Background: Free-living amoeboflagellate, Naegleria fowleri causes acute fulminant primary amoebic meningoencephalitis (PAM). Interaction of N. fowleri with human cells is essential for the cytopathogenic process before phagocytosis and tissue invasion.
Objective: In order to understand the pathogenesis mechanisms of N. fowleri, subtractive cDNA library was used to analyze overall induction in gene expression of N. fowleri during exposure to the human neuroblastoma cells.
Methods: Co-cultivation of N. fowleri and human neuroblastoma SK-N-MC monolayer cultivation was performed. Subtractive cDNA libraries of inoculated N. fowleri at 15, 30, 45, 60, and 120 minutes were constructed. The PCR amplified products were cloned into E. coli. The specific clones were selected and further sequenced. Nucleotide sequences were compared with those deposited in the Genbank using BlastX. Significant probabilities were considered when E-value was less than 10-4. The induction of several gene expressions was validated by real-time RT-PCR.
Results: Extensive changes in gene expression of N. fowleri during the interaction with the human neuroblastoma SK-N-MC in vitro were detected. One hundred twenty clones were obtained. Among these, five clones containing DNA sequence homologue to known genes were identified. These genes included acetyl Co-A synthetase, 18s ribosome RNA, naegleria pore B precursor, isocitrate dehydrogenase, and pyruvate kinase. Real-time quantitative RT-PCR indicated that the expression level of all five genes was up-regulated within 1 hour after exposure. The expression level of acetyl Co-A synthetase increased and reached 7-times significantly greater than that of the control while pyruvate kinase, isocitrate dehydrogenase, naegleria pore B precursor, and 18s ribosome RNA transcripts increased from 2- to 4-fold, respectively.
Conclusions: A defined set of genes in N. fowleri that was differentially transcribed after contacting with the SKN- MC monolayer cells was identified. The transcription profiles unique for amoebic cell may help elucidate the transcriptional framework of N. fowleri pathogenicity and serve as a basis for identifying transcriptional virulence factors.
1. Schuster FL, Visvesvera GS. Free-living amebaee as opportunistic and non- opportunistic pathogens of humans and animals. Int Parasitol. 2004; 34:1001-7.
2. Marciano-Cabral FM, Patterson M, John DT, Bradley SG. Cytopathogenicity of Naegleria fowleri and Naegleria gruberi for established mammalian cell cultures. J Parasitol. 1982; 68:1110-6.
3. John DT, John RA. Use of Vero-cell culture to assess cytopathogenicity of Naegleria species. Proc Okla Acad. 1994; 74:17-20.
4. Cho MS, Jung SY, Park S, Park S, Kim KH, Kim, HI, et al. Immunological characterizations of a cloned 13.1-kilodalton protein from pathogenic Naegleria fowleri. Clin Diagn Lab Immunol. 2003;10: 954-9.
5. Oh YH, Jeong SR, Kim JH, Song KJ, Kim K, Park S, et al. 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; 27:453-9.
6. Kim JH, Kim D, Shin HJ. Contact-Independent Cell Death of Human Microglial Cells due to Pathogenic Naegleria fowleri Trophozoites. Korean J Parasitol. 2008; 46:217-21.
7. Tiewcharoen S , Malainual N , Junnu V, Chetanachan P, Rabablert J. Cytopathogenesis of Naegleria fowleri Thai strains for cultured human neuroblastoma cells. Parasitol Res. 2008; 102:997-1000.
8. Srinivasan P, Arahame E G, Ghosh AK, Valenzuela J, Ribeiro JM, Dimopoulos G, et al. Analysis of the Plasmodium and Anopheles transcriptomes during oocyst differentiation. J Biol Chem. 2004; 279:5581-7.
9. Cao W, Epstein C, Liu H, DeLoughery C, Ge N, Lin J, et al. Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study. BMC Genomics. 2004; 5: 26.
10. Lacrue AN, Jamus AA, Beerntsen BT. The novel Plasmodium gallinaceum sporozoite protein, Pg93, is preferentially expressed in the nucleus of oocyst sporozoites. Am J Trop Med Hyg. 2005; 73:634-43.
