Experimental Parasitology 113 (2006) 221–226 www.elsevier.com/locate/yexpr
Leishmania aethiopica: Strain identiWcation and characterization of superoxide dismutase-B genes Abebe Genetu a,b,¤, Endalamaw Gadisa a, Abraham AseVa a, Steve Barr e, Mekuria Lakew c, Dagim Jirata a, Teklu Kuru a, Dawit Kidane f, MesWn Hunegnaw d, Lashitew Gedamu a,e a Armauer Hansen Research Institute, P.O. Box 1005, Addis Ababa, Ethiopia University of Gondar, College of Medicine and Health Sciences, P.O. Box 196, Gondar, Ethiopia c Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia d All Africa Leprosy Rehabilitation and Training Hospital (ALERT), P.O. Box 165, Addis Ababa, Ethiopia e University of Calgary, Department of Biological Sciences, 2500 University Drive, Calgary, Alta., Canada T2N 1N4 f Albert-Ludwig Universitat, Stefan Meier Strasse17, 79104 Frieburg, Germany b
Received 12 June 2005; received in revised form 12 January 2006; accepted 13 January 2006 Available online 3 March 2006
Abstract This study was performed to characterize the genes that code for superoxide dismutase (SOD) in Leishmania aethiopica. It involved three main steps: specimen collection and parasite isolation, species identiWcation, and molecular characterization of the SOD genes. Out of 20 skin slit specimens cultured and processed from suspected cutaneous leishmaniasis patients enrolled in the study, Wve (25%) were found to be positive for motile promastigotes. Isoenzyme electrophoresis and PCR–RFLP results conWrmed that the isolates were L. aethiopica. Superoxide dismutase-B (SODB) genes were identiWed from L. aethiopica for the Wrst time. Iron superoxide dismutase-B genes ampliWed from promastigotes of L. aethiopica (LaeFeSODB) were similar in size to the SODB genes of other Leishmania species. Nucleotide sequences of LaeFeSODB1 showed 95.4, 93.5, and 97.3% identity with L. donovani SODB1 (LdFeSODB1) L. major SODB1 (LmFeSODB1) and L. tropica SODB1 (LtrFeSODB1), respectively. Similarly, LaeFeSODB2 showed 95.9 and 94.1 and 97.6% identity with LdFeSODB2 and LmFeSODB2 and LtrFeSODB2, respectively. On the other hand, predicted amino acid sequence comparison indicated that LaeFeSODB1 had 91.3, 89.8, and 93.9% identity with LdFeSODB1, LmFeSODB1, and LtrFeSODB1, respectively. The diVerence in nucleic acid sequence of LaeFeSODB from that of LmFeSODB and LtrFeSODB can be utilized to develop speciWc molecular methods that help diVerentiate these species in places where there is an overlap in the distribution of these species. In addition, the data provide information about the situation of L. aethiopica with respect to SODB genes. © 2006 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Leishmania; Leishmania aethiopica; Superoxide dismutase; Gene; Isoenzyme Electrophoresis; Ethiopia
1. Introduction Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania. It has a wide range of clinical manifestations, ranging from a self-healing cutaneous type to the most severe visceral form associated with high morbidity and mortality. *
Corresponding author. E-mail address:
[email protected] (A. Genetu).
