From the
All species of Leishmania express a major surface
protease (Msp or gp63) that facilitates the interactions of the
parasite with its environment at several steps in its life cycle. The
msp gene family in Leishmania chagasi contains three
classes of genes whose mRNAs are differentially expressed during
parasite growth. Logarithmic phase (low infectivity) promastigotes
express only 63-kDa versions of Msp, whereas stationary phase (high
infectivity) promastigotes express both 63- and 59-kDa Msps. The
different migrations of the 59- and 63-kDa proteins on acrylamide gels
are not due to differences in N-linked glycosylation or the
membrane anchor. Plasmid transfections of Leishmania demonstrate that mspS2 of the stationary gene class
encodes a 59-kDa protein. Expression of the 59-kDa protein in
stationary phase promastigotes ceases after about 12 weeks of in
vitro cultivation when the parasites become attenuated. Attenuated
parasites can be stimulated to re-express the 59-kDa Msp by passage
through mice followed by several in vitro passages of
recovered promastigotes. Amastigotes express yet another subset of Msp
proteins. Thus, the 59-kDa product of mspS2 is expressed only
in stationary phase promastigotes and only after recent exposure to
environmental changes encountered in the mammalian host cell.
The Leishmania sp. are digenic protozoa causing a
diverse group of human diseases. The promastigote form of the parasite
develops in the sand fly vector, whereas the amastigote form is present
exclusively in macrophages of the mammalian host. Promastigotes undergo
a developmental process termed metacyclogenesis, during which they
transform to a highly infectious (metacyclic) state in the sand fly gut
(1, 2) . This development can be mimicked in vitro during growth of promastigotes from logarithmic (less infectious)
to stationary (highly infectious) phase in liquid culture medium
(3) . Attachment of the infectious promastigote to a macrophage
and its subsequent uptake by phagocytosis, as well as survival of the
amastigote inside the phagolysosome, are critical steps in establishing
the infection. Two major surface promastigote molecules, the
lipophosphoglycan (reviewed in Ref. 4) and the major surface protease
(Msp
The major surface protease is present in all
Leishmania species investigated to date. Abbreviations for
this molecule include gp63 (for 63-kDa glycoprotein)
(5) , psp
(for promastigote surface protease)
(6) , or Msp (for major
surface protease)
(7) . We refer to this family of surface
proteases as Msp since, as shown here, Leishmania chagasi promastigotes and amastigotes possess different forms of the
protein. Msp has been shown to facilitate promastigote attachment to
macrophages at least in part via the complement receptor CR3, either
directly or indirectly through opsonized C3bi
(5, 8, 9) . It also may protect proteins from
intracellular degradation in the phagolysosome
(10) . Msp
elicits both humoral and T cell responses, and immunization with the
protein yields partial protection of mice against Leishmania
mexicana infections
(11, 12) . Thus, it may
contribute at many levels to the outcome of disease. Msp is a zinc
protease with a broad substrate specificity and a wide pH optimum
(13, 14) . Both the promastigote and amastigote stages
of the parasite express Msp proteins
(10, 15, 16) , but at least in Leishmania
major the promastigote expresses much higher amounts
(17) .
In the promastigote Msp is a dominant surface protein and is anchored
to the membrane by a glycophosphatidyl (GPI) anchor. However, in L.
mexicana amastigotes, Msp appears to be localized predominantly in
an intracellular compartment and lacks a GPI anchor
(16) .
Differences in migration of the Msp proteins on SDS-polyacrylamide
gels have been observed, and it has been suggested that at least some
of the heterogeneity is due to differences in post-translational
modifications, e.g. glycosylation and GPI anchor attachment
(15, 16, 18) . The genomic organization and the
sequences of genes encoding Msp have been reported for several
Leishmania species, including L. major (19) ,
Leishmania donovani (20) , L. mexicana (21) , L. chagasi (7) , and Leishmania
guyanensis (22) . Sequence comparisons show small clusters
of differences among otherwise highly conserved msp coding
regions, suggesting that some of the observed size heterogeneity of Msp
proteins could be due to differences in the amino acid sequences. One
striking example is a gene present in both L. chagasi and
L. mexicana that contains a unique C-terminal coding region,
which may encode a membrane-spanning isoform of Msp
(21, 23) .
L. chagasi contains three classes
of msp genes whose expression as mature mRNAs is
developmentally regulated
(23) . The stationary genes
( mspS) are expressed only in stationary growth phase by
virulent promastigotes, and the log genes ( mspL) are expressed
only in logarithmic growth phase. A third class, the constitutive gene
( mspC), is expressed at low levels throughout promastigote
development. These genes are organized in a tandem array containing
(5`-3`) at least 4 mspSs followed by a minimum of 12
mspLs, 1 mspC, and a final mspS
(7) .
The 3`-untranslated regions and intergenic regions of these genes are
class specific
(7, 23) and are likely to be important
for regulation of gene expression. The coding regions of several
msp genes of L. chagasi have been determined
(7, 23) and display a high degree of homology with
interspersed regions of heterogeneity.
Here, we report that,
consistent with the differential mRNA expression, different Msp
proteins are expressed during L. chagasi development. A novel
59-kDa product of a stationary msp gene is expressed only in
the most virulent form of promastigotes, indicating its potential
importance in the initial stages of infection. L. chagasi amastigotes also express Msp proteins, but at least some of these
isoforms differ from those expressed in stationary phase virulent
promastigotes. Thus, different forms of Msp are present in different
parasite stages, and their expression follows a highly regulated
program during the life cycle of the parasite.
