Expression of a Novel Leishmania Gene Encoding a …iai.asm.org/content/70/12/6976.full.pdf · 0019-9567/02/$04.00 0 DOI: 10.1128/IAI.70.12.6976–6986.2002 ... The membranes were

INFECTION AND IMMUNITY, Dec. 2002, p. 6976–6986 Vol. 70, No. 120019-9567/02/$04.00�0 DOI: 10.1128/IAI.70.12.6976–6986.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Expression of a Novel Leishmania Gene Encoding a Histone H1-LikeProtein in Leishmania major Modulates Parasite Infectivity In Vitro

Fotini T. Papageorgiou and Ketty P. Soteriadou*Department of Biochemistry, Hellenic Pasteur Institute, 115 21 Athens, Greece

Received 22 February 2002/Returned for modification 9 April 2002/Accepted 22 August 2002

We describe identification and characterization of a novel two-copy gene of the parasitic protozoan Leish-mania that encodes a nuclear protein designated LNP18. This protein is highly conserved in the genusLeishmania, and it is developmentally regulated. It is an alanine- and lysine-rich protein with potentialbipartite nuclear targeting sequence sites. LNP18 shows sequence similarity to H1 histones of trypanosomatidsand of higher eukaryotes and in particular with histone H1 of Leishmania major. The nuclear localization ofLNP18 was determined by indirect immunofluorescence and Western blot analysis of isolated nuclei by usingantibodies raised against the recombinant protein as probes. The antibodies recognized predominantly a18-kDa band or a 18-kDa–16-kDa doublet. Photochemical cross-linking of intact parasites followed by Westernblot analysis provided evidence that LNP18 is indeed a DNA-binding protein. Generation of transfectantsoverexpressing LNP18 allowed us to determine the role of this protein in Leishmania infection of macrophagesin vitro. These studies revealed that transfectants overexpressing LNP18 are significantly less infective thantransfectants with the vector alone and suggested that the level of LNP18 expression modulates Leishmaniainfectivity, as assessed in vitro.

Leishmania spp. are obligately intracellular protozoan par-asites that are members of the family Trypanosomatidae, andthey cause a wide spectrum of human diseases of major healthimportance, ranging from self-healing cutaneous lesions to se-vere visceral leishmaniasis or kala-azar with a high fatality rate(13). These parasites have two developmentally distinct stages;the amastigotes are nonmotile forms that survive and replicatewithin mammalian host macrophages, and the motile flagel-lated promastigotes survive and replicate inside the insect vec-tor (13).

During the last several years, considerable interest has beenfocused on an extensive study of the biology of these kineto-plastid protozoans, which are among the most primitive eu-karyotes (48). Leishmania has assumed importance in molec-ular biology by virtue of the unusual nature of its geneorganization and expression (39). The unusual features includethe presence of multiple copies of the same gene organized intandem arrays or adjacent genes encoding different proteins,which are often transcribed as large polycistronic precursors ofmature mRNAs (32, 37), RNA editing, and transsplicing (38,47). Leishmania molecular genetic studies have provided newinsights into the mechanisms of gene expression (reviewed inreference 38). However, very little is known about gene regu-lation in these protozoans.

In all eukaryotic cells, the DNA is highly compacted due toits association with histone proteins (33). The basic structuralunit of chromatin is the nucleosome, which comprises DNAwrapped tightly around an octameric-histone core having atripartite organization consisting of a central (H3-H4)2 tet-ramer flanked by two H2A-H2B dimers (1). Histones, despite

their low sequence homology, have a common motif, the his-tone fold (1, 2), which is considered to be a general proteindimerization motif. Most of the proteins classified in the his-tone fold superfamily are involved in protein-protein and/orprotein-DNA interactions (2, 6). This motif is found in a widevariety of transcriptional activators resembling histones (11),such as the archaeal DNA-binding proteins HMf, HMt, andHMv (54), the CCAAT-specific transcription factor CBF (60),and the TAFII42 and TAFII62 subunits of the TFIID tran-scriptional complex of Drosophila (30). The linker histones H1and H5 are essential for the organization of nucleosomes intoa higher-order structure (43, 55). Like the relationship of corehistones, a relationship between linker histones and transcrip-tion factors has also been suggested (16, 35).

There is great interest in histone genes in trypanosomatidsbecause in these organisms chromatin is not condensed intochromosomes during cell division but remains decondensed asfine fibers. However, the DNA is associated, probably weakly,with all classes of histones and is packed into nucleosomes (4).Characterization and systematic studies of the Trypanosomagenes coding for histones (3, 8, 25, 42), as well as the Leish-mania genes coding for histones (21, 26, 52, 53), have shownthat the sequence similarity with the genes coding for histonesin higher eukaryotes is low (reviewed in reference 24). Inparticular, the H1 histones of trypanosomatids have signifi-cantly lower molecular masses than the H1 histones of highereukaryotes, and there is only 43.6% similarity between theTrypanosoma cruzi and human H1 sequences. Moreover, incontrast to the histone genes of higher eukaryotes, trypanoso-matid histone genes are located on different chromosomes,and their transcripts are polyadenylated. Although histonegenes and their expression in trypanosomatids have been stud-ied extensively, little is known about the regulation of theirexpression (reviewed in reference 24).

In this paper we describe molecular cloning and character-

* Corresponding author. Mailing address: Department of Biochem-istry, Hellenic Pasteur Institute, 127 Vas. Sophias, 115 21 Athens, Greece.Phone: 30-(0)106478841. Fax: 30-(0)106423498. E-mail: [email protected].

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ization of a novel histone H1-like Leishmania nuclear DNA-binding protein, LNP18, and present evidence that LNP18plays a role in Leishmania infectivity.

MATERIALS AND METHODS

In vitro culture of Leishmania. Promastigotes were grown at 26°C in Dulbeccomodified Eagle medium (Gibco) containing 10% heat-inactivated fetal calf se-rum. The following strains were used in this study and in previous studies:Leishmania major LV39 and Leishmania infantum HOM-Gr78L4, isolated inGreece (57, 58); and Leishmania amazonensis LV78, kindly provided by K.-P.Chang. Amastigotes were isolated from lesions (at least 2 months old) of BALB/cmice infected with 1 � 106 stationary-phase L. major promastigotes by usingestablished protocols (28).

Screening of a cDNA library and DNA sequence analysis. An L. major cDNAlibrary was constructed in the lambda Uni-ZAP XR vector as described in thetechnical manual provided by Stratagene Inc. (La Jolla, Calif.). Poly(A)� mRNAfrom L. major promastigotes was used to synthesize cDNAs. A cDNA clone thatwas recognized by affinity-purified antibodies raised against the purified Leish-mania transferrin receptor molecule (59) was isolated and sequenced. Leishma-nia cDNAs in the Uni-ZAP XR vector were subsequently plated on Escherichiacoli XL-1 Blue cells, and the phagemids were excised by the methods describedin the manufacturer’s protocol manual (Stratagene Inc.). Subsequently, plasmidDNA from the phagemids was isolated and sequenced by using standard proce-dures (45).

