Expression and Function of the Trypanosoma brucei Major Surface Protease (GP63) Genes*

Douglas J. LaCount {ddagger} §, Amy E. Gruszynski ¶, Paul M. Grandgenett {ddagger}, James D. Bangs || and John E. Donelson {ddagger} **

From the {ddagger}Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242 and the Departments of Biological Chemistry and ||Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin 53706

Received for publication, February 10, 2003 , and in revised form, April 14, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The genome of the African trypanosome Trypanosoma brucei (Tb) contains at least three gene families (TbMSP-A, -B, and -C) encoding homologues of the abundant major surface protease (MSP, previously called GP63), which is found in all Leishmania species. TbMSP-B mRNA occurs in both procyclic and bloodstream trypanosomes, whereas TbMSP-A and -C mRNAs are detected only in bloodstream organisms. RNA interference (RNAi)-mediated gene silencing was used to investigate the function of TbMSP-B protein. RNAi directed against TbMSP-B but not TbMSP-A ablated the steady state TbMSP-B mRNA levels in both procyclic and bloodstream cells but had no effect on the kinetics of cultured trypanosome growth in either stage. Procyclic trypanosomes have been shown previously to have an uncharacterized cell surface metalloprotease activity that can release ectopically expressed surface proteins. To determine whether TbMSP-B is responsible for this release, transgenic variant surface glycoprotein 117 (VSG117) was expressed constitutively in T. brucei procyclic TbMSP-RNAi cell lines, and the amount of surface VSG117 was determined using a surface biotinylation assay. Ablation of TbMSP-B but not TbMSP-A mRNA resulted in a marked decrease in VSG release with a concomitant increase in steady state cell-associated VSG117, indicating that TbMSP-B mediates the surface protease activity of procyclic trypanosomes. This finding is consistent with previous pharmacological studies showing that peptidomimetic collagenase inhibitors block release of transgenic VSG from procyclic trypanosomes and are toxic for bloodstream but not procyclic organisms.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The major surface protease (MSP)1 of the protozoan parasite Leishmania is a highly abundant zinc metalloprotease on the cell surface that contributes to the ability of Leishmania to foil the mammalian immune system. This protease, previously called GP63 or leishmanolysin, will be referred to here as MSP in accordance with the recommendations for a standardized nomenclature of trypanosomatid proteins (1, 2). Leishmania exists extracellularly in the sand fly vector and intracellularly in the macrophages of mammalian hosts. Leishmania MSPs have been studied extensively in several Leishmania sp. and have been shown (i) to participate in macrophage attachment and entry, (ii) to support survival within the phagolysosome of the macrophage, and (iii) to provide resistance to complement-mediated lysis (313). Studies of this protein also have included the determination of its three-dimensional structure (14) and post-translational modifications (1517), characterization of its protease activity (1820), and elucidation of its regulation and differential expression (2124). The MSP genes (MSPs) are located in tandem arranged multigene arrays in all Leishmania sp. studied (25, 26). Within the gene arrays, individual genes display different life cycle-specific expression patterns (2732). The relative locations of the MSPs in the Leishmania genome do not provide an indication of their differential expression, but the MSPs expressed in different life cycle stages have different 3' untranslated region sequences (24). Experiments with a reporter gene fused to the MSP 3' untranslated regions demonstrate that this life cycle-specific regulation is conferred by specific sequences within the 3' untranslated region (2, 33).

Another protozoan parasite, the African trypanosome, also effectively thwarts the mammalian immune system but does so by a different mechanism. This pathogen is transmitted by tsetse flies and causes African sleeping sickness, a fatal disease unless treated with highly toxic drugs. In contrast to Leishmania, African trypanosomes are exclusively extracellular throughout their life cycle. In the bloodstream of their host, they evade the immune system by undergoing antigenic variation, a phenomenon whereby they periodically switch their major surface protein, the variant surface glycoprotein (VSG). Because antigenic variation is a very efficient immune evasion strategy for an extracellular parasite, we were surprised to discover genes encoding homologues of Leishmania MSPs in the African trypanosome genome (34, 35). The corresponding MSP molecules in extracellular African trypanosomes do not contribute to parasite entry and survival in macrophages as they do for the intracellular Leishmania, so they might perform cellular functions for African trypanosomes that they either do not provide for Leishmania or are functions yet to be discovered in Leishmania. One possible function was suggested by our earlier biochemical studies showing that when cultured trypanosomes in the procyclic stage, i.e. the developmental stage that occurs in the tsetse fly midgut, were engineered to synthesize a transgenic VSG, the procyclic organisms utilized the enzymatic activity of a cell-surface, zinc-dependent metalloprotease to release the VSG molecules from the plasma membrane (36, 37). However, the first TbMSP species that we identified was expressed solely in bloodstream-form parasites and seemed unlikely to account for the procyclic protease activity (35).

Here we document that Trypanosoma brucei has three differentially expressed families of MSP genes, which we call TbMSP-A, -B, and -C. Examination of the T. brucei genomic DNA sequences currently available in the data bases does not reveal any additional TbMSP families, although more could appear when the determination of the T. brucei genome sequence is complete. All three TbMSP families are expressed in bloodstream-stage trypanosomes, but only TbMSP-B is expressed in the procyclic stage. We show here using RNA interference (RNAi) that TbMSP-B can function to release the ectopically expressed VSG from the surface of procyclic trypanosomes. Thus, TbMSP-B imparts a protein-processing function to the surface of African trypanosomes.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Identification of TbMSP-B and -C Genes—The TbMSP-B and -C genes were identified by using the previously determined TbMSP-A amino acid sequence (GenBankTM/EBI accession number U86345 [GenBank] ) (35) to search the genome survey sequences data base of T. brucei bacterial artificial chromosome end sequences (www.ncbi.nlm.nih.gov/blast/) deposited by The Institute for Genomic Research (TIGR). In addition to TbMSP-A, several related but nonidentical sequences were found and arranged into contigs. Gaps in the sequence were closed by PCR amplifying the gap region from genomic DNA and DNA sequencing. The sequences for TbMSP-B and -C matched those deposited subsequently by TIGR and the Sanger Institute, respectively (GenBankTM/EBI accession numbers AC099045 [GenBank] , AY230807 [GenBank] , and TRYP10.0. 000073_31[48784–50556]).