Transfections of L. donovani strain S-1
promastigotes were performed according to a protocol provided by
Stephen Beverley
(32) . Briefly, log phase promastigotes
suspended at 1
An Msp-enriched fraction of
amphipathic membrane molecules was extracted with TX-114
(37) and treated with varying amounts of endoglycosidase F,
which specifically cleaves both complex and high mannose
N-linked oligosaccharides. Both the 59- and 63-kDa Msp
proteins migrated further into an SDS-polyacrylamide gel upon
treatment, indicating that both forms are glycosylated (Fig.
3 A). However, the Msp proteins did not resolve to the same
size even after apparently complete N-deglycosylation. The
latter was demonstrated by loss of binding to the glycoprotein by ConA,
which binds to terminal or subterminal mannose or glucose-containing
oligosaccharides. ConA recognized fully glycosylated Msp proteins
(Fig. 3 B, lane 1). However, binding to
mostly N-deglycosylated proteins was much weaker (Fig.
3 B, lanes 2 and 3), and no ConA
binding to the N-deglycosylated form could be detected
(Fig. 3 B, lane 4). Recently, Funk and
co-workers
(38) reported that enzymatically deglycosylated Msp
of L. major and Leishmania mexicana amazonensis bound
ConA and attributed this binding to mannose residues in the glycan
tail. However, our results indicate that L. chagasi does not
possess terminal or subterminal mannose in the glycan core available
for ConA binding. Anti-LSa reacted with two N-deglycosylated
proteins (Fig. 3 A, lane 4), and the
lower molecular weight protein was not recognized by anti-LSb
(Fig. 3 A, lanes 2-4), suggesting
that the latter is the N-deglycosylated form of the 59-kDa Msp
protein. One of the N-deglycosylated proteins appeared to be
recognized by both peptide-specific antibodies, suggesting that
mspS1 and/or mspS3 might encode this protein since
these genes encode both peptide epitopes (see Fig. 1). This
result suggests that the 59- and 63-kDa proteins are not differentially
N-glycosylated isoforms of the same protein. Interestingly,
anti-LSb also recognized two N-deglycosylated Msp proteins,
and the larger of these proteins was not recognized by anti-LSa
(Fig. 3 A, lanes 4). The mspS4
gene encodes a protein that should be recognized by anti-LSb but not
anti-LSa and therefore is a candidate gene encoding this protein.
L. donovani transfected with pX mspS2 in either the
forward or reverse orientation expressed a 59-kDa Msp protein not
present in untransfected cells or in pX-transfected cells ( L.
donovani (pX)) (Fig. 5). The 59-kDa protein was recognized
by anti-LSa but not by anti-LSb, confirming that it was a product of
transfected mspS2 gene. Promastigotes transfected with
pX mspS1 in either orientation produced a 63-kDa Msp protein
that was not present in the untransfected or in the pX-transfected
control. As predicted, this 63-kDa protein was detected by both
peptide-specific antisera. The L. donovani cell line that is
transfected with pX mspS2 in the reverse orientation expresses
more protein than the cell line transfected with pX mspS2 in
the forward orientation (Fig. 5, third and fourth lanes from the left). A Southern blot
demonstrated that this observation is likely due to a higher copy
number of the plasmid in the pX mspS2.R-transfected strain
(data not shown). When an attenuated L. chagasi strain was
transfected with pX mspS2, mspS2 also encoded a 59-kDa
protein, demonstrating that this size of the mspS2 gene
product is the correct size of MspS2 and not an artifact of expression
in a different Leishmania strain (Fig. 5). Anti-tubulin
antiserum was used to reprobe the filters and demonstrate approximately
equal loading of lanes. The higher molecular weight background band
seen in the anti-tubulin blot on the right is a signal
remaining from anti-LSb binding.
To investigate
whether the 59-kDa Msp protein is produced by these attenuated
promastigotes, we monitored Msp expression during prolonged
cultivation. Fig. 7, A and B, demonstrates a
correlation between Msp protein and mRNA expression in one virulent
( V) and two attenuated cultures (L6 and L5). In logarithmic
growth phase (day 3), the virulent promastigotes expressed mRNA
characteristic of mspL genes (2.7 kb) and a 63-kDa protein,
whereas in stationary phase (day 9), they expressed mRNA characteristic
of mspS genes (3.0 kb) and both 59- and 63-kDa proteins. In
contrast, the two attenuated cultures expressed only mspL mRNA
and a 63-kDa Msp protein throughout log to stationary development.
Attenuated parasites that had been maintained in liquid culture for up
to 4 years consistently expressed the 63-kDa Msp protein
(Fig. 7 C), but they did not re-express the 59-kDa Msp
protein.
Thus, the expression
of the 59-kDa protein correlates with parasite infectivity. It is
expressed in stationary phase, virulent promastigotes, and its
expression is lost or suppressed as the promastigotes become
attenuated.
