In the present paper, we study the mechanism of antibacterial activity of glutathione (GSH) coated silver nanoparticles (Ag NPs) on model Gram negative and Gram positive bacterial strains. Interference in bacterial cell replication is observed for both cellular strains when exposed to GSH stabilized colloidal silver in solution, and microbicidal activity was studied when GSH coated Ag NPs are (i) dispersed in colloidal suspensions or (ii) grafted on thiol-functionalized glass surfaces. The obtained results confirm that the effect of dispersed GSH capped Ag NPs (GSH Ag NPs) on Escherichia coli is more intense because it can be associated with the penetration of the colloid into the cytoplasm, with the subsequent local interaction of silver with cell components causing damages to the cells. Conversely, for Staphylococcus aureus, since the thick peptidoglycan layer of the cell wall prevents the penetration of the NPs inside the cytoplasm, the antimicrobial effect is limited and seems related to the interaction with the bacterial surfaces. Experiments on GSH Ag NPs grafted on glass allowed us to elucidate more precisely the antibacterial mechanism, showing that the action is reduced because of GSH coating and the limitation of the translational freedom of NPs.

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Helicobacter pylori vacuolating toxin (VacA) causes vacuolation in a variety of cultured cell lines, sensitivity to VacA differing greatly, however, among the different cell types. We found that the high sensitivity of HEp-2 cells to VacA was impaired by treating the cells with phosphatidylinositol-specific phospholipase C (PI-PLC) which removes glycosylphosphatidylinositol (GPI)-anchored proteins from the cell surface. Incubation of cells with a cholesterol-sequestering agent, that impairs both structure and function of sphingolipid-cholesterol-rich membrane microdomains ("lipid rafts"), also impaired VacA-induced cell vacuolation. Overexpression into HEp-2 cells of proteins inhibiting clathrin-dependent endocytosis (i.e., a dominant-negative mutant of Eps15, the five tandem Src-homology-3 domains of intersectin, and the K44A dominant-negative mutant of dynamin II) did not affect vacuolation induced by VacA. Nevertheless, F-actin depolymerization, known to block the different types of endocytic mechanisms, strongly impaired VacA vacuolating activity. Taken together, our data suggest that the high cell sensitivity to VacA depends on the presence of one or several GPI-anchored protein(s), intact membrane lipid rafts, and an uptake mechanism via a clathrin-independent endocytic pathway.

In the present work, we describe a simple procedure to produce biomimetically coated silver nanoparticles (Ag NPs), based on the postfunctionalization and purification of colloidal silver stabilized by citrate. Two biological capping agents have been used (cysteine Cys and glutathione GSH). The composition of the capped colloids has been ascertained by different techniques and antibacterial tests on GSH-capped Ag NPs have been conducted under physiological conditions, obtaining values of Minimum Inhibitory Concentration (MIC) of 180 and 15 μg/mL for Staphylococcus aureus and Escherichia coli, respectively. The antibacterial activity of these GSH capped NPs can be ascribed to the direct action of metallic silver NPs, rather than to the bulk release of Ag(+).

Ghrelin is a new gastric peptide involved in food intake control and growth hormone release. We aimed to assess its cell localisation in man during adult and fetal life and to clarify present interspecies inconsistencies of gastric endocrine cell types. A specific serum generated against amino acids 13-28 of ghrelin was tested on fetal and adult gastric mucosa and compared with ghrelin in situ hybridisation. Immunogold electron microscopy was performed on normal human, rat and dog adult stomach. Ghrelin cells were detected in developing gut, pancreas and lung from gestational week 10 and in adult human, rat and dog gastric mucosa. By immunogold electron microscopy, gastric ghrelin cells showed distinctive morphology and hormone reactivity in respect to histamine enterochromaffin-like, somatostatin D, glucagon A or serotonin enterochromaffin cells. Ghrelin cells were characterised by round, compact, electron-dense secretory granules of P/D(1) type in man (mean diameter 147+/-30 nm), A-like type in the rat (183+/-37 nm) and X type in the dog (273+/-49 nm). It is concluded that, ghrelin is produced by well-defined cell types, which in the past had been labelled differently in various mammals mostly because of the different size of their secretory granule. In man ghrelin cells develop during early fetal life.

The Helicobacter pylori (Hp) type IV secretion system (T4SS) forms needle-like pili, whose binding to the integrin-β receptor results in injection of the CagA oncoprotein. However, the apical surface of epithelial cells is exposed to Hp, whereas integrins are basolateral receptors. Hence, the mechanism of CagA delivery into polarized gastric epithelial cells remains enigmatic. Here, we demonstrate that T4SS pilus formation during infection of polarized cells occurs predominantly at basolateral membranes, and not at apical sites. Hp accomplishes this by secreting another bacterial protein, the serine protease HtrA, which opens cell-to-cell junctions through cleaving epithelial junctional proteins including occludin, claudin-8, and E-cadherin. Using a genetic system expressing a peptide inhibitor, we demonstrate that HtrA activity is necessary for paracellular transmigration of Hp across polarized cell monolayers to reach basolateral membranes and inject CagA. The contribution of this unique signaling cascade to Hp pathogenesis is discussed.

Cell internalization and intracellular fate of H. pylori products/virulence factors in vivo by human gastric epithelium, the main target of H. pylori-induced pathologies (i.e., peptic ulcer and cancer), are still largely unknown. Investigating gastric endoscopic biopsies from dyspeptic patients by means of ultrastructural immunocytochemistry, here we show that, in human superficial-foveolar epithelium and its metaplastic or dysplastic foci, H. pylori virulence factors accumulated in a discrete cytoplasmic structure characterized by 13-nm-thick cylindrical particles of regular punctate-linear substructure resembling the proteasome complex in size and structure. Inside this particle-rich cytoplasmic structure (PaCS) we observed colocalization of VacA, CagA, urease and outer membrane proteins with NOD1 receptor, ubiquitin-activating enzyme E1, polyubiquitinated proteins, proteasome components and potentially oncogenic proteins like SHP2 and ERKs in human gastric epithelium. By means of electron and confocal microscopy, we demonstrate that the in vivo findings were reproduced in vitro by incubating human epithelial cell lines with H. pylori products/virulence factors. PaCSs differed from VacA-induced vacuoles, phagosomes, aggresomes or related bodies. Our data suggest that PaCS is a novel, proteasome-enriched structure arising in ribosome-rich cytoplasm at sites of H. pylori products accumulation. As a site of selective concentration of bacterial virulence factors, the ubiquitin-proteasome system and interactive proteins, PaCS is likely to modulate immune-inflammatory and proliferative responses of the gastric epithelium of potential pathologic relevance.