Proc. Natl. Acad. Sci. USAVol. 90, pp. 775-779, January 1993Medical Sciences

Molecular characterization of a kinesin-related antigen ofLeishmania chagasi that detects specific antibody inAfrican and American visceral leishmaniasis

(repetitive epitope/motor protein/parasite/diagnosis)

JAMES M. BURNS, JR.*, WAYNE G. SHREFFLER*, DARIN R. BENSON*, HASHIM W. GHALIBt,ROBERTO BADARO*, AND STEVEN G. REED*§¶*Seattle Biomedical Research Institute, Seattle, WA 98109; tUniversity of Juba, Khartoum, Sudan; tHospital Professor Edgard Santos, Federal University ofBahia, Salvador, Bahia, 40.140, Brazil; and §Division of International Medicine, Department of Medicine, Cornell University Medical College,New York, NY 10021

Communicated by Paul B. Beeson, October 7, 1992

ABSTRACT We report the cloning of a Leishmania cha-gasi antigen gene and an evaluation of leishmaniasis patientantibody responses to the recombinant protein, rK39. rK39contains a 39-amino acid repeat that is part of a 230-kDaprotein predominant in L. chagasi tissue amastigotes. Sequenceanalyses showed this protein, LcKin, to be related to the kinesinsuperfamily of motor proteins. Southern blot analyses demon-strated LcKin-related sequences in seven species of Leish-mania, with conservation of the repeat between L. chagasi andLeishmania donovani. Serological evaluation revealed that 98%(56 of 57) of Brazilian and 100% (52 of 52) of Sudanese visceralleishmaniasis patients have high antibody levels to the rK39repeat. Detectable anti-K39 antibody was virtually absent incutaneous and mucosal leishmaniasis patients and in individ-uals infected with Trypanosoma cruzi. The data show that rK39may replace crude parasite antigens as a basis for serologicaldiagnosis of visceral leishmaniasis.

Protozoan parasites of the genus Leishmania are widelydistributed and transmitted by the bite of sandflies. In thevertebrate host, the infecting promastigotes differentiate intoand replicate as amastigotes within macrophages. Symptomsrange from self-healing skin lesions to diffuse cutaneous andmucosal manifestations, or severe visceral involvement ofthe spleen, liver, and lymph nodes. Visceral leishmaniasis(VL) is generally caused by Leishmania donovani in Africa,India, and southern Europe or Leishmania chagasi in LatinAmerica. In VL, high antibody levels are observed prior tothe detection of parasite-specific T-cell responses (1). Thisantibody response has been exploited for the diagnosis of L.chagasi and L. donovani infection (2-5). The current WorldHealth Organization's estimate of 12 million cases of leish-maniasis and recent epidemics of VL in Sudan and India (6,7) highlight the need for more effective control measures.Diagnostic tests use whole or lysed Leishmania, and a fewstudies have begun to examine patient antibody responses tospecific antigens (2, 8-10). Such studies may improve diag-nostic assays and help to evaluate B-cell responses duringdisease progression.We report the cloning and expression ofan L. chagasi gene

sequence"I and an evaluation of patient antibody responses tothe recombinant product, rK39. The sequence encodes animmunodominant protein with a repetitive epitope closelyconserved between L. chagasi and L. donovani. This repeatis part of a large kinesin-related protein expressed predom-inantly by amastigotes. More than 98% of Brazilian and 100%

of Sudanese VL patients had readily detectable serum anti-body responses to the K39 repeat.

MATERIALS AND METHODSParasites. L. chagasi (MHOM/BR/82/BA-2,C1), L. cha-

gasi (MHOM/BR/84/Jonas), Leishmania amazonensis(IFLA/BR/67/PH8), Leishmania brasiliensis (MHOM/BR/75/M2903; obtained from Diane McMahon-Pratt, Yale Uni-versity), Leishmania guyanensis (MHOM/BR/75/M4147),L. donovani (MHOM/Et/67/HU3), Leishmania infantum(IPT-1; obtained from Lee Schnur, The Hebrew University-Hadassah Medical School, Jerusalem), Leishmania major(LTM p-2, obtained from David Moser, Temple University,Philadelphia), and Trypanosoma cruzi (MHOM/CH/00/Tulahuen C2) were used. Promastigotes and epimastigoteswere cultured in axenic media. L. chagasi and L. amazon-ensis amastigotes were obtained from spleens of Syrianhamsters and footpads of BALB/cByJ mice, respectively,and purified as described (11).

