Functional Identification of Galactosyltransferases (SCGs) Required for Species-specific Modifications of the Lipophosphoglycan Adhesin Controlling Leishmania major-Sand Fly Interactions*

Deborah E. DobsonDagger §, Luella D. ScholtesDagger , Kelli E. ValdezDagger , Deborah R. Sullivan, Brenda J. Mengeling, Salvatore Cilmi||, Salvatore J. Turco, and Stephen M. BeverleyDagger ||

From the Dagger  Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110, the  Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky 40536, and the || Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, February 13, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lipophosphoglycan (LPG) is an abundant surface molecule that plays key roles in the infectious cycle of Leishmania major. The dominant feature of LPG is a polymer of phosphoglycan (PG) (6Galbeta 1,4Manalpha 1-PO4) repeating units. In L. major these are extensively substituted with Gal(beta 1,3) side chains, which are required for binding to midgut lectins and survival. We utilized evolutionary polymorphisms in LPG structure and cross-species transfections to recover genes encoding the LPG side chain beta 1,3-galactosyltransferases (beta GalTs). A dispersed family of six SCG genes was recovered, whose predicted proteins exhibited characteristics of eukaryotic GalTs. At least four of these proteins showed significant LPG side chain beta GalT activity; SCG3 exhibited initiating GalT activity whereas SCG2 showed both initiating and elongating GalT activity. However, the activity of SCG2 was context-dependent, being largely silent in its normal genomic milieu, and different strains show considerable variation in the extent of LPG galactosylation. Thus the L. major genome encodes a family of SCGs with varying specificity and activity, and we propose that strain-specific LPG galactosylation patterns reflect differences in their expression.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The trypanosomatid protozoan parasite Leishmania infects over 12 million people worldwide, causing a variety of diseases that range from mild cutaneous lesions to fatal visceral infections (1). Within vertebrates Leishmania resides within acidified phagosomes of macrophages as the amastigote stage. A key step of the infectious cycle is the ability of the parasite to be transmitted to fresh hosts by an insect vector, phlebotomine sand flies. Several studies have emphasized the importance of lipophosphoglycan (LPG),1 an abundant surface glycolipid of Leishmania promastigotes, in sand fly survival (reviewed in Refs. 2-4). Following a sand fly bite, Leishmania and the blood meal are enclosed by a midgut peritrophic matrix for several days, whereas parasites differentiate to the replicating procyclic promastigote stage. During this period LPG and other phosphoglycans (PGs) contribute to survival in the hydrolytic milieu of the midgut (3). After a few days the matrix is degraded and the remnants of the blood meal are excreted; at this time, promastigotes bind to midgut epithelium through an LPG-dependent interaction to avoid being excreted as well (5). As digestion is completed and the fly prepares to feed again, parasites differentiate to the infectious metacyclic stage, which synthesize a structurally modified metacyclic form LPG that is unable to bind the midgut (5-7). The detached metacyclic parasites are adapted for transmission and establishment of the infection in a new vertebrate host (8).

The basic "backbone" structure in all Leishmania consists of a 1-O-alkyl-2-lyso-phosphatidylinositol lipid anchor and heptasaccharide core, to which is joined a long PG polymer composed of 15-30 (Galbeta 1,4Manalpha 1-PO4) repeating units, terminated by a capping oligosaccharide (Fig. 1). In different species and/or developmental stages a variety of modifications of the LPG backbone have been observed, including changes in the terminal capping oligosaccharide of LPG, the addition of side chain (sc) sugar modifications to the prototypic PG (Gal-Man-P) repeating unit, and increases in the number of PG repeats (reviewed in Refs. 2-4). These modifications contribute in a species-specific manner to the binding and release of Leishmania promastigotes during development in the sand fly. In procyclic Leishmania donovani the PG repeats are unmodified, and midgut binding occurs through the Gal-containing capping oligosaccharide; in metacyclics, the number of PG repeats approximately doubles, resulting in a conformational change that precludes midgut binding (9). In L. major the PG repeats are modified by side chain beta 1,3 galactosyl residues (scbeta Gal); in metacyclics, the number of PG repeats increases and the scbeta Gal residues are further modified by addition of arabinose caps to block midgut binding (6, 7). These species-specific modifications also play important roles in the ability of the natural sand fly vector to transmit Leishmania species (2-4). For example, neither L. donovani nor Leishmania major mutants lacking LPG PG scbeta Gal residues can be maintained in the natural L. major host Phleobotomus papatasi (10, 11).

A common theme in many protozoan parasites is their ability to alter their surface coats to ensure survival in both the insect vectors and mammalian hosts (12). In many respects, LPG functions as a stage- and species-specific adhesin in the sand fly. Adhesins have been extensively studied in bacteria (13) and fungi (14). Typically microbial adhesins are proteins that mediate attachment through interactions with specific carbohydrate, lipid, and/or protein moieties in host receptors. In Leishmania, this relationship is reversed because LPG is a glycolipid adhesin responsible for binding to a putative protein receptor in sand fly midguts (4, 15).

Because of its importance to parasite development and vector transmission in the well characterized L. major-P. papatasi model, we focused on genes affecting the attachment of the side chain Gal residues to the LPG PG repeats (Fig. 1). Previously, we utilized LPG-deficient mutants in transfection-based functional rescue approaches to identify genes affecting synthesis of the LPG backbone (16-19). Here, we used "cross-species" transfections to identify loci mediating LPG scbeta Gal additions. The SCG (side chain galactose) genes identified represent the first "expression cloning" of a glycosylation gene family crucial for mediating midgut attachment of parasites, and suggest an approach for identifying genes involved in midgut attachment of other parasite species. Targeting genes that disrupt normal Leishmania-sand fly interactions may represent a novel approach for interrupting disease transmission and compromising virulence.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Leishmania Culture and Transfection-- L. major strain Friedlin V1 (LmFV1) is a virulent clonal derivative of the Friedlin line (MHOM/IL/80/Friedlin) obtained from D. L. Sacks (National Institutes of Health). L. donovani Sudanese strain 1S2D (Ld) is a virulent clonal derivative (MHOM/S.D./00/1S-2D) obtained from D. Dwyer (National Institutes of Health). Cells were grown in M199 medium containing 10% heat-inactivated fetal bovine serum (20). Procyclic promastigotes were harvested from logarithmically growing (log phase) cultures and metacyclic promastigotes were isolated by the peanut agglutinin method (21) from cultures that had been in stationary growth phase for 2-3 days (stationary phase parasites). Infection of BALB/c mice and recovery and purification of lesion amastigotes were performed as described (22).

