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Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 30–36J P DIsolation and characterization of the paraflagellar rodproteins of Leishmania donovaniA. Lahiri and A. BhattacharyaImmunoparasitology Research Unit, Department of Zoology, University of Calcutta, Kolkata.ABSTRACT. The paraflagellar rod (PFR) is a unique cytoskeletal structure present in thekinetoplastid protozoans but absent from their mammalian hosts. It is a massive network of wovencytoskeletal filaments attached to one face of the common 9+2 axoneme of the flagellum. The PFR isnecessary for proper parasite motility, viability and successful infection. Biochemical studiescarried out on the PFR have revealed that it is composed of both major and minor proteincomponents. In this paper, purification of the PFR proteins of Leishmania donovani has beendescribed, which involves a combination of flagella isolation, non-ionic detergent treatment andrestricted proteolysis. Scanning electron microscopy was done to monitor the process of isolation offlagella from intact cell bodies. The major PFR proteins have been identified as two distinct bandsof mol wt 76 kDa and 68 kDa, by using sodium dodecyl sulphate polyacrylamide gel electrophoresisanalysis. The ultrastructure of the flagellum was studied by using a transmission electronmicroscope.Keywords: axoneme, flagellum, kinetoplastid, paraflagellar rodINTRODUCTIONLeishmania donovani, an intracellular protozoanparasite, causes Kala-azar (visceral leishmaniasis) inhumans. The parasite is transmitted by various speciesof female sandflies (Phlebotomus argentipes). Itexists in two morphological forms: the promastigote,residing in the gut of female sandflies, and theamastigote, living in the reticuloendothelial system ofthe mammalian hosts. The promastigotes possess afull-length, free-flagellum whereas it is rudimentaryin amastigotes.The flagellum of promastigotes helps in motility and isinvolved in hemidesmosomal attachment to thechitinous itima, and maintenance of the parasitewithin the sandfly gut (Killick-Kendrick, 1979;Killick-Kendrick et al., 1974; Walters et al., 1987).Corresponding author: Dr. A. Bhattacharya, Immuno-parasitologyResearch Unit, Department of Zoology, University of Calcutta, 35Ballygunge Circular Road, Kolkata-700 019, W.B., India.The flagellar modes of attachment have been observedwith other kinetoplastids also and appear to beessential for their survival within the insect vector(Killick-Kendrick, 1979; Rowton et al., 1981;Vickerman, 1973; Vickerman and Preston, 1976). Theflagellum of Leishmania is also involved inchemotactic responses (Bray, 1983). Structurally, theflagellum of Leishmania has a typical 9+2microtubular axoneme and also possesses afilamentous, lattice-like structure called theparaflagellar rod (PFR) or paraxial rod. Themicrotubules of the axoneme are arranged in a precisepattern of nine outer double microtubules and twoseparate central ones.The PFR is a unique cytoskeletal structure found inkinetoplastids, euglenoids and some dinoflagellates(Bastin et al., 1996; Hyams, 1982; Cachon et al.,1988). The paraxial rod of kinetoplastida is similar tothat of euglenoids, although apparently, they appear tobe morphologically distinct from each other (Farina et
The PFR proteins of Leishmania donovani31al., 1980; Fuge, 1969; Gallo and Schrevel, 1985). The significant homology has been reported betweenPFR is a massive network of woven cytoskeletal major PFR proteins and other known proteinsfilaments, running alongside the typical eukaryotic (Imbodem et al., 1995).9+2 axoneme of the flagellum (Russell et al., 1983).Recent molecular studies have demonstrated that theThe ultrastructural study of the flagellum includingPFR is necessary for proper promastigote motility andthe PFR and the purification of the PFR proteinsviability (Santrich et al., 1997; Bastin et al., 1998).provides an important guideline to the researchers forAblation of a specific molecule of the PFR resulted inunderstanding the complete biology of this structure.mutant cells that were paralyzed, indicating theThe parasite L. donovani represents a standard modelessential role of PFR in cell motility (Deflorin et al.,for studying unique structures such as the PFR1994). Also, ATPase activity was detected in the PFRbecause it is one of the most common humanof euglenoids (Moreira-Leite et al., 1999). Thepathogens in tropical countries such as India.ultrastructure of kinetoplastid PFR appears largelysimilar in all species of the group (De Souza andMATERIALS AND METHODSSouto-Padron, 1980; Beard et al., 1992). The structure Parasite culture: The promastigotes were cultured athas been divided in three distinct zones in relation to 22-25° C in liquid M-199 medium supplemented withthe axoneme namely, proximal, intermediate and 10% heat-inactivated fetal bovine serum (FBS, Sigmadistal (Freymüller and Camargo, 1981). The proximal Aldrich, St. Louis, MO, USA; Morgan et al., 1950).and distal regions each contain filaments of 7-10 nm The culture medium was filtered under sterilethat intersect at an angle of 100°. The intermediate conditions in a culture room