INFECTION AND IMMUNITY, Feb. 1994, p. 657-664 Vol. 62, No. 20019-9567/94/$04.00+0Copyright C 1994, American Society for Microbiology

Molecular Cloning, Characterization, and Expression inEscherichia coli of Iron Superoxide Dismutase cDNA from

Leishmania donovani chagasiSAID 0. ISMAIL,' YASIR A. W. SKEIKY,2 AJAY BHATIA,' LEVI A. OMARA-OPYENE,

AND LASHITEW GEDAMUl*Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4,'

and Seattle Biomedical Research Institute, Seattle, Washington 981092

Received 18 June 1993/Returned for modification 7 October 1993/Accepted 29 October 1993

A cDNA corresponding to superoxide dismutase (SOD; EC 1.15.1.1.) was isolated from a Leishmaniadonovani chagasi (L. d. chagasi) promastigote cDNA library, using PCR with a set of primers derived fromconserved amino acids of manganese SODs (MnSODs) and iron SODs (FeSODs). Comparison of the deducedamino acid sequences with previously reported SOD amino acid sequences revealed that the L. d. chagasi585-bp open reading frame had considerable homology with FeSODs and MnSODs. The highest homology wasshared with prokaryotic FeSODs. The coding region of L. d. chagasi SOD cDNA has been expressed in fusionwith glutathione-S-transferase, using an Escherichia coli mutant, QC779, lacking both MnSOD and FeSODgenes (sodA4 and sodB). Staining of native polyacrylamide gels for SOD activity of Leishmania crude lysate andthe recombinant SOD revealed that both had SOD activity that was inactivated by 5 mM hydrogen peroxidebut not by 2 mM potassium cyanide, which is indicative of FeSOD. The recombinant enzyme also protected E.coli mutant QC779 from paraquat toxicity. This indicated that the glutathione-S-transferase peptide does notinterfere with the in vivo and in vitro activities of the recombinant SOD. Cross-species hybridization showedthat FeSOD is highly conserved in the Leishmania genus. Interestingly, the hybridization pattern of the FeSODgene(s) coincided with other classification schemes that divide Leishmania species into complexes. The cloningof FeSOD cDNA may contribute to the understanding of the role of SODs in Leishmania pathogenesis.

Leishmanias are obligate intracellular parasites that cause awide spectrum of human diseases grouped under the termleishmaniasis (50). During its life cycle, the parasite passesthrough two developmental stages. Inside the insect vector,Leishmania exists as a motile flagellated form called promas-tigotes that are introduced into the human bloodstream duringthe insect blood meal. In the human body, promastigotesestablish themselves within the macrophage phagolysosomes,where they transform into a nonflagellated form calledamastigotes. Although activated macrophages are efficientkillers of intracellular microorganisms, Leishmania amasti-gotes have evolved to survive and replicate in this harsh andacidic environment.

Activated macrophages have been shown to use oxygen-dependent and oxygen-independent mechanisms to eliminateingested microorganisms (2, 3). The first includes productionof reactive oxygen intermediates (ROI) such as superoxideanion (02-) and hydrogen peroxide (H202), while the latterincludes the production of reactive nitrogen intermediates(RNI) such as nitric oxide. The mechanisms of macrophage-derived ROI and RNI toxicity have been proposed to involvelipid peroxidation, DNA damage, and inactivation of iron-sulfur centers important for mitochondrial respiration (21, 39,47). During Leishmania infection, mechanisms responsible forprotecting intramacrophage amastigotes from killing are notfully understood. However, it is likely that enzymes that canabrogate the effects of the macrophage-produced oxidantswould be beneficial to the parasite.

Superoxide dismutases (SODs) are a group of metalloen-

* Corresponding author. Mailing address: Department of BiologicalSciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4.Phone: (403) 220-5556. Fax: (403) 289-9311.

zymes that have been isolated from diverse organisms (4). Onthe basis of their metal requirement, SODs are classified intothree forms: copper-zinc SOD (Cu/ZnSOD), manganese SOD(MnSOD), and iron SOD (FeSOD) (4). By virtue of theirability to dismutate 02- into H202 and 02, SODs have beenconsidered virulent factors in a number of intracellular patho-gens (6, 14, 20). The role of SODs in Leishmania pathogenicityis unknown. In this study, we report the isolation, character-ization, and expression in Escherichia coli of a cDNA encodingSOD from Leishmania donovani chagasi (L. d. chagasi).

