1. Cholesterol glucosylation by Helicobacter pylori delays internalization and arrests phagosome maturation in macrophages 
Shin-Yi Du, Hung-Jung Wang, Hsin-Hung Cheng, Sheng-De Chen, Lily Hui-Ching Wang, Wen-Ching Wang 
Journal of Microbiology, Immunology and Infection 
Volume 49, Issue 5, Pages (October 2016)
DOI: /j.jmii
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2. Figure 1
Engulfment of Helicobacter pylori by macrophages. (A) J774A.1 cells were plated on glass-bottom dishes and infected with the PKH67-stained H. pylori. Infected live cells were visualized using confocal laser scanning microscopy for time-lapse experiments. Confocal images (488 nm) taken at different times were merged with the corresponding phase-contrast images. The framed region was magnified and is shown below. The arrows indicate bacteria interacting with cells. Scale bars represent 20 μm. (B) Internalization time was measured based on the movement of bacteria and host cells. Each value represents the mean ± standard deviation of data from at least 14 independent experiments. (C) J774A.1 cells were infected with WT, ΔcapJ, or ΔcapJ-in H. pylori for 2 hours, 4 hours, or 6 hours. The viability of H. pylori was calculated using the gentamicin colony-forming unit (CFU) assay. Each value represents the mean ± standard deviation of data from three independent experiments. Statistical significance was evaluated using the Student t test (*p < 0.05; **p < 0.001).
Journal of Microbiology, Immunology and Infection  , DOI: ( /j.jmii )
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3. Figure 2
Colocalization of LysoTracker with Helicobacter pylori in infected macrophages. (A) J774A.1 cells were stained with LysoTracker Red DND-99 (red) to label acidic phagolysosomes and subsequently infected with wild type (WT), ΔcapJ, or ΔcapJ-in H. pylori for 0.5 hours, 1 hour, and 1.5 hours. Infected cells were stained with an antibody against H. pylori, labeled with Cy2-conjugated secondary antibody (green), and observed with confocal fluorescence microscopy. The merged fluorescence picture shows colocalization of LysoTracker with H. pylori in yellow. The framed region was magnified, and the magnified images were shown in the lower right corner. Scale bars represent 20 μm. (B) Quantification of the percentage of cells with yellow organelles was determined by counting at least 200 random cells from each sample. The data represent the mean ± standard deviation of at least three independent experiments. Statistical significance was evaluated using the Student t test (*p < 0.05).
Journal of Microbiology, Immunology and Infection  , DOI: ( /j.jmii )
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4. Figure 3
Colocalization of EEA1 with Helicobacter pylori in infected macrophages. (A) J774A.1 cells were infected with WT, ΔcapJ, or ΔcapJ-in H. pylori for 0.5 hours. Infected cells were stained with antibodies and observed with confocal fluorescence microscopy. The merged fluorescence picture shows co-localization of EEA1 with H. pylori in yellow. Scale bars represent 20 μm. (B) Quantification of the percentage of cells with yellow organelles was determined by counting at least 200 random cells from each sample. The data represent the mean ± standard deviation of at least three independent experiments. Statistical significance was evaluated using Student t test (*p < 0.05). EEA1 = early endosome antigen 1.
Journal of Microbiology, Immunology and Infection  , DOI: ( /j.jmii )
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5. Figure 4
Colocalization of LAMP1 with Helicobacter pylori in infected macrophages. (A) J774A.1 cells were infected wild type (WT), ΔcapJ, or ΔcapJ-in H. pylori for 1 hour. Infected cells were stained with antibodies and observed with confocal fluorescence microscopy. The merged fluorescence picture shows co-localization of LAMP1 with H. pylori in yellow. Scale bars represent 20 μm. (B) Quantification of the percentage of cells with yellow organelles was determined by counting at least 200 random cells from each sample. The data represent the mean ± standard deviation of at least three independent experiments. Statistical significance was evaluated using Student t test (**p < 0.001). LAMP1 = lysosomal-associated membrane protein 1.
Journal of Microbiology, Immunology and Infection  , DOI: ( /j.jmii )
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6. Figure 5
Effect of endocytosis inhibitors on the internalization of Helicobacter pylori into macrophages. J774A.1 cells pretreated with cytochalasin D, nocodazole, genistein, staurosporine, or LY for 30 minutes were infected with (A) wild type (WT), (B) ΔcapJ, or (C) ΔcapJ-in H. pylori for 30 minutes. The viable bacterial uptake was determined by using the gentamicin colony-forming unit (CFU) assay. Control (100%) of WT, ΔcapJ and ΔcapJ-in H. pylori was represented as ∼106 CFU/105 cells, ∼103 CFU/105 cells, and ∼106 CFU/105 cells, respectively. The data represent the mean ± standard deviation of at least three independent experiments. Statistical significance was evaluated using Student t test (*p < 0.05, **p < 0.001).
Journal of Microbiology, Immunology and Infection  , DOI: ( /j.jmii )
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7. Figure 6
Proposed model of how Helicobacter pylori evades eradication in macrophages by delaying internalization and interrupting phagosome maturation. Cholesterol is extracted from macrophage membranes when H. pylori contact host cells [step 1; (1)]. In WT but not ΔcapJ H. pylori, CGs, including αCG, αCAG, and αCGP, are synthesized by membrane-bound CGT. In parallel, the fluctuating CGs in bacteria can drift into the lipid bilayer, which is composed of phospholipids (PL), of macrophage (magnified illustration). Accumulated CGs and cholesterol facilitate selective lateral-phase segregation and induce raft coalescence at host-bacterium contact sites, which may reduce the membrane fluidity and cause wild type (WT) H. pylori to be engulfed more slowly than the ΔcapJ H. pylori [step 2; (2)]. During phagocytosis, WT but not ΔcapJ H. pylori is phagocytized via lipid-raft and PI3K-dependent mechanisms. The CGs may reorganize the macrophage membrane architecture and delay the process of phagosome maturation [step 3; (3)]. In contrast to phagosomes containing WT H. pylori, the majority of phagosomes containing ΔcapJ H. pylori fuse with lysosomes [step 4; (4)], which cause WT H. pylori displays extremely high survival in macrophages. αCAG = cholesteryl-6′-O-tetradecaboyl-α-d-glucopyranoside; CG = cholesteryl glucosides; CGT = cholesterol-α-glucosyltransferase.
Journal of Microbiology, Immunology and Infection  , DOI: ( /j.jmii )
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8. Figure S1
Phagocytosed Helicobacter pylori in macrophages. J774A.1 cells were infected with the wild type (WT) or ΔcapJ H. pylori for 1 hour. The intracellular/extracellular (green) and extracellular (red) H. pylori were determined by differential immunofluorescence staining. The merged fluorescence picture shows intracellular H. pylori in green (not yellow). The framed region was magnified, and the magnified images are shown in the lower panel. Scale bars represent 20 μm.
Journal of Microbiology, Immunology and Infection  , DOI: ( /j.jmii )
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9. Figure S2
Change in phosphorylation of Akt in macrophages infected with Helicobacter pylori. J774A.1 cells were infected with the wild type (WT), ΔcapJ, or ΔcapJ-in H. pylori for 20 minutes. Whole-cell lysates were subjected to immunoblotting assay with anti-Akt-phosphoSer-473 (p-Akt), anti-Akt (Akt), or anti-actin (Actin) antibodies as indicated.2
Journal of Microbiology, Immunology and Infection  , DOI: ( /j.jmii )
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