Southern and Northern blot hybridization analyses. DNAs were isolated frompromastigotes, digested to completion (3 h at 37°C) with endonuclease XbaI (5U/�g of DNA), electrophoresed in 0.8% agarose containing 45 mM Tris, 45 mMboric acid, and 1 mM EDTA (pH 8), and transferred to nylon membranes; theagarose was stained with ethidium bromide. Plasmid DNA in Leishmania cellswas isolated by the alkaline lysis method (51). Total cellular RNA was extractedfrom Leishmania promastigotes by the hot acid phenol method as modified byBrown and Kafatos (10). For Northern blot analysis, total parasite RNA waselectrophoresed through a 1.2% (wt/vol) agarose gel containing formaldehydeand was subsequently transferred to blotting membranes (Zetaprobe; Bio-Rad).

Hybridization and washing for either DNA or RNA analysis were carried outas previously described (51). Filters were hybridized overnight at 65°C with32P-labeled cDNA probes, labeled by the random priming method (22), inhybridization solution containing 1% crystalline grade bovine serum albumin, 1mM Na2 EDTA, 0.5 M NaH2PO4 (pH 7.2), and 7% (wt/vol) sodium dodecylsulfate (SDS). The filters were washed as described by Church and Gilbert (14).The membranes were autoradiographed on X-OMAT AR film (Kodak) at�80°C with two intensifying screens. For rehybridization of a membrane, theprobe was eluted by two washes with 2 mM Tris–EDTA (pH 8.2)–0.1% (wt/vol)SDS for 15 min at 95°C.

The amounts of RNA loaded on filters were estimated by ethidium bromidestaining of agarose gels loaded with equivalent amounts of the samples, as wellas by staining the nitrocellulose filters with methylene blue in order to evaluatethe amounts of transferred rRNAs.

Probes. The following probes were used in this study: pBS10Rb.1, containinga 2.2-kb EcoRI fragment from an L. major gp63 cDNA clone (12), kindlyprovided by L. Button and R. McMaster (Medical Genetics, University of BritishColumbia, Vancouver, Canada); and T11, containing a 2-kb PstI fragment froman L. amazonensis �-tubulin cDNA clone, T11 (23), kindly provided by K.-P.Chang (Department of Microbiology and Immunology, University of HealthSciences, The Chicago Medical School, North Chicago, Ill.).

Production of antibodies. Anti-LNP18 antibodies were obtained from rabbitsimmunized with recombinant LNP18 (rLNP18). rLNP18 was produced by usingthe pRSET-E Echo cloning system (Invitrogen, Groningen, The Netherlands). Inthe Echo cloning system a one-step cloning strategy is utilized for direct insertionof a PCR product into an appropriate plasmid vector, and the classic cloningprocedures are not required. The fusion plasmid was transformed into TOP10cells, and positive clones were analyzed. To confirm the fusion junctions, isolatedplasmid DNA was analyzed by restriction analysis and sequencing. After over-expression in E. coli, rLNP18 was purified by affinity chromatography on Ni2�-nitrilotriacetate resin columns under denaturing conditions by following thesupplier’s instructions (Qiagen). Two New Zealand White rabbits were immu-nized subcutaneously three times every 2 weeks with 50 �g of rLNP18. Affinity-purified anti-LNP18 antibodies were isolated by low-pH elution from immuno-blots of purified rLNP18 as previously described (59).

Gel electrophoresis and immunoblotting. SDS-polyacrylamide gel electro-phoresis (PAGE) was performed on 12.5% polyacrylamide gels by the method of

Laemmli (34). The separated proteins were then transferred to nitrocellulose,and a Western blot analysis (56) was carried out essentially as previously de-scribed (50). The filters were blocked overnight at 4°C with 5% powdered nonfatmilk in Tris-buffered saline. After incubation with horseradish peroxidase-con-jugated secondary antibodies, the filters were developed in diaminobenzamidinewith nickel enhancement (0.03% [wt/vol] DAB and 0.03% [wt/vol] NiCl2 inTris-buffered saline).

Nucleus preparation. Nuclei were prepared as described by Hinterberger et al.(29). Briefly, stationary-phase promastigotes (109 cells) were washed and lysed in6 ml of NB buffer containing 10 mM HEPES, 10 mM MgCl2, 2 mM dithiothre-itol, and 250 mM sucrose supplemented with 0.5% (vol/vol) Triton X-100 in aDounce homogenizer. The lysate was centrifuged (20 min, 1,900 � g), and theresulting pellet was washed in NB buffer and centrifuged at 100,000 � g for 90min at 4°C over a 2 M sucrose cushion in NB buffer containing 0.5% (wt/wt)Triton X-100. The nuclear pellet was subsequently washed and resuspended in 50mM HEPES–5 mM MgCl2–2 mM dithiothreitol–1 mM EDTA containing 40%(vol/vol) glycerol.

Immunofluorescence staining. Promastigotes (106 cells/ml) were washed inphosphate-buffered saline (PBS) and fixed on glass microscope slides as previ-ously described (49). Antibodies were diluted in PBS containing 0.3% (wt/vol)bovine serum albumin. The primary antibody was then applied overnight at 4°C,and the fluorescence-conjugated secondary antibody was applied for 30 min atroom temperature. After immunostaining, coverslips were mounted on the glassslides in 90% glycerol in PBS, and the preparations were viewed either with aZeiss Axiophot photomicroscope or with a Leica laser scanning confocal micro-scope.

In vitro UV cross-linking of nucleic acids to proteins. For in vitro cross-linkingof DNA to proteins, we used the approach used by Pelle and Murphy (41).Briefly, isolated nuclei from approximately 5 � 109 L. major promastigotes werehomogenized on ice in 200 �l of homogenization buffer (25 mM Tris-HCl, 1 mMEDTA; pH 8) in a Dounce homogenizer. The lysate was centrifuged (20 min,1,900 � g), and the resulting nuclear extract was subsequently digested with 5 Uof Sau3AI per �l. Ten microliters of the nuclear extract in 400 �l of 25 mMHEPES–120 mM NaCl–6 mM KCl–2 mM MgCl2 (pH 7.4) was incubated atroom temperature for 20 min before UV irradiation. Irradiation was carried outwith 254-nm UV light for 20 min at 4°C. After UV treatment, 2 volumes of coldethanol was added, and the precipitate was pelleted by centrifugation. Theresulting pellet was dissolved in SDS-PAGE sample buffer, boiled for 5 min, andthen separated by SDS-PAGE and analyzed by Western blotting by using theanti-LNP18 antibodies as probes. For the control we used samples treated with100 U of DNase I per ml before or after UV cross-linking and samples preparedby a procedure from which the irradiation step was omitted.

Plasmid constructs. The pX63-HYG leishmanial expression vector (17) waskindly provided by S. Beverley. The cDNA of LNP18 was end filled with theKlenow fragment of DNA polymerase I (New England Biolabs, Hertfordshire,United Kingdom) and cloned into the SmaI restriction site of the pX63-HYGpolylinker.

Transfection of Leishmania. L. major LV39 stationary-phase parasites weretransfected with approximately 10 �g of pX63-HYG carrying the LNP18 geneand with pX63-HYG alone as a control. Cells were subjected to electroporationwith a Gene Pulser II (Bio-Rad) by using established protocols. After 24 h ofrecovery in hygromycin B-free medium 199, resistant cells were selected in thepresence of increasing concentrations of hygromycin B (up to 500 �g/ml).

