Genome-wide identification of pathogenicity factors of the free-living amoeba Naegleria fowleri

BMC Genomics

Matthias Wittwer²  Bruno Gottstein³  Nadia Schürch²  Manfred Heller¹  Christian Beuret²  Norbert Müller³  Denise C Zysset-Burri³ 

[1] Mass Spectrometry and Proteomics, Department of Clinical Research, University Hospital, Bern CH-3010, Switzerland;Biology Division, Spiez Laboratory, Federal Office for Civil Protection, Austrasse, CH-3700 Spiez, Switzerland;Institute of Parasitology, University of Bern, Länggassstrasse 122, CH-3012 Bern, Switzerland

关键词: Pathogenicity factors;    Comparative proteomics;    RNA sequencing;    Whole genome sequencing;    Primary amoebic meningoencephalitis;    Naegleria fowleri;   

Others  :  857090
DOI  :  10.1186/1471-2164-15-496

 received in 2013-12-04, accepted in 2014-06-11,  发布年份 2014

【 摘 要 】

Background

The free-living amoeba Naegleria fowleri is the causative agent of the rapidly progressing and typically fatal primary amoebic meningoencephalitis (PAM) in humans. Despite the devastating nature of this disease, which results in > 97% mortality, knowledge of the pathogenic mechanisms of the amoeba is incomplete. This work presents a comparative proteomic approach based on an experimental model in which the pathogenic potential of N. fowleri trophozoites is influenced by the compositions of different media.

Results

As a scaffold for proteomic analysis, we sequenced the genome and transcriptome of N. fowleri. Since the sequence similarity of the recently published genome of Naegleria gruberi was far lower than the close taxonomic relationship of these species would suggest, a de novo sequencing approach was chosen. After excluding cell regulatory mechanisms originating from different media compositions, we identified 22 proteins with a potential role in the pathogenesis of PAM. Functional annotation of these proteins revealed, that the membrane is the major location where the amoeba exerts its pathogenic potential, possibly involving actin-dependent processes such as intracellular trafficking via vesicles.

Conclusion

This study describes for the first time the 30 Mb-genome and the transcriptome sequence of N. fowleri and provides the basis for the further definition of effective intervention strategies against the rare but highly fatal form of amoebic meningoencephalitis.

