©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Developmentally Regulated Expression of a Novel 59-kDa Product of the Major Surface Protease (Msp or gp63) Gene Family of Leishmania Chagasi(*)

Sigrid C. Roberts (1), Mary E. Wilson (2), John E. Donelson (1) (3)(§)

From the (1) Departments of Biochemistry and (2) Microbiology and Internal Medicine, University of Iowa, Veterans Administration Medical Center and the (3) Howard Hughes Medical Institute, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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() or gp63) likely contribute to each of these steps.

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.


MATERIALS AND METHODS

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 10parasites/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 neogene are called pX mspS1.F and pX mspS2.F, whereas constructs containing the msp genes in the reverse orientation of neoare termed pX mspS1.R and pX mspS2.R.

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 10/ml in electroporation buffer (21 m M HEPES, pH 7.5, 137 m M NaCl, 5 m M KCl, 0.7 m M NaPO, 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 10parasites/ml. Lysates were analyzed on Western blots.


RESULTS

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.

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.


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).

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.


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.

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.


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.

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.

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.


DISCUSSION

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.


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.

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.() 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.


FOOTNOTES

*
This research was supported in part by National Institutes of Health Research Grants AI32135 and AI30126, a Veterans Administration Merit Review Grant, and an Established Investigator Award from the American Heart Association (to M. E. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 319-335-7889; Fax: 319-335-6764.

The abbreviations used are: Msp, major surface protease; PI-PLC, phosphatidyl-specific inositol phospholipase C; GPI, glycophosphatidyl; ConA, concanavalin A; CRD, cross-reactive determinant; HOMEM, modified minimal essential medium.

J. Streit, personal communication.

R. Ramamoorthy, personal communication.

Mary E. Wilson, unpublished results.


ACKNOWLEDGEMENTS

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.


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