Parasites were transfected by electroporation and clonal lines were obtained by plating on semisolid M199 media (20), containing drugs appropriate for each selective marker (50 µg/ml HYG, 15 µg/ml G418). Agglutination assays were performed as described (23) using WIC79.3 monoclonal antibody (24).

Cosmid Library Transfection and WIC79.3 Monoclonal Antibody Panning-- These studies were approved by the relevant institutional biosafety committees. An LmFV1 genomic DNA library constructed in the cosmid shuttle vector cLHYG (23) was introduced by 30 separate electroporations into Ld; 13,100 independent transfectants were obtained. These were combined into three independent pools, and transfectants bearing Gal-modified LPG PG repeats were isolated by panning with WIC79.3 antibody as described (18, 19). Three successive rounds of WIC79.3 antibody panning were performed, yielding a population that was strongly reactive. After plating to obtain single colonies, cosmid DNAs cSCG1 (laboratory strain B3547), cSCG2 (B3558), cSCG2a (B4876), and cSCG4 (B3559) were recovered by transformation of Escherichia coli (18).

Genomic Cosmid Library Screen for SCG Genes-- The 774-bp radiolabeled SCG universal probe (described below; Fig. 2B) was used to screen the LmFV1 genomic cosmid library (23). Eighteen positive cosmids were identified, including cSCG3 (B3979), cSCG5 (B3985), and cSCG6 (B3971). Mapping and limited sequence analysis showed that in their respective cosmids, SCG1, SCG4, SCG5, and SCG6 are in the same transcriptional orientation as the HYG marker, whereas SCG2 and SCG3 are in the opposite orientation.

Molecular Constructs-- Partial XhoI deletions of cosmids B3547/SCG1 and B3558/SCG2 (Fig. 2A) were generated by limiting digestion followed by circularization with T4 DNA ligase. A 7.6-kb HindIII fragment from B3558 was inserted into HindIII-digested pSNBR (25), yielding pSNBR-SCG2 (B3743). pSNBR-SCG2 was digested with Bse36I, blunted with T4 DNA polymerase, partially digested with SmaI to release a 2.8-kb 5'-flanking region fragment, then religated using T4 DNA ligase to create pSNBR-SCG2del1 (B3899). Digestion of pSNBR-SCG2del1 with XcmI followed by circularization with T4 DNA ligase generated pXK-SCG2 (B3900), containing the 2.4-kb SCG2 coding region plus 48 bp of 5' and 147 bp of 3'-flanking DNA. Relevant sequence of all constructs was verified using standard methods with an Applied Biosystems ABI-373 automated DNA sequencer.

In Vitro Transposon Mutagenesis-- The XhoI deletion cAX21/SCG1 (B3556) (Fig. 2A) was used as a target for mariner mosK transposon (Tn) mutagenesis as described (26). A total of 80 transposon insertions were mapped and eight that fell within the 5.6-kb Leishmania insert were analyzed by transfection into Ld parasites (Fig. 2B and data not shown). We also used pSNBR-SCG2 (B3743) as a target for mosK Tn mutagenesis, mapping a total of 72 Tn insertions. Eight Tn insertions that fell within the 7.6-kb Leishmania insert were analyzed by transfection into Ld parasites (Fig. 2B and data not shown).

DNA Sequencing-- Complete double-stranded DNA sequences were obtained for the 2.4-kb SCG coding and flanking regions from B3547/SCG1 (GenBankTM AY230144), B3558/SCG2 (GenBankTM AY230145), B3979/SCG3 (GenBankTM AY230146), B3559/SCG4 (GenBankTM AY230147), B3985/SCG5 (GenBankTM AY230148), and B3971/SCG6 (GenBankTM AY230149). In some cases, primers were labeled with [33P]ATP (2000-4000 Ci/mmol) or [35S]dCTP (1250 Ci/mmol) and templates sequenced manually. Single strand sequence from both ends of the SCG1-6 cosmids was obtained using cLHYG-specific primers.

Northern and Southern Blot Analyses-- Total Leishmania RNAs were prepared using the Trizol method (Invitrogen). RNA (5 µg) was analyzed by Northern blotting as described (27). RNA loading was normalized to ethidium bromide-stained rRNA. Genomic DNA was isolated and analyzed by Southern blotting as described (27). For molecular karyotype analyses, Leishmania chromosomes were prepared in agarose plugs and stored at 4 °C as described (28). Pulse field gel electrophoresis was performed in a Bio-Rad model CHEF-DR II apparatus using an electrophoresis field program separating 0.4-1.8 megabase DNAs.

SCG Probes-- Probes were generated by PCR amplification in a PTC-200 thermocycler (MJ Research) using 20 pmol of the indicated primers, 50 ng of template, 0.25 mM dNTPs, and 1 unit of TAQ polymerase (Roche Diagnostics) in 50 µl of total volume following the manufacturer's directions. PCR-amplified DNAs were purified on a QIAquick PCR purification column (Qiagen) following the manufacturer's directions and probes were labeled as described above.

The 774-bp SCG "universal" coding region probe (corresponding to amino acids 1-258) was amplified as described above, using primers B890 (5'-CGCCATCGCAAACAGCATC) and B1240 (5'-gcggatccaccATGCGAGAGGAGAACAATGTGCCA; Leishmania sequences in uppercase) and pXK-SCG2 template (B3900), 55 °C annealing temperature, and 2-min elongation time. The PCR product (1 µl) was used as template for an additional round of PCR amplification and purified before probe labeling.