【 授权许可】

   
2014 Zysset-Burri et al.; licensee BioMed Central Ltd.

【 图 表 】

【 参考文献 】

• [1]De Jonckheere J, Voorde H: The distribution of Naegleria fowleri in man-made thermal waters. Am J Trop Med Hyg 1977, 26:10-15.
• [2]De Jonckheere JF: Molecular definition and the ubiquity of species in the genus Naegleria. Protist 2004, 155:89-103.
• [3]Carter RF: Description of a Naegleria sp. isolated from two cases of primary amoebic meningo-encephalitis, and of the experimental pathological changes induced by it. J Pathol 1970, 100:217-244.
• [4]Jarolim KL, McCosh JK, Howard MJ, John DT: A light microscopy study of the migration of Naegleria fowleri from the nasal submucosa to the central nervous system during the early stage of primary amebic meningoencephalitis in mice. J Parasitol 2000, 86:50-55.
• [5]Schuster FL, Visvesvara GS: Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int J Parasitol 2004, 34:1001-1027.
• [6]Debnath A, Tunac JB, Galindo-Gomez S, Silva-Olivares A, Shibayama M, McKerrow JH: Corifungin, a new drug lead against Naegleria, identified from a high-throughput screen. Antimicrob Agents Chemother 2012, 56:5450-5457.
• [7]Vargas-Zepeda J, Gomez-Alcala AV, Vasquez-Morales JA, Licea-Amaya L, De Jonckheere JF, Lares-Villa F: Successful treatment of Naegleria fowleri meningoencephalitis by using intravenous amphotericin B, fluconazole and rifampicin. Arch Med Res 2005, 36:83-86.
• [8]Fritz-Laylin LK, Prochnik SE, Ginger ML, Dacks JB, Carpenter ML, Field MC, Kuo A, Paredez A, Chapman J, Pham J, Shu S, Neupane R, Cipriano M, Mancuso J, Tu H, Salamov A, Lindquist E, Shapiro H, Lucas S, Grigoriev IV, Cande WZ, Fulton C, Rokhsar DS, Dawson SC: The genome of Naegleria gruberi illuminates early eukaryotic versatility. Cell 2010, 140:631-642.
• [9]Herman EK, Greninger AL, Visvesvara GS, Marciano-Cabral F, Dacks JB, Chiu CY: The mitochondrial genome and a 60-kb nuclear DNA segment from Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis. J Eukaryot Microbiol 2013, 60:179-191.
• [10]Burri DC, Gottstein B, Zumkehr B, Hemphill A, Schürch N, Wittwer M, Müller N: Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri. Microbiology 2012, 158:2652-2660.
• [11]Jung SY, Kim JH, Lee YJ, Song KJ, Kim K, Park S, Im KI, Shin HJ: Naegleria fowleri: nfa1 gene knock-down by double-stranded RNAs. Exp Parasitol 2008, 118:208-213.
• [12]Sohn HJ, Kim JH, Shin MH, Song KJ, Shin HJ: The Nf-actin gene is an important factor for food-cup formation and cytotoxicity of pathogenic Naegleria fowleri. Parasitol Res 2010, 106:917-924.
• [13]Jung SY, Kim JH, Song KJ, Lee YJ, Kwon MH, Kim K, Park S, Im KI, Shin HJ: Gene silencing of nfa1 affects the in vitro cytotoxicity of Naegleria fowleri in murine macrophages. Mol Biochem Parasitol 2009, 165:87-93.
• [14]Lee YJ, Kim JH, Jeong SR, Song KJ, Kim K, Park S, Park MS, Shin HJ: Production of Nfa1-specific monoclonal antibodies that influences the in vitro cytotoxicity of Naegleria fowleri trophozoites on microglial cells. Parasitol Res 2007, 101:1191-1196.
• [15]Jeong SR, Lee SC, Song KJ, Park S, Kim K, Kwon MH, Im KI, Shin HJ: Expression of the nfa1 gene cloned from pathogenic Naegleria fowleri in nonpathogenic N. gruberi enhances cytotoxicity against CHO target cells in vitro. Infect Immun 2005, 73:4098-4105.
• [16]Jeong SR, Kang SY, Lee SC, Song KJ, Im KI, Shin HJ: Decreasing effect of an anti-Nfa1 polyclonal antibody on the in vitro cytotoxicity of pathogenic Naegleria fowleri. Korean J Parasitol 2004, 42:35-40.
