Genetic Data Showing Evolutionary Links Between Leishmania And
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 93(5): 677-683, Sep./Oct. 1998 677 Genetic Data Showing Evolutionary Links between Leishmania and Endotrypanum Elisa Cupolillo/+, Luiza OR Pereira, Octávio Fernandes*, Marcos P Catanho*, Júlio C Pereira**, Enrique Medina-Acosta**, Gabriel Grimaldi Jr Laboratório de Leishmaniose, Departamento de Imunologia *Departamento de Medicina Tropical, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil **Laboratório de Biotecnologia, Universidade Estadual do Norte Fluminense, 28015-620 Campos, RJ, Brasil Striking similarities at the morphological, molecular and biological levels exist between many trypanosomatids isolated from sylvatic insects and/or vertebrate reservoir hosts that make the identifi- cation of medically important parasites demanding. Some molecular data have pointed to the relation- ship between some Leishmania species and Endotrypanum, which has an important epidemiological significance and can be helpful to understand the evolution of those parasites. In this study, we have demonstrated a close genetic relationship between Endotrypanum and two new leishmanial species, L. (V.) colombiensis and L. (V.) equatorensis. We have used (a) numerical zymotaxonomy and (b) the variability of the internal transcribed spacers of the rRNA genes to examine relationships in this group. The evolutionary trees obtained revealed high genetic similarity between L. (V.) colombiensis, L. (V.) equatorensis and Endotrypanum, forming a tight cluster of parasites. Based on further results of (c) minicircle kDNA heterogeneity analysis and (d) measurement of the sialidase activity these parasites were also grouped together. Key words: Leishmania colombiensis - Leishmania equatorensis - Endotrypanum - multilocus enzyme electro- phoresis - molecular characterization - numerical analysis - sialidase activity - kDNA Parasitic protozoa of the genus Leishmania human cutaneous and/or mucosal leishmaniasis (Kinetoplastida: Trypanosomatidae) are biologi- (Grimaldi & Tesh 1993). Infections with other bio- cally diverse group of microorganisms. Taxonomic logically distinct groups of trypanosomatid proto- studies of leishmanial isolates from the New World zoa, such as Endotrypanum and Trypanosoma are indicate tremendous diversity within this genus also found in sloths (Deane 1961, Pipkin 1968, (Cupolillo et al. 1995). A number of new Leish- Travi et al. 1989, Shaw 1992). mania species from sylvan areas of the Neotropics In nature, all Leishmania spp. are transmitted are associated with disease in humans; others ap- by the bite of infected phlebotomine sand flies pear to be restricted to lower orders of mammals, (Diptera:Psychodidae). However, many flagellates such as rodents and edentates (Grimaldi et al. other than Leishmania commonly are found in 1989). sand flies in Neotropical forests. Arias et al. (1985) Sloths are reservoir hosts of at least five named identified E. schaudinni and other Endotrypanum Leishmania species of the subgenus Viannia [L. sp. infections in sand flies and sloths captured in guyanensis Floch, 1954; L. panamensis Lainson the Amazon Region of Brazil. Results of kineto- & Shaw 1972; L. shawi Lainson et al. 1989; L. plast DNA probe identifications of promastigotes colombiensis Kreutzer et al. 1991 and L. present in sand flies captured near Manaus, Bra- equatorensis Grimaldi et al. 1992], responsible for zil also demonstrated Endotrypanum infections in Lu. shannoni, as well as in Lu. umbratilis and Lu. anduzei (Rogers et al. 1988). Further evidence for the development of Endotrypanum in This work was supported by grants of the Instituto phlebotomines was obtained by feeding several Oswaldo Cruz-Fiocruz, FAPERJ and CNPq to EC, OF laboratory-reared sand fly species on infected and GG Jr. Research work at the Laboratório de sloths (Christensen and Herrer 1976, 1979, Shaw Biotecnologia-UENF is supported by grants from 1981). FENORTE, FINEP, FAPERJ and WHO/TDR to EM-A. Endotrypanum spp. are digenetic trypa- +Corresponding author. Fax: +55-21-280.1589. E-mail: nosomatids in that they are intraerythrocytic para- [email protected] sites of sloths and are transmitted by phlebotomine Received 15 June 1998 sand flies (Shaw 1992). Endotrypanum shares Accepted 30 July 1998 many other charecteristics with Leishmania. Cul- 678 Evolutionary Links between Leishmania and Endotrypanum Elisa Cupolillo et al. tured-derived promastigotes of parasites in both was performed in automatic sequencing genera are morphologically similar. Studies em- (AbiPrisma, Applied Biosystem). ploying monoclonal antibodies for the analysis of Numerical analysis - The MLEE data was ana- the genus Endotrypanum have shown antigenic lyzed by phenetic methods using