From the 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
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ABSTRACT |
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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) (6Gal 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 (Gal 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 sc 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 LPG Side Chain Isolation of Cosmids Conferring Addition of Gal Side Chains to
LPG--
The LPG PG repeating units from LmFV1 bear
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).
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).
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 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).
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 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).
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
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 L. major SCG Expression Confers LPG sc SCG2 Expression Is Context-dependent and Shows Both
Initiating and Elongating sc
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
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 For L. major Friedlin V1 parasites, the 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 Transfection of the SCG3 cosmid into L. donovani
resulted in the synthesis of an LPG bearing predominantly single Unexpectedly, two of the six SCG genes were inactive in the
sensitive Ld transfection assay and synthesized no detectable Why does the Leishmania major genome encode so many
LPG sc 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
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 sc 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.
1,4Man
1-PO4) repeating
units. In L. major these are extensively substituted with
Gal(
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
1,3-galactosyltransferases (
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
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,4Man
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
1,3 galactosyl
residues (sc
Gal); in metacyclics, the number of PG repeats increases
and the sc
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 sc
Gal residues can be maintained in the
natural L. major host Phleobotomus papatasi (10, 11).
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
1,3-Galactosyltransferase (sc
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 sc
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 sc
GalT activity (10, 31). We
reasoned expression of LmFV1 sc
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( 1,4)Man(
1)-PO4
is represented by circles with curved lines.
The glycan "core" is
Gal(
1,6)Gal(
1,3)Galf(
1,3)[Glc(
1)-PO4
6]Man(
1,3)Man(
1,4)GlcN(
1,6) and is linked to a
1-O-alkyl-2-lyso-phosphatidylinositol anchor. Residues
comprising WIC79.3 epitopes
(Galn-(6Gal(
1,4)Man(
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 sc
GalT
expression in L. donovani LPG. The structures shown were
observed in Ld SCG transfectants and are defined in
panel A.
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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).
Activity of SCG genes in Ld transfection assay
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.
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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 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.
-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 sc
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
GalTs with a lumenal catalytic domain, a conclusion supported by
enzymatic studies of SCG transfectants (below).
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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.
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
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.
Gal side chain (Gal-G-M), as confirmed by their
susceptibility to digestion with
-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
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.
GalT Activity in L. donovani--
An in vitro assay for LPG sc
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 sc
GalT activity relative to Ld
microsomes (Table I) (10). Significantly, LPG sc
GalT activity was
12-fold higher in Ld-cSCG3 cosmid transfectants relative to
L. donovani controls. The microsomal LPG sc
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
1,3-galactosyltransferases themselves.
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).
-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 sc
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.
-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 sc
GalTs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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
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
1,2-arabinosyltransferase.2
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
GalTs comes from
enzymatic assays of Ld SCG transfectants, as some
SCGs conferred synthesis of sc
Gal-modified LPG PG repeats (Figs. 6-8), and showed elevated LPG sc
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
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
-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
Gal
side chain, similar to that of the parental L. major
Friedlin V1 line, and thus displayed initiating sc
GalT activity. In
contrast, LPG synthesized by the pXK-SCG2 Ld transfectants
contained PG side chains of 1-3
Gal residues, and thus displayed
both initiating and elongating sc
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 sc
GalTs in the Leishmania genome.
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 sc
GalT activity, and possess only
elongating sc
GalT activity. Tests of this model will require
expression of these gene products simultaneously with an initiating
sc
GalT such as SCG3.
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 sc
GalT activities as seen for SCG2. Alternatively, perhaps
different SCG sc
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).
-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 sc
GalTs
are expressed (such as that encoded by SCG3), while in the
LV39 strain both initiating and elongating LPG sc
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
-galactosylation pattern.
GalTs.
![]() |
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;
scGalT, side chain
1,3-galactosyltransferases;
Ld, Leishmania
donovani Sudanese strain 1S2D;
ORF, open reading frame;
aa, amino acid(s);
nt, nucleotide(s);
GalT,
1,3-galactosyltransferases;
EST, expressed tag sequence.
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