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Nucleic Acids Res. Jun 2009; 37(10): 3407–3417.
Published online Mar 31, 2009. doi:  10.1093/nar/gkp172
PMCID: PMC2691823

Genomic organization and expression profile of the mucin-associated surface protein (masp) family of the human pathogen Trypanosoma cruzi

Abstract

A novel large multigene family was recently identified in the human pathogen Trypanosoma cruzi, causative agent of Chagas disease, and corresponds to ~6% of the parasite diploid genome. The predicted gene products, mucin-associated surface proteins (MASPs), are characterized by highly conserved N- and C-terminal domains and a strikingly variable and repetitive central region. We report here an analysis of the genomic organization and expression profile of masp genes. Masps are not randomly distributed throughout the genome but instead are clustered with genes encoding mucin and other surface protein families. Masp transcripts vary in size, are preferentially expressed during the trypomastigote stage and contain highly conserved 5′ and 3′ untranslated regions. A sequence analysis of a trypomastigote cDNA library reveals the expression of multiple masp variants with a bias towards a particular masp subgroup. Immunofluorescence assays using antibodies generated against a MASP peptide reveals that the expression of particular MASPs at the cell membrane is limited to subsets of the parasite population. Western blots of phosphatidylinositol-specific phospholipase C (PI-PLC)-treated parasites suggest that MASP may be GPI-anchored and shed into the medium culture, thus contributing to the large repertoire of parasite polypeptides that are exposed to the host immune system.

INTRODUCTION

Trypanosoma cruzi is an important human pathogen and the etiological agent of Chagas disease. It is estimated that there are 15–18 million infected people primarily in Central and South America. Trypanosoma cruzi trypomastigotes are transmitted typically from a reduviid bug to the mammalian host through the vector feces during the insect bite, but also by ingestion of contaminated food, following blood transfusion or organ donation. Trypomastigotes can invade several types of host cells where they differentiate intracellularly into replicative amastigotes. Amastigotes develop into non-dividing trypomastigotes which are released extracellularly upon cell disruption and can initiate another round of host cell infection. They can also infect a reduviid vector during feeding, within which they differentiate into replicative epimastigotes. Acute Chagas disease follows initial infection with T. cruzi and is characterized by high blood parasitaemia and broad tissue parasitism. This phase is usually a mild self-limited systemic illness involving fever and malaise, and in most cases, is not specifically diagnosed. Chronic Chagas disease appears years later and may lead to cardiomyopathy, megaesophagus and/or megacolon. Currently, there are no vaccines available. The drugs used in the treatment are toxic and effective only during the acute phase of the disease (1).

The first draft of the T. cruzi genome was published (2) along with the complete genome sequences of two related trypanosomatid human pathogens, T. brucei (3) and Leishmania major (4). A comparative analysis of gene content of the three parasites has allowed the identification of a conserved core of ~6200 genes and numerous species-specific genes (5). On one hand, these analyses provided the foundation for the development of unexplored chemotherapeutic approaches against these parasites, such as drugs that could be designed against conserved core processes and potentially useful against all three organisms. On the other hand, the characterization of species-specific genes is allowing us to better understand the distinct nature of the disease they cause and may help develop more specific interventions for treatment and prevention.

The T. cruzi diploid genome size is ~100 Mb with an estimated haploid gene number of 12 000 genes in the CL-Brener strain (2). Compared to T. brucei and L. major, a remarkable feature of the T. cruzi genome is the massive expansion of surface protein gene families, which include the previously characterized gp85/trans-sialidase (TS)-like superfamily, mucins and the metalloprotease gp63. A major finding of the T. cruzi genome project was the discovery of a ~1400-member gene family encoding the novel mucin-associated surface protein (MASP). Despite its large size, no member of the masp family has been characterized to date. We report here on the genome organization and expression profile of the family. Gene families encoding MASP and other surface protein gene are clustered in T. cruzi-specific regions of the genome which fall outside of the regions of synteny (i.e. where gene order is conserved) between the three trypanosomatids. Within these large clusters, masp genes and pseudogenes are preferentially located downstream of mucin TcMUCII. MASP members contain N- and C-terminal conserved domains that encode a putative signal peptide and a GPI-anchor addition site. The central region is variable both in length and in sequence and contains a large repertoire of repetitive motifs. In contrast to the highly heterogeneous coding region, masp mRNAs have conserved 5′ and 3′ untranslated regions (UTRs). Western blots of phosphatidylinositol specific phospholipase C (PI-PLC)-treated parasites suggest that MASP is GPI-anchored and is preferentially expressed during the trypomastigote (bloodstream) stage. Interestingly, despite the large number of genes, an examination of the expression profile reveals that a subset of masp members is preferentially expressed in a parasite population. This is the first detailed analysis of the masp gene family of T. cruzi.