RT-PCR. Total RNA was extracted from transfected LV39 promastigostes byusing an RNeasy kit for RNA isolation (Qiagen) according to the manufacturer’sinstructions. cDNA synthesis and a PCR were performed in an one-step reactionby using the Titan one-tube reverse transcription (RT)-PCR system (Roche). ForPCR the following LNP18- and �-actin-derived oligonucleotides were used:LNP18F (5�-AGGTGGCTACGCCGAAGAAGC-3�) and LNP18R (5�-ACTGCGGACGAAGTAGACAC-3�); and �-actinF (5�-GACTCCTATGTGGGTGACGAGG-3�) and �-actinR (5�-GGGAGAGCATAGCCCTCGTAGAT-3�).Each PCR was performed in a 50-�l reaction mixture containing 10 �l ofRT-PCR buffer, 25 pmol of each primer, 5 �l of a solution containing eachdeoxynucleoside triphosphate at a concentration of 2.5 mM (Promega), and 1 �lof enzyme mixture. In addition, 0.5 �l of an RNase inhibitor (RNasin; 40 U/�l;Promega) was added. The amplification cycle (denaturation at 94°C for 30 s,annealing at 60°C for 30 s, and extension at 68°C) was repeated 30 times, and thiswas followed by a 7-min final extension step.

Infection of macrophages. Macrophages of the J774 line (American TypeCulture Collection, Manassas, Va.) were plated in 96-well flat-bottom microcul-ture plates at a concentration of 106 cells/ml of medium 199. Lesion-derived L.major promastigotes (wild type, transfected with vector pX63-HYG alone andwith pX63-HYG carrying the LNP18 cDNA) were used in infection studies at a

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ratio of 10 parasites per cell. The microculture plates were incubated at 37°C inan atmosphere containing 5% CO2 for 5 h. After removal of nonadherentparasites, the macrophages were washed three times and incubated for an addi-tional 72 h. The cultures were then washed with medium, lysed with 100 �l of0.01% SDS/well, and incubated at 37°C for 30 min (62). Schneider’s mediumsupplemented with 10% fetal calf serum was added, and the released parasiteswere incubated at 26°C for 48 h. Cultures were subsequently pulsed with 1 �Ciof [3H]thymidine per well, and the radioactivity incorporated was counted witha �-counter. The results were expressed as means � standard errors of themeans. Data from three infection experiments were pooled.

Flow cytometry. Fluorescence-activated cell sorting analysis was performed for12,000 events, and numeric data were processed with Cellquest software. Ananalysis of infected and noninfected macrophages was performed as described byDi Giorgio et al. (18) by using antibodies generated against LV39 lesion-derivedamastigotes. Cells were incubated with antibodies in permeabilization buffer(0.1% sodium azide, 1% bovine serum albumin, 1% fetal calf serum, and 0.1%saponin in PBS [pH 7.4]) and then with fluorescein isothiocyanate-conjugatedanti-mouse immunoglobulins (1:1,000; Sigma). Cells were washed two times andwere analyzed with a FacSort analytical cytometer (Becton Dickinson).

Nucleotide sequence accession number. The nucleotide sequence reported inthis paper has been deposited in the EMBL, GenBank, and DDBJ databasesunder accession number LMA237814.

RESULTS

Cloning and sequencing of the LNP18 cDNA. In an effort toclone and characterize the Leishmania transferrin receptorgene, a molecule that was first identified in our laboratory (59),a cDNA clone encoding a novel protein, LNP18, was acciden-tally identified and subsequently sequenced. Figure 1 showsthe complete nucleotide sequence of the 0.690-kb cDNA insertand the deduced amino acid sequence. The deduced aminoacid sequence predicts a basic protein (pI 12.2) containing 98amino acids and having a predicted molecular mass of 9.9 kDa.The LNP18 predicted amino acid sequence was compared withthe sequences of proteins listed in SBASE by advancedBLAST. This search revealed that LNP18 belongs to the his-tone H1-histone H5 family. Interestingly, LNP18 shows signif-

icant similarity with H1 histones of members of the Trypano-somatidae. Sequence alignments for LNP18 and the H1histones of T. cruzi and other Leishmania species, obtained byusing CLUSTALW, are shown in Fig. 2A. It should be notedthat H1 histones in eukaryotes are less conserved than the corehistones and exhibit a high degree of variability. Moreover, H1histones in members of the Trypanosomatidae have muchlower molecular masses than their eukaryotic counterparts andexhibit exclusive motifs characteristic of the C-terminal do-main of eukaryotic H1 histones (reviewed in reference 24).Galanti et al. found 43.6% similarity between T. cruzi H1 andthe C-terminal region of human H1 (24). Furthermore, the L.major (21) and T. cruzi H1 histones (24) exhibit 61.79% simi-larity (data not shown). The sequence of LNP18 was comparedwith that of L. major histone H1. This comparison (Fig. 2B)showed that 61% of the residues are either identical (56%) orwell conserved (5%). Thus, LNP18 is a histone H1-like protein.

Analysis of the sites and signatures of the LNP18 amino acidsequence revealed that it is an alanine- and lysine-rich proteinwith eight potential sites of bipartite nuclear localization sig-nals (Fig. 1). Bipartite nuclear localization signals are known tobe necessary for nuclear targeting (5, 19, 44, 63). Interestingly,the same motif is present in almost one-half the nuclear pro-teins in the Swiss protein database but in less than 5% of thenonnuclear proteins, and many of these proteins are secretedor targeted to other organelles. An example of a moleculehaving a relatively low molecular mass (143 residues) that hasa functional nuclear localization signal-like motif is humangamma interferon (63). A hydrophobicity analysis performedby using PC GENE suggests that LNP18 is a globular protein(data not shown).

Genomic organization and expression of the LNP18 gene:Southern and Northern analyses. Southern blot analysis wasperformed to determine the copy number of the LNP18 geneby using genomic DNAs from L. major and L. infantum pro-mastigotes. The DNAs were digested with the following re-striction enzymes, separately or in combination: EcoRI, Hin-dIII, and BglI, which have no internal sites within the cDNA;and Eco0109I and AvaI, which have one internal site. As shownin Fig. 3A, digestion with enzymes having no internal siteswithin either the L. major cDNA (lanes 1 to 3) or the L.infantum genomic DNA (lanes 8 and 9) resulted in two bandswhen the preparations were hybridized (under highly stringentconditions) with a probe containing the complete LNP18cDNA. If the physical maps of sw3.0 and sw3.1 and the physicalmap of LNP18 (Fig. 3B) are taken into account, the possibilityof cross-hybridization of the LNP18 cDNA probe with frag-ments of the two L. major histone H1 genes cannot be ex-cluded. However, it has been reported that when the sw.3 openreading frame is used as a probe in Southern blots, it does notdetect any digests in Leishmania donovani, a species that be-longs to the same complex as L. infantum (7). This findingsupports the assumption that at least in L. infantum the twofragments detected correspond to digests of the LNP18 locus.When the L. major DNA was digested with AvaI (Fig. 3A, lane5), three bands, at1.9, 1.75, and 0.85 kb, were observed; the0.85-kb band probably corresponds to an sw3.1 fragment of theL. major H1 gene (Fig. 3B, map I). BglI digestion resulted intwo bands, at 2 and 1.8 kb (Fig. 3A, lane 3). However, AvaI-BglI double digestion resulted in a 0.85-kb band (Fig. 3A, lane

FIG. 1. Nucleotide sequence of L. major LNP18 cDNA. The de-duced amino acid sequence is indicated below the DNA sequence. Thedomain that contains the eight potential sites of bipartite nuclearsequences (bipartite nuclear localization signals) is underlined. Theamino acid sequence is indicated by the single-letter amino acid code.