MATERIALS AND METHODS

Parasites and culture. L. chagasi (MHOM/BR/84/Jonas), L.amazonensis (IFLA/BR/67?PH8), L. brasiliensis (MHOM/BR/75/M2903), L. donovani (MHOM/Et/67/HU3), L. infantum(IPT-1), L. major (LTM p-2), and L. tropica (1063-C) wereused and have been described previously (11). Promastigotesof L. d. chagasi were cultured at 26°C in RPMI 1640 mediumsupplemented with L-glutamine, sodium pyruvate, minimumessential medium essential and nonessential amino acids, and10% heat-inactivated fetal calf serum. Medium and mediumsupplements were from GIBCO/BRL, Grand Island, N.Y.PCR and screening of L. d. chagasi cDNA library. Two

converging primers based on conserved amino acids ofMnSODs and FeSODs from various organisms were synthe-sized on a Gene Assembler Oligonucleotide synthesizer (Phar-macia LKB, Uppsala, Sweden). The sequences of these prim-ers were as follows: primer 1 (sense), 5' CTGCACCACTCGAAG CACCA 3'; primer 2 (antisense), 5' CAGGTAGTACGCG TGCTCCCA 3'. To check for the presence ofSOD in the L. d. chagasi promastigote cDNA library (kindlyprovided by S. Reed, Seattle Biomedical Research Institute,

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Seattle, Wash.), a 10-iLl aliquot of a X-ZAP library phage lysatewas heated at 70°C for 5 min to denature the phage and wasused for PCR. Conditions for PCR were as follows. A 100-,ureaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mMKCl, 1.5 mM MgCl2, 200 mM each deoxynucleoside triphos-phate, 2.5 U of Taq DNA polymerase (Pharmacia), and 100pmol of each primer were subjected to 30 cycles of amplifica-tion (94°C for 1 min, 42°C for 1 min, and 72°C for 1 min) witha PHC-2 Dri-Block Cycler (Techne, Cambridge, United King-dom). The amplified product was gel purified, ligated intopCR1000, and transformed into competent E. coli INVotF',using a TA cloning kit (Invitrogen, San Diego, Calif.). Recom-binant colonies were verified by sequencing by the dideoxychain termination method (41).

After the presence of SOD in the cDNA library wasconfirmed, a total of 105 PFU was plated and duplicate filterswere lifted. Screening was done with the 426-bp PCR productamplified with primers 1 and 2 and labeled to a specific activityof >109 cpm/,ug with [oa-32P]dCTP (New England Nuclear,Cambridge, Mass.) by the random priming method (19). Inorder to select only the cDNA clones with intact 5' ends, phagelysates from 54 hybridizing plaques were subjected to PCRwith an external 5'-end sense primer derived from the consen-sus sequences of Leishmania spliced leader 5' CGCTATATAAGTATCAGTITCTGTAC 3' and an internal antisenseprimer (primer 2). Of 54 samples which gave expected prod-ucts, 9 samples were considered to have intact 5' ends and weresubjected to a second round of screening to get isolatedplaques. The cDNA inserts of all nine clones were recovered inBluescript plasmid by using an in vivo excision procedureutilizing E. coli XL1-blue and R408 helper phage (Strategene,Palo Alto, Calif.). Both strands of the coding region weresequenced with external vector-based primers and designedprimers.Genomic Southern and Northern (RNA) analyses. Genomic

DNA was isolated from L. d. chagasi and other species byincubating 109 cells in 10 mM Tris-HCl (pH 8.3)-50 mMEDTA-1% sodium dodecyl sulfate (SDS)-100 ,ug of RNase A(Boehringer, Mannheim, Germany) per ml at 37°C for 1 h.Proteinase K (Boehringer) was then added at a final concen-tration of 100 ,ug/ml, and incubation was continued overnightat 42°C. The DNA was further purified by phenol-chloroformextraction and ethanol precipitation. The isolated DNA wasdigested with various restriction enzymes, separated on a 0.8%agarose gel, and blotted onto a Nytran membrane (Schleicher& Schuell, Keene, N.H.). Total RNA was isolated frompromastigotes of L. d. chagasi by the acid guanidinium isothio-cyanate method (15). Ten micrograms of total RNA wasloaded into separate wells, resolved on a 1.5% formaldehyde-agarose gel, and alkali transferred onto Zeta Probe (Bio-RadLaboratories, Richmond, Calif.) with 50 mM NaOH. Filterswere hybridized at 65°C for 18 h with a 32P-labeled, 357-bpPCR product amplified from the cDNA (Fig. 1) with thefollowing primers: SODlB (sense), 5' CTTG CCGACGAGATCAACGC 3'; SODlC (antisense), 5' CGAGAATGATGTGACCC 3'. Conditions for hybridization and posthy-bridization washes were as described previously (44).

Expression and purification of recombinant FeSOD protein.Leishmania FeSOD protein was expressed in E. coli withprokaryotic expression vector pGEX-2T (Pharmacia), whichcontains an isopropyl-o-D-thiogalactoside (IPTG)-inducibletac promoter and a glutathione-S-transferase (GST) codingsequence (45). The whole coding region of FeSOD cDNA wasamplified by PCR, using sense primer GSTSODB, 5' AACAACAGGATCCA ACATGGTCTTCAGCATTCCTCCG 3',which contains a BamHI site upstream of the start codon, and

