During a recent investigation of human gastric mucosa, both H. pylori and its virulence factors were found to accumulate selectively into endosomal vesicles, phagolysosomes and the cytoplasm of dendritic cells, granulocytes, macrophages and mast cells, with or without associated cytotoxic changes [12]. Thus, thorough reinvestigation of H. pylori-infected human gastric epithelium seemed to be warranted so as to gain more evidence on possible cellular accumulation of bacterial products, their intracellular fate and pertinent host cell responses. The following questions were given special consid- eration: 1) Are bacterial products accumulated inside gastric epithelium, and in which cellular compartments? 2) Do ‘‘naked’’ (i.e., devoid of host enveloping membrane) H. pylori occur intracellularly so as to directly contact cytosolic host components? 3) Are bacterial products like VacA and outer membrane components, known to be taken up by endosomal vesicles of infected cells, released to the cytosol where sensors like NODs or UPS may sense them? Here we demonstrate the existence of a novel intracellular structure we named PaCS (for Particle-rich Cytoplasmic Struc- tures) where H. pylori products concentrate in vivo in colonized gastric epithelium as well as in vitro in human epithelial cell lines incubated with H. pylori products. PaCS, characterized by 13-nm- thick cylindrical particles we identified as proteasome complexes, is a distinctive cytoplasmic compartment where both NOD1, a selective H. pylori receptor, and UPS components are co- concentrated. This structure may have a role in bacterial recognition and handling, and may modulate the activity of toxins/virulence factors and induce pertinent immune responses, especially through immunoproteasome. The presence of PaCSs in gastric preneoplastic lesions, and their selective colocalization with putative oncogenic factors like CagA, SHP2 and members of the MAPK/ERK signaling system, may suggest this structure has a role in the regulation of cell growth and neoplastic transformation, also in view of its highly enriched UPS content and the mounting evidence for a role of UPS in cancer origin or progression. Results Identification of a discrete particle-rich cytoplasmic structure Conventional paraffin sections of human gastric mucosa showed 19 patients with gastritis, among the 26 cases investigated, 15 of whom with H. pylori on the luminal side of their superficial-foveolar epithelium. Toluidine blu-stained semithin resin sections from the same cases allowed epithelial degenerative-cytotoxic changes to be identified more easily and revealed small pink-stained cytoplasmic areas inside the blue-stained infra/perinuclear cytoplasm of the foveolar cells (Figure 1A, B and C). Transmission electron microscopy (TEM) of consecutive sections identified such areas with well-defined, discrete cytoplasmic structures, situated in rough endoplasmic reticulum (RER)-rich cytoplasm and charac- terized by the accumulation of moderately osmiophilic barrel-like particles of about the same size as ribosomes. However, unlike ribosomes, the particles showed a distinctive punctate substructure due to minute spots forming parallel lines oriented orthogonally to the particle main axis (Figure 1B1, b2, b3, D, and E). Unlike ribosomes and other phosphonucleoprotein-containing structures, such particles showed no phosphorus signal when analyzed by electron spectroscopy imaging (ESI) (Figure 2A and A1, B and B1). From now on, we will refer to the Particle-rich Cytoplasmic Structures as PaCSs. Common cytoplasmic organelles like mitochondria, Golgi, lysosomes, endosomes, centrosomes or H. pylori and PaCS microtubules were generally excluded from PaCSs or pushed to their borders. H. pylori and their products inside PaCS Sparse intraepithelial, intercellular or intracellular, bacterial bodies or their H. pylori-immunoreactive remnants were detected by TEM in most biopses examined, including specimens from 7 of 11 cases lacking bacteria on the luminal side and diagnosed as H. pylori-negative at light microscopy investigation of paraffin sections. Sometimes, H. pylori bodies or remnants were found inside PaCSs (Figure 2C and D), usually in cells also showing H. pylori outside PaCS, in membrane-enclosed vacuoles of the supranuclear cytoplasm (Figure 2E). Inside PaCS the bacteria, although frequently surrounded by a clear empty space, were devoid of host membrane envelopment, thus being allowed to interact with PaCS components (Figure 2c and d). The H. pylori nature of such bacterial bodies was confirmed by their immunogold reactivity for urease, VacA, and H. pylori OMPs (Figure 2c). In addition, PaCS itself, either with (Figure 2c) or without (Figures 2F and 3A, B and C) inner bacteria, also reacted selectively for bacterial products like VacA, CagA, urease or OMPs. Median immunogold particles counts inside at least 100 mm2 PaCS sections ranged from 3.41 2 (OMPs) to 3.68 (VacA), to 4.33 (urease) per mm , with a ratio between PaCS and surrounding cytoplasm ranging from 8.5 (OMPs) or 8.6 (VacA) up to 27.1 (urease). CagA immunoreactivity of PaCS changed prominently in different biopsies, some of which being unreactive while others showing selective, though variable 2 reactivity (from 1.05 up to 9.90 particles/mm , and a PaCS/ cytoplasm ratio ranging from 4.0 up to 12.8, partly depending on different antisera employed). PaCSs apparently arise in the cytoplasm interposed between RER cisternae by focal, progressive concentration of their distinctive particles at sites where bacterial products accumulate (Figure 2F and f), with subsequent dislocation of surrounding cytoplasm and its organelles. Indeed, thin cytoplasmic remnants were sometimes found inside a PaCS, suggesting it originated from expansion and fusion of smaller foci (Figure 3A, a1, D, d1). PaCSs differ from VacA-induced vacuoles In the majority of cases, H. pylori products stored in PaCS are likely to take origin, as well as from the relatively few intracellular bacteria, from more abundant extracellular bacteria colonizing gastric epithelial cells on their luminal side (Figure 3A and D). Indeed, many membrane invaginations (Figure 4A, a1 and a2), often carrying VacA labelling, were found at sites of bacterial adhesion. They most likely correspond to the ‘‘early VacA carriers’’ seen in vitro to enter cell cytoplasm of VacA-incubated HeLa cells and form tubular early endosomes [8]. Indeed, many tubular vesicles, sometimes VacA-storing, were seen in the apical cytoplasm of colonized superficial-foveolar cells, together with distended round vesicles probably corresponding to late endo- somes (Figure 4A and B). We also observed reciprocal fusion of the latter to form larger VacA-storing vacuoles, still showing remnants of the original endosome membrane inside, especially in endoluminally bulging cells bordered by intercellular luminal clefts (Figure 4B). In addition, autophagic vacuoles storing various cytoplasmic remnants and dense bodies, as well as VacA immunoreactivity were frequently found in the supranuclear cytoplasm (Figure 4C and c). In vitro experiments on HeLa cells incubated for 24 h with H. pylori broth culture filtrate (BCF), besides confirming previous observations on VacA (and OMPs) uptake into endosomal vesicles and vacuoles [7,9], clearly identified typical PaCSs showing VacA immunoreactivity (Figure 4D and d), thus proving their indepen- PLoS ONE | www.plosone.org 2 March 2010 | Volume 5 | Issue 3 | e9716