Reverse Transcriptase-PCR Analyses-- cDNA was prepared using 1 µg of LmFV1 RNA, random primers, and Superscript reverse transcriptase (Invitrogen) in a 20-µl reaction volume, following the manufacturer's directions. PCR reactions (50 µl), containing 1 µl of cDNA, 20 pmol of L. major miniexon (B936, 5'-AACGCTATATAAGTATCAGTTCTGTACTTTA) and common SCG (B871, 5'-GACGATGAGAGCAAGTAAGAC) primers, 0.25 mM dNTPs, and 1 unit of TAQ polymerase (Roche Diagnostics), were performed as described above using 62 °C annealing temperature and 2-min elongation time. Products were analyzed on a 5% acrylamide, 1× TBE (90 mM Tris, 90 mM borate, 2 mM EDTA, pH 8) gel run overnight at 2.3 V/cm. DNA was visualized by staining with SYBR Gold (Molecular Probes).

Purification and Analysis of LPG-- LPG was extracted from logarithmically growing parasites (4-8 × 106 cells/ml; 109 cells) in solvent E and purified by phenyl-coupled Sepharose chromatography (29). PG repeat units were generated by hydrolysis of purified LPG under mild acid conditions, recovered, and separated by HPLC Dionex chromatography (30). Aliquots of the PG repeats were dephosphorylated with E. coli alkaline phosphatase (0.1 unit, 16 h, 37 °C), desalted by passage through a two-layered column of AG 50W-X12 over AG 1-X8, labeled at the reducing ends with 8-aminonaphthalene-1,3,6-trisulfate and analyzed by GLYKO-FACE fluorophore-assisted carbohydrate electrophoresis according to manufacturer's specifications (Glyko Inc., Novato, CA). When [3H]Gal-labeled LPG PG repeats were included, gels were visualized by UV illumination and the radioactivity eluted from excised bands was measured by scintillation counting. The presence of Gal in LPG side chains was shown by digestion with E. coli beta -galactosidase prior to fluorescent labeling (10). Migration distances were compared with oligosaccharide standards. Strong acid hydrolysis (2 N trifluoroacetic acid, 2.5 h, 100 °C) of the repeat units followed by monosaccharide analysis indicated that >95% of the radiolabel remained as [3H]Gal (data not shown).

LPG Side Chain beta 1,3-Galactosyltransferase (scbeta GalT) Assays-- Microsomes from logarithmically growing parasites (4-8 × 106 cells/ml; 2 × 109 cells) were prepared by nitrogen cavitation and differential centrifugation, and LPG scbeta GalT assays were performed (10). Transfer of [3H]Gal from UDP-[3H]Gal to Ld LPG was verified by analysis of PG repeats by thin layer or Dionex HPLC chromatography (30).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES

Isolation of Cosmids Conferring Addition of Gal Side Chains to LPG-- The LPG PG repeating units from LmFV1 bear beta 1,3-linked Gal modifications, which confer reactivity to the antibody WIC79.3 (Fig. 1A) (24). In contrast, the unmodified L. donovani (Ld) LPG is unreactive with WIC79.3 (Fig. 1B). In vitro, Ld LPG can serve as substrate for L. major PG scbeta GalT activity (10, 31). We reasoned expression of LmFV1 scbeta GalTs in L. donovani would confer WIC79.3+ LPG reactivity, and designed a functional rescue strategy to exploit this (Fig. 1C).


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Fig. 1.   Predicted consequence of LPG side chain galactosyltransferase overexpression on L. donovani LPG structure. The structures shown are modified from Refs. 6 and 48. A, L. major FV1 (LmFV1) LPG. The PG repeat unit backbone 6Gal(beta 1,4)Man(alpha 1)-PO4 is represented by circles with curved lines. The glycan "core" is Gal(alpha 1,6)Gal(alpha 1,3)Galf(beta 1,3)[Glc(alpha 1)-PO4 right-arrow 6]Man(alpha 1,3)Man(alpha 1,4)GlcN(alpha 1,6) and is linked to a 1-O-alkyl-2-lyso-phosphatidylinositol anchor. Residues comprising WIC79.3 epitopes (Galn-(6Gal(beta 1,4)Man(alpha 1)-PO4) (24) are shaded. Note that the precise site of attachment of the Gal side chains in the PG repeats is heterogeneous. B, L. donovani (Ld) LPG. The structures shown are modified from Refs. 37 and 48, and abbreviations are defined in panel A. C, predicted LPG structure following LPG scbeta GalT expression in L. donovani LPG. The structures shown were observed in Ld SCG transfectants and are defined in panel A.

An LmFV1 genomic library prepared in the Leishmania shuttle cosmid vector cLHYG (23) was transfected into L. donovani, yielding a library of 13,100 Ld transfectants that provided >10-fold coverage of the ~35 megabase L. major genome. WIC79.3+ Ld transfectants were recovered following 3 rounds of WIC79.3 antibody panning, and clonal lines were obtained by plating. From 24 lines we recovered four different cosmids, which upon retransfection into L. donovani conferred WIC79.3 reactivity. Restriction mapping showed that these contained 3 different loci that we termed SCG: SCG1 (cosmid B3547), SCG2 (cosmids B3558, B4876), and SCG4 (cosmid B3559; Figs. 2 and 3, and data not shown).