Gene Cloning—The coding sequences of TbMSP-A and -B were PCR-amplified from T. brucei genomic DNA with the following primers (XbaI sites are underlined): 5'-GCTCTAGATGGCAGTGATTATGTTCCCTCGT and 5'-GCTCTAGAGCCGTCACACGCATACTACTATC (Tb-MSP-A) and 5'-GCTCTAGACGCGGTTTTCTGTATTGTTTTG and 5'-GCTCTAGAGCATACATTACCATATCAGCGTC (TbMSP-B). The PCR products were digested with XbaI and ligated into the XbaI sites of plasmid p2T7TiA/GFP (42) to generate plasmids p2T7TiA/TbMSP-A and p2T7TiA/TbMSP-B, respectively.

Cell Lines and Transfections—Procyclic T. brucei 29-13 cells (T7RNAP NEO TETR HYG) and the bloodstream T. brucei single marker cell line (T7RNAP TETR NEO) were gifts from G. A. M. Cross (Rockefeller University, New York) (43). The 29-13 cells were maintained in Cunningham's SM medium supplemented with 10% fetal calf serum (FCS) and were transfected with NotI-linearized plasmids (5–10 µg) as described (42). Stable transformants were selected in 15 µg/ml G418, 50 µg/ml hygromycin, and 2.5 µg/ml phleomycin. Clonal lines were established from drug-resistant pooled lines obtained by limiting dilution. The bloodstream T. brucei single marker line was grown in HMI-9 medium (54) supplemented with 10% FCS and transfected as described (43). Transfected cells were transferred to 12 ml of HMI-9 + 10% FCS and distributed among wells in a 24-well tissue culture plate. After recovering overnight, an equal volume of HMI-9 + 10% FCS plus 5 µg/ml G418 and 5 µg/ml phleomycin was added to the wells.

Northern Analysis—Total RNA (5 µg) was subjected to agarose gel electrophoresis and transferred to a nitrocellulose membrane with 20x SSC. Blots were hybridized with 5 x 106 cpm of 32P-random primed probe/ml in 50% formamide, 5x SSC, 5x Denhardt's solution, 0.1% SDS, and 100 µg/ml single-stranded salmon sperm DNA. Final wash conditions were 0.2x SSC and 0.1% SDS at 65 °C.

Expression of VSG117 in TbMSP-B RNAi Cells—The neomycin resistance cassette (NEO) in the plasmid pXS2neo:117wt (36) was replaced with a PCR-generated puromycin resistance cassette (PAC) using flanking AscI and PacI restriction sites. The resultant vector is designed for stable constitutive expression of the VSG117 gene in procyclic trypanosomes following integration into the tubulin gene locus. The BstXI-linearized plasmid was electroporated into procyclic T. brucei 29-13 cells already bearing p2T7Ti/TbMSP-B (clone F2) or p2T7Ti/TbMSP-A (clone A1) and selected with puromycin (5 µg/ml) as described previously (36).

VSG Release Assay—Proteolytic release of VSG from the surface of transgenic procyclic trypanosomes has been described previously (36). Briefly, procyclic RNAi cell lines stably expressing VSG117 were surface-biotinylated with sulfo-N-hydroxysulfosuccinimidobiotin (Pierce) and then incubated for 6 h at 27 °C in culture medium (36). VSG was immunoprecipitated from cell and medium fractions and separated by SDS-PAGE. After electroblotting to membranes, biotinyl-VSG was detected by chemiluminescence with avidin-horseradish peroxidase (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD). Cells displayed excellent motility and morphology throughout the 6-h assay period. To determine the effect of ablation of specific TbMSP genes, the RNAi cell lines were maintained in log phase culture (1 x 105–2 x 106 cells/ml) in the absence or presence of tetracycline (1 µg/ml) for 5 days prior to assay of the VSG release.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Identification of T. brucei Genes Encoding Homologues of Leishmania MSPs—The first leishmanial MSP-like gene to be identified in T. brucei was discovered while sequencing sheared T. brucei genomic DNA fragments, i.e. genomic survey sequences, and the corresponding cDNA was identified in a bloodstream T. brucei cDNA library (34, 35). Subsequent Southern blots and sequence determination of a cloned 14-kb genomic DNA fragment revealed that the T. brucei genome contains at least five closely related copies of the gene encoding this TbMSP, and northern blots showed that they are expressed predominately in bloodstream trypanosomes (35). We have named this gene family TbMSP-A. Inspection of the partially determined chromosomal sequences of T. brucei now available at the web sites of TIGR (tigrblast.tigr.org/er-blast/index.cgi?project=tba1), the Sanger Institute (www.genedb.org/genedb/tryp/index.jsp), and the National Center for Bioinformatics (NCBI; www.ncbi.nlm.nih.gov/blast/) reveals two other gene families encoding proteins with substantial homology to the leishmanial MSPs, and we have named these families TbMSP-B and -C. As described below, the coding regions for these three families do not cross-hybridize on northern blots despite the fact that they encode proteins with about 33% amino acid identity. The TbMSP-B family contains four tandem genes located on chromosome VIII, and the TbMSP-C family is comprised of a single gene at the end of a long gene cluster on chromosome X. The diploid T. brucei genome has 11 megachromosome pairs (chromosomes I–XI), and the chromosomal location of the TbMSP-A family has not yet been identified by either chromosome-specific DNA sequencing or hybridization to chromosomes separated on pulsed field electrophoresis gels. No evidence for the presence of additional TbMSP families is available in the current data bases of the partially determined T. brucei genomic sequence. These data bases are now large enough that any given gene has a >95% chance of being at least partially represented.2 However, the data bases of TIGR and the Sanger Institute contain randomly sheared end sequences and other small genomic sequences (<0.5 kb) encoding segments of TbMSP-like genes, and to date it is not possible to determine whether they all fit into one of the three TbMSP classes. Therefore, it remains possible that additional intact TbMSPs will be found when the T. brucei genomic sequence determination is completed.

