Đặc tính di truyền của các chủng Leishmania amazonensis được phân lập ở miền đông bắc Brazil thông qua giải trình tự DNA, phân tích dựa trên PCR và karyotyping phân tử

Kinetoplastid Biology and Disease - Tập 6 - Trang 1-8 - 2007
João Paulo C de Oliveira1, Flora Fernandes1, Angela K Cruz2, Viviane Trombela2, Elisângela Monteiro3, Anamaria A Camargo3, Aldina Barral1, Camila I de Oliveira1
1Centro de Pesquisas Gonçalo Moniz, FIOCRUZ, Salvador, Brazil
2Faculdade de Medicina de Ribeirão Preto – USP, Ribeirão Preto, Brazil
3Ludwig Institute for Cancer Research, São Paulo, Brazil

Tóm tắt

Nhiễm Leishmania (Leishmania) amazonensis ở người dẫn đến một phổ lâm sàng với các biểu hiện bệnh khác nhau, từ tổn thương da đến tổn thương niêm mạc hoặc nội tạng. Trong nghiên cứu hiện tại, chúng tôi đã điều tra tính biến đổi di truyền của 18 chủng L. amazonensis được phân lập ở miền đông bắc Brazil từ các bệnh nhân có biểu hiện lâm sàng khác nhau của leishmaniasis. DNA ký sinh trùng đã được phân tích qua giải trình tự ITS bao quanh tiểu đơn vị 5.8 S của các gen RNA ribosome, bằng phương pháp RAPD và SSR-PCR và bằng PFGE sau đó là lai với các mồi gen cụ thể. Kết quả giải trình tự ITS và các phương pháp dựa trên PCR cho thấy sự không đồng nhất di truyền giữa các mẫu L. amazonensis được nghiên cứu, và karyotyping phân tử cũng cho thấy sự biến đổi về kích thước nhiễm sắc thể của các mẫu khác nhau. Các cây di truyền không gốc đã tách các chủng thành các nhóm khác nhau. Những kết quả này cho thấy rằng các chủng L. amazonensis được phân lập từ các bệnh nhân leishmaniasis ở miền đông bắc Brazil có tính đa dạng di truyền, tuy nhiên không tìm thấy mối tương quan giữa đa hình di truyền và kiểu hình.