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Fig. 2.   Functional mapping of SCG1 and SCG2 loci. A, functional analyses of SCG1 and SCG2 cosmid deletions. A map of the Leishmania DNA present in cosmids cSCG1/B3547 (top) and cSCG2/B3558 (bottom) is shown. Insets from representative XhoI deletion cosmids are shown as solid lines; dotted lines represent the deleted region. X, XhoI restriction site. WIC79.3 reactivity in agglutination tests of Ld SCG transfectants expressing each cosmid is shown to the right of each construct ("+" = positive; "-" = negative). Predicted gene locations (gray boxes) are based on WIC79.3 reactivity of deletions. B, localization by transposon mutagenesis mapping. The location of the relevant mariner mosK Tn insertion sites (gray triangles with Tn number) within SCG1 deletion cosmid AX21 (top) and SCG2 plasmid pXK-SCG2 (bottom) are shown. WIC79.3 reactivity of Ld SCG transfectants expressing each Tn insertion construct is indicated above the insertion site, as defined in panel A. Open arrow, predicted SCG ORFs; H4, histone H4 homolog. The position of the SCG "universal probe" is marked. The predicted SCG(X) splice acceptor is shown ().


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Fig. 3.   Arrangement of SCG loci in LmFV1. A, SCG gene structures. Genomic DNA from LmFV1 (genomic) and DNAs from LmFV1 SCG1-6 cosmids (cSCG1-6) were digested with XhoI, separated by gel electrophoresis, transferred to GeneScreen Plus membrane, and hybridized to a radiolabeled universal SCG probe (Fig. 2B). Positions of DNA size standards are marked. B, molecular karyotype analysis of SCG genes. Chromosomes of LmFV1 were prepared, separated by pulsed field electrophoresis, transferred to GeneScreen Plus, and hybridized with a radiolabeled universal SCG probe. The approximate size of SCG-hybridizing chromosomes was determined by comparison to mobility of S. cerevisiae chromosomes. The location of each SCG gene was determined by stripping the blot and hybridizing the unique flanking region radiolabeled probes from each SCG cosmid (data not shown).

Identification of SCG Genes-- The active regions within SCG1 and SCG2 cosmids were identified by WIC79.3 reactivity tests of Ld transfectants bearing deletion derivatives, or following mariner Tn insertion mutagenesis (26) (Fig. 2 and data not shown). Analysis of 10 cosmid SCG1/B3547 XhoI deletions identified a 5.6-kb SCG1 active region (gray box, Fig. 2A, and data not shown). Analysis of eight transposon insertions within this region showed that four had lost WIC79.3 reactivity (Tns 45, 31, 63, and 13; Fig. 2B and data not shown). The WIC79.3-unreactive Tn insertion sites were clustered in a 1.4-kb region; sequence analysis out to the nearest flanking WIC79.3+ Tn insertion sites revealed a 2.4-kb open reading frame (ORF) encoding a 814-amino acid protein (Figs. 2B and 4A). Notably, all WIC79.3-unreactive Tn insertions were mapped within the predicted SCG1 ORF.

A similar approach was used to localize the active SCG2 gene to a 7.2-kb region in cosmid B3558 deletion B1X17 (gray box, Fig. 2A). A 7.6-kb HindIII fragment encompassing this region was inserted into the Leishmania shuttle vector pSNBR (25) (pSNBR-SCG2), sequenced, and subjected to Tn mutagenesis as described above. The sequence revealed a 2.4-kb ORF encoding a 814-amino acid protein, and 2.1 kb upstream of this a histone H4 pseudogene with a highly divergent 5' end bearing numerous deletions and frameshifts. As seen for SCG1, the three WIC 79.3-unreactive Tn insertions obtained clustered in an 1.5-kb region within the SCG2 ORF (Fig. 2B). This SCG2 ORF was closely related to the SCG1 ORF and other SCG genes as discussed below. Expression of the SCG2 ORF alone in an expression vector similar to the pX vectors (pXK-SCG2) yielded exclusively WIC79.3+ colonies when transfected into Ld parasites (Table I).


                              
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Table I
Activity of SCG genes in Ld transfection assay

The SCG Family Comprises Six Independent Loci-- Southern blot analysis of LmFV1 chromosomes or digested DNAs with SCG1/SCG2 ORF probes indicated that there were additional SCG loci not recovered in our screen, suggesting it had not reached saturation (Fig. 3A and data not shown). These were recovered from the LmFV1 cLHYG genomic cosmid library by screening with a universal SCG coding region probe (Fig. 2B): from a total of 18 cosmids we obtained new representatives of SCG1, SCG2, and SCG4, as well as three new cosmid loci termed SCG3 (B3979), SCG5 (B3985), and SCG6 (B3971; Fig. 3A and data not shown).

Southern blot analysis of LmFV1 chromosomes separated by pulsed-field electrophoresis revealed six chromosome bands hybridizing to the universal SCG probe (Fig. 3B). To assign each SCG locus to a given chromosome, locus-specific fragments were isolated from each cosmid and hybridized to chromosome blots. This assigned the SCG3, SCG1, SCG2, SCG6, SCG5, and SCG4 loci to chromosomes of ~300, 550, 700, 850, 1600, and 2800 kb, respectively (Fig. 3B and data not shown). These data and comparisons of the XhoI digestion patterns of total LmFV1 genomic DNA against the SCG cosmid panel (Fig. 3A and data not shown) suggested that the entire family of LmFV1 SCG loci had been identified and recovered intact.

WIC79.3 tests of multiple clonal lines for each Ld cosmid SCG transfectant showed that all SCG3/B3979 parasites were reactive, whereas all SCG5/B3985 and SCG6/B3971 transfectants were unreactive. The WIC79.3-unreactive phenotype of SCG5 and SCG6 Ld transfectants was not because of alterations in SCG cosmid DNAs following transfection into Leishmania (data not shown). As a control, the three cosmids identified by functional complementation (SCG1/B3547, SCG2/B3558, and SCG4/B3559) again generated WIC79.3+ Ld transfectants.