Fig. 1 shows a comparison of the deduced amino acid sequences of a representative member of each of the three TbMSP families. In all three sequences the locations of 20 cysteines, 10 prolines, and the metalloprotease catalytic site motif of HEXXH are conserved. These same cysteine and proline positions and the HEXXH catalytic site are conserved in virtually all leishmanial MSPs for which genes have been sequenced to date, suggesting that the TbMSPs have the same general secondary and tertiary structure as do the leishmanial MSPs. The three TbMSP sequences range in size from 591 to 622 amino acids and display about 33% positional identity overall. They differ most at their termini, and their C-terminal differences are particularly apparent in the alignment shown in Fig. 1. Nascent TbMSP-A has an extended C-terminal region, absent in the other two, that is rich in serines and glutamates and culminates in a short hydrophobic segment. Nascent TbMSP-B has a C-terminal hydrophobic tail reminiscent of the hydrophobic C termini of most but not all leishmanial MSPs that are replaced by a glycosylphosphatidylinositol (GPI) anchor to the plasma membrane. Nascent TbMSP-C has a highly hydrophilic C terminus rich in charged amino acids and prolines, indicating that it is not linked to a membrane via a GPI anchor. All three TbMSPs have hydrophobic residues at their N termini that are predicted to serve as signal peptides, although this has yet to be proven.



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FIG. 1.
Comparison of the deduced amino acid sequences of a representative member of three TbMSP families in T. brucei. The sequences were deduced from full-length cDNA sequences containing a 5' spliced leader and 3' poly(A) tail (TbMSP-A) and T. brucei chromosomal DNA sequences available at the web sites of TIGR and the Sanger Institute (TbMSP-B and -C). Asterisks indicate identical residues in at least two of the three sequences. Dots represent gaps inserted into the sequences to maximize alignment. Ovals show 20 conserved cysteines, and inverted triangles show 10 conserved prolines that also occur in Leishmania MSPs. A box surrounds the conserved positions of the zinc binding site HEXXH in zinc metalloproteases.

 

Differential Expression of the Three TbMSP Families— Northern blots of RNAs from the two main developmental stages of T. brucei, the procyclic and bloodstream stages, were probed with 32P-labeled representatives of the three TbMSP families (Fig. 2). Within each of the three TbMSP families the nucleotide sequences of the coding, untranslated, and intergenic regions are highly similar and cross-hybridize. However, despite the amino acid similarities of the TbMSP proteins encoded by the three families, the nucleotide sequences of the three families are sufficiently different (data not shown) that they do not cross-hybridize with each other under high stringency hybridization conditions (Fig. 2). The northern blots in Fig. 2 show that TbMSP-A and -C mRNAs are expressed predominantly in bloodstream trypanosomes, whereas TbMSP-B mRNA is about 2-fold more abundant in procyclic trypanosomes. A reprobe of the blot with 32P-labeled tubulin coding region verified equal loadings of the bloodstream and procyclic RNAs in the blots (data not shown). However, the relative intensities of the signals between the three blots cannot be compared directly because of potential differences in the specific activities of the three radioactive TbMSP probes. To estimate the relative expression levels, 30,000 cDNA clones from a T. brucei bloodstream cDNA library were probed separately with the coding regions for TbMSP-A, -B, and -C, and the ratio of cDNAs encoding TbMSP-A, -B, and -C was found to be about 10:4:1 (not shown). Thus, in bloodstream trypanosomes the mRNA for TbMSP-A is about twice as abundant as the mRNA for TbMSP-B, which in turn is four times more abundant than the mRNA for TbMSP-C. As a further comparison, the ratio of cDNA clones encoding the highly abundant VSG (which comprises 5–10% of the total protein of the organism (38)) compared with those encoding TbMSP-A was found to be about 150:1.



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FIG. 2.
Autoradiograms of Northern blots of total T. brucei RNA showing stage-specific expression of TbMSP mRNAs. RNAs (5 µg/lane) from bloodstream (B) and procyclic (P) T. brucei were probed with 32P-labeled DNAs representing the gene families of TbMSP-A (lane A, a full-length cDNA), TbMSP-B (lane B, a genomic DNA fragment encompassing the 3' 586 bp of one TbMSP-B, the entire intergenic region, and the 5' 896 bp of the next TbMSP-B), and TbMSP-C (lane C, 900 bp from the 3' end of the coding region).

 

RNAi Silencing of the TbMSP-B Family in Procyclic and Bloodstream T. brucei—Because TbMSP-B expression occurs in both procyclic and bloodstream trypanosomes, the resulting protein product likely serves a function(s) needed by trypanosomes during both of these developmental stages. In contrast, TbMSP-A and -C play roles required only during the bloodstream stage because their expression was detected only in bloodstream organisms.