Patient Sera. Brazilian leishmaniasis sera and T. cruziinfection sera were from well-characterized patients in Bahia,Brazil; Sudanese leishmaniasis sera were from Leishmania-positive patients; normal sera were from healthy individualsin Sudan or the United States (2, 4, 12).

Identification and Purification of rK39. A genomic librarywas constructed with sheared DNA of L. chagasi (MHOM/BR/82/BA-2,C1) in Lambda ZAPII (Stratagene) andscreened with preadsorbed serum (13) of a L. donovanipatient. The 39-kDa recombinant antigen of clone K39 (rK39)was purified from a 25-40o ammonium sulfate fraction ofbacterial lysate by preparative isoelectric focusing (IEF) withthe Rotofor IEF cell and 1% 3/10 ampholytes (pH range3.5-9.5; Bio-Rad) in the presence of 8 M urea and 10 mMdithiothreitol. Peak fractions were concentrated by ammo-nium sulfate precipitation and dialyzed against 25 mMTris'HCI, pH 8/150 mM NaCl (TBS). Protein concentrationswere determined by using the Pierce BCA assay, and puritywas assessed by Coomassie blue-staining following SDS/PAGE (14).Immunoblot Analysis. Immunoblots of parasite lysates or

purified rK39 were prepared (11). Filters were blocked withTBS containing 5% nonfat dried milk and probed with patientsera (1:250 dilution) or rabbit sera (1:400 dilution) diluted inTBS containing 0.1% Tween-20 and 1% bovine serum albu-

Abbreviation: VL, visceral leishmaniasis.ITo whom reprint requests should be addressed at: Seattle Biomed-ical Research Institute, 4 Nickerson Street, Seattle, WA 98109-1651.'The sequence reported in this paper has been deposited in theGenBank data base (accession no. L07879).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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97-68- - FIG. 1. Expression and purification43 _ of rK39. Coomassie blue-stained SDS/-3 W w 12% polyacrylamide gel of molecular

- 9 _ ;mass markers (lane 1), Escherichia coli2f: lysates from uninduced (lane 2) and in-

duced (lane 3) cultures of clone K39, and18 - * purified rK39 (2 ,g) (lane 4). Sizes are

shown in kDa.

min. Bound antibody was detected with 125I-labeled proteinA (1 x 106 cpm per blot) followed by autoradiography.Rabbit antiserum was raised by s.c. immunization of an

adult New Zealand White rabbit (R & R Rabbitry, Stanwood,WA) with 200 ,ug of purified rK39 in Freund's incompleteadjuvant (IFA; GIBCO) together with 100 ,ug of muramyldipeptide (Calbiochem) and a booster with 200,ug of rK39 inIFA alone. Three weeks later the rabbit received a boosteri.v. with 25,g ofrK39, and serum was collected 6 days later.K39 Gene Sequence Analysis. The radiolabeled insert of

K39 was used to screen the L. chagasi genomic library toobtain clones flanking the K39 gene fragment. A set ofoverlapping deletions of clones K39 and LcKin were gener-ated by exonuclease III digestion (15) to obtain the completesequence of the coding and noncoding strands. Single-stranded template was prepared (16), and sequence wasobtained by Sanger dideoxynucleotide chain-terminationwith [35S]-labeled dATP (17) or by fluorescence-based se-quencing on the Applied Biosystems Automated Sequencermodel 373A. Sequence comparisons during GenPept (release72.0) and Swiss-Prot (release 22.0) data bank searches weremade with the Lipman/Pearson method (18), and final align-ments were made according to the Needleman/Wunschalgorithm (19). Secondary structural predictions were ac-cording to Garnier-Robson (20) and Chou-Fasman (21).

Parasite DNAs were isolated, digested with Pst I, sepa-rated by agarose gel electrophoresis, and analyzed by South-em blotting (13). Blots were probed with a 2.4-kilobase (kb)HindIII fragment of LcKin derived from the 5' end of thegene (probe A) or the 915-base-pair (bp) insert of clone K39(probe B). Probes were radiolabeled with [a-32PldCTP to aspecific activity of 9 x 108 cpm/,ug ofDNA by using randomoligonucleotide primers (Boehringer Mannheim). Finalwashes were for 1 hr in 0.015 M NaCl/0.0015 M sodiumcitrate, pH 7/0.5% SDS at 68°C. Blots of L. chagasi DNAdigested with BamHI, HindIII, and Pst I were prepared andprobed as above. Probe A contained one BamHI, one Pst I,and no HindIlI sites. Probe B did not contain sites for theseenzymes.K39 Antigen ELISA. rK39 or L. chagasi promastigote

