Hostname: page-component-78c5997874-94fs2 Total loading time: 0 Render date: 2024-11-04T18:08:47.965Z Has data issue: false hasContentIssue false
  • English
  • Français

Genetic fingerprinting and identification of differentially expressed genes in isolates of Leishmania donovani from Indian patients of post-kala-azar dermal leishmaniasis

Published online by Cambridge University Press:  28 August 2007

B. V. SUBBA RAJU ,
R. SINGH ,
G. SREENIVAS ,
S. SINGH  and
P. SALOTRA
B. V. SUBBA RAJU
Affiliation:
Institute of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi-110 029, India
R. SINGH
Affiliation:
Institute of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi-110 029, India
G. SREENIVAS
Affiliation:
Institute of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi-110 029, India
S. SINGH
Affiliation:
Department of Microbiology, Jiwaji University, Gwalior, M.P.India
P. SALOTRA*
Affiliation:
Institute of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi-110 029, India
*
*Corresponding author: Institute of Pathology (ICMR), Safdarjung Hospital Campus, New Delhi-110 029, India. Tel: +91 11 26166124. Fax: +91 11 26166124. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

Post-kala-azar dermal leishmaniasis (PKDL) is an unusual dermatosis that develops as a sequel in 5–15% of cured cases of kala-azar (KA) after months or years of treatment in India. Molecular differences are reported to exist between the KA and PKDL isolates which may underlie the diversity in clinical manifestations of the disease. Here, arbitrary primed-PCR (AP-PCR) has been used for genetic fingerprinting of parasite isolates from dermal lesions of PKDL patients (n=14) and compared with bone-marrow derived parasites from KA patients (n=3). All isolates showed an identical AP-PCR pattern with 4 arbitrary primers. Further, AP-PCR was exploited to identify the stage regulated genes of the parasite. Six polymorphic fragments were identified in PKDL in comparison with KA isolates, and were subjected to Northern blot analysis. Five polymorphic fragments represented transcribed sequences; 4 out of 5 drew differential expression in pro- and amastigote stages, although the expression was comparable between PKDL and KA isolates. The study led to the identification of genes, which exhibit stage-regulated expression in Leishmania donovani derived from PKDL or KA patients, including a putative phosphodiesterase, DEAD box RNA helicase, iron superoxide dismutase b (fesodb) and a hypothetical protein. Demonstration of transcripts of DEAD box RNA helicase in PKDL and KA diseased tissues implicates its role in disease pathogenesis.

Keywords

Leishmania donovaniPKDLvisceral leishmaniasisAPPCRdifferentially expressed genesgenomic fingerprinting
Type
Research Article
Information
Parasitology , Volume 135 , Issue 1 , January 2008 , pp. 23 - 32
Copyright
Copyright © Cambridge University Press 2007

