate supplementation had no influence on either growth rate at pH 5.0 or survival at pH 2.5 when L. monocytogenes 10403S was grown in a chemically defined medium (DM). In response to acidification, cells grown in DM failed to efflux GABA, even when glutamate was added to the medium. In contrast, in brain heart infusion (BHI), the same strain produced significant extracellular GABA (GABAe) in response to acidification. In addition, high levels of GABAi (>80 mM) were found in the cytoplasm in response to low pH in both growth media. Medium-swap and medium-mixing experiments revealed that the GABA efflux apparatus was nonfunctional in DM, even when glutamate was present. It was also found that the GadT2D2 antiporter/decarboxylase system was transcribed poorly in DM-grown cultures while overexpression of gadD1T1 and gadD3 occurred in response to pH 3.5. Interestingly, BHI-grown cells did not respond with upregulation of any of the GAD system genes when challenged at pH 3.5. The accumulation of GABAi in cells grown in DM in the absence of extracellular glutamate indicates that intracellular glutamate is the source of the GABAi. These results demonstrate that GABA production can be uncoupled from GABA efflux, a finding that alters the way we should view the operation of bacterial GAD systems.

Ghrelin/motilin-related peptide is a potent prokinetic to reverse gastric postoperative ileus in rat

A novel peptide called ghrelin or motilin-related-peptide (MTLRP) was found in the stomach of various mammals. We studied its effect on the motor function of the rat gastrointestinal tract. In normal, conscious unoperated animals, ghrelin/MTLRP (5 or 20 μg/kg iv) significantly accelerated the gastric emptying of a methylcellulose liquid solution (gastric residue after 15 min: 57 ± 7, 42 ± 11, 17 ± 4, and 9 ± 3% of the ingested meal with doses of 0, 1, 5, and 20 μg/kg iv, respectively) Transit of the methylcellulose liquid solution was also accelerated by ghrelin/MTLRP in the small intestine but not in the colon. Des-[Gln14]ghrelin, also found in the mammalian stomach, was as potent as ghrelin in emptying the stomach (gastric residue after 15 min: 12 ± 3% at a dose of 20 μg/kg iv). In rats in which postoperative gastrointestinal ileus had been experimentally induced, ghrelin/MTLRP (20 μg/kg iv) reversed the delayed gastric evacuation (gastric residue after 15 min: 28 ± 7% of the ingested meal vs. 82 ± 9% with saline). In comparison, the gastric ileus was not modified by high doses of motilin (77 ± 7%) or erythromycin (82 ± 6%) and was only partially improved by calcitonin gene-related peptide (CGRP) 8–37 antagonist (59 ± 7%). Ghrelin/MTLRP, therefore, accelerates the gastric emptying and small intestinal transit of a liquid meal and is a strong prokinetic agent capable of reversing the postoperative gastric ileus in rat.

Profound increase in viscosity and aggregation of pig gastric mucin at low pH

Epithelial mucins are glycoproteins of very large molecular weight that provide viscoelastic and gel-forming properties to mucus, the jellylike protective layer covering epithelial organs. In the mammalian stomach the mucus gel layer protects the underlying epithelial cells from HCl in the lumen. We report here that pig gastric mucin undergoes a 100-fold increase in viscosity in vitro when pH is lowered from 7 to 2. Sedimentation velocity and dynamic light-scattering measurements revealed the formation of extremely large aggregates at low pH consistent with the observed increase in viscosity. Aggregation of mucin at low pH was prevented by increasing the ionic strength, suggesting the involvement of electrostatic interactions. Trypsin digestion and thiol reduction, but not enzymatic removal of neuraminic acid, prevented aggregation at low pH. This implies that the peptide core rather than the oligosaccharide side chains of the molecule is involved in the aggregation of mucin at low pH. Increased aggregation and viscosity at low pH were also observed in a solvent made to mimic the ionic composition of gastric juice, indicating the physiological relevance of our findings. Our observations suggest that one mechanism of gastric protection may be the ability of gastric mucin to undergo aggregation with a marked increase in viscosity at low pH.

A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates

A method is presented that consistently yields a large number of calcium-tolerant myocytes from mammalian, amphibian, and elasmobranch hearts and from mammalian stomach. The use of incubating solutions or cell harvesting techniques was not required. The time needed to isolate cells was shorter than previously reported values. Action potentials recorded from each cell type appear similar in configuration to that of the intact multicellular tissue. The isolated myocytes appear to tolerate long periods of electrophysiological experimentation using the "giga-seal" suction electrode technique of Hamill et al. (Pfluegers Arch. 391: 85-100, 1981). This method is ideally suited for comparative electrophysiological studies, since the procedure for cell isolation was not seriously modified according to the preparation or species used.

Regulation of gastroduodenal HCO-3 transport by luminal acid in the frog in vitro

Luminal acid (10 mM HCl) is a stimulant of surface epithelial HCO-3 transport in mammalian stomach and duodenum in vivo. To determine whether a humoral mechanism is involved in mediation of this response, amphibian fundic, antral, or proximal duodenal mucosae were mounted in parallel in an in vitro chamber with their nutrient (serosal) surfaces facing a common solution. The mucosal surfaces were bathed by separate solutions and the rate of HCO-3 transport by one mucosa titrated (at pH 7.40) during exposure of the parallel tissue to luminal acid. In studies of fundic HCO-3 transport, H+ secretion was inhibited with the histamine H2-antagonist tiotidine (10(-4) M). Fundic luminal acid stimulated HCO-3 transport by a parallel fundus (27 +/- 6%) or antrum (53 +/- 27%) but had no effect on a parallel duodenum. Antral luminal acid had no effect on a parallel antrum, indicating that the gastric stimulant is of fundic origin. Duodenal luminal acid increased HCO-3 transport by both parallel duodenum (21 +/- 5%) and fundus (109 +/- 32%). Stimulation of HCO-3 transport occurred at higher luminal pH in duodenum (approximately 4.0) than in fundus (approximately 2.0). Thus, exposure to luminal acid releases humoral factor(s) capable of stimulating surface epithelial HCO-3 transport by both stomach and duodenum. The actions of these putative stimulants are in part tissue specific, and they may be important in mediation of mucosal protection against luminal acid.