GGGCATATTTTAAAAAAAATTATCCAAGAAAATAACAACAACAGGAAGAAACGSTSODB

M V F S I p P L P W G Y D G LATG GTC TTC AGC ATT CCT CCG CTC CCA TGG GGC TAC GAT GGG CTT

Primer 1A A K G L S K Q Q V T L H Y DGCG GCA AAA GGC CTC TCA AAG CAG CAG GTG ACG CTC CAC TAC GAC

K H H Q G Y V T K L N A A A QAAG CAC CAT CAG GGG TAT GTG ACG AAA CTC AAC GCT GCG GCG CAG

T N S A L A T K S I E E I I RACA AAC TCC GCG CTT GCA ACG AAG AGC ATC GAG GAG ATC ATC AGG

T E K G P I F N L A A Q I F NACG GAG AAA GGC CCC ATC TTC AAC CTT GCG GCG CAG ATT TTT AAC

H T F Y W E S M C P N G G G ECAC ACG TTC TAC TGG GAG AGC ATG TGT CCT AAT GGC GGT GGC GAG

SODlBP T G k L A D E I N A S F G SCCG ACG GGA AAA CTT GCC GAC GAG ATC AAC GCT TCA TTT GGC AGT

F A K F K E E F T N V A V G HTTT GCG AAG TTC AAG GAG GAG TTT ACA AAC GTG GCT GTG GGC CAC

F G S G L A W L V K D T N S GTTT GGC TCG GGT TTG GCG TGG CTT GTG AAG GAC ACC AAT TCC GGC

K L K V Y Q T H D A G C P L TAAA CTG AAG GTC TAC CAG ACG CAT GAC GCG GGA TGT CCA CTG ACA

Primer 2E P N L K P L L T C D V W E HGAG CCC AAC TTG AAG CCT CTC CTT ACA TGC GAT GTA TGG GAG CAT

A Y Y V D Y K N D L A G Y V QGCG TAC TAC GTG GAC TAC AAG AAC GAC CTG GCG GGA TAC GTG CAG

A F W N V V N W K N V E R Q LGCC TTT TGG AAC GTT GTC AAC TGG AAG AAC GTG GAA CGA CAA CTT

GSTSODE SODlCTGAACACGACTGAGTGAAACGAAAGACACGGAGTGATGGGTCACATCATTCTCGCCCCG

A~~~~~~~~mmtlmmnnvoAA A flf "l-emt AM noR rorsM^^ ^Mf^. M ^ A

CCGAAAGGGTGCATGGAAAACGGTAAGAAAAAGAGCTTGGCAATAATTGGCATGGAAAAAAAGAGAGTAGCATTTAGAGAAAAACC(A) 25

-80-52

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3090

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75225

90270

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120360

135405

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644703762789

FIG. 1. Nucleotide and predicted amino acid sequences of L. d.chagasi FeSOD. Nucleotide and amino acid numbering are withrespect to the first A of the initiation codon. The Leishmania splicedleader sequences are underlined. Amino acid residues characteristic ofFeSODs are shown in boldface. The positions of the primers used inthis study are highlighted by a double underline under the nucleotidesequences. Primer 1 and primer 2 were used to amplify the 426-bpPCR product employed for screening the cDNA library. PrimersSODlB and SODlC were used to amplify the 357-bp fragment used asa probe in Southern and Northern blotting. Primers GSTSODB andGSTSODE were used for construction of the expression plasmidpGSTSOD3.

antisense primer GSTSODE, 5' GTCT'l'GAATTCACTCAGTCGTGTTCAAAGTTGTCG 3', which contains anEcoRI site just downstream of the stop codon. The GSTSODBprimer was designed such that the ATG of the amplifiedproduct will be in frame with the GST. The PCR product wasdigested with BamHI and EcoRI and ligated to pGEX-2Tdigested with the same enzymes, using standard techniques(40). The resulting plasmid (named pGSTSOD3) andpGEX-2T were transformed into competent E. coli DHSo andE. coli double mutant strain QC779 lacking both genes forMnSOD and FeSOD (sodA sodB). QC779 and its wild-typeparent strain, GC4468, were kindly provided by B. Bachmann,E. coli Genetic Stock Center (Yale University, New Haven,Conn.). Fusion protein from 1 liter of IPTG-induced culturewas purified on glutathione-agarose resin (Sigma ChemicalCo., St. Louis, Mo.) as described before (45). The fusionprotein was concentrated by centrifugation in Filtron tubes,and its concentration was determined by using the BCAProtein Assay kit (Pierce Chemical Co., Rockford, 111.). To

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cleave the fusion protein from the GST carrier, it was incu-bated with thrombin (Sigma), 1:500 (wt/wt), in cleavage buffer(50 mM Tris-HCl [pH 8.0], 100 mM NaCl, 2.5 mM CaCl2,0.1% ,B-mercaptoethanol) for 4 h at room temperature.SOD activity assays. Leishmania cells were harvested at

late-log stage (5 to 6 days in culture) by centrifugation at 800x g. Cells were disrupted after incubation in 5 mM Tris-HCl(pH 7.8)-0.1 mM EDTA-5 mM phenylmethylsulfonyl fluoridefor 30 min on ice by passage through a 26.5-gauge needle.Lysed cells were centrifuged at 100,000 x g for 1 h at 4°C, andthe supernatant was kept at - 80°C until used for SOD activityassays. E. coli was grown in LB with or without ampicillin for18 h. Cells were harvested, resuspended in phosphate-bufferedsaline (pH 7.4), and lysed by sonication, using a Virsonic CellDisrupter model 16-850 (The VirTis Co., Gardiner, N.Y.). Tovisualize SOD activity, a 10% native polyacrylamide gel con-taining different lysates was stained with 2 mg of nitrobluetetrazolium (Sigma) per ml for 15 min in the dark and thenimmersed in a solution containing 10 mg of riboflavin (Sigma)per ml with or without 5 mM H202 and 2 mM potassiumcyanide (KCN) for 15 min in the dark. The gel was illuminatedfor 2 h, during which time a uniform blue color developed onthe gel, except in areas containing SOD bands.

