PAGES: 279-284 DOI: Full paper
An Oligonucleotide Probe Derived from kDNA Minirepeats is Specific for Leishmania (Viannia)

Octavio FernandesI +, Marcelo BozzaII, Juan M PascaleIII, Antonio B de Miranda, Ulisses G LopesII, Wim M Degrave

Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil
IDepartamento de Patologia, Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, RJ, Brasil
IIDepartamento de Biofísica, Universidade Federal do Rio de Janeiro, RJ, Brasil
IIIDepartamento de Parasitología, Universidad de Panama, Panama


Sequence analysis of Leishmania (Viannia) kDNA minicircles and analysis of multiple sequence alignments of the conserved region (minirepeats) of five distinct minicircles from L. (V.) braziliensis species with corresponding sequences derived from other dermotropic leishmanias indicated the presence of a sub-genus specific sequence. An oligonucleotide bearing this sequence was designed and used as a molecular probe, being able to recognize solely the sub-genus Viannia species in hybridization experiments. A dendrogram reflecting the homologies among the minirepeat sequences was constructed. Sequence clustering was obtained corresponding to the traditional classification based on similarity of biochemical, biological and parasitological characteristics of these Leishmania species, distinguishing the Old World dermotropic leishmanias, the New World dermotropic leishmanias of the sub-genus Leishmania and of the sub-genus Viannia.

The parasitological diagnosis of leishmaniasis has received, in recent years, a growing attention due to the development of molecular probes, capable both of detecting and typing the infecting parasite (Ramirez & Guevara 1987, Van Eys et al. 1987, 1991, Howard et al. 1991, Degrave et al. 1994a, b). Traditional methods for identification are slow and cumbersome, and suffer from a lack of sensitivity (Weigle et al. 1987). Thus, methods like culture of the protozoa from human samples, microscopic analysis of stained smears or histopathological study of biopsy material might be replaced in the future by fast, sensitive and accurate diagnostic assays, using molecular probes and/or the polymerase chain reaction (PCR)(Barker et al. 1986, Barker 1987, Uliana et al. 1991).

Probes derived from different sequences of the nuclear and mitochondrial genome have been used in an attempt to develop Leishmania detection and typing assays (Wirth & McMahon-Pratt 1982, Van Eys et al. 1992). However, the kinetoplast DNA (kDNA) network, which is comprised of maxi- and minicircle molecules, has been the target of choice in many laboratories (Simpson 1987). Leishmania minicircles show features that make them almost ideal as molecular probes, since they are present in the kinetoplast network in a high copy number and contain a conserved region of at least 120 bp that can be evidenced in every molecule (Rogers & Wirth 1987, 1988). Specific probes based on total kDNA, complete minicircle molecules or restriction fragments, have been generated and used in the identification of different leishmanias from the Old and New World (Lopes & Wirth 1986, Das Gupta et al. 1991, Laskay et al. 1991). This approach proved to be suitable for epidemiological purposes and as a diagnostic method (Wirth et al. 1986, Rogers et al. 1988, Barker 1989).

Detection systems based on PCR are very promising due to their sensitivity and specificity (Saiki et al. 1985). However, secondary detection and specific typing probes for identification of PCR amplified products are highly desirable, since they offer a drastically increased specificity of the PCR assay and raise about ten-fold the sensitivity of the system, allowing for automation of the PCR scoring procedure, concomitantly with the typing of the infecting Leishmania species. Several PCR systems use amplification oligonucleotides complementary to the conserved sequence motifs corresponding to the origins of replication of the minicircle light and heavy strands (Rodgers et al. 1990).

Whole kDNA can be used as a molecular probe to distinguish between the two New World dermotropic Leishmaniagroups: the sub-genus Viannia (braziliensis, guyanensis, panamensis, etc.) and Leishmania (amazonensis, mexicana,etc.) (Wirth & McMahon-Pratt 1982). However, cross hybridization with Old World leishmanias and L. chagasi occurs (Das Gupta et al. 1991). Synthetic oligonucleotides directed to specific sequences can circumvent these problems and can be used in a PCR assay.

We report here the partial nucleotide sequences of L. braziliensis and L. panamensis minicircles and the identification of an oligonucleotide sequence specific to the subgenus Viannia, based on multiple sequence alignments of the minicircles conserved regions (minirepeats) from New World dermotropic leishmanias. Furthermore, a dendrogram reflecting the different minirepeat sequence homologies between these dermotropic leishmanias was constructed.



Parasite culture and kDNA isolation - The Leishmania strains used in this study were: L. braziliensisMHOM/BR/75/M2903, kindly provided by Dr Gabriel Grimaldi, FIOCRUZ, Rio de Janeiro, and L. panamensis IPAN V (Panama University, Panama). Promastigotes were grown at 24°C in Schneider's medium (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum. The taxonomic nomenclature follows the recommendation of the World Health Organization.

The cells were spun down at 5000 X g for 10 min at 4°C, washed with cold saline/EDTA (0,15 M NaCl/10 mM EDTA), centrifuged and resuspended in TE10 (10 mM Tris-HCl pH 7.4 /10 mM EDTA). Proteinase K (Boehringer Mannheim) and sarkosyl were added to final concentrations of 100 mg/ml and 0.5%, respectively. The lysate was incubated at 56°C for 2 hr and submitted to mechanical shear, followed by two ultracentrifugations at 30.000 rpm for 1 hr at 4°C. The pellet was resuspended in 200 ml of TE buffer and extracted with phenol/chloroform (1:1). The DNA was precipitated with ethanol and resuspended in TE1 (10 mM Tris-HCl pH 7.4/ 1 mM EDTA).

