Shin-Yi Du1, Hung-Jung Wang1, Hsin-Hung Cheng2, Sheng-De Chen1, Lily Hui-Ching Wang1, Wen-Ching Wang3. 1. Institute of Molecular and Cellular Biology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan. 2. Institute of Molecular and Cellular Biology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan; Biomedical Science and Engineering Center, National Tsing Hua University, Hsinchu, Taiwan. 3. Institute of Molecular and Cellular Biology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan; Biomedical Science and Engineering Center, National Tsing Hua University, Hsinchu, Taiwan. Electronic address: wcwang@mx.nthu.edu.tw.
Abstract
BACKGROUND/ PURPOSE: Helicobacter pylori colonizes the human stomach and contributes to chronic inflammation of the gastric mucosa. H. pylori persistence occurs because of insufficient eradication by phagocytic cells. A key factor of H. pylori, cholesterol-α-glucosyltransferase encoded by capJ that extracts host cholesterol and converts it to cholesteryl glucosides, is important to evade host immunity. Here, we examined whether phagocytic trafficking in macrophages was perturbed by capJ-carrying H. pylori. METHODS: J774A.1 cells were infected with H. pylori at a multiplicity of infection of 50. Live-cell imaging and confocal microscopic analysis were applied to monitor the phagocytic trafficking events. The viability of H. pylori inside macrophages was determined by using gentamicin colony-forming unit assay. The phagocytic routes were characterized by using trafficking-intervention compounds. RESULTS: Wild type (WT) H. pylori exhibited more delayed entry into macrophages and also arrested phagosome maturation more than did capJ knockout mutant. Pretreatment of genistein and LY294002 prior to H. pylori infection reduced the internalization of WT but not capJ-knockout H. pylori in macrophages. CONCLUSION: Cholesterol glucosylation by H. pylori interferes with phagosome trafficking via a lipid-raft and PI3K-dependent manner, which retards engulfment of bacteria for prolonged intracellular survival of H. pylori.
BACKGROUND/ PURPOSE:Helicobacter pylori colonizes the human stomach and contributes to chronic inflammation of the gastric mucosa. H. pylori persistence occurs because of insufficient eradication by phagocytic cells. A key factor of H. pylori, cholesterol-α-glucosyltransferase encoded by capJ that extracts host cholesterol and converts it to cholesteryl glucosides, is important to evade host immunity. Here, we examined whether phagocytic trafficking in macrophages was perturbed by capJ-carrying H. pylori. METHODS: J774A.1 cells were infected with H. pylori at a multiplicity of infection of 50. Live-cell imaging and confocal microscopic analysis were applied to monitor the phagocytic trafficking events. The viability of H. pylori inside macrophages was determined by using gentamicin colony-forming unit assay. The phagocytic routes were characterized by using trafficking-intervention compounds. RESULTS: Wild type (WT) H. pylori exhibited more delayed entry into macrophages and also arrested phagosome maturation more than did capJ knockout mutant. Pretreatment of genistein and LY294002 prior to H. pyloriinfection reduced the internalization of WT but not capJ-knockout H. pylori in macrophages. CONCLUSION:Cholesterol glucosylation by H. pylori interferes with phagosome trafficking via a lipid-raft and PI3K-dependent manner, which retards engulfment of bacteria for prolonged intracellular survival of H. pylori.
Authors: Weronika Gonciarz; Agnieszka Krupa; Krzysztof Hinc; Michał Obuchowski; Anthony P Moran; Adrian Gajewski; Magdalena Chmiela Journal: PLoS One Date: 2019-08-07 Impact factor: 3.240