Susceptibility of recombinant E. coli to oxidative stress. Theeffect of 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat)on wild-type, mutant strain, and transformed mutant strain E.coli was assayed as described by Carlioz and Touati (13). Anovernight culture was diluted 1:50 with fresh LB broth with orwithout 100 ,ug of ampicillin per ml. After 1 h of growth,paraquat was added (0.05 mM final concentration) and incu-bation was continued. The optical density at 600 nm wasmonitored hourly for 8 h.

Nucleotide sequence accession number. L. d. chagasiFeSOD cDNA has been assigned EMBL/GenBank accessionnumber L20082.

RESULTS

Cloning and sequencing of L. d. chagasi SOD cDNA. Com-parison of SOD protein sequences of various organisms re-vealed certain regions of the protein that are highly conserved(37). By using two such regions and the Leishmania codonpreference, we designed two nondegenerate primers. Thecodon preference of Leishmania spp. was derived from nucle-otide and amino acid sequences of several Leishmania clonedgenes. PCR amplification of an aliquot of the L. d. chagasiX-ZAP cDNA library, using primers 1 and 2, gave a productwith the expected size of 426 bp. Sequence analysis confirmedthat the 426-bp fragment contained amino acid residues knownfor FeSODs and MnSODs. To obtain a full-length cDNAclone(s), we used the 426-bp product as a probe for screening.The primary screening of the cDNA library yielded 54 hybrid-izing plaques which consisted of mixed populations of full-sized and truncated plaques. To obtain only the full-sized ones,we designed an external 5'-end sense primer on the basis of theconsensus nucleotides of the trans-spliced leader sequencefound at the 5' end of all trypanosome transcripts (34). Byusing such a primer with an internal antisense primer, a cDNAclone will give a PCR product only if it has a complete 5' endwhich includes the spliced leader sequences. Of the 54 plaquesthat hybridized, 9 gave PCR products. According to their sizes,the nine clones could be divided into three classes: the firstclass (SODcDNA3) was 590 bp and had five clones; the secondclass (SODcDNA1) was 710 bp and had three clones; andthe third class had only one member and was 500 bp. Theseresults indicated that there is more than one cDNA species in

the library to which the probe can hybridize and one class isrepresented more than the others. One clone from theSODcDNA3 class was subcloned into a Bluescript plasmid(hereafter designated pBSSOD3), and the entire insert wassequenced. The nucleotide sequence of the original 426-bpPCR product exactly matched the sequences obtained for thesame region of pBSSOD3. Depicted in Fig. 1 are the completenucleotide and deduced amino acid sequences of the 869-bpcDNA insert. The cDNA starts with a part of the Leishmaniaspliced leader sequence followed by 56 bp of 5'-untranslatedsequences, 585 bp of the coding region, 204 bp of the 3'-untranslated region, and the poly(A) tail. An open readingframe with a coding capacity for a 195-amino-acid protein witha molecular weight of 21,671 was identified.Comparison of the deduced amino acid sequences with

those from diverse organisms revealed considerable homologywith FeSODs and MnSODs. As shown in Fig. 2, LeishmaniaSOD had a higher ratio of amino acid identity with prokaryoticFeSODs than with prokaryotic MnSODs or eukaryoticFeSODs and MnSODs. Among the prokaryotes, the highestidentity (52 to 55%) was scored with E. coli, Pseudomonasovalis, and Coxiella burnetii (24, 37). Identity scores withFeSOD from Nicotina plumbaginifolia fell to 43% (12), whichwas comparable to ratios obtained by comparing leishmanialSOD with bacterial and human MnSODs (37). These resultsindicate that leishmanial SOD is closer to bacterial FeSODsthan prokaryotic or eucaryotic MnSODs or eukaryotic (plant)FeSOD. This observation is further strengthened by the factthat comparison of hydrophobicity distribution patterns (25) ofL. d. chagasi SOD, E. coli FeSOD, and Bacillus stearother-mophilus MnSOD showed a close structural similarity betweenthe leishmanial SOD and the E. coli FeSOD (Fig. 3). More-over, additional sequence analysis showed that all amino acidresidues which are considered distinctive for FeSOD (37) arepresent in L. d. chagasi SOD. These residues in L. d. chagasiare Ala-70, Gln-71, Tyr-78, Ala-144, and Gly-145. In additionto these residues, Trp-73 has also been proposed to be FeSODspecific and to confer H202 sensitivity to FeSODs (12). In L. d.chagasi, this residue is replaced with Phe without a change inthe sensitivity towards H202.