• [17]Oh YH, Jeong SR, Kim JH, Song KJ, Kim K, Park S, Sohn S, Shin HJ: Cytopathic changes and pro-inflammatory cytokines induced by Naegleria fowleri trophozoites in rat microglial cells and protective effects of an anti-Nfa1 antibody. Parasite Immunol 2005, 27:453-459.
• [18]Kang SY, Song KJ, Jeong SR, Kim JH, Park S, Kim K, Kwon MH, Shin HJ: Role of the Nfa1 protein in pathogenic Naegleria fowleri cocultured with CHO target cells. Clin Diagn Lab Immunol 2005, 12:873-876.
• [19]Reveiller FL, Suh SJ, Sullivan K, Cabanes PA, Marciano-Cabral F: Isolation of a unique membrane protein from Naegleria fowleri. J Eukaryot Microbiol 2001, 48:676-682.
• [20]Baverstock PR, Illana S, Christy PE, Robinson BS, Johnson AM: srRNA evolution and phylogenetic relationships of the genus Naegleria (Protista: Rhizopoda). Mol Biol Evol 1989, 6:243-257.
• [21]De Jonckheere JF: Origin and evolution of the worldwide distributed pathogenic amoeboflagellate Naegleria fowleri. Infect Genet Evol 2011, 11:1520-1528.
• [22]Marciano-Cabral F, Cabral GA: The immune response to Naegleria fowleri amebae and pathogenesis of infection. FEMS Immunol Med Microbiol 2007, 51:243-259.
• [23]Han KL, Lee HJ, Shin MH, Shin HJ, Im KI, Park SJ: The involvement of an integrin-like protein and protein kinase C in amoebic adhesion to fibronectin and amoebic cytotoxicity. Parasitol Res 2004, 94:53-60.
• [24]Brown T: Observations by light microscopy on the cytopathogenicity of Naegleria fowleri in mouse embryo-cell cultures. J Med Microbiol 1978, 11:249-259.
• [25]Marciano-Cabral FM, Patterson M, John DT, Bradley SG: Cytopathogenicity of Naegleria fowleri and Naegleria gruberi for established mammalian cell cultures. J Parasitol 1982, 68:1110-1116.
• [26]Marciano-Cabral FM, Fulford DE: Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells. Appl Environ Microbiol 1986, 51:1133-1137.
• [27]Lowrey DM, McLaughlin J: Activation of a heat-stable cytolytic protein associated with the surface membrane of Naegleria fowleri. Infect Immun 1985, 50:478-482.
• [28]Toney DM, Marciano-Cabral F: Alterations in protein expression and complement resistance of pathogenic Naegleria amoebae. Infect Immun 1992, 60:2784-2790.
• [29]Fritzinger AE, Toney DM, MacLean RC, Marciano-Cabral F: Identification of a Naegleria fowleri membrane protein reactive with anti-human CD59 antibody. Infect Immun 2006, 74:1189-1195.
• [30]Toney DM, Marciano-Cabral F: Membrane vesiculation of Naegleria fowleri amoebae as a mechanism for resisting complement damage. J Immunol 1994, 152:2952-2959.
• [31]Herbst R, Ott C, Jacobs T, Marti T, Marciano-Cabral F, Leippe M: Pore-forming polypeptides of the pathogenic protozoon Naegleria fowleri. J Biol Chem 2002, 277:22353-22360.
• [32]Matsuo H, Chevallier J, Mayran N, Le Blanc I, Ferguson C, Faure J, Blanc NS, Matile S, Dubochet J, Sadoul R, Parton RG, Vilbois F, Gruenberg J: Role of LBPA and Alix in multivesicular liposome formation and endosome organization. Science 2004, 303:531-534.
• [33]Wang L, Zhan Y, Song E, Yu Y, Jiu Y, Du W, Lu J, Liu P, Xu P, Xu T: HID-1 is a peripheral membrane protein primarily associated with the medial- and trans- Golgi apparatus. Protein Cell 2011, 2:74-85.
• [34]Yu Y, Wang L, Jiu Y, Zhan Y, Liu L, Xia Z, Song E, Xu P, Xu T: HID-1 is a novel player in the regulation of neuropeptide sorting. Biochem J 2011, 434:383-390.