We show here that L. chagasi differentially
expresses a mixture of Msp proteins, consistent with our previous
observation that they express different msp mRNAs
(23) . We have focused on those msp products expressed
in stationary phase virulent promastigotes, the most infective form of
the parasite. We demonstrate that one of these stationary Msp proteins,
MspS2, is a protease that migrates as a 59-kDa protein on
SDS-polyacrylamide gels and is found solely in virulent promastigotes
during stationary phase. It does not occur in attenuated promastigotes
or in amastigotes.
All known mspS genes contain 598 or 599
codons
(7) , so it is not clear why there is a difference
equivalent to a molecular mass of about 4 kDa in the migration of MspS2
and other mspS gene products. The putative pre- and
propeptides of all known L. chagasi Msps are identical,
suggesting that N-terminal processing of all Msps is the same.
Likewise, the proposed C-terminal GPI anchor attachment site (DGGN)
encoded by all known members of the mspS gene family is
conserved. Furthermore, we found that removal of the GPI anchor
(Fig. 4) or N-deglycosylation with endoglycosidase F
(Fig. 3) did not resolve the size difference between
MspS2 and MspS1. These observations suggest that the
post-translational modifications most commonly identified in the Msps,
i.e. N-terminal processing, asparagine-linked glycosylation,
and GPI anchor attachment, are not solely responsible for the size
difference between the 59- and 63-kDa proteins. Possible explanations
for the difference in migration include post-translational
modifications not investigated here ( e.g. O-glycosylation, phosphorylation, acetylation,
methylation) or differences in the folding properties of proteins not
fully denatured on SDS-polyacrylamide gels.
Several
attenuated lines of L. chagasi promastigotes do not express
the stationary Msps (Figs. 7 and 8), and Southern blots demonstrated
that their absence was not due to gene deletion or rearrangement (not
shown). Attenuated promastigotes could be stimulated to express the
59-kDa protein by passage through mice (Fig. 9) or through the
human U937 cell line (not shown). In contrast, heat shock or an
artificial stress such as electroporation did not induce its
re-expression (not shown). These observations collectively indicate
that attenuated parasites have not permanently lost the capacity to
express the 59-kDa protein and that the environmental changes
encountered during phagocytosis and residence in macrophage
phagolysosomes can induce re-expression of the stationary Msp proteins.
Consistent with this possibility is the observation that repeated
passage of attenuated Leishmania through mice increases their
infectivity.
We thank Dr. Stephen Beverley at Harvard University
and Dr. Paul Englund at Johns Hopkins University for providing
transfection vector pX and anti-CRD antiserum, respectively. We are
grateful to Dr. K.-P. Chang at Chicago Medical School for advice on
protease activity gels.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
or gp63) likely contribute to each of
these steps.
Parasites
L. chagasi,
originally isolated from a patient in Brazil, and L. donovani Sudanese strain S-3 were kindly provided by R. Pearson (University
of Virginia, Charlottesville, VA). L. donovani Sudanese strain
S-1 was kindly provided by Louis V. Kirchhoff (University of Iowa, IA).
Virulent parasites were maintained by serial passages through male
golden hamsters as described
(24) . Amastigotes convert to
promastigotes when cultivated at 26 °C in HOMEM supplemented with
hemin and heat-inactivated fetal calf serum
(23) . For
experiments with virulent promastigotes, parasites were maintained in
liquid culture no longer than 3 weeks after isolation from a hamster.
For experiments with attenuated promastigotes, parasites were
maintained in liquid culture with weekly passages between 12 weeks and
4 years. When needed, parasites were cloned on semi-solid media
(26) . During most experiments, promastigotes were seeded at 1
10
parasites/ml and harvested in logarithmic to
stationary phases of growth, as defined by morphology and cell
concentration
(27) .
Protein Lysates and Western
Blots
Promastigote cell lysates were prepared as previously
described
(7) . Antibodies were obtained by using synthetic
peptides LSa (HIKRRLGGVDIC, corresponding to amino acid residues
128-139 of MspS1) and LSb (SLGKCGVTRHPDLPPC, corresponding to
amino acid residue 418-432 of MspS1) as described
(7) . In
each case, the C-terminal cysteine was added for use in linking the
peptide to the carrier ovalbumin. SDS-polyacrylamide gels, Western
blotting, and detection by enhanced chemiluminescence were performed as
described
(7) . Wells were loaded either with lysates from
equivalent numbers of cells in the same growth stage or equivalent
amounts of protein from promastigotes in any growth stage. Protein was
measured with a Bio-Rad protein assay. Promastigote membrane molecules
were extracted by Triton X-114 (TX-114) phase separation as previously
described
(14, 28, 29) .
N-Deglycosylation of heat denatured TX-114 preparations was
performed in a sodium acetate buffer (50 m
M sodium acetate, pH
5, 50 m
M EDTA, 0.2% SDS, 1% -mercaptoethanol) using
varying concentrations of endoglycosidase F (Boehringer Mannheim) for 1
h at 37 °C. Concanavalin A (ConA) binding to promastigote membrane
molecules was performed on nitrocellulose membranes blocked with 3%
periodate-treated bovine serum albumin (Sigma) in TBST (Tris-buffered
saline (25 m
M Tris-HCl, pH 7.6, 137 m
M NaCl) with
0.01% Tween 20). The membranes were incubated with 50 µg of ConA/ml
(Sigma) in TBST for 1 h at room temperature and subsequently in 50
µg of horseradish peroxidase/ml (Sigma) in TBST as described
(30) . ConA blots were developed by enhanced chemiluminescence
(Amersham Corp.). Some Western blots were probed with an antibody
against the cross-reactive determinant (CRD) of the GPI anchor, kindly
provided by Paul Englund (Johns Hopkins University, Baltimore, MD),
diluted 1:100 in TBST. For protease activity gels, 2% gelatin was added
to the separating gel, and non-denatured TX-114 preparations were
loaded. After electrophoresis, gels were washed twice in 2.5% Triton
X-100 (Sigma), incubated overnight at 37 °C in 40 m
M Tris-HCl, pH 9.0, and subsequently stained with Coomassie Blue.