Southern and Northern analyses. As shown in Fig. 4,restricted genomic Leishmania DNA probed with the 357-bpPCR fragment produced multiple hybridizing bands. It shouldbe noted that none of the restriction enzymes used has acleavage site inside the L. d. chagasi FeSOD cDNA. Thishybridization pattern is compatible with the presence of morethan one gene for FeSOD in the Leishmania genome. Usingthe same probe, Northern analysis revealed that L. d. chagasiFeSOD is expressed as a single transcript of 1.3 to 1.4 kb.

Expression and purification of recombinant FeSOD. Inorder to express L. d. chagasi FeSOD, the whole codingportion of plasmid pBSSOD3 was cloned into the pGEX-2Tplasmid that expresses foreign sequences as fusion proteinswith 26-kDa GST (45). Figure 5A shows the construction ofplasmid pGSTSOD3, which contains fusion sequences underthe control of the tac promoter. After the fusion protein isobtained, the GST carrier can be cleaved with the site-specificprotease, thrombin.

Expression and purification of Leishmania recombinantFeSOD are shown in Fig. SB. Mutant E. coli QC779 trans-formed with parental vector pGEX-2T and induced with IPTGyielded a 26-kDa protein which corresponds to GST. WhenQC779 was transformed with the constructed plasmid pGST-SOD3 (Fig. SA) and induced with IPTG, a fusion protein wasobtained. This fusion protein has a calculated molecularweight of 47,000, with 26 kDa from the GST and 21 kDa from

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L. chagasi Fe V F S I P P L P W G Y D G L A A K G L S K Q Q V T L H Y D K Hn Q G Y V T KE. coli Fe S F E L P A L P Y A K D A L A P H - I S A E T I E Y H Y GK E E Q T Y V T NC. burnettii Fe A F E L P D L P Y K L N A L E P H - I S Q E T L E Y E H G K H H R A Y V N KP. ovalis Fe A F E L P P L P Y A H D A L Q P H - I S K E T L E Y H H D K H H N T Y V V NN. plumba Fe K F E L Q P P P Y P M D A L E P H - M S S R T F E F E W G K HK R A Y V D NB. stearoth Mn P F E L P A L P Y P Y D A LE P H - I D K E T M N I K H T K K B N T Y V TNHuman Mn K H S L P D L P Y D Y G A LE P H - I N A Q I M Q L B H S K B B A A Y V NN

50L. chagasi Fe L N A A A Q T N S A L A T K S I E E I I R - - - - - - - T E K G P I F N L AE. coli Fe L N N L I K G - T A F E G K S L E E I I R - - - - - - - S S E G G V F N N AC. burnettFiFe L N K L I E G - T P F E K E P L E E I I R - - - - - - - K S D G G I F N N AP. ovalis Fe L N N L V P G T P E F E G K T L E E I I V K - - - - - - S S S G G I F N N AN. plumba Fe L N K Q I D G T - E L D G K T L E D I I L V T Y N K G E P L - P A - F N N AB. stearoth Mn L N A A L E G H P D L Q N K S L E E L L S N L E A L P B S I R T A V R N N GHuman Mn L N V T E E K Y Q E - - - - - - - A L A K G D V T A Q I A L Q P A L K F N G

* * + * 100L. chagasi Fe A Q I F N H T F Y W E S M C P N G G G E P T G K L A D E I N A S F G S F A KE. coli Fe A Q V W N H T P Y W N C L A P N A G G E P T G K V A E A I A A S F GS F A DC. burnettii Fe A Q H W N H T F Y W H C M S P D G 0 G D P S 0 E L A S A I D K T F 0 S L E KP. ovalis Fe A Q V W N H T F Y W N C L S P D G 0 0 Q P T G A L A D A I N A A F 0 S F D KN. plumba Fe A Q A W N B Q F F W E S M K P N G G G E P S G E L L E L I N R D F GS Y D AB. stearoth Mn G G H A N H S L F W T I L S P N G G G E P T G E L A D A I N K K F G S F T AHuman Mn G G H I N H S I F W T N L S P N G G G E P K G E L L E A I K R D F G S F D K

L. chagasi FeE. coli FeC. burnettilP. ovalis FeN. plumba FeB. stearoth MHuman Mn

L. chagasi FeE. coli FeC. burnettiiP. ovalis FeN. plumba FeB. stearoth MHuman Mn

L. chagasi FeE. coli FeC. burnettiIP. ovalis FeN. plumba FeB. stearoth M.Human Mn

150 *F K E E F T N V A V G H F G S G W A W L V K D - T N S G K L A V Y Q T H D AF K A Q F T D A A I K N F G s G W T W L V K N - S D - G K L A I V S T S N A

Fe F K A L F T D S A N N H F G S 0 W A W L V K D - N N - G K L E V L S T V N AF K D E F T K T S V G T F G S G W A W L V K D - - - - G S L A L C S T I G AFVKEFKAAAATQFOSGWAWLAAYKPEEKKLALVKTPNA