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4) that could correspond to the AvaI fragment detected in Fig.3A, lane 5. The possibility that this fragment could be a diges-tion product of the sw3 locus that cross-hybridized with theLNP18 probe, due to the high homology of the two genes,cannot be excluded. However, two of the three bands detectedin Fig. 3A, lane 4 (the 0.75- and 0.7-kb bands), cannot beattributed to fragments of L. major H1, since there are nointernal BglI sites in the AvaI fragments of the two H1 genesequences in the region that shows homology to the LNP18cDNA probe (Fig. 3B). The latter finding is consistent with thehypothesis that L. major LNP18 is a two-copy gene.

Transcription of the gene was confirmed by Northern blotanalysis of total RNAs extracted from diverse species of OldWorld and New World Leishmania promastigotes. As shown inFig. 4A, under highly stringent conditions the probe recog-nized two transcripts (approximately 0.8 and 0.7 kb). The sizesof these transcripts are consistent with the size of the reportedfull-length cDNA. These two transcripts may be due to multi-ple polyadenylation sites or to two closely related genes orcopies.

Subsequently, we examined the level of expression of LNP18in the amastigote stage of L. major parasites (Fig. 4A, lane 4).Estimation of the amount of RNA transferred onto filters, asdescribed in Materials and Methods, showed that the level ofamastigote RNA was at least four times higher than the levelof promastigote RNA. On the other hand, the level of the0.8-kb transcript in L. major promastigotes was 15- to 20-foldhigher than the corresponding level in amastigotes, as deter-mined by scanning densitometry of a blot that was exposed fora shorter period of time (data not shown). This was evaluated

by simultaneously hybridizing the filter with the LNP18 cDNAprobe and a 2.2-kb EcoRI fragment from L. major gp63 cDNA.gp63 is down-regulated at both the mRNA and protein levelsin amastigotes. Interestingly, only one transcript (0.8 kb) wasdetected in L. major amastigotes, but the level was significantlylower (Fig. 4A, lane 4). Most probably the transcript detectedwas due to cross-hybridization of the LNP18 probe with thehistone H1 transcript due to overloading of the gel. HistoneH1 is up-regulated in amastigotes compared to promastigotes(21, 40).

The level of expression of LNP18 in amastigotes was alsoanalyzed by RT-PCR. To do this, RNAs were isolated fromlesion-derived amastigotes and promastigotes and were ampli-fied by using primers designed from the LNP18 open readingframe. Using this approach, we showed that LNP18 expressionwas significantly lower in amastigotes than in promastigotes(Fig. 4B, lanes 4 and 2, respectively). Importantly, LNP18protein was not detected in L. major amastigotes (Fig. 5B).The significance of this finding is discussed below.

Identification and localization of native Leishmania LNP18.Anti-LNP18 antibodies, generated by using rLNP18, recog-nized in Western blots of L. major lysates an 18-kDa protein oran 18-kDa–16-kDa doublet (Fig. 5A, lane 1). The antibodiesrecognized the same bands for different Leishmania species,indicating that LNP18 is a highly conserved protein (Fig. 5A).The LNP18 antibodies do not seem to cross-react with theclosely related H1 protein, since they do not recognize anypolypeptides in lesion-derived amastigotes (Fig. 5B lane 1); ithas been demonstrated in several studies that expression ofH1, at both the mRNA and protein levels, is significantly

FIG. 2. (A) Alignment of amino acid sequences of trypanosomatid histones H1 and LNP18. (B) Alignment of LNP18 and L. major histone H1(19) sequences. The asterisks, colons, and dots indicate aligned residues that are identical, very similar, and similar, respectively. Gaps (dashes)were introduced for optimal alignment. The numbers at the beginning and the end of a line indicate amino acid positions in the parent proteins.L.brazil., L. braziliensis; L.mexic., L. mexicana.



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higher in lesion-derived amastigotes than in L. major promas-tigotes (21, 40). In order to ensure that the amount of amas-tigote protein transferred was sufficient to detect LNP18, blotswere cohybridized with antibodies against �-tubulin, a proteinthat is down-regulated in the amastigote stage of L. major (15).The fact that the intensities of �-tubulin in the two stages werecomparable suggests that the amount of protein loaded in lane

1 (amastigotes) of Fig. 5B was severalfold higher than theamount of protein loaded in lane 2 (promastigotes). Evenunder these experimental conditions LNP18 was not detectedin amastigotes. Therefore, the difference in the level of expres-sion of LNP18 between the two stages is even greater than thedifference shown in Fig. 5B.

The molecular mass of purified rLNP18 was also estimated

FIG. 3. (A) Southern blots of L. major (lanes 1 to 7) and L. infan-tum (lanes 8 to 11) genomic DNAs digested with various restrictionenzymes. Blots were hybridized with LNP18 cDNA. The followingrestriction enzymes were used: HindIII (lanes 1 and 8); HindIII andEcoRI (lane 2); BglI (lanes 3 and 9); BglI and AvaI (lane 4); AvaI (lane5); Eco0109I (lanes 6 and 10); and BglI and Eco0109I (lanes 7 and 11).(B) Map I is a physical map of L. major H1 (copies sw3.0 and sw3.1)and the L. infantum LNP18 gene (EMBL/GenBank/DDBJ accessionno. AJ223860, AJ223861, and AF469106, respectively). Map II is aphysical map of the sequenced gene of L. infantum and of the twocopies of the L. infantum LNP18 gene deduced from both Southernblot analysis (panel A, lanes 8 to 11) and restriction enzyme analysis ofthe sequenced LNP18 DNA gene. Map III is a physical map of the twocopies of L. major LNP18 deduced from Southern blot analysis (panelA, lanes 1 to 7) and restriction enzyme analysis of the sequencedLNP18 cDNA. The cross-hatched boxes indicate the homology ofsw3.1 and sw3.0 to the cDNA of L. major LNP18. The solid boxescorrespond to the LNP18 cDNA sequence in both species. The dashedlines represent DNA that has not been sequenced yet. Enzyme abbre-viations: E, EarI; A, AvaI; B, BglI; Ec, Eco0109I.



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to be 18 kDa (data not shown), a value that is significantlyhigher than the value expected from the deduced amino acidsequence. It should be noted that it is becoming increasinglyevident that very basic or acidic proteins may migrate anoma-lously on SDS-PAGE gels (31). Therefore, the anomalous mi-gration of LNP18 might be due to abnormal binding of SDS tobasic amino acids, since LNP18 is a Lys-rich basic protein,which in turn might lead to an abnormal shape of the SDS-protein complex. It should be pointed out that anomalousmigration of LNP18 in SDS-PAGE gels is a common charac-teristic of histone H1 of L. major. The amino acid sequence ofthe latter protein deduced from the cDNA predicts that theprotein has 105 amino acid residues. However, specific anti-bodies recognize a 17-kDa–19-kDa doublet in different L. ma-jor strains (40). Like H1 polymorphism, LNP18 size polymor-phism may reflect the presence of LNP18 variants possessingcommon epitopes. Moreover, Western blot analysis of thetransfectants overexpressing LNP18 showed that the bandsdetected were derived from the cloned cDNA (Fig. 6C, lane 2).