Properties of the Predicted SCG Proteins-- The regions of the SCG3-6 cosmids that hybridized to the SCG1/2 ORF probes were mapped and sequenced (Fig. 4 and data not shown), revealing the presence of ORFs showing strong homology to those of SCG1/2. The predicted SCG1, -2, -3, and -6 ORFs were 814 amino acids (aa), whereas SCG4 had an N-terminal 32-amino acid extension arising from a single nt change; comparisons among these showed from 92 to 96% aa identity (Fig. 4, A and B). The SCG5 ORF encoded a protein of 816 residues, whose first 596 amino acids were highly homologous (94-97% aa identity) to the remaining SCG ORFs. Thereafter, the presence of numerous nucleotide substitutions/insertions beginning at SCG5 ORF nt 1789 caused higher divergence, resulting in 50% aa and 55% nt identity over the terminal 220 amino acids (Fig. 4, A and B, and data not shown). The 5'-flanking regions of the SCG genes were highly homologous, except for SCG1 that diverged from other SCG genes 122 nt upstream of the predicted conserved start codon. Similarly, the 3'-flanking regions were also highly homologous, except for SCG5 as noted above (data not shown).


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Fig. 4.   Properties of SCG proteins. A, predicted SCG proteins. The conserved ORF identified in each SCG cosmid is represented as an open box. The locations of the predicted cytoplasmic domains (light gray), transmembrane domains (TM, black), galactosyltransferase catalytic domain (DDD/GalT, dark gray), N-glycosylation sites (*), and LmEST 0269 homology on antisense strand (Lm EST, arrow) are shown. Relevant amino acid positions are noted above the box. Positions of divergent regions are shown below the conserved SCG ORF: the C-terminal region of SCG5 (stippled box) and the N-terminal 32 amino acid extension of SCG4 (striped box). B, phylogenetic analysis of SCG ORFs. The amino acid sequence of the predicted SCG ORFs was analyzed using the Clustal method with the identity residue weight table (DNAstar); the percent aa sequence divergence is plotted. The unique N-terminal 32 amino acids in SCG4 were not included for this analysis. C, alignment of GalT domains. Alignment of the GalT homology domain for SCG1-6 (SCG consensus), rat beta 1,3-GalT (GenBankTM AB003478), and GalT family consensus (GenBankTM pfam01762) is presented; numbers refer to the position in the respective proteins. SCG5 differs from the SCG consensus at position 539 (M > T). Identical and conservative residues are in bold and the DDD catalytic motif (32) is underlined.

All six predicted SCG proteins contained a single "DXD" sequence motif (DDD at aa 538-540 in SCG1, -2, -3, -5, -6, or 570-572 in SCG4; Fig. 4, A and C), a motif common to many glycosyltransferases that is implicated in catalytic activity (32). This motif was located within a region of weak homology (45% aa identity, 68% aa similarity) to eukaryotic beta -galactosyltransferases (GalT, Fig. 4, A and C; GenBankTM conserved domain data base pfam01762). As shown below, transfection of SCG cosmids or an SCG2 ORF expression construct into L. donovani confers elevated LPG scbeta GalT activity. All predicted SCG proteins have the topology of type II membrane proteins (33), with a single predicted transmembrane domain (TM, Fig. 4A) preceded by an N-terminal signal anchor sequence of 108-141 aa (34). There were five potential N-linked glycosylation sites conserved in all SCG family members (*, Fig. 4A). These data suggested that the SCG proteins encode beta GalTs with a lumenal catalytic domain, a conclusion supported by enzymatic studies of SCG transfectants (below).

Expression of SCG mRNAs-- In log phase LmFV1 procyclic promastigotes, an SCG universal ORF probe identified a 3.8-kb mRNA, which will be referred to as SCG(X) (Fig. 5A). Lower amounts of a 5.5-kb transcript were also observed, which may reflect a processing intermediate arising from the polycistronic transcriptional mechanism employed by trypanosomatid protozoans (35). The 5' end of the SCG(X) transcripts was mapped by reverse transcriptase-PCR to a position 264-nt upstream of the conserved ATG in the SCG ORFs (Figs. 5B and 2B), and both SCG(X) transcripts were sufficiently large to encode the predicted SCG proteins (Fig. 4A). Relative to logarithmic growth phase procyclic parasites, SCG(X) transcript levels increased slightly in stationary growth phase parasites (about 1.5-fold for both transcripts) and more in metacyclic parasites (3- and 7.4-fold for 3.8- and 5.5-kb transcripts, respectively; Fig. 5A). This may be related to the doubling in the LPG PG repeat number known to occur as parasites differentiate from procyclic to metacyclic promastigotes upon entering stationary growth phase (6, 7). The presence of SCG(X) transcripts in amastigotes (Fig. 5A) may reflect the synthesis of phosphoglycans other than LPG, such as PPGs, that also bear Gal side chain modifications (36).


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Fig. 5.   Structure and expression of SCG(X) transcripts. A, Northern blot analysis. Total LmFV1 RNAs (5 µg) from logarithmicallly growing (L) and stationary phase (S) promastigotes, purified metacyclics (M), and lesion amastigotes (A) were separated by electrophoresis, transferred to GeneScreen Plus, and hybridized with a radiolabeled SCG universal probe (Fig. 2B). Positions of RNA size standards are marked. Ribosomal RNA was used as a loading control (lower panel). B, mapping transcript start site by reverse transcriptase-PCR. cDNA from log-phase LmFV1 promastigotes was subjected to reverse transcriptase-PCR using miniexon and conserved SCG primers, and the products were separated by electrophoresis. Positions of DNA size standards are marked. The data in both panels are representative of three independent experiments.

Searches of the L. major EST data base revealed an EST (lmEST0269; GenBankTM H64199) that was highly homologous (83-85% nucleotide identity) to the antisense strand of the SCG1-6 genes (nt 1465-1162 in the SCG1, -2, -3, -5, -6 ORFs; nt 1561-1258 in SCG4; Fig. 4A). However, searches of the L. major genome did not yield a sequence identical to the EST. Potentially the sequence divergence could arise from technical sources (rapid EST sequencing) or polymorphisms between the Friedlin V1 strain studied here and the LV39 line studied in the EST project. Interestingly, these two strains differ in the degree of LPG PG galactosylation.2 It was also surprising that the EST arose from the antisense strand of the SCG ORFs. Again, this could have a technical origin arising during cDNA library construction. However, antisense transcripts could play a role in SCG regulation through any one of a variety of mechanisms, although Leishmania appear to be deficient in the RNA-interference pathway (38).3 At present we have no data addressing the reality or role of this EST.