the NTSYS soft- similarites between these parasites and some Leish- ware program (version 1.7, exeter software). Prin- mania species (Franco et al. 1997). Furthemore, cipal coordinate analysis was performed based on molecular trees clustered the sandfly-borne dige- Euclidian distance between the samples. The simi- netic parasites Leishmania and Endotrypanum to- larity level between the Leishmania species and gether, sharing a common ancestor and represent- Endotrypanum was calculated using the Jaccard’s ing a relatively recent lineage from the coefficient. The kDNA sequences of the parasites Trypanosomatidae family (Fernandes et al. 1993). were analyzed using the MEGA program (Kumar Results of hybridization using kDNA probes et al. 1993). The number of differences between (Pacheco et al. 1990) support the view that the sequences were calculated and a similarity tree Endotrypanum and the peripylarian leishmanial constructed by the Neighbor-Joining method. Boot- parasites of the subgenus Viannia Lainson & Shaw strap analysis was based on 500 replicates. 1987 are phylogenetically close (Shaw 1992). In RESULTS AND DISCUSSION addition, phylogenetic studies have demonstrated that the most divergent Leishmania species are L. Leishmania and Endotrypanum are very close (L.) hertigi and L. (L.) herreri, claimed to be closer protozoan parasites (Fernandes et al. 1993) com- to Endotrypanum than to the other Leishmania monly found in the same vertebrate and insect (Croan & Ellis 1996, Noyes et al. 1996, 1997, hosts. Recents studies have showing the relation- Croan et al. 1997). ship between Endotrypanum and some New World In this study, we have shown evolutionary links Leishmania species, mainly those from L. (L.) between Endotrypanum and some leishmanial hertigi and L. (L.) herreri complex (Croan & Ellis parasites based on their molecular genetics, as 1996, Noyes et al. 1996, 1997, Croan et al. 1997). characterized using a broad assemblage of meth- Moreover, DNA analysis of phylogenetically in- odologies. The data presented here demonstrate formative RNA polymerase II gene of L. (V.) that E. schaudinni, L. (V.) colombiensis and L. (V.). equatorensis and Endotrypanum demonstrated se- equatorensis form a tight phylogenetic cluster, an quence similarities among these parasites (JJ Shaw, evolutionary linked group that should be explored pers. commun.). Similarly, it appears that a close to understand the origin(s) of neotropical patho- antigenic links may exist between L. (V.) genic Leishmania. colombiensis, L. (V.) equatorensis and Endo- trypanum (Franco et al. 1997, Grimaldi et al. 1992). MATERIALS AND METHODS Leishmania (V.) colombiensis was found infect- Parasites - Leishmania and Endotrypanum ing humans, sloths (Choloepus hoffmanni), (Table I) were cultured in Scheneider’s Drosophila sandflies (Lu. hartmani and Lu. gomezi), and dogs medium (Gibco, Grand Island, NY) supplemented in Colombia, Panama, and Venezuela (Kreutzer et with 10% heat-inactivated FBS (Biolab, Rio de al. 1991, Delgado et al. 1993, unpublished data). Janeiro, Brazil) at 24oC. In the preparation of L. (V.) equatorensis is an enigmatic parasite, which samples, the parasite (promastigotes in the late was isolated from the viscera of a sloth (C. phase of growth cultures) were harvested by cen- hoffmanni) and a squirrel (Sciurus granatensis), trifugation (3,800x g for 15 min at 4oC) and washed captured in humid tropical forest on the Pacific twice in saline pH 8.0, containing the appropriate Coast of Ecuador. Data based on biological and buffer. molecular criteria, as well as numerical Biochemical/Molecular characterization - The zymotaxonomy analysis indicated that both these procedures used for characterizing the parasites parasites are clearly distinguishable from all other (multilocus enzyme electrophoresis - MLEE, mea- known species, but clustered within the L. (V.) surement of the sialidase activity, PCR amplifica- braziliensis complex (Kreutzer et al. 1991, tion and restriction enzyme digestion of the para- Grimaldi et al. 1992). Multilocus enzyme electro- site ITSrRNA, cloning and sequencing of the con- phoresis data and the restriction fragments of the served region of the minicircle kDNA molecules) internal transcribed spacers of the rRNA gene have been described in detail in previous publica- (Cupolillo et al. 1995, 1997) have indicated a close tions (Cupolillo et al. 1994, 1995, Medina-Acosta relationship between L. (V.) equatorensis and L. et al. 1994, Fernandes et al. 1996). Sialidase activ- (V.) colombiensis, as previously demonstrated ity was measured using a single-cell HITACHI F- (Kreutzer et al. 1991, Grimaldi et al. 1992). In or- 4500 spectrofluorometer (350 nm excitation and der to better understand their taxonomic position 460 nm emission wavelengths). The