Silver staining was performed as described
(31) .
Plasmid Constructs and Transfections
The
Leishmania transfection vector pX containing the neomycin
resistance gene ( neo) was kindly provided by
Stephen Beverley (Harvard University, Boston, MA)
(26) . Genomic
DNA fragments of L. chagasi containing the coding regions of
mspS1 plus 4 kb of upstream region and mspS2 plus 1.8
kb of upstream region were excised from genomic DNA clones and
subcloned in both orientations at the XbaI site of pX.
Plasmids containing the msp genes cloned in the same
orientation as the neo
gene are called
pX mspS1.F and pX mspS2.F, whereas constructs
containing the msp genes in the reverse orientation of
neo
are termed pX mspS1.R and
pX mspS2.R.
10
/ml in electroporation buffer (21
m
M HEPES, pH 7.5, 137 m
M NaCl, 5 m
M KCl, 0.7
m
M Na
PO
, 6 m
M glucose) and 5,
10, or 20 µg of the various plasmids were electroporated with 0.45
V delivering a pulse of 2.25 kV/cm in a 0.2-cm cuvette and capacitance
at 500 microfarads in the Gene Pulser (Bio-Rad). Transfected
promastigotes were cultured overnight in drug-free HOMEM and then
plated onto Medium 199 (Life Technologies, Inc.) with 1% agar, 5%
heat-inactivated fetal calf serum, 0.02
M HEPES, pH 7.5, 25
units of penicillin/ml, 25 µg of streptomycin sulfate/ml (Life
Technologies, Inc.), 0.05 m
M adenine, 4 µg of hemin/ml,
0.6 µg biopterin/ml, and 40 µg of G418/ml (geneticin disulfite
salt, Sigma). After 1 or 2 weeks, single colonies were recovered and
adapted to liquid HOMEM containing 200 µg of G418/ml.
Macrophage Infectivity Studies
An in
vitro model for L. chagasi infection of the U937 human
histocyte cell line (American Type Culture Collection) has been
developed.(
)
Briefly, U937 cells were stimulated
to differentiate toward macrophages by culture in RPMI medium (Cancer
Center, University of Iowa) with 10% heat-inactivated fetal calf serum,
2 m
M glutamate, and 50 µg each of penicillin and
streptomycin/ml with 5 ng of phorbol 12-myristate 13-acetate/ml. After
washing, U937 cells were infected with stationary phase promastigotes
at a ratio of 1:20 in medium without phorbol 12-myristate 13-acetate.
After incubation for 2 h at 37 °C in 5% CO
, unattached
promastigotes were removed by two gentle washes twice in Hank's
buffered saline solution (Cancer Center, University of Iowa); fresh
media was added, and the culture was incubated at 37 °C for
1-5 days. U937 cells were scraped from the flasks, washed twice
in Hank's buffered saline solution, and sequentially passed
through 8 and 5 µm polycarbonate filters using positive pressure.
Amastigotes were recovered by pelleting at 3600 rpm, counted, and
resuspended in protease inhibitor buffer mixture
(7) at 5
10
parasites/ml. Lysates were analyzed on Western
blots.
Two Peptide-specific Antibodies Recognize Different
Msp Proteins in Stationary Phase Promastigotes
The coding
regions of several L. chagasi msp genes have been reported
(7, 23) . Fig. 1 shows an abbreviated version of the
major regions of amino acid differences among three Msp proteins
(MspS1, MspS2, and MspS4) whose corresponding RNAs are expressed in
promastigotes during stationary phase. Two additional
stationary-specific genes have been sequenced, mspS3 and
mspS5, whose protein products are highly homologous to MspS1
and MspS4, respectively
(7) . The sequence of one gene expressed
in logarithmic growth phase, mspL1, has been determined, and
its product is identical to MspS1 except for two amino acid
substitutions
(23) . Fig. 1also shows the relative
positions of the proposed macrophage attachment site
(33) , the
consensus sequences for zinc binding in metalloproteases
(6) ,
and the proposed GPI anchor attachment site
(34) . Also
indicated are two peptide sequences against which rabbit polyclonal
antisera were raised. Peptide LSa is present in the predicted sequences
of Msp L1, S1, and S2, whereas peptide LSb is present in the predicted
sequences of Msp L1, S1, and S4. Note that the sequence of LSb is not
present in MspS2.
