title: Proteomic analysis of the Ehrlichia chaffeensis phagosome in cultured DH82 cells.
abstract: Ehrlichia chaffeensis is an obligately intracellular bacterium that resides and multiplies within cytoplasmic vacuoles of phagocytes. The Ehrlichia-containing vacuole (ECV) does not fuse with lysosomes, an essential condition for Ehrlichia to survive inside phagocytes, but the mechanism of inhibiting the fusion of the phagosome with lysosomes is not clear. Understanding the ECV molecular composition may decipher the mechanism by which Ehrlichia inhibits phagosome-lysosome fusion. In this study, we obtained highly purified ECVs from E. chaffeensis-infected DH82 cells by sucrose density gradient centrifugation and analyzed their composition by mass spectrometry-based proteomics. The ECV composition was compared with that of phagolysosomes containing latex beads. Lysosomal proteins such as cathepsin D, cathepsin S, and lysosomal acid phosphatase were not detected in E. chaffeensis phagosome preparations. Some small GTPases, involved in membrane dynamics and phagocytic trafficking, were detected in ECVs. A notable finding was that Rab7, a late endosomal marker, was consistently detected in E. chaffeensis phagosomes by mass spectrometry. Confocal microscopy confirmed that E. chaffeensis phagosomes contained Rab7 and were acidified at approximately pH 5.2, suggesting that the E. chaffeensis vacuole was an acidified late endosomal compartment. Our results also demonstrated by mass spectrometry and immunofluorescence analysis that Ehrlichia morulae were not associated with the autophagic pathway. Ehrlichia chaffeensis did not inhibit phagosomes containing latex beads from fusing with lysosomes in infected cells. We concluded that the E. chaffeensis vacuole was a late endosome and E. chaffeensis might inhibit phagosome-lysosome fusion by modifying its vacuolar membrane composition, rather than by regulating the expression of host genes involved in trafficking.
authors.affiliation: Department of Clinical Microbiology and Immunology, College of Medical Laboratory, Third Military Medical University, Chongqing, China ; Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America ; Bethune International Peace Hospital, Shijiazhuang, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America ; Anhui Province Center for Disease Control and Prevention, Hefei, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America ; Jiangsu Province Center for Disease Control and Prevention, Nanjing, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Wu
authors.affiliation: School of Health Professions, University of Texas Medical Branch, Galveston, Texas, United States of America.
authors.forename: Jian-zhi
authors.initials: JZ
authors.lastname: Zhang
authors.affiliation: Department of Clinical Microbiology and Immunology, College of Medical Laboratory, Third Military Medical University, Chongqing, China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Gu
authors.affiliation: Department of Clinical Microbiology and Immunology, College of Medical Laboratory, Third Military Medical University, Chongqing, China.
authors.forename: Ya-ling
authors.initials: YL
authors.lastname: Liao
authors.affiliation: Bethune International Peace Hospital, Shijiazhuang, China.
authors.forename: Fu-kun
authors.initials: FK
authors.lastname: Wang
authors.affiliation: Department of Clinical Microbiology and Immunology, College of Medical Laboratory, Third Military Medical University, Chongqing, China.
authors.forename: Xu-hu
authors.initials: XH
authors.lastname: Mao
authors.affiliation: Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America ; School of Public Health, Shandong University, Jinan, China.
authors.forename: Xue-jie
authors.initials: XJ
authors.lastname: Yu
mesh_terms: D000135:Acid Phosphatase / Q000737:chemistry; D000818:Animals; D001343:Autophagy; D001692:Biological Transport; D002402:Cathepsin D / Q000737:chemistry; D002403:Cathepsins / Q000737:chemistry; D002460:Cell Line; D004285:Dogs; D018469:Ehrlichia chaffeensis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000201:enzymology; D008264:Macrophages / Q000382:microbiology*; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D009000:Monocytes / Q000382:microbiology*; D010586:Phagocytes / Q000166:cytology; D010588:Phagosomes / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000737:chemistry; D000090802:rab7 GTP-Binding Proteins
chemical_list: D020543:Proteome; D000090802:rab7 GTP-Binding Proteins; D000135:Acid Phosphatase; D002403:Cathepsins; C028217:cathepsin S; D002402:Cathepsin D; D020558:GTP Phosphohydrolases; D020691:rab GTP-Binding Proteins