lysate was diluted in coating buffer (15 mM Na2HCO3/28 mMNaH2CO3, pH 9.6), and microassay plates (Probind; Falcon)were sensitized overnight at 4°C with rK39 (50 ng) or pro-

mastigote lysate (1 ug) followed by blocking with phosphate-buffered saline (PBS) containing 1% Tween-20 for 1 hr atroom temperature. After five washes with PBS containing0.1% Tween-20 (PBS-T), 50 ,ul per well of sera diluted 1:100with PBS-T were incubated for 30 min at room temperature.Wells were washed five times with PBS-T, and bound anti-body was detected by protein A-conjugated horseradishperoxidase (Zymed Laboratories) as described (2). Absor-bance values are relative to the mean of five control seraassayed on each plate.

RESULTSIdentification of L. chagasi Antigen Genes. To characterize

Leishmania antigens recognized by VL patients, a L. chagasiexpression library was screened with sera from a L. donovanipatient. From =32,000 recombinants, 7 clones were selectedthat contained inserts of 0.9-2.6 kb and produced immuno-reactive recombinant proteins of 35-100 kDa. The recombi-nant antigen of clone K39 (rK39) was exceptionally reactivewith test serum. rK39 migrated as a 39-kDa protein in inducedbacterial extracts (Fig. 1, lane 3) and was purified (Fig. 1, lane4) with a yield of 25-30 mg per liter.Both L. chagasi and L. donovani VL Sera Recognize rK39.

The reactivity of patient sera with rK39 was evaluated byimmunoblot. Both rK39 and L. chagasi promastigote lysatewere strongly recognized by L. chagasi (Fig. 2, lanes A-C)and L. donovani (Fig. 2, lanes D-F) infection sera but not bypools of sera from mucosal (Fig. 2, lanes G) or cutaneous(Fig. 2, lanes H) leishmaniasis patients or with Chagasdisease patient sera (Fig. 2, lanes I). All sera reacted stronglywith promastigote lysates. These results indicate that K39may be specific to L. chagasi and L. donovani and/or K39induces a strong antibody response only in VL patients.Sequence Analysis of the K39 Gene. The DNA and deduced

amino acid sequences of clone K39 were determined (Fig.3A, nucleotides 2426-3319) revealing a single open readingframe encoding 298 amino acids with a predicted molecularmass of 32.7 kDa and pI of 4.4. rK39 contains an additional6.2 kDa of plasmid fusion sequences. Of particular interestwere 6.5 copies of a tandemly arrayed 39 amino acid repeat(Fig. 3B).Clones containing sequences flanking the K39 gene frag-

ment were isolated from the L. chagasi library. Sequenceanalysis of one overlapping clone, LcKin (Fig. 3A, nucleo-tides 1-3109), showed that the open reading frame extended1971 bp in the 5' direction, encoding 657 nonrepetitive aminoacids; 5' to the putative ATG initiation codon, 454 bp ofsequence were obtained with multiple termination codons ineach reading frame. Partial characterization of clones con-taining 3' flanking sequences indicated that the repeat domainextended -3-4 kb.Data bank searches revealed sequence similarity between

LcKin and members of the superfamily of kinesin-relatedproteins, particularly in the N-terminal motor domain. Fig. 4

A B C D E F G H I J K1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 3 1 2 1 2

97-98-i j j1 ;43- Amok

29- low.

18- *

14-;3

FIG. 2. Reactivity of patient sera with rK39. Blots containing 10 ,ug of L. chagasi promastigote lysate (lanes 1), 50 ng of purified rK39 (lanes2), and 10 ,ug of T. cruzi epimastigote lysate (lane 3) were probed with individual L. chagasi (lanes A-C) or L. donovani (lanes D-F) VL sera,pooled mucosal (lane G, n = 4) or cutaneous (lanes H, n = 4) leishmaniasis sera, or T. cruzi infection (lane I, n = 5) sera. Pooled normal humansera (n = 3) and no primary antibody controls are shown in lanes J and K, respectively. Sizes are shown in kDa.