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In Ethiopia, visceral and cutaneous leishmaniases are caused mainly by Leishmania donovani and Leishmania aethiopica, respectively. Cutaneous leishmaniasis (CL) ranges from a localized self-healing type to the disWguring mucocutaneous and diVuse cutaneous types (Hailu and Frommel, 1993). To date, no eVective drug or vaccine is available for Ethiopian cutaneous leishmaniasis. The drugs currently in use are not eYcacious and/or induce severe complications (Sarojini et al., 1984). Moreover, there is no simple and
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speciWc molecular diagnostic tool such as PCR that can diVerentiate L. aethiopica from other species. Thus, the need to develop an eVective drug or vaccine as well as a speciWc diagnostic method is of paramount importance. To escape from the host immune response and survive inside host cells, Leishmania utilizes several evasion mechanisms. Among these is the detoxiWcation of oxygen radicals produced by the immune cells. The metalloenzyme, superoxide dismutase (SOD) is used in the Wrst stage of this process. The enzyme converts superoxide to hydrogen peroxide (Bogdan et al., 1990). A number of studies have been performed on superoxide dismutase to understand the role of this enzyme in the survival of Leishmania and to determine whether it could be a possible target for new antileishmanial drugs. These studies have conWrmed that superoxide dismutase plays an important role in host-parasite relationship in leishmaniasis suggesting its possible use as target for intervention (Bogdan et al., 1996; Paramchuk et al., 1997; Plewes et al., 2003). The whole genome of L. aethiopica in general and the LaeFeSOD genes in particular have not been characterized. This, in eVect, will limit the understanding of the biology of L. aethiopica during pathogenesis thereby aVecting the development of new drugs or vaccines and the use of newly emerging ones which could be useful against CL in Ethiopia. This work, therefore, aimed at characterizing L. aethiopica with respect to the genes encoding superoxide dismutase. 2. Materials and methods 2.1. Study population and parasite isolation The materials were obtained from suspected cutaneous leishmaniasis patients attending the Dermatology Clinic of ALERT Hospital, Addis Ababa. The study was started after obtaining institutional and national ethical clearance, and only those patients who gave written informed consent were included. Skin slit was made from the lesions following standard procedures and the Xuid was immediately inoculated into biphasic, Novy–MacNeal–Nicolle (NNN) medium in duplicate. Five samples positive for promastigotes designated 1093/02, 1184/02, 1185/02, 1118/02, and 1181/02 were obtained. The promastigote stage of the parasite was then isolated following the standard protocol (Evans, 1989). Reference strains of diVerent Leishmania species were used as controls for strain typing of the experimental isolates. These were L. aethiopica MHOM/ET/72/L-100, L. donovani MHOM/IN/80/DD8, Leishmania infantum MHOM/FR/LEM-75, Leishmania major MHOM/SU/73/ 5ASKH, and Leishmania tropica K-27. 2.2. Species typing Species typing of the isolates was done using isoenzyme electrophoresis and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP).
Isoenzyme electrophoresis was performed on two isolates (1093/02 and 1184/02) using glucose-phosphate-isomerase (GPI EC 5.3.1.9) and glucose-6-phosphate dehydrogenase (G6PD). In brief, the crude protein was extracted using lysis reagent (0.5 ml Triton X-100 (Sigma), 0.825 g sucrose in 10 ml sterile distilled water) and subjected to electrophoresis on 11% starch gel. The protein was then stained following standard protocols (Rioux et al., 1990). PCR–RFLP was also performed on the same isolates (1093/02 and 1184/02) following the modiWed protocol of Minodier et al. (1997). The procedure involved PCR-ampliWcation of a repetitive genomic DNA of 250 and 350 bp using T2 (5⬘- CGG CTT CGC ACC ATG CGG TG-3⬘), B4 (5⬘- ACA TCC CTG CCC ACA TAC GC-3⬘) and LITSR (5⬘-CTG GAT CAT TTT CCG ATG-3⬘), L5.8S (5⬘-AAG TGC GAT AAG TG GTA-3⬘) primers, respectively. The PCR products were subjected to restriction digestion by HaeIII and HhaI, respectively (Pharmacia). 