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Addy, M. and Nandy, A. (1992). Ten years of kala-azar in west Bengal, Part I. Did post-kala-azar dermal leishmaniasis initiate the outbreak in 24-Parganas? Bulletin of the World Health Organization 70, 341–346.Google ScholarPubMed
Akopyants, N. S., Matlibs, R. S., Bukanova, E. N., Smeds, M. R., Brownstein, B. H., Stormo, G. D. and Beverley, S. M. (2004). Expression profiling using random genomic DNA microarray identifies differentially expressed genes associated with three major developmental stages of the protozoan parasite Leishmania major. Molecular and Biochemical Parasitology 136, 71–86.CrossRefGoogle ScholarPubMed
Almeida, R., Norrish, A., Levick, M., Vetrie, D., Freeman, T., Vilo, J., Ivens, A., Lange, U., Stober, C., McCann, S. and Blackwell, J. M. (2002). From genomes to vaccines: Leishmania as a model. Philosophical Transactions of the Royal Society of London. Series B, 357, 5–11.CrossRefGoogle ScholarPubMed
Bellatin, J. A., Murray, A. S., Zhao, M. and McMaster, W. R. (2002). Leishmania mexicana: identification of genes that are preferentially expressed in amastigotes. Experimental Parasitology 100, 44–53.CrossRefGoogle ScholarPubMed
D'Angelo, M. A., Sanguineti, S., Reece, J. M., Birnbaumer, L., Torres, H. N. and Flawia, M. M. (2004). Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi. The Biochemical Journal 378, 63–72.CrossRefGoogle ScholarPubMed
Desjeux, P. (2004). Leishmaniasis: current situation and new perspectives. Comparative Immunology, Microbiology and Infectious Diseases 27, 305–318.CrossRefGoogle ScholarPubMed
Dey, A. and Singh, S. (2006). Genetic heterogeneity among visceral and post-kalaazar dermal leishmaniasis strains from eastern India. Infection, Genetics and Evolution: Journal of Molecular Epidemiology and Evolutionary Genetics in Infectious Diseases 7, 219–222.CrossRefGoogle Scholar
El-Hassan, A. M., Ghalib, W. H., Zijlstra, E. E., Eltoum, I. A., Satti, M., Ali, M. S. and Ali, H. M. A. (1992). Post kala-azar dermal leishmaniasis in the Sudan: clinical features, pathology and treatment. Transactions of the Royal Society of Tropical Medicine and Hygiene 86, 245–248.CrossRefGoogle Scholar
Gong, K. W., Kunz, S., Zoraghi, R., Kunz, R. C., Brun, R. and Seebeck, T. (2001). cAMP-specific phosphodiesterase TbPDE1 is not essential in Trypanosoma brucei in culture or during midgut infection of tsetse flies. Molecular and Biochemical Parasitology 116, 229–232.CrossRefGoogle ScholarPubMed
Ghosh, S., Goswami, S. and Adhya, S. (2003). Role of superoxide dismutase in survival of Leishmania within the macrophage. The Biochemical Journal 369, 447–452.CrossRefGoogle ScholarPubMed
Heard, P. L., Lewis, C. S. and Chaudhuri, G. (1996). Leishmania mexicana amazonensis: differential display analysis and cloning of mRNAs from attenuated and infective forms. Journal of Eukaryotic Microbiology 43, 409–415.CrossRefGoogle ScholarPubMed
Jaffe, C. L., Bennettm, E., Grimaldi, G. Jr. and McMahon-Pratt, D. (1984). Production and characterization of species-specific monoclonal antibodies against Leishmania donovani for immunodiagnosis. Journal of Immunology 133, 440–447.CrossRefGoogle ScholarPubMed
Joshi, M., Dwyer, D. M. and Nakhasi, H. L. (1993). Cloning and characterization of differentially expressed genes from in vitro grown amastigotes of Leishmania donovani. Molecular and Biochemical Parasitology 58, 345–354.CrossRefGoogle ScholarPubMed
Liu, K., Zinker, S., Arguello, C. and Salgado, L. M. (2000). Isolation and analysis of a new developmentally regulated gene from amastigotes of Leishmania mexicana mexicana. Parasitology Research 86, 140–150.CrossRefGoogle ScholarPubMed
McNicoll, F., Drummelsmith, J., Muller, M., Madore, E., Boilard, N., Ouellette, M. and Papadopoulou, B. (2006). A combined proteomic and transcriptomic approach to the study of stage differentiation in Leishmania infantum. Proteomics 6, 3567–3581.CrossRefGoogle Scholar
Neto, E. D., deSouza, P., Rollinson, D., Katz, N., Pena, S. D. J. and Simpson, A. J. G. (1993). The random amplification of polymorphic DNA allows the identification of strains and species of Schistosome. Molecular and Biochemical Parasitology 57, 83–88.CrossRefGoogle Scholar
Noyes, H. A., Belli, A. A. and Maingon, R. (1996). Appraisal of various random amplified polymorphic DNA-polymerase chain reaction primers for Leishmania identification. American Journal of Tropical Medicine and Hygiene 55, 98–105.CrossRefGoogle ScholarPubMed
Paramchuk, W. J., Ismail, S. O., Bhatia, A. and Gedamu, L. (1997). Cloning, characterization and over-expression of two iron superoxide dismutase cDNAs from Leishmania chagasi: role in pathogenesis. Molecular and Biochemical Parasitology 90, 203–221.CrossRefGoogle Scholar
Plewes, K. A., Stephen, D. B. and Gedamu, L. (2003). Iron superoxide dismutases targeted to the glycosomes of Leishmania chagasi are important for survival. Infection and Immunity 7, 5910–5920.CrossRefGoogle Scholar
Pogue, G., Koul, S., Lee, N. S., Dwyer, D. M. and Nakhasi, H. L. (1995 a). Identification of intra- and inter-specific Leishmania genetic polymorphisms by arbitrary primed PCR and use of polymorphic DNA to identify differentially regulated genes. Parasitology Research 81, 282–290.CrossRefGoogle Scholar