LPG Side Chain Galactosylation in SCG Cosmid L. donovani Transfectants-- The WIC79.3 reactivity of cSCG1-4 Ld transfectants suggested that they synthesized LPGs with PG repeats containing beta Gal side chains (Fig. 1). Purified LPGs were subjected to mild acid hydrolysis and dephosphorylation, and the PG repeats were separated by Dionex HPLC chromatography (Fig. 6 and data not shown). As expected, Ld LPG yielded primarily the unsubstituted Gal-Man PG repeat (peak G-M in Fig. 6) (6, 29), whereas the majority of LmFV1 PG repeat units were substituted with 1-3 Gal residues (peaks G2-4-M in Fig. 6). Ld-cSCG3 cosmid transfectants showed significant levels of Gal-substituted PG repeat units (~77% of total repeat units), as expected given their strong reactivity with WIC79.3 (Table I). In contrast, only trace levels of Gal-substituted PG repeats were evident in the cSCG1, cSCG2, or cSCG4 transfectants (1-4% of the total PG repeat units were so modified), and none were evident in the SCG5 and SCG6 cosmid transfectants (Fig. 6, Table I). Whereas the results with cSCG5 and cSCG6 were expected given their WIC79.3-negative phenotype, those for cSCG1, cSCG2, and cSCG4 were surprising given their clear reactivity with WIC79.3 (Table I). Because LPG is highly abundant (>106 molecules/cell), presumably even a low level of beta Gal side chain addition to PG repeats is sufficient to confer strong reactivity with multivalent antibodies or agglutinins.


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Fig. 6.   Effect of SCG expression in L. donovani on LPG structure. PG repeating units from LPG isolated from untransfected L. donovani (Ld), Ld SCG-transfectants, or LmFV1 were fractionated by Dionex HPLC. The elution positions of unsubstituted PG repeats (G-M) and PG repeats containing one (G2-M), two (G3-M), or three (G4-M) Gal side chains are marked. An arrow denotes the position of PG repeats containing a single Gal side chain (G2-M). The abundance of each PG repeat species was quantitated by ED40 electrochemical detection (nC). Unlabeled peaks are derived from the LPG cap (Fig. 1). Two independent experiments gave similar results.

The Dionex HPLC results were confirmed by analysis of the LPG repeating unit structures by fluorophore labeling and electrophoresis (Fig. 7). LPG samples from Ld and Ld-vector control transfectants exhibited a single band corresponding to unsubstituted PG repeats (G-M). Ld-cSCG1, -cSCG3, and -cSCG4 cosmid transfectant samples showed an additional band corresponding to PG repeats containing a single beta Gal side chain (Gal-G-M), as confirmed by their susceptibility to digestion with beta -galactosidase (data not shown) and reactivity with WIC79.3 (Table I). In contrast, Ld4-cSCG5 and -cSCG6 samples showed only a single band corresponding to unsubstituted PG repeats (Fig. 7). Thus, SCG5 and SCG6 were unable to add either beta Gal or any other side chain sugar to LPG PG repeats.


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Fig. 7.   Electrophoretic analysis of LPG repeat units. Dephosphorylated PG repeat units from Ld and transfectants (vector, cLHYG; c, cosmid; pSCG2, pXK-SCG2) were fluorophore-labeled, separated by electrophoresis, and visualized with UV light. LPG repeat side chain structures corresponding to each major band are noted, with G-M = unsubstituted PG repeat. Two independent experiments gave similar results.

L. major SCG Expression Confers LPG scbeta GalT Activity in L. donovani-- An in vitro assay for LPG scbeta GalT activity (10) was used to test whether the SCG genes exhibited the predicted enzymatic activities. Parasite microsomes were incubated with the nucleotide sugar donor UDP-[3H]Gal and purified unsubstituted L. donovani LPG acceptor, and the transfer of [3H]Gal to LPG was measured. As expected, LmFV1 microsomes showed 13-fold more LPG scbeta GalT activity relative to Ld microsomes (Table I) (10). Significantly, LPG scbeta GalT activity was 12-fold higher in Ld-cSCG3 cosmid transfectants relative to L. donovani controls. The microsomal LPG scbeta GalT activity in the other SCG cosmid transfectants was lower (1-6-fold above the Ld background; Table I), consistent with the low levels of galactosylation observed in purified LPGs (Figs. 6 and 7). In combination, the sequence, structural, and enzymatic data suggest the active SCG genes likely encode the LPG side chain beta 1,3-galactosyltransferases themselves.

SCG2 Expression Is Context-dependent and Shows Both Initiating and Elongating scbeta GalT Activity-- Given the high sequence similarity among the predicted SCG proteins, the variation in PG repeat galactosylation in the SCG cosmid transfectants was unexpected (Figs. 6 and 7). One explanation was that the context of each SCG gene relative to the cosmid vector backbone led to differences in expression. However, restriction mapping and end sequencing of these cosmids showed that each contained all flanking sequences necessary to generate the 3.8-kb SCG mRNA (Fig. 5A), with the C terminus of each SCG ORF located 2.1-4.1 kb from the cloning site (data not shown). Although SCG genes in these cosmids were not all in the same orientation with respect to the selectable HYG marker (SCG1, 4-6 = same, SCG2,3 = opposite orientation), there was no correlation between SCG orientation and activity. Studies of other genes have shown that the effect of the cosmid vector orientation has little effect (39).