2.3. DNA extraction Genomic DNA was extracted from promastigotes using modiWed protocol of Paramchuk et al. (1997). Five hundred microliters of lysis buVer (10 mM Tris–HCl, pH 8.3, 50 mM EDTA, pH 8.0, and 1% SDS) was added to the promastigote pellet and incubated in a boiling water bath for 15 min. Twenty-Wve microliters of 2 mg/ml RNAase A (Pharmacia) was added to the suspension and incubated at 37 °C for 1 h. This was followed by addition of 5 l of 10 mg/ml proteinase K (GIBCO) and incubation at 42 °C overnight. The next day, phenol-chloroform-isoamyl alcohol (PCIA) extraction and ethanol precipitation were done. 2.4. Polymerase chain reaction PCR was done to amplify open reading frames (ORFs) of LaeFeSODB1 and LaeFeSODB2 genes following the protocol of Paramchuk et al. (1997). The primers used were those previously designed to amplify LcFeSOD genes. The nucleotide sequence of the primers were (1) FeSODB1 ATG/ BamHI 5⬘-TTT CCC GGG GGG ATC CAT GCC GTT CGC TGT TCA GCC-3⬘, (2) FeSODB1TAA/ BamHI 5⬘-TCG CAG GGA TCC TTA AAG CTG GCT AGT GGC-3⬘, and (3) FeSODB2TAA/ BamHI 5⬘-TCC TCC CGG GGG ATC CTT ACA GAT CAC TGT TG-3⬘. FeSODB1 was ampliWed using primers 1 and 2 while LaeFeSODB2 gene was ampliWed using 1 and 3 as forward and reverse primers, respectively. Promastigote genomic DNA was subjected to PCR in a total reaction volume of 25 l PCR mix with Ready-to-Go PCR beads (Pharmacia P-L Biochemicals, Upsala, Sweden). To these beads were added 1 l of 50 pmol/l of each primer, 3 l of 0.1 g/l of promastigote DNA and sterile distilled water. The ampliWcation reaction involved denaturation at 94 °C for 1 min, primer annealing at 50 °C for 30 s and extension at 72 °C for 1 min for a total of 30 cycles in a thermocycler (Hybaid). In the reaction, both positive and negative
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(blank) controls were included. The positive control was L. donovani genomic DNA previously used to amplify LdFeSOD genes. In the blank reaction tube, all the ingredients of PCR were added except the genomic DNA. The PCR products were then visualized by electrophoresis on 1.5% (w/v) agarose (Sigma) in the presence of ethidium bromide. 2.5. Sequencing The open reading frames of LaeFeSODB1 and LaeFeSODB2 genes from two isolates (1093/02 and 1184/02) were sequenced at the DNA sequencing facility, University of Calgary, Canada. The PCR products were subjected to puriWcation using QIAquick PCR puriWcation kit (Qiagen). The puriWed PCR products were sequenced from both strands. 3. Results Of the total of 20 skin slit specimens collected and cultured, only 5 (25%) were found to be positive for motile promastigotes. 3.1. Species typing 3.1.1. Isoenzyme electrophoresis (IE) Isoenzyme electrophoresis of GPI and G6PD from isolates 1093/02 and 1184/02 gave distinct bands equal in migration with that of L. aethiopica L-100. However, L. major, L. tropica, and L. infantum showed diVerent band patterns from those of the L. aethiopica reference strain and the isolates (Fig. 1). L. aethiopica L-100 and the isolates showed equivalent zymograms (50%) and which diVered from that of other species (120, 80, and 85% for A
GPI
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L. infantum LEM 75, L. major 5ASKH, and L. tropica K27, respectively). 3.1.2. PCR–RFLP T2 and B4 and LITSR and L5.8S primers ampliWed repetitive genomic DNA of 250 and 350 bp in size, respectively, from isolates 1093/02 and 1184/02 as well as from the reference strains. Restriction digestion of the above PCR products with HaeIII and HhaI further conWrmed the similarity of our isolates to the L. aethiopica reference strain. As shown in Fig. 2A, restriction digestion with HaeIII indicated that both the reference strain (L. aethiopica L-100) (lane 3) and the isolates (1093/02 and 1184/02) (lanes 7 and 8) resulted in two bands of about 215 and 35 bp in size, whereas, L. infantum produced only one band of about 250 bp in size (lane 6) and L. major (lane 2) resulted in 95 and 35 bp fragments. On the other hand L. tropica (lane 4) resulted in bands similar in size to that of L. aethiopica. AmpliWcation of ITS-1 gene followed by restriction digestion with HhaI strengthened the identiWcation procedure and conWrmed that our isolates are L. aethiopica. As shown in Fig. 2B, L. tropica, L. donovani, and L. infantum (lanes 1, 2, and 5, respectively) gave a single band of 350 bp size and L. major gave bands of 260 and 90 bp in size (lane 4). L. aethiopica reference strain and the isolates resulted a band of size 162 bp (lane 3 and lanes 6 and 7, respectively). 3.2. Characterization of SODB genes 3.2.1. Polymerase chain reaction Total genomic DNA was extracted and open-reading frames (ORFs) of LaeSODB1 and LaeSODB2 were 250 bp
A
215 bp 95 bp
1
2
3
4
5
6
7
180 bp 70 bp
35 bp 1
B
2
3
4
G6PD
5
6
7
8
260bp
B 350 bp
162 bp 1
1
2
3
4
5
6
7
Fig. 1. Isoenzyme electrophoresis. (A) Glucose phosphate isomerase. Lanes: 1, Isolate 1093/02; 2, Isolate 1184/02; 3, L. donovani DD8; 4, L. tropica K-27; 5, L. aethiopica L-100; 6, L. infantum LEM-75; and 7, L. major 5ASKH. (B) Glucose-6-phosphate dehydrogenase. Lanes: 1, L. major 5ASKH; 2, L. infantum LEM-75; 3, L. aethiopica L-100; 4, L. tropica K27; 5, L. donovani DD8; 6, isolate 1093/02; and 7, isolate 1184/02.