Figure 1:
Summary of the main regions
of heterogeneity among Msp proteins S1, S2, and S4 expressed during
stationary phase of L. chagasi promastigotes. The complete
sequences of S1, S2, and S4 (598 or 599 amino acids each) are given in
Ref. 7. Open boxes represent clusters of differences
among S1, S2, and S4 ( top, middle, and bottom rows, respectively), dashes indicate the same
amino acid as shown for S1, and horizontal arrows show two synthetic peptides that were used to raise polyclonal
antiserum (LSa, HIKRRLGGVDI; LSb, SLGKCGVTRHPDLPP). Black boxes show functionally important regions: Mø, the
predicted macrophage attachment site; Zn, the conserved
sequence motifs for the zinc binding associated with protease activity;
GPI, the proposed GPI anchor attachment site. Vertical arrows indicate positions of cleavage for the signal
peptide and pro-peptide.
The mspS1 gene was subcloned into the
Escherichia coli pET-3 expression vector
(35, 36) , and both the anti-LSa and anti-LSb antisera
specifically recognized the recombinant MspS1 protein (data not shown).
The antisera were then used to analyze the expression of Msp proteins
in virulent promastigotes. Parasites were harvested from days 2-8
as they developed from logarithmic to stationary phase. Anti-LSa and
anti-LSb both recognized a 63-kDa protein(s) present throughout growth
(Fig. 2 A). Anti-LSa also recognized a 59-kDa protein expressed
only in stationary phase promastigotes (days 5-8). Western blots
using a general anti-Msp serum recognized the same protein bands as
anti-LSa (not shown). We have shown previously that log phase parasites
express 2.7-kb mspL mRNAs, whereas stationary phase
promastigotes express 3.0-kb mspS mRNAs
(23, 24) . Here, Northern blot analysis of RNAs from the
same cultures that were used for Western blot analysis showed that the
switch from the 2.7-kb to the 3.0-kb msp mRNA occurred on
about day 5 (Fig. 2 B), correlating with the onset of
expression of the 59-kDa protein. Therefore, the 59-kDa protein may be
a product of an mspS gene. The differential recognition by
anti-LSa and anti-LSb suggest that mspS2 is one possibility
for this gene.
Figure 2:
Expression of different Msp proteins by
promastigotes as they develop from logarithmic to stationary phase
during in vitro cultivation. A, Western blots of
lysates of promastigotes harvested from days 2-8 and probed with
anti-LSa and anti-LSb. The locations of the 59- and 63-kDa Msp proteins
are indicated by arrows. B, Northern blot of RNA from
promastigotes harvested from days 2-8 and probed with a 1-kb
polymerase chain reaction product of the msp coding region.
The 2.7- and 3.0-kb msp RNAs are indicated by
arrows.
The Size Difference between the 59- and the 63-kDa
Msp Proteins Is Not Due Solely to Differences in N-Linked Glycosylation
or GPI Anchor Attachment
Size heterogeneity of the Msp
proteins has been observed in other Leishmania species and
attributed at least in part to differences in N-linked
glycosylation or membrane anchor attachment
(15, 16, 18) . The deduced amino acid sequences
of the Msp proteins show different numbers of potential
N-linked glycosylation sites. Thus, experiments were conducted
to determine whether differences in N-linked glycosylation are
responsible for the difference in migration between the 59- and 63-kDa
Msp proteins on SDS-polyacrylamide gels.
Figure 3:
The difference in migration of Msp
proteins on Western blots is not due exclusively to differential
N-linked glycosylation. A, Western blot showing
denatured TX-114 preparations of virulent promastigotes that were
treated with 0, 0.09, 0.225, and 0.5 units of endoglycosidase F
( Endo F) ( lanes 1-4,
respectively). One-half of the blot was probed with anti-LSa and the
other half with anti-LSb. B, binding of ConA to denatured
TX-114 preparations of virulent promastigotes that had been treated
with different amounts of endoglycosidase F.
In
several Leishmania species, Msp is anchored to the membrane by
a GPI anchor
(37) . We examined whether both the 59- and the
63-kDa Msp proteins of L. chagasi are GPI anchored. For this
analysis, the Msp proteins were again enriched and partially purified
by a TX-114 phase separation. During this procedure, amphipathic
molecules partitioning into the detergent phase are separated from
hydrophilic molecules, which are found in the aqueous phase. Due to the
GPI moiety, the anchored protein is amphipathic and will partition in
the detergent phase. Cleavage of the GPI anchor with
phosphatidylinositol phospholipase C (PI-PLC) releases the lipid
anchor, resulting in a hydrophilic Msp protein, which partitions in the
aqueous phase. The untreated 59- and 63-kDa Msp proteins of L.
chagasi partitioned into the detergent phase and thus are
amphipathic (Fig. 4). After PI-PLC treatment, both proteins migrated
with the aqueous phase, indicating they now were hydrophilic and
suggesting that both contain a GPI membrane anchor. As expected,
removal of the anchor resulted in proteins that did not migrate as far
into gels
(37) . Cleavage of the GPI anchor with PI-PLC is known
to expose a new antigenic epitope, the CRD in Msp and other
GPI-anchored proteins
(39) . Western blots of PI-PLC-treated Msp
proteins show that, as expected, only PI-PLC-treated proteins, present
in the aqueous phase, were recognized by the anti-CRD antibody (Fig.
4). These results demonstrate that the 59- and the 63-kDa Msp proteins
are both GPI-anchored proteins and demonstrate that the differences in
migration of the 59- and 63-kDa Msp proteins are not due to
differential processing by N-glycosylation or GPI anchor
attachment.
