Abstract
Uptake, intracellular trafficking and pathologic effects of VacA toxin from Helicobacter pylori have been widely investigated in vitro. However, no systematic analysis investigated VacA intracellular distribution and fate in H. pylori-infected human gastric epithelium in vivo, using ultrastructural immunocytochemistry that combines precise toxin localization with analysis of the overall cell ultrastructure and intercompartimental/interorganellar relationships. By immunogold procedure, in this study we investigated gastric biopsies taken from dyspeptic patients to characterize the overall toxin’s journey inside human gastric epithelial cells in vivo. Endocytic pits were found to take up VacA at sites of bacterial adhesion, leading to a population of peripheral endosomes, which in deeper (juxtanuclear) cytoplasm enlarged and fused each other to form large VacA-containing vacuoles (VCVs). These directly opened into endoplasmic reticulum (ER) cisternae, which in turn enveloped mitochondria and contacted the Golgi apparatus. In all such organelles we found toxin molecules, often coupled with structural damage. These findings suggest direct toxin transfer from VCVs to other target organelles such as ER/Golgi and mitochondria. VacA-induced cytotoxic changes were associated with the appearance of auto(phago)lysosomes containing VacA, polyubiquitinated proteins, p62/SQSTM1 protein, cathepsin D, damaged mitochondria and bacterial remnants, thus leading to persistent cell accumulation of degradative products.
Highlights
Helicobacter pylori is a Gram-negative bacterium that colonizes the stomach of about half the global population, being one of the most common bacterial infections worldwide[1,2,3,4,5]
A prominent finding of this investigation of H. pylori-infected human gastric epithelium in vivo was the detection of a population of small subluminal vesicles, interposed with or overlying mucin granules, which were essentially lacking in non-infected epithelium
The non-coated tubular nature of these endocytic pits suggests that the clathrin-independent non-caveolar pinocytic mechanism[31] of VacA internalization documented in experimental models in vitro[9,32] may have an in vivo counterpart in H. pylori-infected patients. Given their abundance in the infected epithelium, especially in association with H. pylori intimately adhering to surface epithelial cells, it seems likely that the endocytic-endosomal vesicles represent the main route of VacA cellular uptake in vivo
Summary
Helicobacter pylori is a Gram-negative bacterium that colonizes the stomach of about half the global population, being one of the most common bacterial infections worldwide[1,2,3,4,5]. An increasing body of evidence indicates that a functional crosstalk exists between VacA and another key virulence factor of H. pylori, the oncoprotein CagA (which is directly injected into host cells by the bacterium through a type IV secretion system) (reviewed in[2,6,7]). VacA and CagA would act synergistically, for instance in providing H. pylori with specific nutrients (e.g., iron) required for its growth This functional crosstalk would be finalized to achieve an optimal fitness of H. pylori with its ecological niche (i.e., the hostile gastric environment), limiting the overall cell damage caused and improving the infection efficiency of the bacterium[2,6,7]. The possibility arises that VCVs may exert a specific functional role in the intoxication process of the toxin, acting as a platform to trigger specific trafficking and signaling pathways exploited or influenced by VacA8
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