2
3
4
5
6
7
90 bp
Fig. 2. PCR–RFLP of 250 (A) and 350 (B) bp PCR products. HaeIII digest. (A) Lanes: 1, Negative control; 2, L. major 5ASKH; 3, L. aethiopica L-100; 4, L. tropica K-27; 5, L. donovani DD8; 6, L. infantum LEM-75; 7, Isolate 1093/02; and 8, Isolate 1184/02. (B) HhaI digest. Lanes: 1, L. donovani DD8; 2, L. tropica K-27; 3, L. aethiopica L-100; 4, L. major 5ASKH; 5, L. infantum LEM-75; 6, Isolate 1093/02; and 7, Isolate 1184/02.
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ampliWed from both the isolates and the positive control (L. donovani). The open reading frame (ORF) encoding LaeSODB1 was ampliWed from the isolates 1093/02, 1184/02, and 1185/02 as well as from the positive control (L. donovani). This gene has a molecular size of about 600 bp. PCR ampliWcation of the
ORF of LaeSODB2 resulted in a fragment of about 650 bp in size. In both ampliWcations, control failed to give any band. 3.2.2. DNA sequencing Nucleic acid sequencing showed that LaeSODB1 and LaeSODB2 ORFs are 588 and 627 bp long, respectively.
Fig. 3. Multiple amino acid sequence alignment of Leishmania iron SODs. The Wve boxes in the multiple amino acid sequence alignment above indicate the conserved regions of iron and manganese SODs as deWned by Heinzen et al. (1992). Asterisk represents the invariant amino acids involved in coordinating the metal ion.
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The nucleotide and predicted amino acid sequences were aligned and the identity was determined using ClustalW provided by Bioedit sequence alignment editor program. Nucleic acid sequence of LaeSODB1 (1093/02) demonstrated higher identity to LdFeSODB1 of L. donovani (95.4%) and LtrFeSODB1 of L. tropica (97.3%) than LmFeSODB1 of L. major (93.5%). The amino acid sequence analysis also showed that LaeSODB1 is more closely related to L. tropica (93.9%) than to L. major (89.8%) (Fig. 3). Comparison of the predicted amino acid sequence of LaeSODB1 and LaeSODB2 (from 1093/02 and 1184/02) with that of LcFeSODBs of L. chagasi (Paramchuk et al., 1997) and manganese SOD of the bacterium, Thermus thermophilus (Ludwig et al., 1991) indicated that LaeSODBs fall within the category of iron SOD. The amino acid sequence of LaeFeSODB1 from 1093/02 showed 89.8% identity to LcFeSODB1 and 40.6% to manganese SOD of T. thermophilus (Ludwig et al., 1991). The invariant amino acid residues involved in Fe and Mn metal binding, three histidines and one aspartate residue (Parker and Blake, 1988), are conserved in LaeFeSODB1 and LaeFeSODB2. The histidine residues are found at amino acid positions 28,76, and 165 and the aspartate residue is located at position 161 (Fig. 3). The two LaeFeSODs (LaeFeSODB1 and LaeFeSODB2) showed less identity with each other (86.6%) than each enzyme separately with other organisms. Furthermore, LaeFeSODB2 contains a 13 amino acid extension at the carboxyl terminus that is absent in LaeFeSODB1. 4. Discussion The objectives of this study were (i) to identify L. aethiopica strains and (ii) to isolate and characterize SODB genes in L. aethiopica and to compare their similarities and diVerences with those of other Leishmania species. To address these objectives, isolation of the parasites from cutaneous leishmaniasis patients and species typing of the isolates were undertaken as a Wrst step, and both wild type parasites and their SOD genes were then characterized. We preferred working with clinical isolates as these are more representative of Weld material and could be validated against recognized laboratory isolates. According to the clinico-epidemiological data, the clinical isolates obtained from cutaneous leishmaniasis patients were representative of circulating strains. The age, sex, geographic location of the place where the patients came, duration of lesion as well as site of lesion Wts with known pattern in the literature (data not shown). Species identiWcation of the clinical isolates was done using two techniques: isoenzyme electrophoresis (IE) and PCR–RFLP. Although isoenzyme electrophoresis has been considered as a gold standard for typing Leishmania world wide (Rioux et al., 1990), it is labor-intensive and time consuming. On the other hand molecular techniques such as PCR or PCR–RFLP are relatively rapid and easier to perform (Minodier et al., 1997).