MATERIALS AND METHODS

In silico analysis of MASP sequences

The figures depicting the genome organization of MASP family were generated by in-house PERL (Practical Extraction and Report Language) scripts taking advantage of the Bio::Graphics module, part of Bioperl toolkit (http://www.bioperl.org). The frequency distribution of genes in the vicinity of masp loci was computed by PERL and AWK scripts from the data stored in our local T. cruzi database and the result exported to excel to generate the graphs. To identify MASP conserved regions, the coding and flanking regions of the 771 MASP genes annotated in the T. cruzi genome containing both the N- and C-terminal conserved domains were aligned using the ClustalW algorithm (6). A consensus sequence was generated and the percentage identity for each position across the entire sequence was calculated using an in-house PERL script. The cDNA sequences were analyzed using Phred-Phrap-Consed package (7,8). To identify the MASP expressed members, the cDNA sequences were searched against a local database of the T. cruzi coding sequences and contigs using the BLASTN algorithm (9). MASP repetitive motifs were identified using the algorithm SSRIT (Simple Sequence Repeats Identification Tool) (10). N- and O-glycosylation and phosphorylation predictions were performed using the algorithms NetNGlyc 1.0, NetOGlyc 3.1 (11) and NetPhos 3.1 (12), respectively. Predictions for signal peptide and GPI-anchor addition sites were performed using SignalP (13) and GPI-SOM (14) algorithms, respectively. Multidimensional scaling was performed using XGobi/XGvis (15). The dataset consisted of a pairwise distance matrix of the first 60 nt encoding the N-terminal and the last 60 nt encoding the C-terminal of MASP (771 genes), serine-alanine-proline rich protein (SAP) (35 genes), TcSMUGS (eight genes), TcSMUGL (seven genes), TcMUC I (53 genes), TcMUC II (550 genes) and TcMUC III (two genes). The pairwise distance matrix was computed using Kimura-2p (16) implementation of MEGA2 (17).

Parasites

Epimastigote forms of CL-Brener strain of T. cruzi were maintained in logarithmic growth phase at 28°C in supplemented liver digested-neutralized tryptose (LDNT) medium (18) containing 10% fetal bovine serum. Amastigote and trypomastigote forms were obtained from infected Vero cells grown at 37°C or 34°C, respectively and in 5% CO2 in RPMI medium supplemented with 5% or 1% of fetal bovine serum, respectively. Only preparations of the amastigote and trypomastigote forms having >90% purity were used.

Generation of antisera

A MASP peptide was selected based on MEME-identified motifs (2) using the following criteria: (i) the most conserved motifs present in the predicted protein; (ii) predicted peptide immunogenicity; (iii) specificity of peptide to MASP. Using the above criteria, we have synthesized peptide 7 (Cys-QHQQHEH-(P/S)-AENGEESAKDK) (Pacific Immunology Corp., San Diego, CA). A cysteine residue was added to the N-terminal of the peptide to allow conjugation to the keyhole limpet hemocyanin (KLH) carrier protein. To generate affinity purified anti-MASP antibodies, two rabbits were immunized with 100 μg of the degenerate peptide. MASP antibodies present in the antiserum were purified by affinity chromatography using CNBr-activated sepharose coupled to purified MASP peptide.

Southern and northern blot analyses

Total RNA was isolated from parasite cultures using the RNeasy Kit (Qiagen). For northern blot analysis, 10 μg of total RNA were separated in 1.2% agarose/MOPS/formaldehyde gel. The RNA was blotted onto a Hybond N+ membrane (Amersham-Pharmacia Biotech) and fixed through UV irradiation using UV linker 2400 (Stratagene). The MASP probes were labeled with [α-32P]dCTP using the Megaprime DNA-labeling protocol from Amersham-Pharmacia Biotech. The membranes were hybridized in a 50% formamide buffer for 18 h at 42°C as previously described (19), washed twice with 2× SSC/0.1% SDS at 65°C for 30 min each, and exposed to X-ray films (Kodak). The T. cruzi chromosomes blots were kindly provided by Dr Andersson (Karolinska Institute) using the pulse-field gel electrophoresis conditions previously described (20). The hybridization of the chromosome blots was performed as previously described (21).

Construction and screening of a trypomastigote cDNA library

A unidirectional trypomastigote expression library of the T. cruzi CL Brener strain was constructed in the lambda-ZAPII vector using procedures provided by the supplier of the kits for cDNA synthesis and in vitro phage packing (Stratagene). It was generated from poly-A+ RNA isolated from tissue culture purified trypomastigotes obtained from Vero host cell. The primary library, which has the titer of 1.6 × 106 pfu/ml, was amplified to a titer of 109/ml. A total of ~15 000 clones were screened with a 32P-labeled fragment derived from the MASP 3′ UTR conserved region as previously described (19). Plaques yielding positive signals in the first screen were purified by successive rounds of screening until isolated positive clones were obtained.

cDNA characterization

Recombinant phagemids were excised by co-infecting Escherichia coli XL-1 Blue cells with the selected λZAPII phage and R408 helper phage as described by the commercial supplier of the phage (Stratagene). Plasmid DNAs were prepared using standard protocols (22) and the cDNA inserts sequenced. The nucleotide sequence for the cDNAs clones has been deposited in the GenBank database under GenBank accession number EU825796-EU825847.