Từ khóa

#Leishmania amazonensis; đa dạng di truyền; miền đông bắc Brazil; phân lập; PCR

Tài liệu tham khảo

Barral A, Pedral-Sampaio D, Grimaldi Junior G, Momen H, McMahon-Pratt D, Ribeiro de Jesus A, Almeida R, Badaro R, Barral-Netto M, Carvalho EM, et al: Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease. The American Journal of Tropical Medicine and Hygiene. 1991, 44 (5): 536-546. Barral A, Costa JM, Bittencourt AL, Barral-Netto M, Carvalho EM: Polar and subpolar diffuse cutaneous leishmaniasis in Brazil: clinical and immunopathologic aspects. Int J Dermatol. 1995, 34 (7): 474-479. 10.1111/j.1365-4362.1995.tb00613.x. Costa JML, Cunha AK, Gama MEA, Saldanha ACR: Leishmaniose cutânea difusa: revisão. An Bras Derm. 1998, 73: 565–576- Marsden PD: Mucocutaneous leishmaniasis. Bmj. 1990, 301 (6753): 656-657. Miles MA, Povoa MM, de Souza AA, Lainson R, Shaw JJ: Some methods for the enzymic characterization of Latin-American Leishmania with particular reference to Leishmania mexicana amazonensis and subspecies of Leishmania hertigi. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1980, 74 (2): 243-252. 10.1016/0035-9203(80)90253-9. Tibayrenc M, Neubauer K, Barnabe C, Guerrini F, Skarecky D, Ayala FJ: Genetic characterization of six parasitic protozoa: parity between random-primer DNA typing and multilocus enzyme electrophoresis. Proceedings of the National Academy of Sciences,USA. 1993, 90 (4): 1335-1339. 10.1073/pnas.90.4.1335. Jackson PR, Wohlhieter JA, Jackson JE, Sayles P, Diggs CL, Hockmeyer WT: Restriction endonuclease analysis of Leishmania kinetoplast DNA characterizes parasites responsible for visceral and cutaneous disease. The American Journal of Tropical Medicine and Hygiene. 1984, 33 (5): 808-819. Macedo AM, Melo MN, Gomes RF, Pena SD: DNA fingerprints: a tool for identification and determination of the relationships between species and strains of Leishmania. Molecular and Biochemical Parasitology. 1992, 53 (1-2): 63-70. 10.1016/0166-6851(92)90007-7. Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV: DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 1990, 18 (22): 6531-6535. 10.1093/nar/18.22.6531. Zietkiewicz E, Rafalski A, Labuda D: Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics. 1994, 20 (2): 176-183. 10.1006/geno.1994.1151. Rubinsztein DC, Amos W, Leggo J, Goodburn S, Jain S, Li SH, Margolis RL, Ross CA, Ferguson-Smith MA: Microsatellite evolution--evidence for directionality and variation in rate between species. Nat Genet. 1995, 10 (3): 337-343. 10.1038/ng0795-337. Gomes RF, Macedo AM, Pena SD, Melo MN: Leishmania (Viannia) braziliensis: genetic relationships between strains isolated from different areas of Brazil as revealed by DNA fingerprinting and RAPD. Experimental Parasitology. 1995, 80 (4): 681-687. 10.1006/expr.1995.1084. Volpini AC, de Azeredo Passos VM, Romanha AJ: Attempt to differentiate Leishmania (Leishmania) amazonensis, L. (L.) chagasi, Leishmania (Viannia) braziliensis and L. (V.) guyanensis using the SSR-PCR technique. Parasitolology Research. 2001, 87 (12): 1056-1059. Schriefer A, Schriefer AL, Goes-Neto A, Guimaraes LH, Carvalho LP, Almeida RP, Machado PR, Lessa HA, de Jesus AR, Riley LW, Carvalho EM: Multiclonal Leishmania braziliensis population structure and its clinical implication in a region of endemicity for American tegumentary leishmaniasis. Infect Immun. 2004, 72 (1): 508-514. 10.1128/IAI.72.1.508-514.2004. Cupolillo E, Grimaldi Junior G, Momen H, Beverley SM: Intergenic region typing (IRT): a rapid molecular approach to the characterization and evolution of Leishmania. Molecular and Biochemical Parasitology. 1995, 73 (1-2): 145-155. 10.1016/0166-6851(95)00108-D. Davila AM, Momen H: Internal-transcribed-spacer (ITS) sequences used to explore phylogenetic relationships within Leishmania. Ann Trop Med Parasitol. 2000, 94 (6): 651-654. 10.1080/00034980050152085. Pages M, Bastien P, Veas F, Rossi V, Bellis M, Wincker P, Rioux JA, Roizes G: Chromosome size and number polymorphisms in Leishmania infantum suggest amplification/deletion and possible genetic exchange. Mol Biochem Parasitol. 