Cloning and sequence analysis of -tubulin gene of Trypanosoma evansi isolates from Indian dromedaries (Camelus dromedarius)

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1 Indian J. Anim. Res., 49 (5) 2015 : Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Cloning and sequence analysis of -tubulin gene of Trypanosoma evansi isolates from Indian dromedaries (Camelus dromedarius) M. Ashraf*, Meera Srivastava, S.K. Ghorui, G. Nagarajan, Shelesh kumar Swami and Sanjay Kumar National Research Centre on Camel, Bikaner , India Received: Accepted: DOI: /ijar.5573 ABSTRACT Trypanosomosis caused by Trypanosoma evansi is a disease of economic significance of domestic livestock, causing trypanosomiasis, commonly known as Surra. In the present study, gene of Trypanosoma evansi have beer cloned and sequenced. Beta-tubulin is important for cellular structure and physical functions. Sequence analysis of this gene will be useful for the vaccine studies. The open reading frame of -tubulin of Trypanosoma evansi is 1329 bp in length, encoding 442 amino acid polypeptide. The sequence analysis revealed that the ß tubulin gene of Trypanosoma evansi from India was having the identity of 99% with T.equiperdum, T.evansi (China isolate), T.evansi (Indian isolates), T.brucei and 88% with T.cruzi. The stable biological characteristics of Trypanosomatidae beta-tubulin make it an ideal target of many anti-parasitic drugs that block tubulin function. Key words: ß tubulin gene, Dromedarian camel, Phylogenetic analysis, T. evansi INTRODUCTION Trypanosomiasis is the most important and serious pathogenic protozoan disease of camel caused by T. evansi. This parasite has a wide range of distribution throughout tropical and sub-tropical regions of the world. T. evansi was reported originally from India, where the term Surra is used to describe the disease. In Africa, south of the Sahara (inside the tsetse fly belt), another form of Trypanosomiasis, due to other trypanosome species, like T. brucei, T. congolense and T. vivax, is prevalent which is known as Nagana. T. evansi probably evolved from T. brucei, when camels entered the tsetse fly belt and acquired infection. Then, leaving the tsetse area the disease was maintained via mechanical transmission by biting flies: notably, Tabanus striatis, Haematobia, Stomoxys, Chrysops and Lyperosia, Pangonia and spread to the Northern Africa, Middle East, India and the Far East Asian countries ( Luckins, 1992, Jensen et al., 2008 and Lun et al., 2010). Trypanosomes have the capacity for antigenic variation, which is the basis of their ability to escape the host immune response and because of this; prospects for the development of a vaccine against trypanosomiasis have been considered poor. However, the most effective and sustainable way of controlling trypanosomiasis should be a safe and costeffective vaccine. Therefore, it would be necessary to develop a vaccine against the infection of trypanosomes based on the *Corresponding author s aicrp@rediffmail.com. other potential target proteins. Microtubules are cytoskeletal proteins that are present in all eukaryotic cells and are especially abundant in the Trypanosomatidae (Rasooly, and Balaban, N. 2004). They are composed of tubulin heterodimers and of microtubule-associated proteins (Kohl and Gull, 1998). Beta-tubulin is an important member of the tubulin family in Trypanosoma species. It is a single isoform and it is distributed beneath the surface membrane, the flagellum, paraflagellar rod and mitotic spindle apparatus of dividing nuclei (Lubega et al., 2002). The properties of betatubulin in lower eukaryotes, such as protozoa and helminths are significantly different from those of higher ones, such as mammals. Beta-tubulin is important for cellular structure and physical functions. The stable biological characteristics of Trypanosomatidae beta tubulin make it an ideal target of many anti-parasitic drugs that block tubulin function. Therefore, microtubule components have also been suggested as an effective vaccine candidate to protect from trypanosome infections. However, thus far recently, beta tubulin gene of many protozoan parasites have been cloned including T.rangeli (Amorim et al., 1993), T. cruzi (Gonzalez-Pino et al., 1997), African trypnosomes (Wu and Yarbrough, 1987), Leishmania (Jackson et al., 2006). In the present study, the full-length -tubulin cdnas of Trypanosome evansi isolates from dromedarian camel was cloned and