• [35]Lynch EC, Rosenberg IM, Gitler C: An ion-channel forming protein produced by Entamoeba histolytica. EMBO J 1982, 1:801-804.
• [36]Que X, Reed SL: Cysteine proteinases and the pathogenesis of amebiasis. Clin Microbiol Rev 2000, 13:196-206.
• [37]Juarez-Hernandez LJ, Garcia-Perez RM, Salas-Casas A, Garcia-Rivera G, Orozco E, Rodriguez MA: Entamoeba histolytica: the over expression of a mutated EhRabB protein produces a decrease of in vitro and in vivo virulence. Exp Parasitol 2013, 133:339-345.
• [38]Hernandes-Alejandro M, Calixto-Galvez M, Lopez-Reyes I, Salas-Casas A, Cazares-Apatiga J, Orozco E, Rodriguez MA: The small GTPase EhRabB of Entamoeba histolytica is differentially expressed during phagocytosis. Parasitol Res 2013, 112:1631-1640.
• [39]Bosch DE, Siderovski DP: G protein signaling in the parasite Entamoeba histolytica. Exp Mol Med 2013, 45:e15.
• [40]Brown T: Observations by immunofluorescence microscopy and electron microscopy on the cytopathogenicity of Naegleria fowleri in mouse embryo-cell cultures. J Med Microbiol 1979, 12:363-371.
• [41]Marciano-Cabral F, John DT: Cytopathogenicity of Naegleria fowleri for rat neuroblastoma cell cultures: scanning electron microscopy study. Infect Immun 1983, 40:1214-1217.
• [42]Kim JH, Lee SH, Sohn HJ, Lee J, Chwae YJ, Park S, Kim K, Shin HJ: The immune response induced by DNA vaccine expressing nfa1 gene against Naegleria fowleri. Parasitol Res 2012, 111:2377-2384.
• [43]Kim JH, Sohn HJ, Lee J, Yang HJ, Chwae YJ, Kim K, Park S, Shin HJ: Vaccination with lentiviral vector expressing the nfa1 gene confers a protective immune response to mice infected with Naegleria fowleri. Clin Vaccine Immunol 2013, 20:1055-1060.
• [44]Song KJ, Song KH, Kim JH, Sohn HJ, Lee YJ, Park CE, Shin HJ: Heat shock protein 70 of Naegleria fowleri is important factor for proliferation and in vitro cytotoxicity. Parasitol Res 2008, 103:313-317.
• [45]Gumbiner BM: Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 1996, 84:345-357.
• [46]Bretscher A: Microfilament structure and function in the cortical cytoskeleton. Annu Rev Cell Biol 1991, 7:337-374.
• [47]Voigt H, Guillen N: New insights into the role of the cytoskeleton in phagocytosis of Entamoeba histolytica. Cell Microbiol 1999, 1:195-203.
• [48]Gonzalez-Robles A, Castanon G, Hernandez-Ramirez VI, Salazar-Villatoro L, Gonzalez-Lazaro M, Omana-Molina M, Talamas-Rohana P, Martinez-Palomo A: Acanthamoeba castellanii: identification and distribution of actin cytoskeleton. Exp Parasitol 2008, 119:411-417.
• [49]Chesarone MA, DuPage AG, Goode BL: Unleashing formins to remodel the actin and microtubule cytoskeletons. Nat Rev Mol Cell Biol 2010, 11:62-74.
• [50]Higgs HN: Formin proteins: a domain-based approach. Trends Biochem Sci 2005, 30:342-353.
• [51]Matsudaira P, Janmey P: Pieces in the actin-severing protein puzzle. Cell 1988, 54:139-140.
• [52]Pavlov D, Muhlrad A, Cooper J, Wear M, Reisler E: Actin filament severing by cofilin. J Mol Biol 2007, 365:1350-1358.
• [53]Bamburg JR: Proteins of the ADF/cofilin family: essential regulators of actin dynamics. Annu Rev Cell Dev Biol 1999, 15:185-230.
• [54]Khurana S, George SP: Regulation of cell structure and function by actin-binding proteins: villin’s perspective. FEBS Lett 2008, 582:2128-2139.
• [55]Gakovic M, Shu X, Kasioulis I, Carpanini S, Moraga I, Wright AF: The role of RPGR in cilia formation and actin stability. Hum Mol Genet 2011, 20:4840-4850.