Another explanation involves location of cosmid-borne SCG genes within their normal genomic context. As described above, we expressed the SCG2 ORF using an expression vector similar to the widely used pX vectors (40). Unlike the cSCG2 transfectants, the pXK-SCG2 plasmid transfectants showed extensive beta -galactosylation of LPG in both Dionex HPLC and electrophoretic analyses of fluorophore-labeled repeat units (Figs. 7 and 8), comparable or greater than the most active cSCG3 cosmid Ld transfectant (Figs. 6 and 7; Table I). Similarly, the LPG scbeta GalT activity of the pXK-SCG2 Ld transfectants was 35-fold higher than untransfected L. donovani, or nearly 3-fold higher than the most active cSCG3 cosmid transfectants (Table I).


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Fig. 8.   Context-dependent activity of SCG2. LPG PG repeat units isolated from SCG2 cosmid (Ld-cSCG2) and pXK-SCG2 plasmid (Ld-pXKSCG2) Ld transfectants were analyzed by Dionex HPLC as described in the legend to Fig. 6. Two independent experiments gave similar results.

A second finding from this study was that unlike the cosmid transfectants, the LPG synthesized in the pXK-SCG2 Ld transfectant contained PG repeats bearing both single and oligo-Gal side chains (Figs. 7 and 8). Approximately 80% of the LPG PG repeats bore Gal modifications with about 46% consisting of oligo-Gal substitutions (Figs. 7 and 8; Table I). Because the pXK-SCG2 plasmid and cSCG3 cosmid Ld transfectants had similar overall levels of LPG beta -galactosylation substitution (Fig. 7 and Table I, LPG side chain profile), the differences in side chain length must reflect inherent differences in the specificity of these two LPG scbeta GalTs.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

For L. major Friedlin V1 parasites, the beta 1,3-Gal side chains on the prominent LPG PG repeats (Fig. 1) are recognized by lectins in the P. papatsi midgut, and participate in parasite binding and survival in its sand fly host. We exploited a species-specific LPG structural polymorphism recognized by WIC79.3 antibodies (24) to isolate the LmFV1 SCG gene family that mediates addition of beta Gal side chains to LPG PG repeats. Three SCG genes were identified using functional genetic complementation of WIC79.3-negative L. donovani parasites (SCG1, -2, -4; Figs. 1-3), and the SCG family was completed by homology/library screening (SCG3, -5, -6; Fig. 3). This functional approach has great potential for identifying genes involved in other species-, strain-, or stage-specific LPG structural polymorphisms that are distinguishable by lectin or antibody reactivity. For example, we have adapted this protocol to recover a candidate LPG PG side chain capping beta 1,2-arabinosyltransferase.2

Although SCG genes were dispersed on distinct chromosomes (Fig. 3B), they exhibited 82-96% overall amino acid identity (Fig. 4, A and B). The predicted SCG proteins show features expected for eukaryotic beta GalTs, including a short conserved GalT region including a potential "DDD" catalytic motif, and have a type II membrane protein topology with a large lumenal domain (Fig. 4, A and C). Functional evidence that many SCGs encode PG side chain beta GalTs comes from enzymatic assays of Ld SCG transfectants, as some SCGs conferred synthesis of scbeta Gal-modified LPG PG repeats (Figs. 6-8), and showed elevated LPG scbeta GalT activity when expressed in L. donovani (Table I) and when expressed using baculovirus vectors in insect cells.4 The SCG proteins are most likely associated with the parasite Golgi apparatus, because other LPG proteins involved in PG synthesis and side chain modification are found in this compartment (17).2

Transfection of the SCG3 cosmid into L. donovani resulted in the synthesis of an LPG bearing predominantly single beta Gal modifications on PG repeats, whereas the remaining SCG cosmid Ld transfectants did not show significant levels of activity, as monitored by PG repeat structures characterized by HPLC or fluorophore labeling and electrophoresis (Figs. 6 and 7). However, expression of the SCG2 coding region alone using a standard Leishmania expression vector approach yielded transfectants that synthesized an LPG with abundant PG side chain beta -galactosylation. This suggests that expression of the individual SCG genes is context-dependent, which is discussed more extensively below. Notably, whereas the overall degree of PG repeat galactosylation was similar in the transfectants bearing cosmid SCG3 or the SCG2 expression vector, the pattern differed markedly (Figs. 6-8, Table I). The LPG synthesized by Ld cSCG3 transfectants bore predominantly a single beta Gal side chain, similar to that of the parental L. major Friedlin V1 line, and thus displayed initiating scbeta GalT activity. In contrast, LPG synthesized by the pXK-SCG2 Ld transfectants contained PG side chains of 1-3 beta Gal residues, and thus displayed both initiating and elongating scbeta GalT activities. Because our ability to detect PG polygalactosylation is compromised by low levels of activity (as shown in comparisons of SCG2 cosmid versus expression vector patterns; Fig. 8), determination of the exact specificities of SCG1 and SCG4 will require similar ORF expression vector tests. These studies, however, establish for the first time the presence of at least two classes of LPG scbeta GalTs in the Leishmania genome.

Unexpectedly, two of the six SCG genes were inactive in the sensitive Ld transfection assay and synthesized no detectable beta Gal-modified LPG PG repeats (SCG5 and -6; Table I, Fig. 6). We can exclude the possibility that these mediated transfer of sugars other than Gal by the fluorophore labeling experiment (Fig. 7). Although the molecular basis underlying this heterogeneity was not pursued, the SCG ORFs and flanking regions showed numerous polymorphisms, which may contribute to differences in mRNA and/or protein expression, activity, specificity, or regulation. For example, the C-terminal region of SCG5 is highly divergent from the other SCGs, and the predicted transmembrane domain of SCG6 contains a charged aspartyl residue at position 113. Another contributing factor may be the use of a heterologous Leishmania species for these assays. Whereas in general Leishmania signals are recognized across species, some species specificity has been found (41). However, preliminary studies where L. major strains or mutants lacking Gal-modified LPG were transfected with the SCG cosmids have yielded similar results to those obtained with Ld.5 An attractive possibility is that SCG5 and SCG6 (and possibly other SCGs) lack significant initiating LPG scbeta GalT activity, and possess only elongating scbeta GalT activity. Tests of this model will require expression of these gene products simultaneously with an initiating scbeta GalT such as SCG3.