Pogue, G. P., Lee, N. S., Koul, S., Dwyer, D. M. and Nakhasi, H. L. (1995 b). Identification of differentially expressed Leishmania donovani genes using arbitrarily primed polymerase chain reactions. Gene 165, 31–38.CrossRefGoogle ScholarPubMed
Procunier, J. D., Fernando, M. A. and Barta, J. R. (1993). Species and strain differentiation of Eimeria spp. of deomestis fowl using DNA polymorphisms amplified by arbitrary primers. Parasitology Research 79, 98–102.CrossRefGoogle ScholarPubMed
Ramesh, V. (1993). Post-kala-azar dermal leishmaniasis. Australian Journal of Dermatology 34, 35.CrossRefGoogle ScholarPubMed
Ramesh, V. and Mukherjee, A. (1995). Post kala-azar dermal leishmaniasis. International Journal of Dermatology 34, 85–91.CrossRefGoogle ScholarPubMed
Remme, J. H., Blas, E., Chitsulo, L., Desjeux, P. M., Engers, H. D., Kanyok, T. P., Kayondo, J. F., Kioy, D. W., Kumaraswami, V., Lazdins, J. K., Nunn, P. P., Oduola, A., Ridley, R. G., Toure, Y. T., Zicker, F. and Morel, C. M. (2002). Strategic emphases for tropical diseases research: a TDR perspective. Trends in Parasitology 10, 421–426.CrossRefGoogle ScholarPubMed
Saha, S., Mazumdar, T., Anam, K., Ravindran, R., Bairagi, B., Saha, B., Goswami, R., Pramanik, N., Guha, S. K., Kar, S., Banerjee, D. and Ali, N. (2005). Leishmania promastigote membrane antigen-based enzyme linked immunosorbent assay and immunoblotting for differential diagnosis of Indian post-kala-azar dermal leishmaniasis. Journal of Clinical Microbiology 43, 1269–1277.CrossRefGoogle ScholarPubMed
Salotra, P., Raina, A. and Ramesh, V. (1999). Western blot analysis of humoral immune response to Leishmania donovani antigens in patients with post kala-azar dermal leishmaniasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 93, 98–101.CrossRefGoogle ScholarPubMed
Salotra, P., Sreenivas, G., Pogue, G. P., Lee, N., Nakhasi, H. L., Ramesh, V. and Negi, N. S. (2001). Development of a species-specific PCR assay for detection of Leishmania donovani in clinical samples from patients with kala-azar and post-kala-azar dermal leishmaniasis. Journal of Clinical Microbiology 39, 849–854.CrossRefGoogle ScholarPubMed
Salotra, P., Duncan, R. C., Singh, R., Subba Raju, B. V., Sreenivas, G. and Nakhasi, H. L. (2006). Upregulation of surface proteins in Leishmania donovani isolated from patients of post kala-azar dermal leishmaniasis. Microbes and Infection 8, 637–644.CrossRefGoogle ScholarPubMed
Schönian, G., Schweynoch, C., Zlateva, K., Oskam, L., Kroon, N., Gräser, Y. and Presber, W. (1996). Identification and determination of the relationship of species and strains within the genus Leishmania using single primers in the polymerase chain reaction. Molecular and Biochemical Parasitology 77, 19–29.CrossRefGoogle ScholarPubMed
Selvapandiyan, A., Duncan, R., Debrabant, A., Bertholet, S., Sreenivas, G., Negi, N. S., Salotra, P. and Nakhasi, H. L. (2001). Expression of a mutant form of Leishmania donovani centrin reduces the growth of the parasite. Journal of Biological Chemistry 276, 43253–43261.CrossRefGoogle ScholarPubMed
Singh, R., Kumar, D., Ramesh, V., Singh, S. and Salotra, P. (2006). Visceral leishmaniasis, or kala azar (KA): High incidence of refractoriness to antimony is contributed by anthroponotic transmission via post-KA dermal leishmaniasis. Journal of Infectious Diseases 194, 302–306.CrossRefGoogle ScholarPubMed
Sreenivas, G., Subba Raju, B. V., Singh, R., Selvapandiyan, A., Duncan, R., Sarkar, D., Nakhasi, H. L. and Salotra, P. (2004 a). 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 42, 1739–1741.CrossRefGoogle ScholarPubMed
Sreenivas, G., Singh, R., Selvapandiyan, A., Negi, N. S., Nakhasi, H. L. and Salotra, P. (2004 b). Arbitrary-primed PCR for genomic fingerprinting and identification of differentially regulated genes in Indian isolates of Leishmania donovani. Experimental Parasitology 106, 110–118.CrossRefGoogle ScholarPubMed
Srividya, G., Duncan, R., Sharma, P., Subbaraju, B. V., Nakhasi, H. and Salotra, P. (2007). Transcriptome analysis during the process of in vitro differentiation of Leishmania donovani using genomic microarrays. Parasitology (in the Press).CrossRefGoogle ScholarPubMed
Tibayrenc, M., Neubauer, K., Barnabe, C., Guerrine, F., Skarecky, D. and Ayala, F. J. (1993). Genetic characterization of six parasitic protozoa: parity between random primer DNA typing and multi locus enzyme electrophoresis. Proceedings of the National Academy of Sciences, USA 90, 1335–1339.CrossRefGoogle Scholar
Waitumbi, J. N. and Murphy, N. B. (1993). Inter-and intra-specific differentiation of trypanosomes by genomic fingerprinting with arbitrary primers. Molecular and Biochemical Parasitology 58, 181–186.CrossRefGoogle Scholar
Wu, Y., El Fakhry, Y., Sereno, D., Tamar, S. and Papadopoulou, B. (2000). A new developmentally regulated gene family in Leishmania amastigotes encoding a homolog of amastin surface proteins. Molecular and Biochemical Parasitology 110, 345–357.CrossRefGoogle ScholarPubMed
Zijlstra, E. E., Musa, A. M., Khalil, E. A., El-Hassan, I. M. and El-Hassan, A. M. (2003). Post-kala-azar dermal leishmaniasis. Lancet Infectious Diseases 3, 87–98.CrossRefGoogle ScholarPubMed