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To type a strain and give it an international code, IE is expected to be done for at least 13 enzymes (Le Blanq et al., 1986; Rioux et al., 1990). However, in this study only GPI and G6PD were analyzed. This was because the parasite number at the time of typing was insuYcient to perform IE for 13 or more enzymes. GPI together with G6PD are able to diVerentiate L. aethiopica from other cutaneous leishmaniasis causing species. According to Le Blanq et al. (1986), out of 13 isoenzymes analyzed, L. aethiopica had only one isoenzyme pattern (esterase) in common with L. tropica and had none with L. major. These data justify the use of isoenzyme electrophoresis (IE) of only GPI and G6PD for species identiWcation of local Leishmania isolates. As shown in Fig. 1A, GPI from the isolates 1093/02 and 1184/02 (lanes 1 and 2) gave bands at similar position to that of GPI from L. aethiopica L-100 (lane 5) but diVered from other Leishmania reference strains tested (lanes 3, 4, 6, and 7). Only L. aethiopica strains showed a zymogram of 53%, while no other species gave less than 55% (Rioux et al., 1990). In our hands, IE for GPI resulted in a zymogram of 50% (for L. aethiopica L-100 and the isolates), 80% (for L. major 5ASKH), 85% (for L. tropica K-27), and 120% (for L. infantum LEM-75). Similar results were obtained for G6PD (Fig. 1B). The isolates (lanes 6 and 7) were similar to L. aethiopica reference strain (lane 3) but diVered from other species (lanes 1, 2, 4, and 5). PCR–RFLP was done to supplement the IE results. It involved PCR ampliWcation of 250 and 350 bp repetitive genomic DNAs followed by restriction digestion with HaeIII and HhaI, respectively. As shown in Fig. 2A, the Wrst one resulted in only one band of 250 bp for L. infantum (lane 6), two bands of 95 and 35 bp for L. major (lane 2), two bands of 180 and 70 bp for L. donovani (lane 5) and two bands of 215 and 35 bp for L. tropica (lane 4) and L. aethiopica (lane 3) strains (Minodier et al., 1997). In our experiment, two bands of 215 and 35 bp were obtained from the isolates 1093/02 and 1184/02 (lanes 7 and 8) as well as from L. aethiopica L-100 thus conWrming that the isolate is not L. major. As shown in Fig. 2B, HhaI digestion of a 350 bp fragment gave bands of size 350 and 162 bp for L. tropica (lane 2) and L. aethiopica (lane 3) reference strains, respectively. The isolates 1093/02 and 1184/02 (lanes 6 and 7) also gave bands equal in size with that of L. aethiopica reference strain. The results of IE and PCR–RFLP showed that our isolates could be used as reference strains for clinical as well as epidemiological studies in Ethiopia. However, the isoenzyme electrophoresis needs further analysis using other enzyme systems. The genes that encode superoxide dismutase-B1 and -B2 were characterized by sequence analysis of the PCR product. PCR ampliWcation of LaeFeSODB1 and LaeFeSODB2 gave bands of about 600 and 650 bp, respectively. Upon sequencing of these PCR products, the size was found to be 588 and 672 for LaeFeSODB1 and LaeFeSODB2, respectively.
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One of the major objectives of this work was to compare the similarity of L. aethiopica with other Leishmania species with respect to superoxide dismutase genes. Thus, antileishmanial drugs that might be developed targeting these proteins in other Leishmania species could be used in L. aethiopica. Furthermore, the variations in the nucleotide sequences might help in the development of species-speciWc probes. Studies on Leishmania chagasi, L. major, and L. tropica have indicated that SODs and the genes encoding SOD are conserved across diVerent Leishmania species (Paramchuk et al., 1997; Ghosh et al., 2003). In agreement with these data, LaeFeSODB amino acid sequences showed high degree of identity with LcFeSODB (89.8%), LdFeSODB (91.3%), and LmFeSODB (90.8%). However, small variations were found in the predicted amino acid sequences of LaeFeSODs. For example, as shown in Fig. 3, valine is substituted for methionine at position 38, and aspartate for glutamate at position 57. A variation (aspartate is substituted for asparagine at position 187) was even seen in a region (region-5, Fig. 3) that is conserved in several Fe and MnSODs (Heinzen et al., 1992). It was also found out that the invariant amino acids that are thought to be involved in coordinating the metal ion at the active site of SODs are conserved in LaeFeSODBs. These are three histidine residues and one asparate residue at positions 28, 76, 165, and 161, respectively (Fig. 3). Human superoxide dismutases are either Cu/Zn SODs or Mn SODs while Leishmania SODs are FeSODs. This, together with, the high degree of homology of L. aethiopica SODs with that of other species is important in the development and use of SOD targeting antileishmanial drugs. The data generated by this study could also contribute to the development of antileishmanial vaccines, using SOD as a candidate. The relatively higher degree of variation in the nucleic acid sequences of LaeFeSODBs and LmFeSODBs (93.5%) can also be used to develop a molecular diagnostic tool. Variation has also been found in other L aethiopica genes (e.g., cysteine proteases, Kuru, et al., personal communication), from which primers could be developed to selectively amplify species-speciWc segments of SOD and other genes. This could help us to diVerentiate between these species in places like Ethiopia where both L. aethiopica (greater than 95%) and L major infections occur. Acknowledgments The study was carried out at the Armauer Hansen Research Institute (AHRI), Addis Ababa, Ethiopia.