Sequence analysis of L. infantum LNP18 genomic clones andcomparison with the L. major LNP18 cDNA sequence revealeda putative initiation codon 39 nucleotides immediately up-stream of the codon proposed for L. major LNP18. Since theopen reading frames of the two genes are identical, we cannoteliminate the possibility that this in-frame ATG could be thetranslation start codon for both genes. In such a case themolecular mass of the deduced amino acid sequence would beslightly larger (11 kDa; 111 amino acids with a pI of 11.74) thanthe predicted Mr of L. major (9.9 kDa). This analysis furthersupports the hypothesis that the proteins detected are theproducts of the LNP18 gene.

LNP18 expression was also investigated during in vitrogrowth of promastigotes from the logarithmic phase to thestationary phase (that is, as promastigotes differentiate from anoninfective stage to an infective stage) (Fig. 5C). An �-tubu-lin antibody was used to examine whether LNP18 is differen-tially expressed during parasite differentiation. The house-keeping gene is down-regulated twofold in stationary-phasepromastigotes compared to log-phase promastigotes (15).Densitometry analysis of both �-tubulin bands and LNP18

bands for the two developmental stages revealed ratios of 3.5(lane 2 versus lane 1) and 1.7 (lane 1 versus lane 2), respec-tively. These data, combined with the known down-regulationof the �-tubulin protein, suggest that the LNP18 level increasesthreefold during promastigote maturation in culture.

The observed sequence similarity of LNP18 and H1 histonesprompted us to investigate the possible nuclear localization ofLNP18. Western blot analysis of Leishmania isolated nuclei(Fig. 5D) and staining of whole promastigotes by indirect im-munofluorescence (Fig. 5E), when the anti-LNP18 antibodieswere used as probes, showed that the protein is located exclu-sively in the nucleus (Fig. 5D, lane 1). To obtain a better ideaof the LNP18 localization, we performed confocal microscopy(Fig. 5F). No protein was detected in the cytoplasmic extracts(Fig. 5D, lane 2) or the cytoplasm of whole parasites (Fig. 5Eand F) with either approach. Ponceau S staining of membranesafter protein transfer confirmed that the amounts of proteintransferred to nitrocellulose were equivalent (data not shown).

In vitro UV-cross-linking hybridization: detection of anLNP18-DNA complex. In order to demonstrate that LNP18,which has characteristics of a DNA-binding protein, does in-teract with Leishmania DNA, we used the UV-cross-linkinghybridization technique (41). Nuclear extracts were irradiatedas described in Materials and Methods. UV irradiation inducescross-links between nucleic acids and proteins in close contact.This method has an additional advantage, its specificity. Thus,boiling extracts from irradiated cells in the presence of SDScauses the dissociation of any nonspecific nucleic acid-proteincomplexes. After UV treatment, samples were separated bySDS-PAGE and analyzed by Western blotting. Hybridizationof the blot with the anti-LNP18 antibodies revealed an approx-imately 94-kDa band (Fig. 7, lane 4). In contrast, the 94-kDaband was not detected when samples were treated with DNaseI prior to UV treatment and when nonirradiated nuclear con-trol samples were used (Fig. 7, lanes 2 and 1, respectively).Treatment with DNase I after UV cross-linking slightly af-fected the migration of the 94-kDa complex. Most probably theresulting 90-kDa band (lane 3) corresponds to a DNA-proteincomplex. Hence, by using this simplified method which induces

FIG. 4. (A) Northern blot analysis of total RNAs extracted from promastigotes of different species of Leishmania (L. amazonensis, L. tropica,L. donovani, and L. major [lanes1 to 3 and 5, respectively]) and L. major amastigotes (lane 4), separated on a 1.2% (wt/vol) agarose–formaldehydegel. After blotting, the filters were hybridized either with LNP18 cDNA and the �-tubulin probe (lanes 1 to 3) or with LNP18 cDNA and the gp63probe (lanes 4 and 5) and autoradiographed. The marker on the right indicates the size and mobility of 28S� rRNA. (B) RT-PCR analysis ofamastigotes and promastigotes. Total RNA isolated from promastigotes (lanes 1 and 2) or amastigotes (lanes 3 and 4) was reversed transcribed,and primers derived from LNP18 (lanes 2 and 4) and �-actin (lanes 1 and 3) were used. The positions of size markers are indicated on the left.



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FIG. 5. (A) Gel electrophoresis and immunoblotting of L. major, L. infantum, and L. amazonensis promastigotes (lanes 1 to 3, respectively)probed with antibodies raised against the recombinant protein (anti-rLNP18). The positions of molecular size standards are indicated on the left.(B) Western blot analysis of lysates from lesion-derived amastigotes (lane 1) and promastigotes (lane 2). Lysates were separated by SDS-15%PAGE, electroblotted, and probed with anti-rLNP18 and �-tubulin antibodies (NeoMarkers). The positions of molecular size standards areindicated on the left. (C) Western blot analysis of L. major promastigotes grown to the early logarithmic phase (day 3) (lane 2) and the stationaryphase (day 5) (lane 1). The blot was probed with anti-rLNP18 and �-tubulin antibodies. (D) L. major isolated nuclei and cytoplasmic extracts fromthe same number of parasites (lanes 1 and 2, respectively). SDS-PAGE was performed under reducing conditions on 12.5% polyacrylamide gels.The positions of molecular size standards are indicated on the left. (E and F) Immunofluorescence labeling of promastigotes when the anti-rLNP18antibodies were used as probes. L. major promastigotes (1.5 � 10 6 cells/ml of PBS) were fixed on glass microscope slides. Binding of anti-rLNP18in the nucleus was visualized by using a secondary antibody conjugated with fluorescein isothiocyanate (FITC) and either phase-contrastmicroscopy performed with a photomicroscope (E) or confocal laser microscopy (F). For confocal microscopy an overlay image is also shown.



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cross-links between nucleic acids and proteins in close contact,we showed that LNP18 is a potential DNA-binding protein.

Modulation of LNP18 level. Together, our data indicate thatLNP18 has interesting features that make its function implicit.Therefore, we developed L. major transfectants in which theendogenous level of LNP18 was modulated. This was accom-plished by transfecting L. major promastigotes with a plasmidconstruct developed by cloning the cDNA of LNP18 in thesense orientation into the SmaI restriction site of the pX63-HYG polylinker. Transfectants with the LNP18 cDNA andtransfectants with the vector alone, developed in parallel, wereselected in medium 199 with hygromycin B at a final concen-tration of up to 500 �g/ml. The integrity of the plasmids inthese transfectants was confirmed by restriction mapping andsequencing, as well as by Southern blot analysis with specificprobes (Fig. 6A). As expected, semiquantitative RT-PCR per-formed with RNA isolated from the transfectants showed sig-nificantly higher levels of LNP18 RNA in the pX63-HYG–LNP18 transfectants than in the transfectants with the vectoralone (Fig. 6B, lanes 2 and 4, respectively). The level of LNP18protein was significantly increased (approximately four- to five-fold) in the pX63-HYG–LNP18 transfectants compared to thecontrol (Fig. 6C). This was estimated by densitometry analysisof the intensity of the LNP18 18-kDa–16-kDa doublet in con-trols and transfectants overexpressing LNP18 compared withthe intensity of �-tubulin.