Proc. Natl. Acad. Sci. USA 90 (1993)

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A

1 * h p s t v r r e a e r v k v

GGTCCTGGGAGGCAGCAACAACAGCGGCGCCGCCGAGTCGATGCGGACT50 v l g g s n n s g a a e s g t

CCGGACGATCGGGTACCCGCTGGTGCAGCACGCGTTCGACGGGTTCAAC100 r t i g y p l

GCGGATCTGCCTGUAGATCTT1150 r i c L e i f

CGAGGAGGTGTACGTGGACGTG200 e v y v d v

CGACCGGAGCAGCCGGAGCCAC250 d r s s r s h

Proc. Natl. Acad. Sci. USA 90 (1993) 777

:150300

:450600

s v r v r p l n e r e n n a p e g t k v t v a a k q a a a v v t v k

a r r v a q d f q f d h v f w s v e t p d a c g a t p a t q a d v f

v q h a f d g f n s c l f a y g q t g a g k t y t m m g a d v s a L s g e g n g v t p

TaCGCGraa6GCvA6CeTGAG6CGCAGh66CACTCrC ivTeyATCvTvyAnCTervGTAkrTkkgGTvkACgggCGCGT6TC6GACCT6CTTG6MCCAAT6T6AAGCGGC66a r k a s v e a q g h s r v i v e l g y v e v y n e r v s d l l g k r k k g v k g g g

GCGC6AGCACCC6A6CCGC66CGT6TTCCTlliGGCA6C66CT66T66A66TT66GA6CCTGSiAC6AT6TT6T6C66CT6ATCGAGATC6GCAAC666GCT6C6CACACC6CTTC6AC6AAGAT6AAr e h p s r 9 v f t e g q r l v e v 9 s l d d v v r l i e i g n g v r h t a s t k m n

750

900

1050

1200

1350a 1 i * t L l r r t m t t k g e t i r t a g k s a r m n L v d L a g s e r v a q

GTCGCA66T6GAG6GGCAGCAGTTCMGAGG GCGACGCACATCAACCT6TCGCT6AC6AC6CTC666C6CGT6ATC6AC6TGCTCGC66ACAT66C6AC6AAG66TGC6AAGGCGCAGTACAGCGTT6C6CCGTTCCGCGACTCGAAGCT 1500300 s q v e g q q

GACGTTCATCCTGAAGGACTCG350 t f i l k d s

CGAGGACCCGCGCGCACGCG6G400 e d p r a r r

CCTSCAGGCGCTGGAGAGGGAS450 L q a L e r e

GGCGCTGAACCTTCGGCTGAAG500 a l n l r L k

GGCGCAGCTGGAGCGT6AGCA6I550 a q L e r e q

TGAGCGTACGCAGCTATCACAG(600 e r t q l s q

TGTGTTTCACCTGCAAACGCTC'650 v f h l q t l

GCGGCTAACCAGCCTTGAGCAG(700 r l t s l e a

ATCCGAGGCGCGCGCTGCGGAG4

f k e a t h 1 n L s l t t l g r v i d v l a d m a t k g a k a q y s v a p f r d 3 k l

L g g n s k t f m 1 a t v s p s a l n y e e t l s t l r y a s r a r d 1 v n v a q v n

i r e l e e q m e d m r q a m a g g d p a y v s e l k k k la* l e s e a q k r a a d

r e h n q v q e r L L r a t e a e k s e Le s r a a a L q e e t a t r r q a d k m q

e e q a r k e r e L l k e m a k k d a a L s k v r r r k d e a s e r e k l e s t v

r e r e v a l d a l q t h q r k l q e a l e s s e r t a a e r d q tl q q l t e l q s

vvTTdTdACCGACCGCGAGCGGCrTACACGCtACTTqCAGCGTATTCAtTACGAGTACqGrGAACC6AGCTCGCaCrAGACGTGGCGCT6T6CGCCGCGCAqeAeATGGAGrChCGCTACCACGCTGCv v t d r e r l t r d l q r i q y e y g e t e l a r d v a l c a a q e * e a r y h a a

l e l a t e w e d l r e r l a e r d e a a e l d a a as t s q n a r e a c e

1650

1800

1950

2100

2250

2400

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iCAGCTTCGC6AATCC6AGGAGCGCGCTGC66A6CT66CGA6CCA6CT66AG6CCACTGCTGCTGC6MGTCGTC66CG6tiACAGGACC6C6A6AACACGA66CCACGCTA6A6CAGCAGCTTCGCGA 2700

q l r e s e e r a a e L a s q Le a t a a a ks s a ae9 d r e n t r a t l e q Ll r eiCTGGCGAGCCAGCTGGAGGCCACTGCT6CTGCGAAGATGTCAGCGG6CAGGACCGC6AGAACAC6A666CCACGCTA6AGCAGCAGCTTC6T6ACTCC6A66AGC6CGCTGC66AGCTG6C6A6CCA 2850