In F K D E F S K A A A G R F G s G W A W L V V N - - - N G E L E I T S T P N QF K E K L T A A S V G V Q G S G W G W L G F N - K E R G H L Q I A A C P N Q

* + +G C P L T E P N L K-P L L T C D V W E3HA Y Y V D Y K N D L A GG T P L T T D A -T - - - - - - P L L T V D V W E A Y Y I D Y R N A R P G

Fe R N P M T E G K -K - - - - - - P L M T C D V W E A Y Y I D T R N D R P KG A P L T S G D -T - - - - - - P L L T C D V W E A Y Y I D Y R N L R P KE N P L V L G Y -T - - - - - - P L L T I D V W E A Y Y L D F Q N R R P D

In D S P I M E G K T - - - - - - - P I L G L D V W E1 A Y Y L K Y Q N R R P ED - P - - - - - L Q G T T G L I P L L G I D V W E H A Y Y L Q Y K N V R P D

200Y V Q A F W -N V V N W K N V E R Q L - - - - - - - -

Y L E H F W -A L V N W E F V A K N L A A - - - - - -

Fe Y V N N F W - Q V V N W D F V M K N F K S - - - - - -

Y V E A F W -N L V N W A F V A E E G K T F K A - - -Y I S I F M E K L V S W E A V S S R L - - - K A A T A

n Y I A A F W N V V N W D E V A K R Y S E A K A K - -

Y L K A I W -N V I N W E N V T E R Y M A C K K - - -

FIG. 2. Comparison of amino acid sequences from L. d. chagasi FeSOD (L. d. chagasi Fe) from this study, E. coli FeSOD (E. coli Fe) (37), C.burnettii FeSOD (C. burnettii Fe) (24), P. ovalis FeSOD (P. ovalis Fe) (37), N. plumbaginifolia FeSOD (N. plumba Fe) (12), B. stearothermophilusMnSOD (B. stearoth Mn) (37), and human MnSOD (human Mn) (37). Amino acid residues common to both FeSODs and MnSODs are inboldface. The metal binding residues are conserved between FeSODs and MnSODs and are marked by pluses on the top. Residues which areprimary candidates for distinguishing FeSODs from MnSODs (according to reference 37) are indicated by asterisks on the top.

Leishmania FeSOD as predicted from the cDNA. However,the GSTSOD fusion protein runs on the SDS-polyacrylamidegel as a 43-kDa band, and the Leishmania FeSOD obtainedfrom cleavage of the fusion protein with thrombin runs as a 24-to 25-kDa band instead of the calculated 21-kDa band. Thesechanges in the migration of these proteins could be due tostructural problems inherent in the GST expression systemand/or in the Leishmania recombinant FeSOD. A similar kindof discrepancy between calculated and apparent molecularweights using a GST expression system have been observedbefore (45).

Functional assays of L. d. chagasi- and E. coli-expressedFeSOD. Enzymatic SOD activity of L. d. chagasi lysates andpurified recombinant SOD was assessed by the direct stainingmethod of Beauchamp and Fridovich (7). As shown in Fig. 6A,total Leishmania lysate contained a SOD activity band whichmigrated between the standard E. coli MnSOD and FeSOD.Recombinant GSTSOD expressed in E. coli and cleaved withthrombin to release pure SOD exhibited a band of SODactivity that migrated slightly slower than the Leishmania band.It should be noted that proteolytic digestion of the GSTSODfusion protein leaves four extra amino acids, including the first

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FIG. 3. Hydropathy plots of L. d. chagasi FeSOD (upper plot), E.coli FeSOD (middle plot), and B. stearothermophilus MnSOD (lowerplot). Numbers on the x axis indicate the number of amino acids fromthe amino terminus. Numbers on the y axis are hydropathy valuescalculated by the method of Kyte and Doolittle (25). Positive scores

indicate a hydrophobic segment, whereas negative scores are indicativeof a hydrophilic segment of the protein.

methionine, at the amino terminus of the recombinant protein.Two of these amino acids are from sequences that follow thethrombin cleavage site (45), while the other two are from theGSTSODB primer sequences. The GSTSOD fusion proteinalso had a band of SOD activity that was the least mobile, as

expected (data not shown). Treatment of Leishmania andQC779/pGSTSOD3 SODs with inhibitors that distinguish be-tween different classes of SOD revealed that both the Leish-mania and the QC779/pGSTSOD3 bands were resistant to 2mM KCN (Fig. 6B) but sensitive to 5 mM H202 (Fig. 6C),which is indicative of FeSOD. Neither the GST protein northrombin showed any SOD activity on the 10% native gel.

Sensitivity of recombinant E. coli to paraquat. E. coli-expressed Leishmania FeSOD was tested for its ability toconfer resistance to QC779 against paraquat-induced oxidativedamage. Earlier, Carlioz and Touati (13) had shown thatQC779 was very sensitive to treatment with paraquat. Asshown in Fig. 7, QC779/pGSTSOD3 and wild-type E. coliGC4468/pGEX-2T grew well in LB broth containing 0.05 mMparaquat, whereas QC779/pGEX-2T harboring only the plas-mid vector grew at a slower rate under the same conditions. Inthe absence of paraquat, none of the E. coli transformantswere affected.

