title: Kinetics and strain variation of phagosome proteins of Entamoeba histolytica by proteomic analysis.
abstract: The protozoan parasite Entamoeba histolytica ingests and feeds on microorganisms and mammalian cells. Phagocytosis is essential for cell growth and implicated in pathogenesis of E. histolytica. We report here the dynamic changes of phagosome proteins during phagosome maturation by proteomic analysis using reversed-phase capillary liquid chromatography and ion trap tandem mass spectrometry. Phagosomes were isolated at various intervals after internalization of latex beads. Immunoblot analysis and electron microscopy verified successful isolation of phagosomes. A total of 159 proteins were identified from the reference strain HM1 at different stages of phagosome maturation. Approximately 70% of them were detected in a time-dependent fashion, suggesting dynamism of phagosome biogenesis. The kinetics of representative proteins were verified by immunoblots and also by video microscopy of live transgenic amebae expressing green fluorescent protein-fused EhRab7A. Furthermore, we observed significant differences in phagosome profiles between HM1 and two recent clinical isolates. Approximately 60% of 229 proteins detected in at least one of these three strains were identified only in one strain, while approximately 20% of these proteins were detected in all three strains. These data should provide significant insights into molecular characterization of phagosome biogenesis, and help to elucidate the pathogenesis of this important infection.
authors.affiliation: Department of Parasitology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan.
authors.forename: Mami
authors.initials: M
authors.lastname: Okada
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Huston
authors.forename: Miho
authors.initials: M
authors.lastname: Oue
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Mann
authors.forename: William A
authors.initials: WA
authors.lastname: Petri
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Kita
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Nozaki
mesh_terms: D000818:Animals; D019677:Artificial Gene Fusion; D002458:Cell Fractionation; D002853:Chromatography, Liquid; D004748:Entamoeba histolytica / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology; D004749:Entamoebiasis / Q000469:parasitology; D020558:GTP Phosphohydrolases / Q000032:analysis / Q000235:genetics; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D013058:Mass Spectrometry; D018715:Microscopy, Video; D010587:Phagocytosis; D010588:Phagosomes / Q000737:chemistry*; D040901:Proteomics; D015800:Protozoan Proteins / Q000032:analysis* / Q000235:genetics; D013997:Time Factors; D000090802:rab7 GTP-Binding Proteins
chemical_list: D015800:Protozoan Proteins; D000090802:rab7 GTP-Binding Proteins; D049452:Green Fluorescent Proteins; D020558:GTP Phosphohydrolases; C464925:Rab7 protein, Entamoeba histolytica