1989, 36 (2): 161-168. 10.1016/0166-6851(89)90188-6. Britto C, Ravel C, Bastien P, Blaineau C, Pages M, Dedet JP, Wincker P: Conserved linkage groups associated with large-scale chromosomal rearrangements between Old World and New World Leishmania genomes. Gene. 1998, 222 (1): 107-117. 10.1016/S0378-1119(98)00472-7. Iovannisci DM, Beverley SM: Structural alterations of chromosome 2 in Leishmania major as evidence for diploidy, including spontaneous amplification of the mini-exon array. Mol Biochem Parasitol. 1989, 34 (2): 177-188. 10.1016/0166-6851(89)90009-1. Hasegawa M, Kishino H, Yano T: Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J Mol Evol. 1985, 22 (2): 160-174. 10.1007/BF02101694. Bittencourt A, Barral A, de Jesus AR, de Almeida RP, Grimaldi Junior G: In situ identification of Leishmania amazonensis associated with diffuse cutaneous leishmaniasis in Bahia, Brazil. Memórias do Instituto Oswaldo Cruz. 1989, 84 (4): 585-586. Hillis DM, Dixon MT: Ribosomal DNA: molecular evolution and phylogenetic inference. Q Rev Biol. 1991, 66 (4): 411-453. 10.1086/417338. von Haeseler A, Strimmer K: Phylogeny inference based on maximum-likelihood methods with TREE-PUZZLE. The Phylogenetic Handbook: a pratical approach to DNA and protein phylogeny. Edited by: Salemi M, Vandamme AM. 2003, Cambridge , Cambridge University Press El Tai NO, El Fari M, Mauricio I, Miles MA, Oskam L, El Safi SH, Presber WH, Schonian G: Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing. Exp Parasitol. 2001, 97 (1): 35-44. 10.1006/expr.2001.4592. Schonian G, Akuffo H, Lewin S, Maasho K, Nylen S, Pratlong F, Eisenberger CL, Schnur LF, Presber W: Genetic variability within the species Leishmania aethiopica does not correlate with clinical variations of cutaneous leishmaniasis. Molecular and Biochemical Parasitology. 2000, 106 (2): 239-248. 10.1016/S0166-6851(99)00216-9. Berzunza-Cruz M, Cabrera N, Crippa-Rossi M, Sosa Cabrera T, Perez-Montfort R, Becker I: Polymorphism analysis of the internal transcribed spacer and small subunit of ribosomal RNA genes of Leishmania mexicana. Parasitolology Research. 2002, 88 (10): 918-925. 10.1007/s00436-002-0672-x. Ishikawa EA, Silveira FT, Magalhaes AL, Guerra junior RB, Melo MN, Gomes R, Silveira TG, Shaw JJ: Genetic variation in populations of Leishmania species in Brazil. Transactions of the Royal Society of Tropical Medicine and Hygiene. 2002, 96 Suppl 1: S111-21. 10.1016/S0035-9203(02)90061-1. Cupolillo E, Brahim LR, Toaldo CB, de Oliveira-Neto MP, de Brito ME, Falqueto A, de Farias Naiff M, Grimaldi G: Genetic polymorphism and molecular epidemiology of Leishmania (Viannia) braziliensis from different hosts and geographic areas in Brazil. Journal of Clinical Microbiology. 2003, 41 (7): 3126-3132. 10.1128/JCM.41.7.3126-3132.2003. Zemanova E, Jirku M, Mauricio IL, Miles MA, Lukes J: Genetic polymorphism within the leishmania donovani complex: correlation with geographic origin. The American Journal of Tropical Medicine and Hygiene. 2004, 70 (6): 613-617. Berzunza-Cruz M, Bricaire G, Romero SZ, Perez-Becker R, Saavedra-Lira E, Perez-Montfort R, Crippa-Rossi M, Velasco-Castrejon O, Becker I: Leishmania mexicana mexicana: genetic heterogeneity of mexican isolates revealed by restriction length polymorphism analysis of kinetoplast DNA. Experimental Parasitology. 2000, 95 (4): 277-284. 10.1006/expr.2000.4541. Sreenivas G, Raju BV, Singh R, Selvapandiyan A, Duncan R, Sarkar D, Nakhasi HL, Salotra P: DNA polymorphism assay distinguishes isolates of Leishmania donovani that cause kala-azar from those that cause post-kala-azar dermal Leishmaniasis in humans. Journal of Clinical Microbiology. 2004, 42 (4): 1739-1741. 10.1128/JCM.42.4.1739-1741.2004. Mahdi M, Elamin EM, Melville SE, Musa AM, Blackwell JM, Mukhtar MM, Elhassan AM, Ibrahim ME: Sudanese mucosal leishmaniasis: isolation of a parasite within the Leishmania donovani complex that differs genotypically from L. donovani causing classical visceral leishmaniasis. Infection, Genetics and Evolution. 