We conducted the experiments described here to examine the function(s) of the constitutively expressed TbMSP-B family. To test the possibility that this function(s) is needed for the growth of procyclic and bloodstream trypanosomes, we employed the technique of RNA interference to deplete both developmental stages of TbMSP-B mRNA. RNAi is a potent method for "knocking down" expression of specific genes in African trypanosomes and other eukaryotes (3941). The method relies on the presence of double-stranded RNA bearing at least part of the targeted mRNA sequence, which in turn triggers a cascade of events still being elucidated that results in degradation of mRNA (41). A 1-kb segment of the 5' coding region of a representative TbMSP-B was PCR-amplified and inserted between opposing T7 promoters in the plasmid p2T7Ti (42). Two tetracycline operator sequences located downstream of the T7 promoters confer tetracycline-responsive double-stranded RNA expression. The resulting plasmid p2T7Ti/TbMSP-B was linearized with NotI and was transfected into the cultured T. brucei procyclic cell line 29-13 and the cultured T. brucei bloodstream single marker line, both of which constitutively expressed T7 RNA polymerase and the tetracycline repressor (43). NotI linearization of the plasmid exposes ribosomal DNA spacer sequences at each end, facilitating integration of the plasmid at a ribosomal DNA spacer region of the genome. Transfected cells bearing the integrated plasmid were selected based upon resistance to bleomycin, and cell clones from each transfection were obtained by serial dilution.

Fig. 3 shows Northern blots of RNAs from two cloned procyclic cell lines (clones F2 and F6) and two cloned bloodstream cell lines (clones A1 and A4) before and after tetracycline induction of TbMSP-B RNAi. As a control, RNAs also were examined from procyclic and bloodstream trypanosome clones containing integrated p2T7Ti/GFP, from which RNAi directed against the green fluorescent protein (GFP), a non-trypanosome protein, can be induced (clone D2). The results show that tetracycline induction of TbMSP-B RNAi diminishes the amount of TbMSP-B mRNA in both procyclic clones F2 and F6 and in bloodstream clones A1 and A4. As expected, tetracycline induction of GFP RNAi in the procyclic and bloodstream D2 clones does not significantly affect the TbMSP-B mRNA level. The filters were stripped and reprobed with the tubulin coding region to confirm that the same amounts of RNA were loaded in each lane.



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FIG. 3.
Use of RNA interference to diminish expression of TbMSP-B mRNA. Autoradiograms of Northern blots of RNAs from procyclic and bloodstream T. brucei bearing integrated p2T7Ti/GFP (GFP and clone D2) or p2T7Ti/TbMSP-B (B, procyclic clones F2 and F6, and bloodstream clones A1 and A4). Prior to RNA isolation the cells were grown in culture for 24 h in the absence (–) or presence (+) of 1 µg/ml tetracycline, which induces expression of double-stranded RNA from the integrated plasmid. The blots were probed with 32P-labeled TbMSP-B coding region (top) and then were stripped and reprobed with 32P-labeled tubulin coding region (bottom).

 

The growth of the cloned TbMSP-B and GFP RNAi cell lines in culture in the absence or continuous presence of tetracycline was examined over a 6- to 7-day period (Fig. 4). Tetracycline induction of RNAi against TbMSP-B or GFP (control) had no significant effect on the growth rate in culture of either procyclic or bloodstream trypanosomes. Thus, diminishing the amount of TbMSP-B mRNA does not affect the growth of either procyclic or bloodstream T. brucei in culture, suggesting that TbMSP-B protein is not essential for cultured growth of either developmental stage.



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FIG. 4.
Growth of procyclic and bloodstream T. brucei with diminished TbMSP-B expression. Growth curves were determined for procyclic (panel A) and bloodstream (panel B) T. brucei bearing integrated p2T7Ti/TbMSP-B (procyclic clones F2 and F6 and bloodstream clones A1 and A4) or p2T7Ti/GFP (clone D2). Procyclic clones F2 and F6 in panel A are depicted with squares and triangles, respectively, as are bloodstream clones A1 and A4 in panel B. Clone D2 is represented with circles. The trypanosomes were grown in culture in the absence (open symbols) or presence (closed symbols) of 1 µg/ml tetracycline, which induces expression of double-stranded RNA from the integrated plasmid. Procyclic and bloodstream cells were diluted to 1 x 105 and 8 x 104 cells/ml, respectively. Cell densities were determined at 12–24 h intervals by counting cells with a hemocytometer. Duplicate counts were performed at each time point. Cultures were diluted as necessary to maintain the cells in log phase. Cell densities shown were corrected to take into account this dilution. After the last procyclic time point, total RNA was prepared and subjected to Northern analysis using the 5' 745 bp of TbMSP-B as a probe.

 

Inhibition of VSG Release from Transgenic Procyclic T. brucei after Induction of RNAi against TbMSP-B—A distinctive feature of T. brucei is that its procyclic and bloodstream stages are each coated with about 107 copies of a GPI-anchored protein that is the predominant protein exposed on the organism's surface at that developmental stage. The outer membrane of procyclic trypanosomes is covered with an invariant protein, of which there are two forms, EP-procyclin and GPEET-procyclin, named for amino acid repeats in their C termini (EP-procyclin has 22–30 Glu-Pro repeats, and GPEET-procyclin has 5–6 Gly-Pro-Glu-Glu-Thr repeats followed by 3 EP repeats (4446)). In contrast, the outer membrane of bloodstream trypanosomes is covered with a VSG that periodically switches as individual trypanosomes within the trypanosome population endeavor to keep one step ahead of the host's immune system (for review, see Ref. 47). In both life cycle stages, the GPI-anchored surface protein constitutes as much as 5–10% of the total cellular protein, placing a large burden on the cell's mechanisms for protein secretion and surface protein turnover. We have developed previously a model system for the study of these secretory and turnover pathways in which procyclic trypanosomes constitutively express a full-length transgenic VSG (36). The transgenic VSG is transported to the cell surface, where it is released to the medium in a truncated form by an endoproteolysis that is inhibited by several known metalloprotease inhibitors, consistent with the possibility that a TbMSP might be involved in the process (37).