Minicircle cloning and nucleotide sequence determination - Total L. braziliensis kDNA was digested with Msp I and minicircles were cloned in the Acc I site of M13mp18. L. panamensis was digested with Dra I and cloned in the Hinc II site of pBluescript® KS-. Nucleotide sequencing was done by the chain termination method using either USB®Sequenase kit and a-32P-dATP labeling or the Taq Dye Primer Cycle Sequencing Kit on a 373A Automated DNA Sequencer (Applied Biosystems), according to the instructions of the manufacturer.

Computer analysis and oligonucleotide synthesis - Nucleotide sequences were analyzed using software from the Genetics Computer Group (GCG) on a VAX computer. Oligonucleotides were synthesized on an Applied Biosystems 381A DNA synthesizer and purified by thin layer chromatography (TLC).

Cell dot experiments and hybridization assays - Leishmania cells were pelleted, washed with TE10 and counted. 105cells were applied on nylon filters (Zetaprobe, Biorad) treated with 10mM Tris-HCl pH 8.0, 10 mM EDTA, 0.1% Triton X-100, 100 mg/ml proteinase K at 56°C during 1 hr. The filter was transferred to 0.4 N NaOH for 5 min and then rinsed with 2xSSC and dried. Hybridization conditions and washing stringencies are detailed below in results.



Minicircle cloning and sequencing - One L. panamensis, one L. amazonensis and several cloned L. braziliensisminicircles were partially sequenced and the data were submitted to GenBank (Fig. 2Table). The molecules apparently belong to different minicircle classes, as judged from the extensive heterogeneity of the minirepeat neighboring sequences (data not shown).

Multiple sequence alignment of minirepeats - We performed a multiple alignment analysis of the conserved minirepeats from New and Old World dermotropic Leishmania minicircles (Fig. 2), using the Pileup program from the GCG package. Regions with higher or lower conservation become evident when comparing the alignment with a deduced consensus sequence. A dendrogram reflecting the homologies between these sequences is shown in Fig. 3. Although this dendrogram does not necessarily reflect a phylogenetic relationship between the different Leishmania species, it does cluster the Leishmania species in groups that are coherent with previous classifications based upon biological, parasitological and biochemical observations (WHO 1980).

Oligonucleotide design - The multiple alignment of the minirepeats regions has enabled us to evidence a region, adjacent to the 5' end of the canonical twelvemer sequence, that seems highly specific to leishmanias of the sub-genus Viannia.An oligonucleotide (5'-TAATTGTGCACGGGGA GGCCA-3' - OLB) corresponding to this region was synthesized and used in hybridization experiments as a probe, confirming the specificity suggested by the sequence analysis. Using stringent hybridization conditions and washing in 0.5xSSC at 37°C, we demonstrated that this oligonucleotide is specific to the sub-genus Viannia (Fig. 1A), in contrast to hybridization with an oligonucleotide complementary to the minicircle "universal 12-mer sequence" (5'-GGGGTTGGTGTAAT/AATAGG/TGGC-3'- OLT) (Fig. 1B) or with L. brasiliensis total kDNA (Fig. 1C). 



Leishmania species belonging to the sub-genus Viannia are the main agents of cutaneous leishmaniasis in Latin America. However, there are other leishmanias from the sub-genus Leishmania, which are also responsible for human cutaneous infections (Grimaldi & Tesh 1993). In some areas there is an overlapping of different dermotropic or viscerotropic leishmanias, creating a complex epidemiologic pattern of transmission. Moreover, the prognosis of human leishmaniasis seems also to depend on the species involved in the infection. It is thus highly desirable to develop molecular probes that are able to identify and type the different leishmanias (Barker 1989).

Since the use of oligonucleotides as molecular probes is inexpensive, more specific, faster and more practical as compared to whole kDNA, we cloned and partially sequenced five different minicircles from L. (V.) braziliensis, searching for short sequences specific to minicircles from this relevant human pathogenic parasite group.

Based on the multiple alignment of the obtained sequences with those of the conserved regions of other Leishmaniaminicircles from different species, we identified a region of 16 nucleotides (OLB, Fig. 2), that proved to be specific for the sub-genus Viannia in cell dot hybridization experiments (Fig. 1), demonstrating its use as a typing probe. This region is 5' adjacent to the conserved block of 12 nucleotides, found in all Leishmania minicircles, for which another oligonucleotide was synthesized (OLT, Fig. 1BFig. 2). Apart from its use as a typing probe in cell-dot experiments, the OLB oligoprobe proved to be very useful for the detection and typing of PCR products (Degrave et al. 1994a), generated using oligonucleotides complementary to the minicircle light and heavy strand origins of replication.

Moreover, using oligonucleotides directed outwardly from the Viannia specific region and from the origin of replication of the minicircle heavy strand (12mer sequence), as primers in a PCR reaction, it is possible to amplify the whole minicircle molecule. This method may be useful in typing experiments in a schizodeme-like analysis after full length minicircle amplification (Degrave et al. 1994b). De Bruijn and Barker (1992) have previously used such amplification of whole minicircles for diagnostic purposes.

The L. Viannia minirepeat sequences demonstrate extensive sequence conservation. Moreover, point mutations are clustered in a few regions and do not distinguish between the different species, as shown by the clustering produced by the computer analysis of the Leishmania minirepeats. All the Viannia species are grouped together, with a near homogeneity inside the sub-genus taxon in Leishmania species when considering the conserved region of the minicircle molecule. Species of the sub-genus Leishmania are also grouped, and separated from the Viannia cluster.



To Dr Gabriel Grimaldi for strains donations and culture facilities.



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+Corresponding author. Fax: 55-21-590.3495
Received 8 February 1995
Accepted 1 February 1996

Sponsored by the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR), the International Atomic Energy Agency, the FIOCRUZ PAPES Program and CNPq.


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