2005, 5 (1): 29-33. 10.1016/j.meegid.2004.05.008. Indiani de Oliveira C, Teixeira MJ, Teixeira CR, Ramos de Jesus J, Bomura Rosato A, Santa da Silva J, Brodskyn C, Barral-Netto M, Barral A: Leishmania braziliensis isolates differing at the genome level display distinctive features in BALB/c mice. Microbes and Infection. 2004, 6 (11): 977-984. 10.1016/j.micinf.2004.05.009. Momen H, Grimaldi G, Pacheco RS, Jaffe CL, McMahon-Pratt D, Marzochi MC: Brazilian Leishmania stocks phenotypically similar to Leishmania major. The American Journal of Tropical Medicine and Hygiene. 1985, 34 (6): 1076-1084. Blaineau C, Bastien P, Rioux JA, Roizes G, Pages M: Long-range restriction maps of size-variable homologous chromosomes in Leishmania infantum. Mol Biochem Parasitol. 1991, 46 (2): 292-302. 10.1016/0166-6851(91)90053-9. Kebede A, De Doncker S, Arevalo J, Le Ray D, Dujardin JC: Size-polymorphism of mini-exon gene-bearing chromosomes among natural populations of Leishmania, subgenus Viannia. Int J Parasitol. 1999, 29 (4): 549-557. 10.1016/S0020-7519(99)00010-7. Antoniazi S, Lima HC, Cruz AK: Overexpression of miniexon gene decreases virulence of Leishmania major in BALB/c mice in vivo. Mol Biochem Parasitol. 2000, 107 (1): 57-69. 10.1016/S0166-6851(99)00232-7. Dujardin JC, Gajendran N, Arevalo J, Llanos-Cuentas A, Guerra H, Gomez J, Arroyo J, De Doncker S, Jacquet D, Hamers R, et al: Karyotype polymorphism and conserved characters in the Leishmania (Viannia) braziliensis complex explored with chromosome-derived probes. Ann Soc Belg Med Trop. 1993, 73 (2): 101-118. Dujardin JC, Dujardin JP, Tibayrenc M, Timperman G, De Doncker S, Jacquet D, Arevalo J, Llanos-Cuentas A, Guerra H, Bermudez H, et al: Karyotype plasticity in neotropical Leishmania: an index for measuring genomic distance among L. (V.) peruviana and L. (V.) braziliensis populations. Parasitology. 1995, 110 ( Pt 1): 21-30. Castilho TM, Shaw JJ, Floeter-Winter LM: New PCR assay using glucose-6-phosphate dehydrogenase for identification of Leishmania species. J Clin Microbiol. 2003, 41 (2): 540-546. 10.1128/JCM.41.2.540-546.2003. McMahon-Pratt D, Bennett E, David JR: Monoclonal antibodies that distinguish subspecies of Leishmania braziliensis. The Journal Immunology. 1982, 129 (3): 926-927. Phred/Phrap/Consed. [http://www.phrap.org] BlastN. [http://www.ncbi.nlm.nih.gov] Hall A: BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. 1999, 41: 46-49. Swofford DL: 2003. PAUP* beta version. Phylogenetic AnalysisUsing Parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, Massachusetts, Huelsenbeck JP: Is the Felsenstein zone a fly trap?. Syst Biol. 1997, 46 (1): 69-74. 10.2307/2413636. Posada D, Crandall KA: MODELTEST: testing the model of DNA substitution. Bioinformatics. 1998, 14 (9): 817-818. 10.1093/bioinformatics/14.9.817. Schmidt HA, Strimmer K, Vingron M, von Haeseler A: TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics. 2002, 18 (3): 502-504. 10.1093/bioinformatics/18.3.502. Strimmer K, von Haeseler A: Likelihood-mapping: a simple method to visualize phylogenetic content of a sequence alignment. Proc Natl Acad Sci U S A. 1997, 94 (13): 6815-6819. 10.1073/pnas.94.13.6815. Page RD: TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci. 1996, 12 (4): 357-358. Wu KS, Jones R, Danneberger L, Scolnik PA: Detection of microsatellite polymorphisms without cloning. Nucleic Acids Res. 1994, 22 (15): 3257-3258. 10.1093/nar/22.15.3257. Sneath PH, Sokal RR: Numerical taxonomy. 1973, San Francisco , Freeman Pavlicek A, Hrda S, Flegr J: Free-Tree--freeware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of the tree robustness. Application in the RAPD analysis of genus Frenkelia. Folia Biol (Praha). 1999, 45 (3): 97-99. Cruz A, Beverley SM: Gene replacement in parasitic protozoa. Nature. 1990, 348 (6297): 171-173. 10.1038/348171a0.