The mspS2 Gene Encodes a 59-kDa
Protein
The mspS2 gene is the only known gene
encoding epitopes recognized by LSa but not by LSb (Fig. 1). The
mspS1 gene contains peptides recognized by both LSa and LSb
and therefore may encode one of the proteins migrating at 63 kDa on an
SDS-polyacrylamide gel. To formally test these possibilities, a DNA
fragment containing the mspS2 coding region plus 1.8 kb
upstream, derived from a genomic DNA clone, was cloned in both
orientations into the XbaI site of the Leishmania transfection vector pX
(26) approximately 626 base pairs
downstream of the neomycin resistance gene ( neo)
(pX mspS2.F and pX mspS2.R). Another fragment
containing the mspS1 coding region plus 4 kb upstream (the
complete intergenic region between the adjacent mspS2 and
mspS1) was also introduced in both orientations into the same
site of the pX vector (pX mspS1.F and pX mspS1.R). It
has been shown that a
-galactosidase gene inserted at this site is
expressed
(26) . The upstream region was included to provide a
216-base pair region common to all msp genes, which has been
found to be important for 5`-splice leader processing of the
pre-mRNA.
(
)
The plasmids were transfected into an
attenuated strain of L. donovani, chosen because anti-LSa and
anti-LSb show no reactive bands on Western blots of an L. donovani extract (Fig. 5). Thus L. donovani provided a clean
background in which to study the expression of mspS2 with the
peptide-specific antibodies. A general L. chagasi Msp
antiserum showed weak hybridization to a triplet around 63 kDa in
lysates of the attenuated L. donovani strain, demonstrating
that endogenous Msp proteins were present (data not shown and Ref. 24).
Figure 5:
The
mspS2 gene encodes a 59-kDa protein. Lysates of stationary
phase promastigotes of attenuated strains of L. donovani ( L. don) and L. chagasi ( L.
chagasi L5) or virulent L. chagasi ( L. chagasi V), either untransfected or
transfected with the plasmid indicated within the parentheses,
were analyzed on Western blots by probing with anti-LSa and anti-LSb.
The same blots were probed with antiserum against tubulin to
demonstrate approximately equivalent loading of
proteins.
The Product of mspS2 Has Proteolytic
Activity
The transfected L. donovani cell lines
L. donovani (pX) and L. donovani (pX mspS2.R)
and virulent L. chagasi promastigotes (used as control) were
examined for protease activity. Fig. 6 A shows a gelatin
substrate gel of TX-114 preparations of these strains with clearing
zones due to protease activity. All three cell lines showed a tight
band of clearing near the top of the gel and a broader cleared zone
migrating further into the gel ( arrows in Fig. 6 A).
The broad clearing zone was more pronounced in the pX mspS2
transfectant than in the pX transfectant and was similar in intensity
to the virulent L. chagasi control. A silver stain of the same
protein preparations as in Fig. 6 A demonstrated similar
amounts of proteins in the TX-114 preparations
(Fig. 6 B). In these substrate gels Msp migrated over a
wide region since Msp proteins were recognized by a polyvalent Msp
antiserum in gel slices corresponding to molecular masses between 50
and 80 kDa (data not shown). These data strongly suggest that
transfected MspS2 is proteolytically active, although we cannot
formally rule out the possibility that transfected MspS2 induces
protease activity in another protein.
Figure 6:
A
L. donovani strain transfected with the mspS2 gene
has more protease activity than the control strain. A,
nondenatured TX-114 preparations of L. donovani containing
plasmid pX or plasmid pX mspS2.R and untransfected virulent
L. chagasi were analyzed for protease activity on a 2% gelatin
activity gel. The clear zones show the locations of protease activities
associated with proteins of high ( top arrow) and
intermediate ( bottom arrow) molecular mass.
B, silver stain of an SDS-polyacrylamide gel containing
aliquots of the same TX-114 preparations described for panel A. The sizes in kDa of protein standards in the marker
lane are indicated.
The 59-kDa Msp Protein Is Not Expressed in Attenuated
Promastigotes
Virulent L. chagasi promastigotes
are more infective at stationary phase than during logarithmic growth
phase
(27) . The expression of the 59-kDa Msp protein therefore
correlates with parasite virulence. We previously demonstrated that
long term cultivation of virulent L. chagasi promastigotes in
liquid culture consistently results in their conversion to attenuated
parasites that have 5-30% of the infectivity at stationary phase
compared with virulent parasites (Ref. 24 and data not shown). For this
study, we define virulent promastigotes as those that have been
passaged in culture for 3 weeks or less and attenuated promastigotes as
those that have passaged for 12 or more weeks.
Figure 7:
Attenuated promastigotes express the
2.7-kb log msp RNA and the 63-kDa protein throughout
development. Lysates were prepared from logarithmic (day 3) or
stationary phase (day 9) promastigotes at 4 weeks ( V), 12
weeks ( L6), or 16 weeks ( L5) after isolation from an
infected hamster. A, Western blot of lysates from
promastigotes probed with anti-LSa. The locations of the 59- and 63-kDa
Msp proteins are indicated. B, Northern blot of RNA from
promastigotes harvested at logarithmic (day 3) or stationary phase (day
9) and probed with the msp coding region probe. The locations
of the 2.7-kb mspL and 3.0-kb mspS RNAs are
indicated. C, Western blot of lysates harvested from virulent
promastigotes and attenuated promastigotes that had been maintained in
liquid culture for 6 months, 1 year, 2 years, and 4 years. The blot was
probed with anti-LSa.