DISCUSSION

In this study we have cloned and characterized an L. d.chagasi cDNA which encodes an FeSOD. This is the first SODof this type to be cloned from a nonphotosynthetic eukaryote.The Leishmania cDNA contains an open reading frame of 585nucleotides, encoding a 195-amino-acid protein with a molec-ular weight of 21,800. The deduced amino acid sequencereveals strong overall homology of about 66 to 74% withprokaryotic FeSODs, whereas the overall homology with plantFeSODs is 59%. Using direct staining of the native gel forSOD activity, we have demonstrated that the proteolytically

FIG. 4. Southern analysis of genomic DNA from L. d. chagasi andother species of Leishmania digested to completion with the enzymesshown and probed with a 32P-labeled 357-bp fragment amplified frompBSSOD3 with primers SODlB and SODIC. The first seven lanes on

the left contain DNA from L. d. chagasi digested with the enzymesshown. DNAs from L. donovani (Ld), L. infantum (Li), L. tropica (Lt),L. major (Lm), L. amazonensis (La), and L. braziliensis (Lb) were

digested with PstI only. (B) Northern analysis of L. d. chagasi RNA.The same 357-bp fragment was labeled with 32P and used to hybridizewith 10 p.g of L. d. chagasi total RNA. Molecular weight markers, 103,are shown on the left-hand side.

cleaved recombinant SOD protein expressed in E. coli isenzymatically active in vitro. Moreover, the protection againstparaquat toxicity of pGSTSOD3-transformed QC779 also in-dicates that the recombinant fusion protein is biologicallyfunctional in vivo. The fact that the fusion GSTSOD proteinshowed both in vivo and in vitro enzymatic activity indicatesthat the GST peptide does not interfere with the SOD activityof the recombinant protein. Similar results have been obtainedby other workers using the GST expression system for a varietyof eukaryotic proteins (9, 35). To characterize the isoenzymetype of the L. d. chagasi SOD we have cloned, total lysatesfrom L. d. chagasi and the bacterially expressed enzyme were

treated with inhibitors that distinguish between the variousisoenzymes of SODs. Our results demonstrate that extractsfrom both L. d. chagasi and the bacterially expressed enzymeexhibit sensitivity patterns towards KCN and H202 that are

compatible with FeSODs. However, the recombinant SODactivity band and the band from Leishmania lysate migrateddifferently on the native gel. This differential mobility could bedue to the presence of the extra amino-terminal amino acids inthe recombinant protein. Alternatively, the native Leishmaniaenzyme might have been posttranslationally modified. Previ-ously, Le Trant et al. (28) and Meshnick and Eaton (33) haveshown that extracts from L. tropica contained SOD activitywhich was inhibited by H202 but not by cyanide. These earlierobservations are consistent with the results from SOD activityassays of the present study.Although FeSODs and MnSODs share identical amino acid

sequences in the regions used for making primers, neither PCRnor the screening of the L. d. chagasi cDNA library detectedMnSOD sequences. Furthermore, under the conditions used,

IL1-

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AThrombin cleavage site

pGSTSOD3:

ttac promoter

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GST SOD cDNAI

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FIG. 5. (A) Construction of bacterial expression plasmidpGSTSOD3. Schematic drawing of pGSTSOD3 showing GST, FeSODcDNA, and the thrombin cleavage site. The expression of GSTSODfusion is under control of the tac promoter, which is IPTG inducible.(B) SDS-polyacrylamide (10%) gel stained with Coomassie blue oflysates from uninduced QC779/pGEX-2T (lane 1), QC779/pGEX-2Tinduced with IPTG (lane 2), QC779/pGEX-2T lysates purified througha glutathione-agarose column (lane 3), lysates from uninduced QC779/pGSTSOD3 (lane 4), QC799/pGSTSOD3 induced with IPTG (lane 5),QC799/pGSTSOD3 lysates purified through a glutathione-agarosecolumn (lane 6), and the purified GSTSOD3 fusion protein cleavedwith thrombin (lane 7). Molecular weight markers, 10 , are shown onthe left-hand side.

SOD activity on the native gel did not reveal a band whichcould be classified as MnSOD (resistant to both H202 andKCN). However, the absence of MnSOD from both the cDNAlibrary and the native gel cannot rule out its presence at thegenomic level. In E. coli, the two genes, sodA and sodB, whichencode MnSOD and FeSOD, respectively, respond in a verydifferent way to environmental and growth conditions (18, 22,38). The MnSOD is markedly induced by oxidative stress

A B1 2 4 5 BI C I

FIG. 6. (A) Nondenaturing 10% polyacrylamide gel stained forSOD activity. Lanes: 1, GSTSOD fusion protein from QC779/pGSTSOD3 purified through a glutathione-agarose column andcleaved with thrombin (10 jxg); 2, total lysates from L. d. chagasi (1.7x 108 cells); 3, total lysates (150 ,ug) from QC779/pGEX-2T; 4,Cu/ZnSOD (2 R,g) from bovine erythrocytes (Sigma); 5, 2 ,ug each ofE. coli MnSOD (top) and FeSOD (bottom) (Sigma). (B) Gel identicalto that in panel A except that it was treated with 2 mM KCN. (C) Gelidentical to that in panel A except that it was treated with 5 mM H202.