with laminar flow. Theregion contains thin (5 nm) filaments that intersect at medium was kept for 48 h at room temperature toan angle of 45° and connect the proximal and distal check for any contamination, and then stored at -20° C.regions (Maga and LeBowitz, 1999). Attachment Aliquots of the above culture of L. donovani were usedfilaments extending from axoneme microtubule in all the experiments.doublets 4-7 connect the proximal region to theIsolation of flagella: The culture medium containingaxoneme (Ismach et al., 1989). In Leishmania, thepromastigotes of L. donovani was centrifuged at 2100promastigotes possess a full-length flagellum with ax g for 20 min (Cunha et al., 1984). The pelletPFR, whereas amastigotes contain only an attenuated,containing the cells was washed twice in phosphatenon-emergent flagellum completely lacking a PFR.buffered saline (PBS; 10 mM, pH 7.2; Sigma) and thenThe biochemical composition of the PFR is quitein buffer A (25 mM TRIS-HCl , 0.2 mM EDTA, 5 mMcomplex and includes both major and minor PFRMgCl 2, 12 mM β-mercaptoethanol, 0.32 M sucroseproteins. Among them, the major PFR proteins havebeen extensively studied in the parasitic(all from Sigma; pH 7.4) and resuspended in buffer Asupplemented with 1% bovine serum albuminhaemoflagellates Trypanosoma cruzi, T. brucei, L.(Sigma), 0.1 mM CaCl 2 (Sigma), 0.5 mM phenylmexicana and L. amazonensis. In these organisms, themajor PFR proteins migrate in two bands on sodium methyl sulfonyl fluoride (Sigma) and 5 µg/mldodecyl sulphate polyacrylamide gel electrophoresis leupeptin (Sigma; Moreira-Leite et al., 1999). The(SDS-PAGE) with mol wt of about 68-75 kDa, and cells were subjected to three different centrifugationappear to be present in approximately equimolar speeds: 2600 x g, 3200 x g and 3700 x g, and theamounts (Deflorin et al., 1994; Schlaeppi et al., 1989).flagella were isolated from the cell bodies byThe major PFR components are conserved in thecentrifugation at an optimum speed of 3200 x g for 30trypanosomatidae family and form a doublet ofmin. The supernatant containing the flagella washomologous proteins in most species of the familyseparated from the pelleted and deflagellated cellbodies. This supernatant was then concentrated by(Araujo and Morein, 1991; Saborio et al., 1989). Bycentrifugation at 6780 x g for 20 min. The aboveSDS-PAGE analysis, PFR1 of L. amazonensisprocedure was carried out at 0-4° C. The pelletsmigrates at 74 kDa and PFR2 at 69 kDa (Bastin et al.,containing the flagella were finally suspended in1996). The PFR1 and PFR2 genes from T. cruzi, T.buffer A.brucei and L. mexicana are highly conserved acrossspecies (over 80% amino acid homology; Maga and Scanning electron microscopy: A scanning electronLebowitz, 1999). More complex patterns have been microscope (SEM) uses a fine beam of electrons todescribed in T. cruzi in which four major PFR proteins scan back and forth across the metal-coated surface.have been identified (Fouts et al., 1998). No The principal application of SEM is in the study of
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Page 18 and 19: 12 Soodconcentration (Kocher et al.
Page 20 and 21: 14 SoodKapur J and Sood ML. 1984b.
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Page 24 and 25: 18 Banyal and Elangbamin vitro. The
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Page 28 and 29: 22 Banyal and Elangbamand presence
Page 30 and 31: 24 Banyal and ElangbamTable I: Mala
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Page 34 and 35: 28 Banyal and ElangbamSchneider J,
Page 38 and 39: 32 Lahiri and Bhattacharyasurfaces
Page 40 and 41: 34 Lahiri and Bhattacharya9+2 micro
Page 42 and 43: 36 Lahiri and BhattacharyaKillick-K
Page 44 and 45: 38Edward and Bernardtopographical f
Page 46 and 47: 40Edward and Bernardfour villages b
Page 48 and 49: 42Sangwan and Sangwanvaccinated aga
Page 50 and 51: 44Sangwan and Sangwanpathogenic mec
Page 52 and 53: 46Ravindran and WilliamsMATERIALS A
Page 54 and 55: 48Ravindran and WilliamsTable I. Mo
Page 56 and 57: 50Ravindran and WilliamsTable IV. D
Page 58 and 59: 52Ravindran and WilliamsACKNOWLEDGE
Page 60 and 61: 54 Jamali et al.(vaginal discharge,
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Page 66 and 67: 60 Gupta et al.therefore, the prese
Page 68 and 69: 62 Gupta et al.Table II: Dimorphic
Page 72 and 73: 66 Jayraw and RaoteTable I. Effect
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Page 84 and 85: 78 Ghosh and GayenDescription of th
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80 Ghosh and Gayen30 µm30 µmCBCBC
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82Manjula and Janardananmicrometres
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84Manjula and JanardananMiracidiumF
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86 Hirani et al.haemoglobin concent
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88Hirani et al.ACKNOWLEDGEMENTSJosh
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90 Gupta and SinghOvary rounded, en
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96 Rajendran and DubeOptiMAL and IC
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