750 s e a r a a e L a s a l e a t a a a k a s a e a d r e n t r a t l e a a L r d a e e r a a e l a s a6CT66AGTCCACTACTGCTGCGAAGATGTCAGCGGAGCA6MACC6C66A6CACGA66GCCACGCTA6A6CA6CA6CTTCGTGACTCC6A666GCGCGCT6C666GCT66C6A6CCAGCTGGA6TCCACTACTGCTGC6AA6ATGTCAGC 3000

800 L e s t t a a k * 3 a e d r e s t r a t l e q q l r d s e e r a a e l a s a l e s t t a a km s a

850 e a d r e s t r a t l ea a l r e s e e r a a e l a s a t e s t t a a k * a a e a d r e s t r a t lA6A6CAGCAGCTTCGTGACTCCGAG6AGCGCGCTGCG6A6CTG6CGAGCCA6CTGGAGGCCACT6CT6CTGCG^AATCGTCGGC6G6CAG6ACCGCGAGAACACGA666CCGCGrTT6AGCAGCAGCTTCGTGACTCCGAGGAGCGCGC 3300

900 e a l r d s

CGCGGAGCTGGCGAGCCAG950 a e l a s q

B L E Q Q L R D S E

1-

e e r a a e L a sa L e a t a a a k s s a e a d r e n t r a a l e a l r d s e e r a

3319

9 R A A E L A S Q LA

A T A a a Ns A E Q D R E T R A AT S

FIG. 3. Sequence analysis of clones LcKin and K39. (A) DNA and deduced amino acid sequence in single-letter code of overlapping clonesLcKin (nucleotides 1-3109) and K39 (nucleotides 2426-3319) with residues doubly underlined to indicate each repetitive segment. (B) Consensussequence of the 39-amino acid repeat of K39 with degeneracies indicated.

shows a comparison between the N-terminal domain ofLcKin and the motor domain of the Caenorhabditis eleganskinesin (22), with 43.8% identity over 349 amino acids. Strongsequence conservation was observed in the putative ATP andmicrotubule binding domains (23). The remaining 500 resi-dues showed limited similarity to the tail regions of kinesinand myosin. Secondary structure analyses predicted thisportion of LcKin (amino acids 426-955) to contain >90%helical structure characteristic of the coiled-coil tail regionsof several motor proteins. Thus, the repetitive rK39 epitopeappears to present in L. chagasi as part of the tail region ofa Leishmania kinesin-related protein.Southern Blot Analysis of LcKin Gene Sequences. Genomic

DNAs from several Leishmania species were analyzed bySouthern blot by using probes of LcKin derived from thekinesin homology domain (probe A, Fig. 5A) or from the K39repetitive domain (probe B, Fig. SB). Probe A hybridizedstrongly to multiple Pst I restriction fragments of all Leish-mania spp. tested (Fig. SA, lanes 3-9), indicating conserva-tion in the kinesin homology domain. Polymorphisms in thesize and number of hybridizing fragments were noted. Lessconservation in the repetitive domain of the LcKin gene wasobserved as probe B hybridized with varying intensity to PstI restriction fragments of L. chagasi, L. amazonensis, L.brasiliensis, L. donovani, L. infantum, and L. major but not

L. guyanensis (Fig. SB, lanes 3-9). The K39 repeat appearedmost closely related between L. chagasi and L. donovani(Fig. 5B, lanes 3 and 7). No hybridization with either probewas observed with T. cruzi DNA (Fig. 5, lane 10).When using L. chagasi digested DNA, two Pst I fragments

were detected, with probe B indicating the presence of a

second copy of the LcKin gene or polymorphism in restric-tion sites present in the 3' repetitive sequences (Fig. 5B, lane3). Probe A hybridized to three fragments in each of theBamHI, HindIII, and Pst I digests of L. chagasi DNA (Fig.SA, lanes 1-3). One BamHI, one Pst I, and no HindIII sitesare present within the probe A sequence. Taken together, theSouthern blot data show that the LcKin gene is present in a

minimum oftwo or three copies in the L. chagasi genome andthat related sequences are present in seven species of Leish-mania examined.