Infection of mouse macrophages. Macrophages (cell lineJ774) cultured in 96-well plates were infected at a ratio of 10parasites per cell for 5 h with stationary-phase wild-type strainLV39 promastigotes and with LNP18-overexpressing andcontrol plasmid-containing transfectants. The growth rate ofamastigotes within macrophages was subsequently monitored.There was no difference in the ability to invade macrophages,the number of infected macrophages, or the number of intra-cellular parasites during the first 24 h of infection. This was

evaluated by cytospin and Giemsa staining of cells (data notshown). However, as shown in Fig. 8A, the number of amas-tigotes released from macrophages (transformed into promas-tigotes when they were incubated at 26°C for 48 h) infectedwith the LNP18 transfectants was significantly lower (sevenfoldlower) than the number of parasites released from macro-phages infected with promastigotes containing the controlplasmid and with wild-type promastigotes. Similar results wereobtained in three separate experiments performed with para-sites generated in two transformation experiments.

It should be noted that amastigotes released from macro-

FIG. 6. Monitoring LNP18 expression in transfected LV39 parasites. (A) Southern blot analysis of extrachromosomal DNA isolated fromparasites transfected with pX63–HYG and from parasites transfected with pX63-HYG–LNP18 (lanes 1 and 3 and lanes 2 and 4, respectively). TheDNA (5 �g per lane) was digested with BglI and probed with the BamHI-EcoRI fragment of the HYG coding region (lanes 1 and 2). Themembrane was stripped and reprobed with the LNP18 coding region (lanes 3 and 4). The positions of size markers are indicated on the left.(B) RT-PCR analysis of the transfectants. Total RNA extracted from parasites transfected with either pX63-HYG–LNP18 (lanes 1and 2) orpX63-HYG (lanes 3 and 4) was reversed transcribed by using the LNP18-derived primers (lanes 2 and 4) and the �-actin-derived primers (lanes1 and 3), which were used as a control. The positions of size markers are indicated on the left. (C) Immunoblot analysis of transfected LV39 totallysates (lane1, pX63-HYG; lane 2, pX63-HYG–LNP18). The blot was probed with anti-LNP18 and anti-�-tubulin antibodies (used as a loadingcontrol). The positions of standard size markers are indicated on the left.

FIG. 7. In vitro UV-cross-linking analysis of nuclear L. major ex-tracts. UV irradiation and Western blot analysis of the extracts wereperformed as described in Materials and Methods. Lane 1, DNase Itreatment prior to UV treatment; lane 2, nontreated extracts used as acontrol; lane 3, UV treatment followed by DNase I treatment; lane 4,UV treatment.



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phages transformed into promastigotes at 26°C grew in thepresence of hygromycin B, indicating that they retained theplasmid. These results suggest that LNP18 affects intramac-rophage survival and/or replication of these parasites.

The infection rates of the transfectants were also evaluatedby flow cytometry. Amastigote-containing macrophages weredetected with a polyclonal mouse antibody raised againstamastigotes isolated from lesions of infected BALB/c mice andpurified by low-pH elution of antibodies from strips blottedwith amastigote lysates. By using this approach the infectionrates in L. major-infected macrophages were measured with ahigh degree of specificity and reproducibility. The histogram inFig. 8B clearly shows two well-defined populations correspond-ing to non-amastigote-containing cells and infected cells. Theflow cytometric assay could not separate cells containing asingle parasite from cells containing more than one amastigote.The accuracy and reproducibility of flow cytometry for detec-tion of intracellular amastigotes, as well as tests for the efficacyof drugs against intracellular parasites, were recently examinedin two independent studies (18, 27). The results of four inde-

pendent experiments showed that the infectivity of the trans-fectants overexpressing LNP18 was significantly lower thanthat of the control plasmid-containing transfectants; the num-ber of infected cells differed by threefold. The infectivity of thelatter (approximately 35 to 40% of macrophages contained atleast one amastigote) was comparable to that of wild-typeparasites. The results obtained with both approaches stronglysuggest that the level of expression of LNP18 modulates par-asite infectivity.

DISCUSSION

In this paper we describe identification and characterizationof a novel two-copy gene of Leishmania that encodes a devel-opmentally regulated nuclear protein, designated LNP18.LNP18 is a DNA-binding protein that shows sequence similar-ity with H1 histones of trypanosomatids and, in particular, withhistone H1 of L. major. It is recognized as a 18-kDa–16-kDaprotein doublet which is highly conserved in the genus Leish-mania. Both polypeptides appear to be related to LNP18 since

FIG. 8. In vitro infection assays. (A) Macrophages were infected with wild-type lesion-derived promastigotes and LV39 transfected withpX63-HYG and with pX63-HYG–LNP18 for 5 h, washed, and incubated for 72 h (lanes 1 to 3, respectively). The cultures were then washed withmedium and lysed with 0.01% (wt/vol) SDS to release the surviving amastigotes, which were cultured for an additional 48 h. Cultures weresubsequently pulsed with 1 �Ci of [3H]thymidine per well, and incorporation of radioactivity was counted with a �-counter. Data from threeindependent experiments (12 wells per transfectant or wild-type promastigotes) were pooled, and the standard errors are indicated by error bars.(B) Fluorescence-activated cell sorter histograms acquired with noninfected macrophages (histogram 1) and macrophages infected with LV39 wildtype (histogram 2) and LV39 transfected with either pX63-HYG (histogram 3) or pX63-HYG–LNP18 (histogram 4). The macrophages wereinfected for 5 h, washed, and incubated for 72 h. The cultures were washed again and processed for fluorescence-activated cell sorter analysis asdescribed in Materials and Methods.



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analysis of transfectants overexpressing the gene showed thatboth polypeptides were up-regulated. Furthermore, the possi-bility that anti-LNP18 antibodies cross-react with the closelyrelated L. major H1 protein must be excluded since the LNP18antibodies do not seem to cross-react with the histone H1protein in lesion-derived amastigotes (Fig. 5B, lane 1), in whichthe expression of H1, at both the mRNA and protein levels, issignificantly higher than the expression of H1 in L. majorpromastigotes (21, 40). Like histone H1 polymorphism in L.major, LNP18 polymorphism may reflect the presence of vari-ants possessing common epitopes, possibly as a result of post-transcriptional modification, a feature seen in H1 histones ofhigher eukaryotes (46).

Several lines of evidence suggest that functional signifi-cance underlies the heterogeneity of the H1 class of his-tones. In mice there are at least seven different H1 variants,which display different patterns of expression during devel-opment and differentiation (20). Thus, we investigated theexpression profile of LNP18 during parasite growth anddifferentiation in vitro. The LNP18 level appeared to in-crease threefold as promastigotes differentiated from a non-infective stage to an infective stage and decreased several-fold in the amastigote stage, as assessed by Northern andWestern blot analyses.

Infection of macrophages in vitro with transfectants overex-pressing LNP18 showed that they are significantly less infectivethan the transfectants with the vector alone. These resultssuggest that the level of LNP18 expression modulates Leish-mania infectivity. The mechanism by which the level of LNP18expression affects Leishmania infectivity is not known yet.However, there is considerable evidence that histone H1 func-tions as a nonspecific repressor of transcription in higher eu-karyotes and in Saccharomyces cerevisiae (36, 61). Similar ob-servations have been made for the protozoan Tetrahymena,which is evolutionarily close to Leishmania. It has been shownthat histone H1 is involved in the modulation of gene expres-sion. Therefore, it is possible that LNP18 is involved in controlof the expression of genes modulating Leishmania infectivity.