2 characterized. The deduced nucleotide sequences were then used for phylogenetic relationship analyses by comparing them with published sequences from other common trypanosome species. MATERIALS AND METHODS Animals and trypanosome strains: A camel suffering from Surra was identified in the National Research Centre on Camel (NRCC), Bikaner, (India). A Giemsa stained blood film was prepared and examined under the microscope to confirm the infection of T. evansi in the camel. After confirmation Trypanosoma evansi isolates were separated from the blood of the infected host. For this 5 ml blood were collected from the jugular vein using a 9 ml vacutainer tube with EDTA (ethyl diamine tetra acetic acid). From this 0.9 ml blood was taken by insulin syringe containing 0.1 ml heparin solution. This was then inoculated into the experimental animals which were swiss albino mice for propagation of trypanosomes. The blood of mice was collected from tail region using 5 ml disposable syringe containing 0.1 ml heparin solution. DEAE-cellulose chromatography method was used for purification of trypanosomes. RNA isolation and RT-PCR: Total RNA was isolated from purified trypanosomes using the RNA isolation kit (Banglore Genie Pvt Ltd. Bangalore, India)). An aliquot of the total RNA (5µg) was reverse-transcribed using GeNei TM M-MuLV RT-PCR Kit (Bangalore Genie Pvt Ltd., India) in a 20 µl volume reaction mixture according to the manufacturer s instructions. -tubulin gene was amplified from the c-dnas of T. evansi by PCR using primers designed based on - tubulin sequence of T. evansi (China) reported in the GenBank (Table 1). Cycling conditions for PCR were 40 cycles of 45 seconds at 94 C, 45 seconds at annealing temperatures given in (Table 1) and 60 seconds at 72 C, followed by a final extension for 10 min at 72 C. The PCR amplified products were checked in 1.2 % agarose gel and shown in Fig. 1. Cloning and sequencing of -tubulin gene: The PCR products were purified using Wizard plus minipreps DNA purification kit (Promega, USA), digested with StuI and XhoI enzymes and cloned into EcoRV and XhoI sites of pbluescript KS+ vector with T4 DNA ligase (Promega, U.S.A.). The plasmids were transformed into E.coli DH5a. Colonies harbouring the recombinant plasmid were inoculated into LB broth and incubated at 37 C overnight with horizontal shaking. The plasmids DNA were extracted from culture using Volume 49 Issue 5 (October 2015) 619 FIG.1:- Amplification of -tubulin gene (Lane 1 Kb Ladder, Lane 2-4: Amplicons -tubulin gene, Lane 5: Negative control) a Wizard plus minipreps plasmid DNA purification kit (Promega, USA). The positive clone was confirmed by restriction analysis with SalI and XhoI. The positive clones were sequenced in both directions at DNA sequencing facility, Delhi University (South campus), Delhi, India. Sequence and phylogenetic analysis: The determined - tubulin gene nucleotide sequences of the dromedarian camel was analyzed with the BLAST program (NCBI) search of GenBank. The sequence data herein have been submitted to GenBank and assigned Accession numbers GQ for -tubulin gene. The resultant nucleotide and amino acid sequences were assembled and analysed with that of other common trypanosomes species available in the GenBank (Table 2) using computer software BIOEDIT Version These sequences were compared in Clustal X (Stevens et al., 1999a) and phylogenetic tree was constructed in Treeview by neighbour joining method (Maslov et al., 1996). RESULTS AND DISCUSSION Three clones were chosen and a consensus -tubulin sequence was determined. The complete sequence of -tubulin of Trypanosoma evansi and its comparision to ten other Trypanosoma sequences are shown in Fig. 2. The open reading frame of -tubulin of Trypanosoma evansi is 1329 bp in length, encoding 442 amino acid polypeptide. Nucleotide sequence homologies of -tubulin of Trypanosoma evansi and other ten trypanosome species TABLE 1:- Primer sequences used to clone â-tubulin gene of Trypanosoma evansi isolates from camel dromedaries. Gene Primer sequences (5-3 Predicted size(base pairs) Annealing temperature( C) Accession No. -tubulin (F) 5¹ GC GG AGG CCT ATG CGT GAG ATT GTG TGC 3¹ o GQ tubulin (R) 5¹ GC GG CTC GAG CTA GTA TTG CTC CTC CTC 3