11. Florent I, Porcel BM, Guillaume E, Silva CD, Artiguenave F, Maréchal E, et al. A Plasmodium falciparum FcB1-schizont-EST collection providing clues to schizont specific gene structure and polymorphism. BMC Genomics. 2009; 10:235.
12. Tiewcharoen S, Rabablert J, Chetanachan P, Junnu V, Worawirounwong D, Malainual N. Scanning electron microscope of Naegleria fowleri Thai strain during cytopathogenesis in human neuroblastoma cells. Parasitol Res. 2008; 103:1119-23.
13. Lee SH, Jo SH, Lee SM, Koh HJ, Song H, Park J, et al. Role of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) on cellular defence against oxidative injury by gamma-rays. Int J Radiat Biol. 2004; 80: 635-42.
14. Pieulle L, Stocker P, Vinay M, Nouailler M, Vita N, Brasseur G, et al. Study of the thiol/disulfide redox systems of the anaerobe Desulfovibrio vulgaris points out pyruvate:ferredoxin oxidoreductase as a new target for thioredoxin 1. Biol Chem. 2011; 286: 7812-21.
15. Tiewcharoen S, Komalamisra N, Junnu V. Zymogram patterns of Naegleria spp isolated from natural water sources in Taling Chan district, Bangkok. Southeast Asian J Trop Med Public Health. 2004; 35:275-80.
16. Zemanova E, Jirkii M, Mauricio IL, Hora’ A, Miles MA, Lukes J. The Leishmania donovani complex: Genotypes of five metabolic enzymes (ICD, ME, MPI, G6PDH, and FH), new targets for multilocus sequence typing. Int J Parasitol. 2006; 18:149-60.
17. Kabir MM, Shimizu K.Metabolic regulation analysis of icd-gene knockout base on 2D electrophoresis with MALDI-TOF mass spectrometry and enzyme activity measurements. Appl Microbiol Biotechnol. 2004; 65:84-96.
18. Minich T, Yokota S, Dringen R. Cytosolic and mitochondrial isoforms of NADP+-dependent isocitrate dehydrogenases are expressed in cultured rat neurons, astrocytes, oligodendrocytes and microglial cells. J Neuronchem. 2003; 86:605-14.
19. Bakstzr R, Amy WA, Allali-Hassani A, Mok MW, Hills T, Hui R, et al.The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1. PLoS One. 2010; 5:e12736.
20. Feksa L R, Cornelio A, Dutra-Filho CS, Wyse AT, Wajner M, Wannmacher CM. The effects of the interactions between amino acids on pyruvate kinase activity from the brain cortex of young rats. Int J Dev Neurosci. 2005; 23:509-14.
21. Hu G, Cheng PY, Sham A, Perfect JR, Kronstad JW. Metabolic adaptation in Cryptococcus neoformans during early murine pulmonary infection. Mol Microbiol. 2008; 69:1456-75.
22. Rivierre L, Morea P, Allmann S, Hahn M, Biran M, Plazolles N, et al. Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes. Proc Natl Acad Sci USA 4. 2009;12694-99.
23. 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; 277:22353-60.
24. Herbst R, Marciano-Cabral F, Leippe M. Antimicrobial and Pore-forming peptides of Free-living and potentially highly pathogenic Naegleria fowleri are released from the same precursor molecule. J Biol Chem. 2004; 279:25955-8.
25. Leippe M, Herbst R. Ancient weapons for attack and defense: the pore-forming polypeptides of pathogenic enteric and free-living amoeboid protozoa. J Eukaryot Microbiol. 2004; 51:516-21.
26. Schroeder JM, Booton GC, Niszl IA, Seal DV, Markus MB, Fuerst PA, et al. Use of Subgenic 18S Ribosomal DNA PCR and Sequencing for genus and genotype identification of Acanthamoebae from humans with keratitis and from sewage sludge. J Clinic Microbiol. 2001; 39:1903-11.
27. Zhang Y, Sun X, Wang Z, Li R, Luo S, Jin X, et al. Identification of 18S ribosomal DNA genotype of Acanthamoeba from patients with keratitis in North China. Invest Ophthalmol Vis Sci. 2004; 45:1904-7.