Ultrastructure of Helicobacter pylori in human gastric mucosa and H. pylori-infected human gastric mucosa using transmission electron microscopy and the high-pressure freezing–freeze substitution technique

Abstract

Conventional ultrastructural analyses of Helicobacter pylori and H. pylori-infected gastric mucosa by transmission electron microscopy (TEM) have limitations because of structural artifacts introduced during fixation. We used high-pressure freezing (HPF) followed by freeze substitution for TEM to investigate the ultrastructure of H. pylori and H. pylori-infected gastric mucosa. For HPF-freeze substitution, human gastric biopsy specimens were placed in the HPF instrument at a pressure of approximately 2,000 atm at the temperature of liquid nitrogen. Specimens were then transferred to an instrument for freeze substitution. Specimens were first placed in acetone containing 2% OsO4 at -85 degrees C. The temperature was increased to -3 degrees C, followed by embedding in Quetol 812. Ultrathin sections were double-stained by uranium acetate/lead nitrate. HPF and conventionally prepared samples were examined by TEM. The H. pylori envelope was clearly seen to consist of an outer membrane, periplasmic space, and plasma membrane. The periplasmic space was filled with electron-dense materials. A peptidoglycan layer was only occasionally visible. A thick, very fine filamentous or reticular fringe corresponding to the bacterial glycocalyx was seen surrounding the H. pylori cells. At the adhesion loci of H. pylori to gastric epithelial cells, H. pylori was connected to the epithelial cells by very fine, thickly arranged filaments or more closely, with a contact zone. The epithelial cells showed indentations or pedestals. The well-developed, thick bacterial glycocalyx of H. pylori appears to strongly interact with external cellular components and may play an important role in the adhesion of H. pylori to epithelial cells.