Recent findings also support the linkage of H1 expressionwith the cell cycle. In Trypanosoma brucei, H1 variants havebeen postulated to participate in the regulation of cell cycleprogression and differentiation (3), as has been postulated forhigher eukaryotes (9). Expression of LNP18, an H1-like pro-tein, could be also linked to the cell cycle and gene expressionand thus could control parasite differentiation and/or replica-tion. Studies are in progress to define the functional role of thisgene and protein in parasite growth and/or development.Moreover, the ability of transfectants to establish infections invivo is currently being investigated.

ACKNOWLEDGMENTS

We thank D. Smirlis for critically reviewing the manuscript.This work was supported by the Greek General Secretariat of Re-

search and Technology and by the EU (INCO-DC contract ERBIC 18CT 97-0252).

REFERENCES

1. Arents, G., R. W. Burlingame, B.-C. Wang, M. E. Love, and E. Moudri-anakis. 1991. The nucleosomal core histone octamer at 3.1 A resolution: atripartite protein assembly and a left-handed superhelix. Proc. Natl. Acad.Sci. USA 88:10148–10152.

2. Arents, G., and E. N. Moudrianakis. 1995. The histone fold: a ubiquitousarchitectural motif utilized in DNA compaction and protein dimerization.Proc. Natl. Acad. Sci. USA 92:11170–11174.

3. Aslund, L., L. Carisson, J. Henriksson, M. Rydaker, C. Toro, N. Galanti, andU. Pettersson. 1994. A gene family encoding heterogeneous histone H1proteins in Trypanosoma cruzi. Mol. Biochem. Parasitol. 65:317–330.

4. Astolfi, F. S., C. Martins de Sa, and E. S. Gander. 1980. On the chromatinstructure of Trypanosoma cruzi. Mol. Biochem. Parasitol. 1:45–53.

5. Bader, T., and J. Wietzerbin. 1994. Nuclear accumulation of interferongamma. Proc. Natl. Acad. Sci. USA 91:11831–11835.

6. Baxevanis, A. D., G. Arents, E. N. Moudrianakis, and D. Landsman. 1995. Avariety of DNA-binding and multimeric proteins contain the histone foldmotif. Nucleic Acids Res. 23:2685–2691.

7. Belli, S., A. Formenton, T. Noll, A. Ivens, R. Jacquet, C. Desponds, D. Hofer,and N. Fasel. 1999. Leshmania major histone H1 gene expression from thesw3 locus. Exp. Parasitol. 91:151–160.

8. Bontempi, E. J., B. M. Porcel, J. Henriksson, L. Carisson, M. Rydaker, E. L.Segura, A. M. Ruiz, and U. Pettersson. 1994. Genes for histone H3 inTrypanosoma cruzi. Mol. Biochem. Parasitol. 66:147–151.

9. Brown, D. T., B. T. Alexander, and D. B. Sittman. 1996. Differential effect ofH1 variant overexpression on cell cycle progression and gene expression.Nucleic Acids Res. 24:486–493.

10. Brown, N. H., and F. C. Kafatos. 1988. Functional cDNA libraries fromDrosophila embryos. J. Mol. Biol. 203:425–430.

11. Burley, S. K., X. Xie, K. L. Clark, and F. Shu. 1997. Histone-like transcrip-tion factors in eukaryotes. Curr. Opin. Struct. Biol. 7:94–102.

12. Button, L. L., W. R. McMaster. 1988. Molecular cloning of the major surfaceantigen of Leishmania. J. Exp. Med. 167:724–729.

13. Chang, K.-P. 1983. Cellular and molecular mechanisms of intracellular sym-biosis in leishmaniasis. Int. Rev. Cytol. 14(Suppl.):267–305.

14. Church, G. M., and W. Gilbert. 1984. Genomic sequencing. Proc. Natl. Acad.Sci. USA 81:1991–1995.

15. Coulson, R., V. Connor, J. Chen, and J. W. Ajioka. 1996. Differential ex-pression of Leishmania major �-tubulin genes during acquisition of promas-tigote infectivity. Mol. Biochem. Parasitol. 82:227–236.

16. Croston, G. E., L. A. Kerrigan, L. M. Lira, D. R. Marshak, and J. T.Kadonaga. 1991. Sequence-specific antirepression of histone H1-mediatedinhibition of basal RNA polymerase II transcription. Science 251:643–649.

17. Cruz, A., C. M. Coburn, and S. M. Beverley. 1991. Double targeted genereplacement for creating null mutants. Proc. Natl. Acad. Sci. USA 88:7170–7174.

18. Di Giorgio, C., O. Ridoux, F. Delmas, N. Azas, M. Gasquet, and P. Timon-David. 2000. Flow cytometric detection of Leishmania parasites in humanmonocyte-derived macrophages: application to antileishmanial-drug testing.Antimicrob. Agents Chemother. 44:3074–3078.

19. Dingwall, C., J. Robbins, and S. M. Dilworth. 1989. Characterisation of thenuclear location sequence of Xenopus nucleoplasmin. J. Cell Sci. 11(Suppl.):243–248.

20. Doenecke, D., W. Albig, H. Bouterfa, and B. Drabent. 1994. Organizationand expression of H1 histone and H1 replacement histone genes. J. CellBiochem. 54:423–431.

21. Fasel, N. J., D. C. Robyr, J. Mauel, and T. A. Gaser. 1993. Identification ofa histone H1-like gene expressed in Leishmania major. Mol. Biochem. Para-sitol. 62:321–324.

22. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNArestriction endonuclease fragments to high specific activity. Anal. Biochem.132:6–13.

23. Fong, D., and B. Lee. 1988. Beta tubulin gene of the parasitic protozoanLeishmania mexicana. Mol. Biochem. Parasitol. 31:97–106.

24. Galanti, N., M. Galindo, V. Sabaj, I. Espinoza, and G. C. Toro. 1998. Histonegenes in trypanosomatids. Parasitol. Today 14:64–70.

25. Garcia-Salcedo, J., J. L. Olivier, R. P. Stock, and A. Gonzalez. 1994. Mo-lecular characterization and transcription of the histone H2B gene from theprotozoan parasite Trypanosoma cruzi. Mol. Microbiol. 13:1033–1043.

26. Genske, J. E., B. R., Cairns, S. P. Stack, and S. M. Landfear. 1991. Structureand regulation of histone H2B mRNAs from Leishmania enriettii. Mol. Cell.Biol. 11:240–249.

27. Guinet, F., A. Louise, H. Jouin, J. C. Antoine, and C. W. Roth. 2000.Accurate quantitation of Leishmania infection in cultured cells by flow cy-tometry. Cytometry 39:235–240.

28. Hart, D. T., K. Vickerman, and G. H. Coombs. 1981. A quick simple methodfor purifying Leishmania mexicana amastigotes in large numbers. Parasitol-ogy 8:345–355.

29. Hinterberger, M., I. Pettersson, and J. A. Steiz. 1982. Isolation of smallnuclear ribonucleoproteins containing U1, U2, U4, U5, and U6 RNAs.J. Biol. Chem. 258:2604–2613.

30. Hoffmann, A., C.-M. Chiang, T. Oelgeschlager, X. Xie, S. K. Burley, Y.Nakatani, and R. G. Roeder. 1996. A histone octamer-like structure withinTFIID. Nature 380:356–359.