To test the possibility that TbMSP-B, i.e. the only TbMSP expressed in procyclic cells, contributes to this release process, a procyclic TbMSP-B RNAi cell line (clone F2) was engineered to express constitutively full-length VSG117 (see "Experimental Procedures"). An equivalent procyclic TbMSP-A RNAi cell line (clone A1) expressing VSG117 was generated as a specificity control. Induction of TbMSP-A RNAi has no effect on procyclic TbMSP-B mRNA levels, cell viability, or growth.3 These two cell lines were maintained in logarithmic phase growth in the absence or presence of tetracycline for 5 days and then tested for VSG release using the surface biotinylation assay described previously (for review, see Refs. 36 and 37 and "Experimental Procedures"). As the half-life of TbMSP protein is not known, the long incubation time of 5 days in the presence of tetracycline was chosen to ensure that the RNAi depletion of TbMSP-B mRNA seen after 24 h (Fig. 3) was likely to be accompanied by a corresponding loss of TbMSP-B protein. Fig. 5 (top) shows the results of TbMSP-B RNAi. Cells incubated in the absence of tetracycline show the typical pattern of VSG release described previously (36, 37). Immediately after surface biotinylation (Fig. 5, top, lane 1), these non-induced cells contain both full-length and truncated biotinylated VSG, whereas after 6 h of incubation, all of the biotinyl-VSG has been released into the medium by truncation (lane 4). This pattern of release is due to the homodimeric structure of native VSG, whereby sequential cleavage of each subunit first will produce a heterodimer anchored to the membrane by one full-length monomer followed by the appearance of a soluble truncated homodimer (36). The tetracycline-induced cells also have both full-length and truncated biotinyl-VSG immediately after biotinylation (Fig. 5, top, lane 5), but the level of cell-associated VSG is significantly elevated in the induced cells relative to the controls (compare lanes 1 and 5). Six h later, however, only some of this biotinyl-VSG has been released into the medium (Fig. 5, top, lane 8); most remains on the cell surface in the same ratio of full-length and truncated forms as at time 0 (lane 7). Identical analyses were performed with the TbMSP-A RNAi cell line, but no effects on VSG levels or release were observed (Fig. 5, bottom). Thus, depletion of TbMSP-B mRNA and, by inference, depletion of TbMSP-B protein specifically inhibit release into the medium, leading to an increase in the steady state level of cell-associated VSG. These results are consistent with the inhibition of VSG release observed earlier with selective metalloprotease inhibitors (36, 37).



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FIG. 5.
Inhibition of VSG release from transgenic procyclic T. brucei after RNAi silencing of TbMSP-B. Procyclic trypanosomes bearing integrated p2T7/TbMSP-B (top, clone F1) or p2T7/TbMSP-A (bottom, clone A1) and stably expressing full-length VSG117, T7 RNA polymerase, and the tetracycline repressor were cultured for 5 days in the absence (–) or presence (+) of 1 µg/ml tetracycline (Tet). The trypanosomes then were surface-biotinylated and cultured for either 0 or 6 h at 27 °C, and aliquots of cells (5 x 106) were separated into cell (c) and medium (m) fractions. The VSG was immunoprecipitated from each sample, subjected to SDS-PAGE, and electrotransferred to nitrocellulose. Biotinyl-VSG was detected by blotting with streptavidin-horseradish peroxidase conjugate and was visualized by chemiluminescence. The positions of full-length (F) and proteolytically truncated (T) VSG are indicated.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The MSPs of Leishmania contribute to the parasite's entry and survival in macrophages as well as its resistance to complement (313). Leishmania chagasi and several other Leishmania species have three well characterized classes of MSPs, one of which is constitutively expressed throughout the life cycle; the other two classes are differentially expressed in the various life cycle stages (24, 25). As shown here, T. brucei also has three classes of TbMSPs that exhibit a similar expression pattern in which one class is constitutively expressed in bloodstream and procyclic forms and the other two are only expressed in the bloodstream form. However, T. brucei does not invade macrophages, so the three TbMSP classes do not play a role in that process. Moreover, the procyclic form does not confront the mammalian immune system; therefore, TbMSP-B, the only TbMSP expressed in procyclic organisms, does not need to participate in complement resistance. Thus, the TbMSPs likely provide different functions for African trypanosomes than do the MSPs for Leishmania.

The TbMSPs and the Leishmania MSPs share about 33% sequence identity and positional conservation of 20 cysteines and 10 prolines (Fig. 1), suggesting that they also share common three-dimensional features. Consistent with this possibility, when the amino acid sequences of the TbMSPs are compared with the known sequence and three-dimensional structure of a Leishmania major MSP (14) by a computer program designed to predict three-dimensional structures (www.expasy.org/spdbv/), the TbMSPs are predicted to fold into three-dimensional shapes similar to that of the L. major MSP (data not shown). Although caution must be exercised in interpreting the predicted TbMSP structures, it is striking that the conformation of {alpha}-helices and {beta}-pleated sheets surrounding the zinc binding and protease sites of the MSP is predicted to be highly conserved in the three TbMSPs. The least conserved regions among the three TbMSP classes are their C termini. Nascent TbMSP-A and -B have C-terminal hydrophobic tails reminiscent of GPI-anchor addition signals, whereas TbMSP-C has a hydrophilic C terminus that is unlikely to be a substrate for GPI addition. These differences suggest that TbMSP-A and -B are membrane-associated and that TbMSP-C is not.