Funding was provided from AHRI core budget (NORAD, SIDA, and Government of Ethiopia) and by the grants from The Canadian Institute of Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada to Lashitew Gedamu. We acknowledge The National Center for Tropical Medicine, Madrid, Spain for allowing us to use the Leishmania strain-typing facility of the institute. References Bogdan, C., Gessner, A., Solbach, W., RollinghoV, M., 1996. Invasion, control and persistence of Leishmania parasite. Current Opinion in Immunology 3, 517–525. Bogdan, C., RollinghoV, M., Solbach, W., 1990. Evasion strategies of Leishmania parasites. Parasitology Today 6, 183–186. Evans, D., 1989. Handbook on isolation, characterization and cryopreservation of Leishmania. UNDP/World Bank/ WHO. Ghosh, S., Goswami, S., Adhya, S., 2003. Role of superoxide dismutase in survival of Leishmania within the macrophage. Biochemical Journal 369, 447–452. Hailu, A., Frommel, D., 1993. Leishmaniasis. In: Kloos, H., Ahmed Zein, Z. (Eds.), The Ecology of Health and Disease in Ethiopia. Westview press, Boulder, San Francisco, Oxford, pp. 375–388. Heinzen, R.A., Frazier, M.E., Mallavia, L.P., 1992. Coxiella burnetii superoxide dismutase gene: cloning, sequencing and expression in Escherichia coli. Infection and Immunity 60, 3814–3823. Le Blanq, S.M, Belehu, A., Peters, W., 1986. Leishmania in the Old world: 3 the distribution of L. aethiopica zymodemes. Transactions of the Royal Society of Tropical Medicine and Hygiene 80, 360–366. Ludwig, M.L., Metzeger, A.L., Pattridge, K.A., Stallings, W.C., 1991. Manganese superoxide dismutase from Thermus thermophilus. Journal of Molecular Biology 219, 335–358. Minodier, P., Piarroux, R., Gambarelli, F., Joblet, C., Dumon, H., 1997. Rapid identiWcation of causitive species in patients with Old world leishmaniasis. Journal of Clinical Microbiology 35, 2551–2555. Paramchuk, W.J., Ismail, S.O., Bhatia, A., Gedamu, L., 1997. Cloning, characterization and overexpression of two iron superoxide dismutase cDNAs from Leishmania chagasi: role in pathogenesis. Molecular Biochemical Parasitology 90, 203–221. Parker, M.W., Blake, C.C., 1988. Iron- and manganese-containing superoxide dismutases can be distinguished by their primary structure. FEBS Lett. 229, 377–382. Plewes, K.A., Barr, D.S., Gedamu, L., 2003. Iron superoxide dismutases targeted to the glycosomes of L. chagasi are important for survival. Infection and Immunity 71, 5910–5920. Rioux, J.A., Lanotte, G., Serres, E., Pratlong, F., Bastien, P., Perieres, J., 1990. Taxonomy of Leishmania. Use of isoenzymes. Suggestions for a new classiWcation. Annales de Parasitologie Humaine et Comparee 65, 111–125. Sarojini, P.A., Humber, D.P., Yemane-Berhan, T., Fekede, E., Belehu, A., Mock, B., WarndorV, J.A., 1984. Cutaneous leishmaniasis cases seen in two years at the All African Leprosy Rehabilitation and Training Center Hospital. Ethiopian Medical Journal 22, 7–11.