Identification of the Native LcKin Antigen. Rabbit anti-rK39serum was used to probe SDS/PAGE blots of L. chagasipromastigote and tissue amastigote lysates. The antiserumbound specifically to purified rK39 (Fig. 6A, lane 2) and to an-230-kDa L. chagasi antigen present in amastigotes (Fig. 6B,lane 6) and to a lesser degree in promastigotes (Fig. 6B, lane5). No reactivity with this serum was detected in promasti-gote and amastigote lysates ofL. amazonensis (Fig. 6B, lanes7-8). Comparable amounts of lysate were loaded in all lanes

. . . . - - .1 . . . . - -. = . .- - . I - - - - - - - - - - - -. . - - - - =-

6GA6CAGGACCGCGAGAGCACGAGGCCACGCTA6A6CA6CAGCTTCGC6AATCATCC6A6CGCGCTGCGGA6CT66CGA6CCA6CTG6^TCCACTACTGCT6CGA6ATGTCA6CGaGCAGACC6GCGAGA6CACGAG6GCCACGCT 3150

GCTCCCACGGCGCTACCCCCTTTCCCGCATGTGCGACAGTTTCACGCGTACAAACGTCTTTCTCTCTCCTTCGCGCGTGTCGCTATGGGCGGCGGCGCGTCGGTGTCMSATTGCACAGCTCACCGCCTCGCCATATMCGTCGT6GCCACGCGACCCCCCGACCTTCCCCTCCTCCGCCCCCAAAGACAAGCCAGACATACCGACCATGCCGTCTGCCCGCGTCTCTGCTTACCAAGCGCGCCACGCACCCCTTCCTCGGCCCTGMTCTTTCGCGCGGCGCCATACATTSCATGCACGTCACTACGCCTGTACACCTTACACCTCCTCTTGCCCACCCCTTTCCCCTTCTACACGCCTAACTACACACACATATATATATATATATATAAAGCGCTCAACGCACACATACTGTGGCCASTATTACTGCACCAACGTCTGCCTCTTCCAGGATGCACCCTTCCACTGTGCGGCGTGAGGCGGAGCGGGTGAAGGTGTCGGTGCGCGTGCGCCCCCTAAACGAACGTGAAAACAATGCCCCGGAAGGGACGAAASTGACCGTTGCGGCGAAACAGGCGGCCGCCGTGGTGACGGTCAA

TGCAAGGCGGGTAGCGCAGGACTTTCAGTTCGACCACGTGTTCTGGTCTGTGGAGACGCCraGACGCGTGCGGCGCGACCCCCGCGACGCAGGCASACGTGTT

CTCGTGCTTGTTTGCGTACGGGCAGACAGGGAGCGGGAASACSTACACGAT6ATGGGCGCGGACGTGAGCGCGCTTAGTOGTGAGGGCAACGGCGTGACGCC

CGCGATCATCATGCTGCTGCTGCGCGAGGA6CGGACGATGACGACGAAGMCOGGGAGACGATCCOTACTGCC66CAASASCAOCCGCATGMCCTTGTG"CCTTGCGWGTCTSAGCGCGTGGCGCA

SCTTGGCGGGAACTCSAAGACGTTCATGATCGCGACTGTGAGCCC6A6CGCGCTGAACTAC6AG6A6ACGCTGAGCAC6CTSC66TACGCGTCGCGC6CGCGC6ACATT6TSAATGTTGCGCA66TGAA

SATCCGCGAGCTGGAGMCAGATOGAGBACATGCGGCAGGCGATGGCTGGCGGCGACCCCGCGTACGTGTCTGAGCTGAAGAASAAGCTTGCGCTGCTGGAGTC66AGGCGCAGAAGCGTGCGGCGGA

iCGGGAGCACAACCAGGTGCAGSASCGGCTGCTSCGCGCGACGUGGCGGAGAAGMCGMCTGGASTCGCGTGCGGCTGC6CTGCASSAGGAGATGACCGCGACTCGACGGCAGSCSGACAA6ATSCA

raMGAGCAGGCGCGCAAGGMCGCGAGCTGCTSAAA6AGATGSC6AAGAA66ACGCCGCGCTCTCGAAGGTTCGGCGAC6CAAA6ACGCCUUTASCAAGCGMCGC6AGAAGCTGSAGTCraACCGT

iCGCGA6C6C6A66T66CTCTGGACraCATTGCASACGCACCAGA6AAAGCTGCAGSMGCGCTCGAGAGCTCTGAGCG6ACAGCCGCGGAAA66SACCAGCTGCT6CASCAGCTAACAGAGCTTCAGTC