Why does the Leishmania major genome encode so many LPG scbeta GalT genes, given that expression of SCG3 alone in L. donovani is apparently sufficient to generate an LPG side chain Gal modification pattern similar to that of the parental L. major Friedlin V1 strain (Fig. 6)? One possibility invokes differences in developmental expression. Whereas gene specific probes for SCG2, -3, -4, and -6 are unavailable, preliminary data suggest this may be the case for SCG1 and SCG5.5 Interestingly, L. major amastigote LPG (which is present at very low levels) contains long polymeric Gal side chains (42, 43), which would require both initiating and elongating LPG scbeta GalT activities as seen for SCG2. Alternatively, perhaps different SCG scbeta GalTs show differences in PG acceptor specificity, which include LPG- and PG-modified proteins such as PPG and secreted acid phosphatase (which is a substrate for PG galactosylation when expressed in L. major) (44, 45). However, preliminary Western blot analyses do not provide support for differential modification of LPG relative to other PGs in the Ld transfectants studied here (data not shown).

A second class of models (not exclusive from those above) considers the role of LPG PG repeat side chain modifications and polymorphisms in parasite biology. Significant variation exists in the degree of LPG side chain galactosylation in L. major; whereas the Freidlin V1 strain shows primarily single Gal LPG side chains, strain LV39 clone 5 synthesizes LPG bearing polygalactosyl modifications,2 and the Seidman strain lacks Gal modifications (46). Notably, LPG side chain galactosylation is associated with the ability of L. major to survive in its sand fly vector P. papatasi (10, 47). Thus, intraspecific LPG polymorphic modifications play important roles in parasite-sand fly interactions, as seen previously for inter-specific LPG differences (3, 4).

We propose that strain-specific patterns in LPG side chain beta -galactosylation depend on the pattern of expression and specificity of SCG genes, which in turn play key roles in sand fly survival. Thus in the Friedlin V1 strain only initiating LPG scbeta GalTs are expressed (such as that encoded by SCG3), while in the LV39 strain both initiating and elongating LPG scbeta GalTs are expressed (such as that encoded by SCG2), and Seidman does not express any SCG activity. Because the number of SCG genes appears to be comparable in most L. major strains,5 there must be mechanisms for regulating the expression and/or activity of the SCG gene repertoire during evolution (supported by the context-dependent activity of SCG2). Note that this model does not necessarily imply expression of only one SCG gene at a time, only that those expressed collectively yield the final LPG side chain beta -galactosylation pattern.

Closely related strains of the other Leishmania species also show differences in LPG side chain modification. In Leishmania tropica and Leishmania aethiopica, more than 10 different patterns have been found involving PG repeat side chain modifications other than galactose (48) and the Indian strains of L. donovani bear sc-glucosyl modifications similar to those described in L. mexicana (30). Whereas the gene(s) and mechanisms responsible for LPG modifications in these species have not been identified, it seems likely that variation in the expression of the relevant glycosyltransferases is responsible, as proposed here for L. major LPG scbeta GalTs.

In many respects the model for differential LPG side chain modifications during evolution is reminiscent of other systems of antigenic variation in microorganisms such as Trypanosoma brucei, Plasmodium falciparum, or Borrelia (49-51). A significant difference is that in these organisms variation concerns expression of a family of surface protein antigens, rather than glycosyltransferases responsible for glycocalyx synthesis, and that variation is induced in response to antigenic pressure. In Leishmania, it seems more likely that variation occurs because of changes in the sand fly population, perhaps in response to selective pressures exerted by Leishmania or other microbes, or because of transport of Leishmania into regions with differing sand fly fauna by mobile vertebrate hosts. However, a role for species-specific PG modifications in vertebrate infections has also been suggested (52). A unique feature of LPG PG modifications is that by simultaneously expressing glycosyltransferases with differing specificities, combinatorial diversity can be generated. In the microbial antigenic variations systems, members of the surface antigen family are encoded at many places within the genome (as for the SCGs), and expression is controlled by mechanisms leading to the exclusive transcription of a single active mRNA encoding each antigen. How this is accomplished in Leishmania remains to be determined, as context-dependent expression of SCG2 activity could be controlled at the levels of transcript abundance and/or translation. The molecular, biochemical, and biological predictions of this model are testable, and future studies will focus on the mechanism(s) of differential regulation and activity of SCG expression.

    ACKNOWLEDGEMENTS

We thank Ashley Bray Mahoney, David Sacks, and members of the Beverley laboratory for discussions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants AI31078 (to S. J. T. and S. M. B.) and AI20941 (to S. J. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: 660 S. Euclid Ave., Box 8230, St. Louis, MO 63110.

Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M301568200

2 D. E. Dobson, B. Mengeling, S. Cilmi, S. Hickerson, S. Turco, and S. M. Beverley, manuscript in preparation.

3 K. Robinson and S. M. Beverley, submitted for publication.

4 D. R. Sullivan, S. M. Beverley, and S. J. Turco, manuscript in preparation.

5 D. E. Dobson, L. D. Scholtes, P. Myler, S. J. Turco, and S. M. Beverley, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: LPG, lipophosphoglycan; PG, phosphoglycan; sc, side chain; Tn, transposon; SCG, side chain galactose; LmFV1, Leishmania major strain Friedlin V1; HPLC, high performance liquid chromatography; scbeta GalT, side chain beta 1,3-galactosyltransferases; Ld, Leishmania donovani Sudanese strain 1S2D; ORF, open reading frame; aa, amino acid(s); nt, nucleotide(s); beta GalT, beta 1,3-galactosyltransferases; EST, expressed tag sequence.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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