In Vivo Accumulation of Helicobacter pylori Products, NOD1, Ubiquitinated Proteins and Proteasome in a Novel Cytoplasmic Structure Vittorio Necchi1, Patrizia Sommi2,3, Vittorio Ricci2*, Enrico Solcia1,3 1 Department of Human Pathology, University of Pavia Medical School, Pavia, Italy, 2 Department of Physiology, University of Pavia Medical School, Pavia, Italy, 3 Pathologic Anatomy Service, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy Abstract 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. Citation: Necchi V, Sommi P, Ricci V, Solcia E (2010) In Vivo Accumulation of Helicobacter pylori Products, NOD1, Ubiquitinated Proteins and Proteasome in a Novel Cytoplasmic Structure. PLoS ONE 5(3): e9716. doi:10.1371/journal.pone.0009716 Editor: Stefan Bereswill, Charite´-Universita¨tsmedizin Berlin, Germany Received February 11, 2010; Accepted February 25, 2010; Published March 16, 2010 Copyright: 2010 Necchi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This investigation was supported in part by grants from the Italian Ministry of Health to the Fondazione IRCCS Policlinico San Matteo and from the University of Pavia (Fondo di Ateneo per la Ricerca). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: vricci@unipv.it Introduction Gastric superficial-foveolar epithelium is the main site of host/ bacterial interaction in H. pylori infection. H. pylori contacts and adheres to the luminal surface of the epithelium, may enter intercellular lateral spaces and may even penetrate inside the cell cytoplasm [1,2]. At this intracellular site, the bacteria are usually enveloped by a host cell membrane preventing direct contact with the cell cytoplasm and related organelles [2,3]. Coculture of H. pylori with epithelial cell lines showed, in addition to bacterial uptake into the cells, the capacity of H. pylori to deliver toxins and other products into the cells, either by a type IV secretion system directly injecting CagA and peptidoglycan into the cytoplasm [4– 6] or by the autotrasporter mechanism, as seen for VacA which then undergoes internalization through endocytosis [7,8]. Endo- cytosis may also internalize outer membrane vesicles (OMVs) carrying several proteins (OMPs), lipopolysaccharides (LPS), and peptidoglycans (PGs) [9,10]. Urease, a major component of H. pylori bodies, is known to be internalized by lamina propria cells [11] as well as luminal and intraepithelial granulocytes [12], while scarce evidence is available concerning epithelial cells. Although it seems clear, at least from in vitro studies, that H. pylori products can enter epithelial cells, at present there is limited in vivo evidence concerning their internalization and intracellular fate in human gastric epithelium. In addition, it is not clear whether intracellular H. pylori may lose their enveloping host membrane, as it has been shown to occur for other bacteria [13], thus allowing free intracellular release of their virulence factors and direct contact with cytosolic components like NODs or the ubiquitin-proteasome system (UPS), known to be involved in intracellular bacterial recognition and management [6,14]. Intracellular NOD receptors play an important role in H. pylori sensing by epithelial cells [6,10] which respond to the bacterium or its virulence factors by releasing cytokines and chemokines which recruit and activate immune-inflammatory cells [15] or through enhanced production of HLA peptides, costimulatory molecules and cathepsins known to take part in antigen processing and presentation [16–18]. In addition, increased epithelial cell proliferation [19], altered apoptosis [20–22], and a variety of ‘‘cytotoxic changes’’ [1,17] have been described, which may play a role in the genesis of the main pathologic sequelae of chronic H. pylori gastritis, such as peptic ulcer and cancer. However, several intracellular processes linking H. pylori virulence factors to the epithelial response in the infected human mucosa remain to be clarified, especially concerning type of cells and organelles or subcellular compartments involved. PLoS ONE | www.plosone.org 1 March 2010 | Volume 5 | Issue 3 | e9716