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LCKIN

CELKIN

LCKIN

CELKIN

LCKIN

CELKIN

LcKIN

CELKIN

NUSlSRARRVKIIVBVVVCaAVSV~h^ TARVQ QV QDC SJpVIGTPwllW ill23* . 2 . 2 :Q . K N.:.: X .: . .FPN MWS . D: TO :V .20 :::3Am

NSSVKVAVRVRPFWQREISNTSKCVLQVNGNTTTINGHS!K33IFSFIFDRSYW8FARNDP---UFITQ!KQVYBULGVKLAF 82ATP

BINDING

DGF118CLFAYGQTGWKTY OnceDV CLBII^ SV8ALabGVtPIFWIVXLOR C VMUNWSAQAFL LV 222:0:3 C:FAYGQTGSGKtYTNUG A . :.G.PR:C ::FAR .t 3.2 2. V::3Y:TY URV.DLL . ... : VR3HP S 2233EGYNVCIFAYGQTG8GKSYTbNG---KANDPD3N0IIPRLCNDLFARIDK-NNDKDVQY3V3V3YI6IYC3RVKDLL--------NPNSGGULRVRBEPMlGPYVDETIK 181

NVG8LDDVVRLIFZOMG HTJT N R8SR8AIIMXz~ Nofr IRTAaK8xsgRLVDKAQ8SZVAQ8Q meQQ E~I IIS IDVLJUMULX.V 8 3D: L:: W.ON.AA:T:. SSRSEMA: ::L 2.R ..2X .X.K S:::LVDLAGBU.. : .39Q::K :::N3 SLTfl.@ VI. LA: :TK"CAVDC8YXDROKARTVAT8T88R81AVFTIV VLTQXl---- ---ZLVD8KI8LSTGBGROKBANgINKLVTII8LV

I MICROTUBULEBINDING

GAKAQYSVAPFRDS T7ILDSLGMSKT RIATVSP IVN3DP3A1RIK:: :V P:RDS LT.:L:::LGGUSKT N:A::SP:.:N::ZTLSTLRYA.RA::IV A VU3DP.A: IRIL.::: .21 2 2

KSNIKGVIPYRDSVLTWILR8LATGGSKTAIMUALSPADIWFDTLSTLRYADRAKQIVCQVVNEDPMAKLIRN151EVIUlRNILKDKGI

333

288

426

381

43.8% identity in 349 a. overlap

FIG. 4. Protein sequence comparison between LcKin (amino acids 1-426) and the motor domain of the kinesin related protein of C. elegans(22) in single-letter code. Identical residues are indicated by letter; colons and periods represent conservative and neutral amino acidsubstitutions, respectively. Putative ATP and microtubule binding domains are overlined (23).

as shown by the reactivity ofa rabbit antiserum raised againsta constitutively expressed L. chagasi ribosomal phospho-protein, LcPO (Fig. 6C) (24).

Reactivity of Patient Sera with rK39. The reactivity ofpatient sera with rK39 was evaluated by ELISA. Among VLpatients, 98.2% of Brazilian sera (56 of 57) and 10%/o ofSudanese sera (52 of 52) were positive on rK39 (absorbancevalues were >3 standard deviations above the mean ofnormal controls). Values ranged from 0.05 to >2.0 (mean =1.38) among Brazilian VL sera and from 0.094 to >2.0 (mean= 1.60) among Sudanese VL sera (Fig. 7B). Detectableantibody to rK39 was restricted to VL patients, as little or noanti-rK39 response was observed in mucosal or cutaneousleishmaniasis sera or T. cruzi infection sera, despite reactivitywith crude L. chagasi lysate (Fig. 7A).

DISCUSSIONWe have identified a 230-kDa antigen of L. chagasi, LcKin,with homology to the kinesin superfamily of motor proteins.LcKin is predominant in amastigotes, is present in diversespecies of Leishmania, and contains an extensive repetitive

A1 2 3 4 5 6 7 8910

B1 2 3 4 5 6 7 8 910

domain. Southern analyses showed the repeat of LcKin to bevariable among species, but closely related in L. chagasi andL. donovani. We demonstrated high antibody titers in Bra-zilian and Sudanese VL patients to rK39, which containsseveral 39-amino acid repeats, indicative of the conservationof the repeat between L. chagasi and L. donovani.