31. Hu, C. C., and S. A. Ghabrial. 1995. The conserved, hydrophilic and argin-ine-rich N-terminal domain of cucumovirus coat proteins contributes to their



http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

anomalous electrophoretic mobilities in sodium dodecyl sulfate-polyacryl-amide gels. J. Virol. Methods 55:367–379.

32. Johnson, P. J., J. M. Kooter, and P. Borst. 1987. Inactivation of transcriptionby UV irradiation of T. brucei provides evidence for a multicistronic tran-scription unit including a VSG gene. Cell 51:273–281.

33. Kornberg, R. D., and J. O. Thomas. 1974. Chromatin structure; oligomers ofthe histones. Science 184:865–868.

34. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680–685.

35. McPherson, C. E., E.-Y. Shim, D. S. Friedman, and K. S. Zaret. 1993. Anactive tissue-specific enhancer and bound transcription factors existing in aprecisely positioned nucleosomal array. Cell 75:387–398.

36. Miloshev, G., P. Venkov, K. van Holde, and J. Zlatanova. 1994. Low levels ofexogenous histone H1 in yeast cause cell death. Proc. Natl. Acad. Sci. USA91:11567–11570.

37. Muhich, M. L., and J. C. Boothroyd. 1988. Polycistronic transcripts in try-panosomes and their accumulation during heat shock: evidence for a pre-cursor role in mRNA synthesis. Mol. Cell. Biol. 8:3837–3846.

38. Myler, P. J., and K. D. Stuart. 2000. Recent developments from the Leish-mania genome project. Curr. Opin. Microbiol. 3:412–416.

39. Nilsen, T. W. 1994. Unusual strategies of gene expression and control inparasites. Science 264:1868–1869.

40. Noll, T. M., C. Desponds, S. I. Belli, T. A. Glaser, and N. J. Fasel. 1997.Histone H1 expression varies during the Leishmania major life cycle. Mol.Biochem. Parasitol. 84:215–227.

41. Pelle, R., and N. B. Murphy. 1993. In vivo UV-cross-linking hybridization: apowerful technique for isolating RNA binding proteins. Application to try-panosome mini-exon derived RNA. Nucleic Acids Res. 21:2453–2458.

42. Puerta, C., J. Martin, C. Alonso, and M. C. Lopez. 1994. Isolation andcharacterization of the gene encoding histone H2A from Trypanosoma cruzi.Mol. Biochem. Parasitol. 64:1–10.

43. Ramakrishnan, V., J. Finch, V. M. Grazziano, P. L. Lee, and R. Sweet. 1993.Crystal structure of globular domain of histone H5 and its implications fornucleosome binding. Nature 362:219–223.

44. Robbins, J., S. M. Dilworth, R. A. Laskey, and C. Dingwall. 1991. Twointerdependent basic domains in nucleoplasmin nuclear targeting sequence:identification of a class of bipartite nuclear targeting sequence. Cell 64:615–623.

45. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing withchain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463–5467.

46. Schulze, E., S. Nagel, K. Gavenis, and U. Grossbach. 1994. Structurallydivergent histone H1 variants in chromosomes containing highly condensedinterphase chromatin. J. Cell Biol. 127:1789–1798.

47. Simpson, L., and D. A. Maslov. 1994. RNA editing and the evolution ofparasites. Science 264:1870–1871.

48. Sogin, M. L., H. J. Elwood, and J. H. Gunderson. 1986. Evolutionary diver-sity of eukaryotic small-subunit rRNA genes. Proc. Natl. Acad. Sci. USA83:1383–1387.

49. Soteriadou, K., A. Tzinia, E. Panou-Pomonis, V. Tsikaris, M. Sakarellos-Daitsiotis, C. Sakarellos, G. Papoulou, and R. Matsas. 1996. Antigenicityand conformational analysis of the Zn(2�)-binding sites of two Zn(2�)-metalloproteases: Leishmania gp63 and mammalian endopeptidase-24.11.Biochem. J. 313:455–466.

50. Soteriadou, K. P., M. S. Remoundos, M. C. Katsikas, A. K. Tzinia, V.Tsikaris, C. Sakarellos, and S. J. Tzartos. 1992. The Ser-Arg-Tyr-Aspregion of the major surface glycoprotein of Leishmania mimics the Arg-Gly-Asp-Ser cell attachment region of fibronectin. J. Biol. Chem. 267:13980–13985.

51. Soteriadou, K. P., A. K. Tzinia, A. Mamalaki, M. Phelouzat, F. Lawrence,and M. Robert-Gero. 1994. Expression of the major surface glycoprotein ofLeishmania, gp63, in wild-type and sinefungin-resistant promastigotes. Eur.J. Biochem. 223:61–68.

52. Soto, M., J. M. Requena, L. C. Gomen, I. Navarette, and C. Alonso. 1992.Molecular characterization of a Leishmania donovani infantum antigen iden-tified as histone H2A. Eur. J. Biochem. 205:211–216.

53. Soto, M., J. M. Requena, G. Morales, and C. Alonso. 1994. The Leishmaniainfantum histone H3 possesses an extremely divergent N-terminal domain.Biochim. Biophys. Acta 1219:533–535.

54. Tabassum, R., K. M. Sandman, and J. N. Reeve. 1992. HMt, a histone-related protein from Methanobacterium thermoautotrophicum delta H. J.Bacteriol. 174:7890–7895.

55. Thomas, F., R. Losa, and A. Klug. 1979. Involvement of histone H1 in theorganization of the nucleosome and of the salt-dependent superstructures ofchromatin. J. Cell Biol. 83:403–427.

56. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets: procedure andsome applications. Proc. Natl. Acad. Sci. USA 76:4350–4354.

57. Tzinia, A. K., and K. P. Soteriadou. 1991. Substrate-dependent pH optima ofgp63 purified from seven strains of Leishmania. Mol. Biochem. Parasitol.47:83–90.

58. Voyiatzaki, C. S., and K. P. Soteriadou. 1990. Evidence of transferrin bindingsites on the surface of Leishmania promastigotes. J. Biol. Chem. 265:22380–22385.

59. Voyiatzaki, C. S., and K. P. Soteriadou. 1992. Identification and isolation ofthe Leishmania transferrin receptor. J. Biol. Chem. 267:9112–9117.

60. Vuorio, T., S. N. Maity, and B. de Combrugghe. 1990. Purification andmolecular cloning of the “A” chain of a rat heteromeric CCAAT-bindingprotein. Sequence identity with the yeast HAP3 transcription factor. J. Biol.Chem. 265:22480–22486.

61. Weintraub, H. 1985. Assembly and propagation of repressed and depressedchromosomal states. Cell 42:705–711.

62. Yang, D. M., and F. Y. Liew. 1993. Effects of qinghaosu (artemisinin) and itsderivatives on experimental cutaneous leishmaniasis. Parasitology 106:7–11.

63. Zu, X. W., and F. T. Jay. 1991. The E1 functional epitope of the humaninterferon gamma is a nuclear targeting signal-like element. Mapping of theE1 epitope. J. Biol. Chem. 266:6023–6026.

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Expression of a Novel Leishmania Gene Encoding a …iai.asm.org/content/70/12/6976.full.pdf · 0019-9567/02/$04.00 0 DOI: 10.1128/IAI.70.12.6976–6986.2002 ... The membranes were