0.5 -

0.0 L0 2 4 6 8

Hours in CultureFIG. 7. Susceptibility to oxidative stress of E. coli transformants

monitored by the effect of paraquat (PQ) on the growth curves ofbacteria grown in the presence of 0.05 mM paraquat (open symbols) orin its absence (closed symbols). OD 600, optical density at 600 nm.

conditions (22), whereas the FeSOD is expressed constitutively(38). Therefore, it is possible that under the conditions usedLeishmania FeSOD might have been preferentially expressed.Because of the lack of homology between Cu/ZnSOD and Fe-and MnSODs, the presence of Cu/ZnSOD could not bedetected by PCR, screening, or Southern blot. We also couldnot detect any band with the Cu/ZnSOD property (sensitive toboth H202 and KCN) by staining the native gel for SODactivity.As shown by Southern hybridization (Fig. 4), FeSOD is

highly conserved among members of the Leishmania genus. Itis interesting that the three species L. d. chagasi, L. d.donovani, and L. d. infantum, which are grouped under thesame complex as L. donovani (26), showed identical PstIhybridization patterns. Except for L. major and L. tropica,which are in the same complex, other species examinedrepresenting different complexes displayed different PstI hy-bridization patterns. It was reported earlier (8) that membersof the L. tropica complex had the most distant intracomplexdivergence compared with members of other complexes.Therefore, our results are in agreement with other classifica-tion schemes in which Leishmania species were grouped intocomplexes according to their nuclear DNA restriction frag-ment patterns (8) or isoenzyme content (32).SODs have been implicated in the pathogenicity of a num-

ber of intracellular pathogens such as Listeria monocytogenes(14), Nocardia asteroides (6), Shigella flexneri (20), and Rickett-sia rickettsii (42). The role of SODs in the intramacrophagesurvival of Leishmania spp. has not been investigated. Acti-vated macrophages usually kill ingested intracellular patho-gens via two separate oxidative pathways involving ROI andRNI (3, 36, 46). The role of each of these oxidants in theintracellular killing of Leishmania spp. is not fully understood.Recently, there have been reports indicating that, because oftheir simultaneous generation, products of the two pathwayssuch as 02- and nitric oxide can actually interact with eachother to form peroxynitrite, which is a stronger oxidant thanthe reactants (1, 17, 29, 31). Thus, by removing the 02-

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component, SODs can protect the intracellular pathogensfrom the deleterious effects of peroxynitrite (39). On the otherhand, the ability of macrophages to control the replication ofintracellular pathogens has been shown to be influenced bydiverse cytokines and growth factors that are produced bydifferent immunocompetent cells (5, 27, 30, 43). It has beenshown that gamma interferon (IFN-y) and tumor necrosisfactor alpha (TNF-ot) synergize each other to induce theleishmanicidal action of macrophages (10, 29). The mecha-nisms by which IFN-y and TNF-ox mediate intramacrophagekilling of Leishmania spp. is unknown, but it is highly suspectedto be mediated by the generation of an ROI and/or RNIspecies (29, 49). Therefore, if IFN-y and TNF-o. cytotoxicity ismediated by these agents, then susceptibility of Leishmaniaspp. to killing by activated macrophages might be influenced byits content of ROI- and RNI-scavenging systems such as SODs.Recently, evidence which shows that both IFN-y and TNF-oinduce SOD expression in different cells, including macro-phages, has been provided (23, 48, 49). This evidence suggestsa possible role of SOD in Leishmania spp. and other intracel-lular pathogens.The finding of FeSOD in Leishmania spp. is also interesting

from an evolutionary perspective. To date, FeSODs have beenisolated from prokaryotes and certain plants. This distinctivedistribution of FeSODs prompted the hypothesis that genetransfer between endosymbiotic organisms might have beenresponsible for FeSOD's distribution. However, the finding ofFeSOD in a nonphotosynthetic eukaryote questions this the-ory. In conclusion, the cloning of FeSOD cDNA from L. d.chagasi will provide an opportunity to study the functional roleof SODs in Leishmania infection. With the recent developmentof technology to inactivate repeated genes of L. enrietta (16), itwill be feasible to determine the role of FeSOD by reversegenetics.

ACKNOWLEDGMENTS

This investigation received financial support from the UNDP/WorldBank/WHO Special Programme for Research and Training in TropicalDiseases (TDR). Y.A.W.S. is a fellow of the Medical Research Councilof Canada.We are greatly indebted to Steven G. Reed (Seattle Biomedical

Research Institute) for providing us with the L. d. chagasi cDNAlibrary. We are also grateful to Sui-Lam Wong (University of Calgary)for his advice.

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