Characterization of a gene encoding a protozoan motorprotein, represented here by the cloning of LcKin, has beenpreviously unreported to our knowledge. Members of thekinesin superfamily share on average 40%o sequence identityin the N-terminal 350-400 amino acids composing the motordomain (25). These microtubule-based motors are involved insuch varied intracellular processes as organelle and synapticvescicle transport, chromosome segregation, and spindlepole body separation, nuclear fusion, protein sorting, andflagellar beating (25, 26). Although they share little sequencesimilarity outside of the motor domain (26), the nonmotor taildomain is predominantly a-helical in structure and likelyforms a coiled-coil interacting with different intracellularligands. The LcKin gene product is similar to members of thisfamily in primary sequence, particularly in the putative ATPand microtubule binding domains, as well as in predictedsecondary structure, although the specific cellular processesinvolving LcKin are not yet known.

A2

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FIG. 5. Southern blot analysis of LcKin gene sequences. Ge-nomic DNA (2.5 I&g per lane) from L. chagasi digested with BamHI(lane 1), HindIll (lane 2), and Pst I (lane 3) or Pst I-digested DNAfrom L. amazonensis (lane 4), L. braziliensis (lane 5), L. guyanensis(lane 6), L. donovani (lane 7), L. infantum (lane 8), L. major (lane 9),or T. cruzi (lane 10) were analyzed by Southern blotting. Blots wereprobed with a 2.4-kb HindIII fragment from the LcKin kinesinhomology domain (A) or with the 915-bp repetitive insert of K39 (B).Sizes are shown in kb.

FIG. 6. Reactivity of rabbit anti-rK39 antiserum on rK39 andLeishmania lysates. (A) Immunoblot of purified rK39 (50 ng) trans-ferred from SDS/12% polyacrylamide gels and probed with preim-mune rabbit serum (lane 1) or rabbit anti-rK39 (lane 2). (B) Immu-noblot of 10 ,ug of L. chagasi promastigote (lanes 1 and 5) andamastigote (lanes 2 and 6) lysates or 10 ,Ag of L. amazonensispromastigote (lanes 3 and 7) and amastigote (lanes 4 and 8) lysatestransferred from SDS/7.5% polyacrylamide gels and probed withpreimmune rabbit serum (lanes 1-4) or rabbit anti-rK39 (lanes 5-8).(C) Reactivity of rabbit antisera raised against L. chagasi ribosomalprotein PO (24) with material in lanes 1-4 of B. Sizes are shown inkDa.

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Proc. Natl. Acad Sci. USA 90 (1993)

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Proc. Natl. Acad. Sci. USA 90 (1993) 779

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FIG. 7. ELISA evaluation of patient seroreactivity on L. chagasi promastigote lysate (A) or purified rK39 (B). Absorbance values (mean+ SEM) of Brazilian VL (VL-B, n = 57), Sudanese VL (VL-S, n = 52), T. cruzi infection (Tc, n = 35), Brazilian cutaneous leishmaniasis (CL-B,n = 13), Sudanese cutaneous leishmaniasis (CL-S, n = 13), mucosal leishmaniasis (ML, n = 15), and normal (n = 15) sera.

A striking feature of LcKin was the high prevalence ofantibody to the K39 repeat in VL patients from geographi-cally distinct regions. This response was restricted to VLpatients, reflecting the relatedness among members of the L.donovani complex (27-29). The characterization of patientB-cell responses to defined Leishmania antigens has beenminimal (2, 8-10). Our studies are unique in showing VL-restricted antibody responses to a recombinant antigen of L.chagasi and showing a marked restriction of this response toL. chagasi- and L. donovani-infected patients.

Repetitive amino acid domains have been observed inmany parasitic protozoan antigens, often being immunodom-inant B-cell epitopes that detect high levels of antibody ininfected patients (16, 30-34). It is unclear if such responsescontribute to the development of protective immunity or areimmunological "smokescreens" limiting the development ofprotective responses (30, 35). In preliminary studies, 10 of 25asymptomatic or subclinical patients infected with L. chagasihad elevated levels of anti-K39 antibody (unpublished data).As only 30-35% of such individuals develop acute VL (36),the fate of those with a positive anti-K39 titer may beinformative. Such prospective studies should help to deter-mine whether the anti-K39 response is associated with theprogression or resolution of VL. Nevertheless, from a prac-tical standpoint, the data presented here as well as data fromfield studies in epidemic regions of VL in Sudan (H.W.G.,W.G.S., D.R.B., J.M.B., S.G.R., unpublished data) showthat rK39 may be useful in the serological diagnosis of acuteVL.

We thank Dr. Marilyn Parsons for helpful discussions and criticalreview of the manuscript and Karen Kinch for assistance withmanuscript preparation. This work was supported by Grants Al-25038 and AI-16282 from the National Institutes of Health. H.W.G.is supported by a Biotechnology Career Fellowship from the Rock-efeller Foundation.

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