To investigate whether attenuated cultures lose their
expression of the 59-kDa Msp protein abruptly or gradually, lysates of
several cloned virulent L. chagasi lines were prepared weekly
from stationary phase promastigotes. The lysates were analyzed by
Western blots with anti-LSa (a representative blot is shown in Fig.
8 A) and antiserum against tubulin (Fig. 8 B) to
demonstrate loading of similar amounts of protein. For 7-8 weeks
after isolation from a hamster, stationary promastigotes continued to
express the 59-kDa Msp protein. Subsequently, its expression decreased,
and after some fluctuation it disappeared completely after 12-14
weeks (week 13 for this example). The identity of the
intermediate-sized Msp protein band that can be seen on this particular
Western blot is not known. Since this weak protein band was recognized
by both peptide-specific antibodies (data not shown) and does not
appear to be developmentally regulated, we have not investigated it
further. A similar time course performed with an uncloned virulent
population also showed a gradual decrease in the expression of the
59-kDa protein. We have not determined whether the decreasing and
fluctuating expression was due to fewer parasites of the whole
population expressing the 59-kDa protein or to all parasites in the
culture expressing the protein at lower levels.
The Expression of the 59-kDa Msp Protein Can Be
Induced in Attenuated Promastigotes
We investigated whether
attenuated parasites permanently lose the ability to express the 59-kDa
protein or whether they could be stimulated by environmental changes to
re-express this protein. Parasites were recovered from the spleens of
mice infected with virulent or attenuated promastigotes and allowed to
convert to promastigotes in HOMEM. The promastigote cultures were
passaged weekly, and protein lysates were prepared each week after the
parasites had reached stationary phase. Western blots of these lysates
were probed with anti-LSa to examine the expression of the Msp proteins
(Fig. 9). Promastigotes that were virulent (expressing the 59- and the
63-kDa Msp protein) prior to mouse infection expressed both forms of
the Msp proteins at all times in stationary phase after recovery from
the mouse spleen. In contrast, attenuated promastigotes, which
expressed only the 63-kDa Msp protein before infection, express only
the 63-kDa protein during the first two weeks after recovery from the
mouse spleen (experiment performed in duplicate, Atten. 1 and 2 in Fig. 9). However, these recovered
attenuated promastigotes expressed a small amount of the 59-kDa protein
after about 3 weeks of cultivation in vitro and, by 6 weeks,
possessed as much of the 59-kDa protein as the recovered virulent
parasites. Results similar to those shown in Fig. 9were obtained
in three independent experiments. Thus, expression of the 59-kDa
protein occurs only in parasites that have recently experienced a
mammalian cell environment, but its re-expression in attenuated
parasites is intimately linked with cultivation of the promastigote
stage of the parasite. The combined results of Figs. 8 and 9 indicate
that after attenuated parasites are passaged through mice, a delay of
at least three promastigote growth cycles in culture is required before
the 59-kDa Msp protein appears at stationary phase, and it will
disappear again after about 12 weeks of promastigote culture as the
parasites once again become attenuated.
Figure 9:
The expression of the 59-kDa Msp protein
can be stimulated in attenuated promastigotes by passage through mice.
Lysates were prepared from stationary phase L. chagasi promastigotes that had been recovered from mice after 6 weeks of
infection. Parasites used to infect the mice were either virulent or
attenuated ( Atten. 1 and Atten. 2).
Samples were prepared weekly between 1 and 6 weeks after recovery from
mouse spleens. The Western blots were probed with anti-LSa and an
antiserum against tubulin to demonstrate that similar amounts of
proteins were loaded.
L. chagasi Amastigotes Express Different Msp Proteins
than Promastigotes
To examine the expression of Msp
proteins by the intracellular amastigote form of L. chagasi,
we stimulated conversion of promastigotes to amastigotes by their
cultivation in the human macrophage-like cell line U937. We used a
general Msp antiserum and anti-LSa and anti-LSb on Western blots to
analyze corresponding promastigote and amastigote lysates (Fig. 10).
Virulent, stationary phase promastigotes were harvested at day 7, and
amastigotes were harvested 1 or 5 days after infection of U937 cells
with the same promastigote culture. Microscopic analysis showed that
1-day amastigote preparations contained primarily small, round
amastigote forms with few promastigote forms, whereas 5-day amastigote
preparations contained only amastigote forms. The general Msp antiserum
reacted with the 59- and 63-kDa proteins in promastigote lysates.
However, after 1 day of U937 infection, a novel protein(s) of
approximately 64 kDa, in addition to the 59-60- and 63-kDa
proteins, was detected in amastigote lysate. The 5-day amastigote
preparations contained the 64- and the 59-60-kDa Msp proteins but
appeared to lack the 63-kDa Msp protein(s). As previously shown,
anti-LSa recognized the 59- and 63-kDa Msp proteins in promastigote
lysates, and anti-LSb recognized the 63-kDa Msp protein. However, these
peptide-specific sera did not recognize any proteins in amastigote
lysates. Northern blots showing the absence of mspS mRNA in
L. chagasi amastigotesare consistent with this
result. Thus, the 64- and 59-60-kDa Msp proteins expressed by
L. chagasi amastigotes do not contain the Msp epitopes
recognized by the LSa and LSb antisera and may represent novel proteins
yet to be studied.