3 620 INDIAN JOURNAL OF ANIMAL RESEARCH TABLE 2:-Accession numbers of nucleic acid sequences of â -tubulin gene used for phylogenetic analysis No. Beta tubulin gene for species Accession No. Collection country Reference 1 Trypanosoma equiperdum DQ China Li (2006) et al. 2 T.evansi DQ China Li (2006) et al. 3 T.evansi EU India Kurup (2008) et al. 4 T. brucei gambiense FN United Kingdom Jackson (2009) et al. 5 T. brucei AL United Kingdom Hall (2008) et al. 6 T.brucei rhodesiense K02836 Africa Kimmel (1993) et al. 7 T.cruzi AF Brazil Bartholomeu (2004) et al. 8 T.danilewskyi DQ Canada Plouffe (2006) et al. 9 Leishmania infantum XM_ United Kingdom Peacock (2007) et al Trypanosoma equiperdum [China] DQ T.evansi [China] DQ T.evansi [India] GQ T.evansi [India] EU T.brucei gambiense [Uk] FN T.brucei [Uk] AL T.brucei rhodesiense [Africa] K02836 T.cruzi [Brazil] AF T. danilewskyi [Canada]DQ Leishmania infantum [Uk] XM FIG. 2:- Phylogenetic tree based on nucleotide sequences of â- tubulin genes from different Trypanosoma species, constructed using the neighbour-joining method in Clustal X and Tree view with bootstrap replicates. [complete sequence available] are shown in Table 3. The nucleotide sequences of -tubulin of Trypanosoma evansi from India showed a higher homology to T. equiperdum, T.evansi (China isolate), T.evansi (Indian isolates), T.brucei. These results were further confirmed by phylogenetic analysis [Fig. 2]. The phylogenetic analysis based on the nucleotide sequences of -tubulin of Trypanosoma evansi and other Trypanosoma species showed the expected clustering of all trypanosomes species. In this study, the -tubulin of Trypanosoma evansi was cloned and sequenced for the first time in Indian dromedaries. Comparison of nucleotide sequence homologies of -tubulin revealed 99.6% homology towards T. equiperdum (China, GenBank Accession No. DQ786575) and T.evansi (China, GenBank Accession No.DQ786574), 99.4% similarities were noted between our isolated gene and T.evansi (India, GenBank Accession No.EU483116) and T. brucei gambiense (UK, GenBank Accession No. FN554964). Similarly, a slightly lower homology was documented between the gene obtained during the present study and T.brucei (UK, GenBank Accession No. AL929603) and T. brucei rhodesiense (Africa, GenBank Accession No. K02836), while, T.cruzi (Brazil, GenBank Accession No. AF455116) and T. danilewskyi (Canada, GenBank Accession No. DQ080028) showed only 88.4% and 87.4% similarités. The comparison with Leishmania infantum (UK, GenBank Accession No. XM_ ) a member belonging to Trypanosomatidae family showed 85.2% homology only. Based on the above homology, L. infantum (UK, GenBank Accession No. XM_ ) in the phylogenetic tree is placed as an outgroup. T.cruzi (Brazil, GenBank Accession No. AF455116) and T. danilewskyi (Canada, GenBank Accession No. DQ080028) as two sub clusters of one mega cluster; the other mega clusters comprising of rest of the species. The earlier reports of T.evansi also showed 99.6% homology with T. equiperdum (China, GenBank Accession No. DQ786575) and T.evansi (China, GenBank Accession No.DQ786574). A cent per cent similarity between this gene and T.b. gambiense was observed by them. The studies of kinetoplastid evolution, so long based on morphological and transmission characteristics (Baker, 1963, Hoare, 1972), appear to finally have been overcome. Since the first broad molecular study of eukaryotic evolution, which included only a single representative of the genes Trypanosoma (Sogin et al., 1986), phylogenetic analysis of TABLE 3:-Percent identity of nucleotide sequences of various â-tubulin genes from various Trypanosomatids Identity of T.eq. T. evansi T. evansi T.b. T.b. [uk] T.b.rh. T.c. T.dan. L.i. β-tubulin gene for [China] [China] [India] gam [uk] [Africa] [Brazil] [Canada] [UK] Trypanosoma evansi β-tubulin