• [56]Diakonova M, Bokoch G, Swanson JA: Dynamics of cytoskeletal proteins during Fcgamma receptor-mediated phagocytosis in macrophages. Mol Biol Cell 2002, 13:402-411.
• [57]Araki N, Hatae T, Furukawa A, Swanson JA: Phosphoinositide-3-kinase-independent contractile activities associated with Fcgamma-receptor-mediated phagocytosis and macropinocytosis in macrophages. J Cell Sci 2003, 116:247-257.
• [58]Coluccio LM: Myosin I. Am J Physiol 1997, 273:C347-C359.
• [59]De Jonckheere JF: Variation of electrophoretic karyotypes among Naegleria spp. Parasitol Res 1989, 76:55-62.
• [60]Clark CG, Cross GA, De Jonckheere JF: Evaluation of evolutionary divergence in the genus Naegleria by analysis of ribosomal DNA plasmid restriction patterns. Mol Biochem Parasitol 1989, 34:281-296.
• [61]Dolezel J, Bartos J: Plant DNA flow cytometry and estimation of nuclear genome size. Ann Bot 2005, 95:99-110.
• [62]Ronildo Clarindo W, Roberto Carvalho C: Flow cytometric analysis using SYBR Green I for genome size estimation in coffee. Acta Histochem 2011, 113:221-225.
• [63]McArthur AG, Morrison HG, Nixon JE, Passamaneck NQ, Kim U, Hinkle G, Crocker MK, Holder ME, Farr R, Reich CI, Olsen GE, Aley SB, Adam RD, Gillin FD, Sogin ML: The Giardia genome project database. FEMS Microbiol Lett 2000, 189:271-273.
• [64]Zubacova Z, Cimburek Z, Tachezy J: Comparative analysis of trichomonad genome sizes and karyotypes. Mol Biochem Parasitol 2008, 161:49-54.
• [65]Hare EE, Johnston JS: Genome size determination using flow cytometry of propidium iodide-stained nuclei. Methods Mol Biol 2011, 772:3-12.
• [66]Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A: Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 2011, 29:644-652.
• [67]Halary S, McInerney JO, Lopez P, Bapteste E: EGN: a wizard for construction of gene and genome similarity networks. BMC Evol Biol 2013, 13:146.
• [68]Cline MS, Smoot M, Cerami E, Kuchinsky A, Landys N, Workman C, Christmas R, Avila-Campilo I, Creech M, Gross B, Hanspers K, Isserlin R, Kelley R, Killcoyne S, Lotia S, Maere S, Morris J, Ono K, Pavlovic V, Pico AR, Vailaya A, Wang PL, Adler A, Conklin BR, Hood L, Kuiper M, Sander C, Schmulevich I, Schwikowski B, Warner GJ, et al.: Integration of biological networks and gene expression data using Cytoscape. Nat Protoc 2007, 2:2366-2382.
• [69]Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol 1990, 215:403-410.
• [70]Al Kaabi A, Traupe T, Stutz M, Buchs N, Heller M: Cause or effect of arteriogenesis: compositional alterations of microparticles from CAD patients undergoing external counterpulsation therapy. PLoS One 2012, 7:e46822.
• [71]Gluck F, Hoogland C, Antinori P, Robin X, Nikitin F, Zufferey A, Pasquarello C, Fetaud V, Dayon L, Müller M, Lisacek F, Geiser L, Hochstrasser D, Sanchez JC, Scherl A: EasyProt–an easy-to-use graphical platform for proteomics data analysis. J Proteomics 2013, 79:146-160.
• [72]Heller M, Schlappritzi E, Stalder D, Nuoffer JM, Haeberli A: Compositional protein analysis of high density lipoproteins in hypercholesterolemia by shotgun LC-MS/MS and probabilistic peptide scoring. Mol Cell Proteomics 2007, 6:1059-1072.
• [73]Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004, 5:R80.
• [74]Kasprzyk A: BioMart: driving a paradigm change in biological data management. Database (Oxford) 2011, 2011:bar049.
• [75]Zhang J, Haider S, Baran J, Cros A, Guberman JM, Hsu J, Liang Y, Yao L, Kasprzyk A: BioMart: a data federation framework for large collaborative projects. Database (Oxford) 2011, 2011:bar038.