The RNAi depletion experiments depicted in Fig. 5 indicate that the constitutively expressed TbMSP-B provides the previously detected zinc metalloprotease activity that releases a transgenic VSG from procyclic cells (36, 37). When the TbMSP-B mRNA level is reduced by RNAi (Fig. 3), truncation and release of the biotinylated VSG from the trypanosome surface is greatly diminished. The reason that the VSG truncation and release process is not completely blocked by TbMSP-B RNAi is unknown, but the simplest explanation is that the RNAi has depleted but not completely eliminated all of the TbMSP-B molecules. This explanation is supported by Fig. 3, which shows that TbMSP-B mRNA indeed is degraded after RNAi induction but that the full-length mRNA molecules may not be completely eliminated. An alternative explanation for why truncation/release is not completely abolished is that another procyclic protease may inefficiently release the transgenic VSG in the absence of TbMSP-B. A third possibility is that some cells of the original clonal TbMSP-B RNAi line may have lost the ability to express TbMSP-B double-stranded RNA in response to tetracycline; we occasionally have observed such a reversion in other T. brucei clonal lines engineered to generate RNAi against unrelated genes.

VSG molecules are not thought to be normally expressed on the surface of procyclic T. brucei; instead, the surface is covered with EP- and GPEET-procyclin, which are innately protease-resistant (45, 46). Why then is TbMSP-B present in procyclic cells, surely not to release transgenic VSG? One possibility is that there might be leaky expression of VSGs in procyclic trypanosomes, requiring a mechanism to eliminate the corresponding VSG proteins before they disrupt the integrity of the procyclin coat. This possibility is supported by the fact that the transcription of endogenous VSG and procyclin genes is regulated much differently than all other protein-encoding genes in T. brucei. Both are transcribed at a high rate by an {alpha}-amanitin-insensitive, RNA polymerase I-like activity that is virtually indistinguishable from the RNA polymerase I activity that transcribes the rRNA genes (46, 48). In addition, VSG transcription occurs from 1 of about 20 potential telomere-linked VSG expression sites that is uniquely located in an extranucleolar compartment of bloodstream-trypanosome nucleus (49). It is not known if the procyclin genes likewise are transcribed in an extranucleolar compartment. However, in a given trypanosome the "silent" VSG expression sites have been shown to be leaky, and mRNAs from the (non-VSG) expression site-associated genes (ESAGs) have been detected in procyclic trypanosomes (50, 51). Thus, TbMSP-B might be present in procyclic cells to ensure that VSG molecules and other unnecessary, non-procyclin proteins do not accumulate on the procyclic cell surface. Alternatively, TbMSP-B may serve another as of yet undefined role for procyclic cells. This other function, if it exists, is not essential for procyclic growth in culture because cultured procyclic trypanosomes induced to generate RNAi against TbMSP-B have the same growth rate as uninduced cells or cells expressing RNAi against GFP (Fig. 4). It might, however, be required for procyclic cell survival in the midgut environment of the tsetse fly.

TbMSP-B mRNA is also present in bloodstream trypanosomes, and its depletion in these cells likewise has no obvious effect on their growth in culture (Fig. 4). Although TbMSP-B may have essential roles during growth in the mammalian host, a more intriguing question is why TbMSP-B is expressed in bloodstream trypanosomes in the first place. Given that TbMSP-B can cleave and release transgenic VSG from the surface of procyclic cells and that bloodstream trypanosomes are covered with endogenous VSG, why does TbMSP-B not cleave the endogenous VSG? Although it is possible that TbMSP-B is in fact cleaving off VSG at a low level, proteolytically cleaved VSG has not been detected in the culture medium from bloodstream cells (53). More likely, TbMSP-B is being regulated in bloodstream cells at the level of translation, localization, or processing to prevent it from removing VSG. In fact, it is possible that the only reason for TbMSP-B expression in bloodstream cells is to ensure that it is present when bloodstream trypanosomes differentiate into the procyclic form. During differentiation, preadapted short stumpy bloodstream trypanosomes quickly replace the 107 VSG molecules on their surface with an equivalent amount of procyclin. VSG release during differentiation occurs via a combination of endoproteolysis and cleavage of the GPI anchor on the VSG by GPI-specific phospholipase C (52, 53). Furthermore, the proteolytic mode of release is selectively inhibited by the same compounds that block release of transgenic VSG from procyclics (37, 53). TbMSP-B, which is up-regulated during the differentiation process, is the most obvious candidate metalloprotease, although an accessory role for the other TbMSPs cannot be excluded. Whether the metalloprotease activity is essential for differentiation is not yet known. Nevertheless, one possible scenario is that TbMSP-B is up-regulated during differentiation to release the old VSG coat and then is maintained in procyclics for some other important function, perhaps to ensure that deleterious effects of any lingering or leaky VSG transcription are squelched at the protein level.

The roles of the other TbMSPs in bloodstream trypanosomes are also unclear. One or more of the TbMSPs are likely to be important in bloodstream cells given that peptidomimetic metalloprotease inhibitors are toxic in vitro in the low micromolar range (37). Another consideration is that bloodstream trypanosomes are resistant to complement-mediated lysis via the alternate pathway, and one of the roles of MSPs in Leishmania is to bind complement component C3 and proteolytically convert C3b to an inactive form, thus interrupting the complement cascade (13). The dense VSG coat of bloodstream trypanosomes is one factor contributing to their complement resistance (55, 56), but one or more of the three TbMSPs also might be involved. As inhibiting TbMSP-B expression in bloodstream cells had no observable effect and TbMSP-B is the only MSP expressed in complement-sensitive procyclic cells, it seems unlikely that TbMSP-B can account for either of these observations. Experiments are underway to determine whether the other TbMSPs are involved in these processes.


    FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AC099045 [GenBank] , AY230807 [GenBank] , and TRYP10.0.000073_31[48784–50556].

* This work was supported in part by National Institutes of Health Grants AI32135 (to J. E. D.) and AI35739 (to J. D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Dept. of Genome Sciences, University of Washington, Seattle, WA 98195. Back

** To whom correspondence should be addressed. Tel.: 319-335-7934; Fax: 319-333-4204.

1 The abbreviations used are: MSP, major surface protease of Leishmania; TbMSP, MSP of Trypanosoma brucei; GFP, green fluorescent protein; RNAi, RNA interference; GPI, glycosylphosphatidylinositol; VSG, variant surface glycoprotein; FCS, fetal calf serum; TIGR, The Institute for Genomic Research. Back

2 N. El-Sayed, personal communication. Back

3 D. J. LaCount and J. E. Donelson, unpublished observations. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Clayton, C., Adams, M., Almeida, R., Baltz, T., Barrett, M., Bastien, P., Belli, S., Beverley, S., Biteau, N., Blackwell, J., Blaineau, C., Boshart, M., Bringaud, F., Cross, G., Cruz, A., et al. (1998) Mol. Biochem. Parasitol. 97, 221–224[CrossRef][Medline] [Order article via Infotrieve]
  2. Myung, K. S., Beetham, J. K., Wilson, M. E., and Donelson, J. E. (2002) J. Biol. Chem. 277, 16489–16497[Abstract/Free Full Text]
  3. Chang, C. S., and Chang, K. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 100–104[Abstract]
  4. Bordier, C. (1987) Parasitol. Today 3, 151–153[Medline] [Order article via Infotrieve]
  5. Russell, D. G., and Wilhelm, H. (1986) J. Immunol. 136, 2613–2620[Abstract/Free Full Text]
  6. Wilson, M. E., and Hardin, K. K. (1988) J. Immunol. 141, 265–272[Abstract/Free Full Text]
  7. Chaudhuri, G., Chaudhuri, M., Pan, A., and Chang, K.-P. (1989) J. Biol. Chem. 264, 7483–7489[Abstract/Free Full Text]
  8. Seay, M. B., Heard, P. L., and Chaudhuri, G. (1996) Infect. Immun. 64, 5129–5137[Abstract]
  9. Joshi, P. B., Sacks, D. L., Modi, G., and McMaster, W. R. (1998) Mol. Microbiol. 27, 519–530[CrossRef][Medline] [Order article via Infotrieve]
  10. Ahmed, A. A., Wahbi, A., Norlind, K., Kharazmi, A., Sundqvist, K.-G., Mutt, V., and Lidén, S. (1998) Scand. J. Immunol. 48, 79–85[CrossRef][Medline] [Order article via Infotrieve]
  11. Garcia, M. R., Graham, S., Harris, R. A., Beverley, S. M., and Kaye, P. M. (1997) Eur. J. Immunol. 27, 1005–1013[Medline] [Order article via Infotrieve]
  12. Corradin, S., Ransijn, A., Corradin, G., Roggero, M. A., Schmitz, A. A. P., Schneider, P., Mauel, J., and Vergeres, G. (1999) J. Biol. Chem. 274, 25411–25418[Abstract/Free Full Text]
  13. Brittingham, A., Morrison, C. J., McMaster, W. R., McGwire, B. S., Chang, K. P., and Mosser, D. M. (1995) J. Immunol. 155, 3102–3111[Abstract]
  14. Schlagenhauf, E., Etges, R., and Metcalf, P. (1998) Structure 6, 1035–1046[Medline] [Order article via Infotrieve]
  15. Button, L. L., and McMaster, W. R. (1988) J. Exp. Med. 167, 724–729[Abstract]
  16. Schneider, P., Ferguson, M. A., McConville, M. J., Mehlert, A., Homans, S. W., and Bordier, C. (1990) J. Biol. Chem. 265, 16955–16964[Abstract/Free Full Text]
  17. Ferguson, M. A. (1999) J. Cell Sci. 112, 2799–2809[Abstract/Free Full Text]
  18. Chaudhuri, G., and Chang, K. P. (1988) Mol. Biochem. Parasitol. 27, 43–52[Medline] [Order article via Infotrieve]
  19. Bouvier, J., Bordier, C., Vogel, H., Reichelt, R., and Etges, R. (1989) Mol. Biochem. Parasitol. 37, 235–246[CrossRef][Medline] [Order article via Infotrieve]
  20. Ip, H. S., Orn, A., Russell, D. G., and Cross, G. A. M. (1990) Mol. Biochem. Parasitol. 40, 163–172[Medline] [Order article via Infotrieve]
  21. McGwire, B. S., and Chang, K.-P. (1996) J. Biol. Chem. 271, 7903–7909[Abstract/Free Full Text]
  22. Roberts, S. C., Wilson, M. E., and Donelson, J. E. (1995) J. Biol. Chem. 270, 8884–8892[Abstract/Free Full Text]
  23. Voth, B. R., Kelly, B. L., Joshi, P. B., Ivens, A. C., and McMaster, W. R. (1998) Mol. Biochem. Parasitol. 93, 31–41[CrossRef][Medline] [Order article via Infotrieve]
  24. Ramamoorthy, R., Donelson, J. E., Paetz, K. E., Maybodi, M., Roberts, S. P., and Wilson, M. E. (1992) J. Biol. Chem. 267, 1888–1895[Abstract/Free Full Text]