4 kinetoplastid flagellates has become successively more focused. Initial studies concentrated on the origin of parasitism in the group (Lake et al., 1988 & Fernandes et al., 1993) and latter on detailed analysis of evolutionary relationship among Trypanosoma and Lieshmania species (Maslov et al., 1996, Croan et al., 1997, Lukes et al., 1997, Haag et al., 1998 and Stevens et al., 1999a and 1999b). Earlier 18s rrna genes studies were summarized by Maslov and Simpson in a phylogenetic tree which included three trypanosme species T.brucei, T.cruzi and a third species from a fish. In common with other earlier studies (Gomez et al., 1991, Fernandes et al., 1993 and Landweber and Gilbert 1994), this tree indicates the genus Tryaposoma to be paraphyletic. Subsequently, Maslov et al., (1996) reported the number of species to seven; however, this still left T.brucei outside both from the main trypanosome clade and the trypanosomatidae family containing Leishmania and Crithidia. The inclusion by Lukes et al (1977) of four more trypanosome species suggested that the genus Trypanosoma might infact be monophyletic and the addition of more outgroup taxa considerably strengthened this result. Subsequently, trees including 24 trypanosome species (Haag et al., 1998) and 47 trypanosme taxa (Stevens et al., 1999a) have both supported monophyly of trypanosomes. Early trees which showed trypanosomes to be paraphyletic suggested that parasitism and the digenetic life cycle had arisen more than once in the trypanosome lineage. The unequivocal evidence of monophyly revealed by more recent trees clearly contradicts this, still support the latter view. According to the reports of Amorinm and his team (Amorim et al., 1993), the sequence analysis of beta tubulin Volume 49 Issue 5 (October 2015) 621 gene of T. rangeli suggested it to be closer to T.brucei rather than to T. cruzi. Cloning and sequence analysis of T.cruzi alpha tubulin c-dna done by Gonzalez-pino and his coworkers (Gonzalez-Pino et al., 1997) revealed 87.79% and 85.36% identity with the DNA sequence of T.brucei and Leishmania respectively. The present findings therefore suggest that the identified beta tubulin gene showed a close homology with T. equiperdum (China, GenBank Accession No. DQ786575), T.evansi (China, GenBank Accession No.DQ786574) and is also quite similar to T.evansi (India, GenBank Accession No.EU483116), T. brucei gambiense (UK, GenBank Accession No.FN554964), T.brucei (UK, GenBank Accession No. AL929603) and T. brucei rhodesiense (Africa, GenBank Accession No. K02836). It could therefore be hypothesized and suggested that vaccine with beta tubulin protein of trypanosomatidae parasite as the antigen could be effective against not only different strains within one trypanosome species but also against other species of the same genus. ACKNOWLEDGEMENTS The authors are grateful to Prof. K.M.L. Pathak, DDG (Animal Sciences), ICAR for the permission to carry out the work at NRC on Camel,. Bikaner. The authors are thankful to Dr. P.N. Sivalingam, Scientist, CIAH, Bikaner, India, for critical reading and preparation of the manuscript. The help rendered by Mr. Jeetendar Kumar, Technical Incharge and M.L. Kiradoo, Lab Attendant, NRC on Camel, Bikaner, in the collection of blood sample from the camels is also gratefully acknowledged. The help rendered by Mr. Harish Kumar Changal, SRF, CIAH, Bikaner and Mrs. Charu Pathak, in the provision of computer assistance is gratefully acknowledged. REFERENCES Amorim M.I., Momen H. and Traub-Cseko Y.M. (1993). Trypanosoma rangeli: sequence analysis of beta-tubulin gene suggests closer relationship to Trypanosoma brucei than to Trypanosoma cruzi. Acta Trop, 53: Baker J.R.(1963). Speculations on the evolution of the family Trypanosomatidae, Experimental Parasitology. 13: Croan, D. G., Morrison, D.A. and Ellis J.T. (1997). Evolution of the genus Leishmania by comparison of DNA and RNA polymerase gene sequences. Molecular Biochemical Parasitol, 89: Fernandes A.P., Nelson K. and Beverley S.M. (1993). Evolution of nuclear ribosomal RNAs in Kinetoplastid protozoa: perspectives on the age and origins of parasitism. Proc NatAcad Sci USA, 90: Gomez E., Valdes A.M., Pinero D. and Hernandez R. (1991). What is a genus in the Trypanosomatidae family? Phylogenetic analysis of two small rrna sequences. Molecular Biology Evoltion, 8: Gonzalez-Pino M.J., Rangel-Aldao R. and Slezynger T.C. (1997). Cloning and sequence analysis of a Trypanosoma cruzi alpha-tubulin cdna. Biol Res, 30: Haag J., O Huigin C. and Overath P. (1998). The molecular phylogeny of trypanosomes: Evidence for an early divergence of the Salivaria. Molecular Biochem Parasitol, 91: Hoare C.A. (1972). The trypanosomes of mammals. Blackwell Scientific Publications., Oxford, 750 pp.

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