Disruption of gastric mucous granule exocytosis by Helicobacter pylori virulence factor CagA

Helicobacter pylori infection is the strongest known risk factor of stomach cancer. Strains harboring the virulence factor CagA (cytotoxin-associated gene A) significantly stimulate host inflammatory response, which increases the risk of ulceration and cancer. However, the mechanisms by which CagA triggers prolonged inflammation with mucosal damage remain elusive. Based on a large-scale genetic screen using Drosophila, we identified a novel CagA target Synaptotagmin-like protein 2-a, Slp2-a, an effector of small GTPase Rab27. Using gastric organoid-derived monolayers of polarized mucous cells, we demonstrated that CagA inhibited Slp2-a-mediated docking of mucous granules to the plasma membrane by direct binding to Slp2-a. We further observed aberrant cytoplasmic retention of mucus in human gastric mucosa infected with CagA-expressing strains. These results suggest that CagA could be disrupting the protective mucous barrier by inhibiting Slp2-a-mediated mucous granule exocytosis, which may lead to mucosal damage from luminal acid and pepsin to promote inflammation leading to cancer.

Absence of CFTR is associated with pleiotropic effects on mucins in mouse gallbladder epithelial cells

Mucus of cystic fibrosis patients exhibits altered biochemical composition and biophysical behavior, but the causal relationships between altered cystic fibrosis transmembrane conductance regulator (CFTR) function and the abnormal mucus seen in various organ systems remain unclear. We used cultured gallbladder epithelial cells (GBEC) from wild-type and Cftr(−/−) mice to investigate mucin gene and protein expression, kinetics of postexocytotic mucous granule content expansion, and biochemical and ionic compositions of secreted mucins. Muc1, Muc3, Muc4, Muc5ac, and Muc5b mRNA levels were significantly lower in Cftr(−/−) GBEC compared with wild-type cells, whereas Muc2 mRNA levels were higher in Cftr(−/−) cells. Quantitative immunoblotting demonstrated a trend toward lower MUC1, MUC2, MUC3, MUC5AC, and MUC5B mucin levels in Cftr(−/−) cells compared with cells from wild-type mice. In contrast, the levels of secreted MUC1, MUC3, MUC5B, and MUC6 mucins were significantly higher from Cftr(−/−) cells; a trend toward higher levels of secreted MUC2 and MUC5AC was also noted from Cftr(−/−) cells. Cftr(−/−) cells demonstrated slower postexocytotic mucous granule content expansion. Calcium concentration was significantly elevated in the mucous gel secreted by Cftr(−/−) cells compared with wild-type cells. Secreted mucins from Cftr(−/−) cells contained higher sulfate concentrations. Thus absence of CFTR is associated with pleiotropic effects on mucins in murine GBEC.

Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine.

The parasympathetic control of goblet cell secretion and the membrane events accompanying accelerated mucus release were studied in large intestinal mucosal biopsies maintained in an organ culture system. The secretory response of individual goblet cells to 10(-6) M acetylcholine chloride with 3 x 10(-3) M eserine sulfate (a cholinesterase inhibitor) was assessed by light microscopy and autoradiography, by scanning and transmission electron microscopy, and by freeze-fracture. Goblet cells on the mucosal surface are unaffected by acetylcholine. In crypt goblet cells acetylcholine-eserine induces rapid fusion of apical mucous granule membranes with the luminal plasma membrane (detectable by 2 min), followed by sequential, tandem fission of the pentalaminar, fused areas of adjacent mucous granule membranes. These events first involve the most central apical mucous granules, are then propagated to include peripheral granules, and finally spread toward the most basal granules. By 60 min, most crypt cells are nearly depleted. The apical membrane, although greatly amplified by these events, remains intact, and intracellular mucous granules do not coalesce with each other. During rapid secretion membrane-limited tags of cytoplasm are observed attached to the cavitated apical cell surface. These long, thin extensions of redundant apical membrane are rapidly lost, apparently by being shed into the crypt lumen.