  25. Roberts, S. C., Swihart, K. G., Agey, M. W., Ramamoorthy, R., Wilson, M. E., and Donelson, J. E. (1993) Mol. Biochem. Parasitol. 62, 157–172[CrossRef][Medline] [Order article via Infotrieve]
  26. Button, L. L., Russell, D. G., Klein, H. L., Medina-Acosta, E., Karess, R. E., and McMaster, W. R. (1989) Mol. Biochem. Parasitol. 32, 271–284[CrossRef][Medline] [Order article via Infotrieve]
  27. Ramamoorthy, R., Swihart, K. G., McCoy, J. J., Wilson, M. E., and Donelson, J. E. (1995) J. Biol. Chem. 270, 12133–12139[Abstract/Free Full Text]
  28. Webb, J. R., Button, L. L., and McMaster, W. R. (1991) Mol. Biochem. Parasitol. 48, 173–184[CrossRef][Medline] [Order article via Infotrieve]
  29. Medina-Acosta, E., Beverley, S. M., and Russell, D. G. (1993) Infect. Agents Dis. 2, 25–34[Medline] [Order article via Infotrieve]
  30. Medina-Acosta, E., Karess, R. E., and Russell, D. G. (1993) Mol. Biochem. Parasitol. 57, 31–46[CrossRef][Medline] [Order article via Infotrieve]
  31. Steinkraus, H. B., Greer, J. M., Stephenson, D. C., and Langer, P. J. (1993) Mol. Biochem. Parasitol. 62, 173–186[CrossRef][Medline] [Order article via Infotrieve]
  32. Wilson, M. E., Hardin, K. K., and Donelson, J. E. (1989) J. Immunol. 143, 678–684[Abstract/Free Full Text]
  33. Kelly, B. L., Nelson, T. N., and McMaster, W. R. (2001) Mol. Biochem. Parasitol. 116, 101–104[CrossRef][Medline] [Order article via Infotrieve]
  34. El-Sayed, N. M., and Donelson, J. E. (1997) Mol. Biochem. Parasitol. 84, 167–178[CrossRef][Medline] [Order article via Infotrieve]
  35. El-Sayed, N. M. A., and Donelson, J. E. (1997) J. Biol. Chem. 272, 26742–26748[Abstract/Free Full Text]
  36. Bangs, J. D., Ransom, D. M., McDowell, M. A., and Brouch, E. M. (1997) EMBO J. 16, 4285–4294[Abstract/Free Full Text]
  37. Bangs, J. D., Ransom, D. M., Nimick, M., Christie, G., and Hooper, N. M. (2001) Mol. Biochem. Parasitol. 114, 111–117[CrossRef][Medline] [Order article via Infotrieve]
  38. Turner, M. J. (1985) Br. Med. Bull. 41, 137–143[Medline] [Order article via Infotrieve]
  39. Ngo, H., Tschudi, C., and Ullu, E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14687–14692[Abstract/Free Full Text]
  40. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Nature 391, 806–811[CrossRef][Medline] [Order article via Infotrieve]
  41. Hannon, G. J. (2002) Nature 418, 244–251[CrossRef][Medline] [Order article via Infotrieve]
  42. LaCount, D. J., Barrett, B., and Donelson, J. E. (2002) J. Biol. Chem. 277, 17580–17588[Abstract/Free Full Text]
  43. Wirtz, E., Leal, S., Ochatt, C., and Cross, G. A. M. (1999) Mol. Biochem. Parasitol. 99, 89–101[CrossRef][Medline] [Order article via Infotrieve]
  44. Roditi, I., and Clayton, C. (1999) Mol. Biochem. Parasitol. 103, 99–100[CrossRef][Medline] [Order article via Infotrieve]
  45. Roditi, I., Furger, A., Ruepp, S., Schurch, N., and Butikofer, P. (1998) Mol. Biochem. Parasitol. 91, 117–130[CrossRef][Medline] [Order article via Infotrieve]
  46. Hotz, H. R., Biebinger, S., Flaspohler, J., and Clayton, C. (1998) Mol. Biochem. Parasitol. 91, 131–143[CrossRef][Medline] [Order article via Infotrieve]
  47. Borst, P., and Ulbert, S. (2001) Mol. Biochem. Parasitol. 114, 17–27[CrossRef][Medline] [Order article via Infotrieve]
  48. Kooter, J. M., and Borst, P. (1984) Nucleic Acids Res. 12, 9457–9472[Abstract]
  49. Navarro, M., and Gull, K. (2001) Nature 414, 759–763[CrossRef][Medline] [Order article via Infotrieve]
  50. Alarcon, C. M., Pedram, M., and Donelson, J. E. (1999) J. Biol. Chem. 274, 16884–16893[Abstract/Free Full Text]
  51. Ansorge, I., Dietmar, S., Melville, S., Hartmann, C., and Clayton, C. (1999) Mol. Biochem. Parasitol. 101, 81–94[CrossRef][Medline] [Order article via Infotrieve]
  52. Ziegelbauer, K., Stahl, B., Karas, M., Steirhof, Y.-D., and Overath, P. (1993) Biochemistry 32, 3737–3742[Medline] [Order article via Infotrieve]
  53. Gruszynski, A. E., DeMaster, A., Hooper, N. M., and Bangs, J. D. (2003) J. Biol. Chem. 278, 24665–24672
  54. Hirumi, H., Hirumi, K., Doyle, J. J., and Cross, G. A. (1980) Parasitol. 80, 371–382
  55. Russo, D. C., Williams, D. J., and Grab, D. J. (1994) Parasitol. Res. 80, 487–492[Medline] [Order article via Infotrieve]
  56. Frevert, U., and Reinwald, E. (1990) Eur. J. Cell Biol. 52, 264–269[Medline] [Order article via Infotrieve]