Figure 4:
The Msp proteins recognized by anti-LSa
and anti-LSb are anchored to the membrane by a GPI anchor. Lysates of
virulent promastigotes were enriched for membrane proteins by a TX-114
preparation. A subsequent TX-114 phase separation was performed on
PI-PLC treated ( +P) or untreated fractions
( -P), and the aqueous and detergent phases were analyzed
on a Western blot. The blots were probed with anti-LSa, anti-LSb, and
anti-CRD.
Transfection of specific
msp genes into attenuated Leishmania strains
expressing only small amounts of the endogenous Msps is useful for
investigating the properties of specific msp gene products.
Liu and Chang
(40) characterized the product of an L. major
msp gene transfected into an attenuated strain of L. mexicana and found it was an active protease that facilitated attachment to
the J774 macrophage cell line. This transfected L. mexicana strain showed a 2-fold increase in macrophage attachment compared
with the untransfected parental strain. We chose to transfect
mspS2 and mspS1 into an attenuated L. donovani strain because its endogenous Msp proteins are expressed at a low
level and are not recognized by the two peptide-specific antisera
(Fig. 5). Transfections of the cloned L. chagasi mspS2
and mspS1 into this L. donovani strain clearly
demonstrated that the S2 gene encodes a 59-kDa product and the S1 gene
specifies a 63-kDa product. Transfectants expressing MspS2 had more
protease activity than the parental strain (Fig. 6). Preliminary
experiments also showed a 1.5-2-fold increase in binding of
mspS2 transfectants to human monocyte-derived macrophages over
that of a control strain transfected with the parental plasmid pX (data
not shown), consistent with the results of Liu and Chang
(40) .
Thus, it is possible that MspS2 is one of the molecular constituents
that contribute to the infectivity of L. chagasi. Attempts to
support this interpretation by eliminating the two copies of the
mspS2 gene in the diploid Leishmania cells by
targeted gene replacement have not been successful to date.
(
)
Curiously, after passage of the
attenuated L. chagasi through mice, the recovered
promastigotes required at least three cycles of logarithmic to
stationary phase growth in vitro before re-expression of the
59-kDa protein was detected in stationary phase (Fig. 9). This
delay indicates that passage through mice or U937 cells was not simply
a selection for a few remaining virulent parasites in the attenuated
population, since a similar delay was not observed when virulent
parasites were passaged through mice. Although the reason for the delay
in re-expression is not known, there appear to be signals that occur
within the mammalian cell and signals that occur during promastigote
growth to stationary phase, both of which are required for optimal
expression of the 59-kDa Msp. The simplest interpretation of these
results is that these signals act at the level of pre-RNA transcription
or processing or mspS mRNA stability, since the presence of
the 59-kDa protein on Western blots always correlates with presence of
the 3.0-kb mspS mRNA as detected on Northern blots (Figs. 2
and 7). Finally, continued growth of the promastigotes in vitro for several additional weeks results in the loss of the 59-kDa
protein at stationary phase (Fig. 8) and a concommitant decline
in infectivity (data not shown). In contrast, the 63-kDa Msps were not
lost even after 4 years of promastigote growth in culture (Fig.
7 C), suggesting but not proving that at least one 63-kDa
species contributes an essential function for this growth.
Investigation of the Msp protein expression in the obligate
intracellular amastigote form of the parasite demonstrated that a still
different set of Msp isoforms is expressed in this stage
(Fig. 10).
Figure 8:
Expression of the 59-kDa Msp protein
ceases during prolonged in vitro cultivation. Lysates were
harvested from cloned stationary phase promastigotes cultured for
6-20 weeks after isolation from a hamster. A, Western
blot probed with anti-LSa. The location of the 59- and 63-kDa Msp
proteins are indicated. B, the Western blot was washed and
probed with anti-tubulin antiserum. The locations of the tubulin
proteins and the now weaker bands of the 59- and 63-kDa Msp proteins
are indicated.
Figure 10:L. chagasi amastigotes do not
express the Msp proteins recognized by anti-LSa and anti-LSb. Lysates
were prepared from virulent, stationary phase promastigotes at day 7
( P, d7) and from amastigotes harvested from U937
macrophages 1 day or 5 days after infection ( A, d1 and d5). The Western blot was probed with anti-Msp,
anti-LSa, and anti-LSb.
In the Trypanosomatid protozoa,
many highly expressed proteins are encoded by tandemly linked families
of genes. An example is the msp gene family of L.
chagasi, investigated here, which contains at least 18 tandem
genes
(7) . To our knowledge, the above data are the first
demonstration that protein products of individual genes within such a
family are differently expressed. The surprising finding that these
gene products are expressed at distinct and predictable times in the
parasite's growth cycle might reflect functional differences. For
instance, the areas of sequence heterogeneity in Msps may affect
properties such as pH optimum, protease specificity, or macrophage
binding. Given the multiplicity of functions attributed to Msp proteins
and the diversity of environments encountered by the parasite during
its life cycle, these differences would explain why the parasite
retains and differentially expresses subtly different isoforms of these
very similar proteins.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.