Protein extracts preparation and western blot

Total parasite proteins were extracted from epimastigotes, amastigotes and trypomastigotes by directly suspending 108 cells in 100 ml of Laemmli's sample buffer. Volumes corresponding to 10 μg of total parasite protein extracts were loaded onto a 12.5% SDS–PAGE gel. For western blot analyses, total protein extracts were transferred to polivinylidene difluoride (PVDF) membranes after electrophoresis (Bio Rad trans-blot transfer medium), stained with Ponceau S (Sigma) and blocked by incubation with 5.0% non-fat dry milk. MASP proteins were detected on western blots using affinity purified anti-MASP antibody diluted at 1:300 in PBS 0.1% Tween-20 and 1.0% non-fat dry milk. The blots were revealed with peroxidase conjugated anti-rabbit IgG at a dilution of 1:5000 and ECL Plus (GE HealthCare).

Immunofluorescence assay

The cells were fixed in 2% paraformaldehyde/PBS and kept at 4°C. To proceed with the IFA, the cells were washed twice in PBS/50 mM NH4Cl, then once in PBS, spotted on a clean glass slide and allowed to dry overnight. The fixed cells were re-hydrated with PBS and blocked for 1 h with 30 μl 5% fetal bovine serum in PBS. Slides were incubated with the affinity-purified anti-MASP antibody or pre-immune serum for 30 min with a 1:250 dilution. The slides were washed five times with blocking solution and incubated with the FITC-conjugated anti-rabbit IgG antibody (Molecular Probes) at the 1:500 dilution for 30 min and then with DAPI at 1:1000 dilution for 1 min. After washing with the blocking solution, the slides were mounted and examined by microscopy.

RESULTS

Genomic context of the masp genes

We have previously reported the discovery of the MASP proteins encoded by the second largest multigene family in the T. cruzi genome (2). Of the 1377 masp genes identified, 814 are full-length and 563 are partial genes located on contig ends and/or pseudogenes. Of the 814 full-length genes, 771 encode proteins containing both N- and C-terminal MASP conserved domains and 43 are chimeric sequences containing the N- or the C-terminal MASP conserved domain. Genes for the MASP and mucin families are interspersed within large non-tandem arrays of genes which encode other surface proteins including mucin TcMUC, gp85/TS-like, GP-63, serine-alanine-proline rich protein (SAP) and dispersed gene family 1 (DGF-1) (Figure 1). Those same regions are enriched with T. cruzi retroelements, such as L1Tc, NARTc, DIRE, VIPER and SIRE and members of the retrotransposon hot spot (RHS) family (2); (23). Because a large number of those genes are located in close proximity of T. cruzi mucins (TcMUC), we have named the family of encoded proteins ‘mucin-associated surface proteins’ (MASPs). Here we have conducted a through inspection of the genes in the vicinity of all masp intact genes and pseudogenes. We observe a strong bias for the occurrence of TcMUCII upstream of masp intact genes or pseudogenes (Figure 2A and B). The frequency of downstream genes differs for masp intact and pseudogenes. Genes more frequently found downstream of intact masp copies correspond to those encoding hypothetical proteins (Figure 2A) whereas those more frequently located downstream of masp pseudogenes correspond to members of the gp85/TS-like superfamily (Figure 2B).

Figure 1.
Genomic organization of the masp family. Two selected T. cruzi genomic scaffolds depicting typical regions containing masp genes and pseudogenes within arrays of surface protein-encoding genes. The colored arrows indicate the position and coding strand ...
Figure 2.
Genes in the vicinity of MASP. Frequency of occurrence of genes up to five positions upstream and downstream to masp intact genes (A) and pseudogenes (B). The values in the graph represent the frequency distribution of gene families independently computed ...

The masp genes are distributed over 210 scaffolds ranging in length from 5.3 to 799 kb. A 796-kb scaffold 1047053516725 (Genbank accession NW_001849575, Supplementary Figure S1) contains the largest number of masp genes, 79 in total. None of the intact masp genes were associated with the 47 subtelomeric regions that are represented in the assembled genome data (2). T. cruzi subtelomeric regions were defined here as sequences that extend from the telomeric hexamer repeats to the first two-way or three-way cluster of orthologous genes (COGs) across the T. cruzi, T. brucei and L. major genomes. In the T. cruzi genome, these telomeric regions are enriched with RHS genes (119 copies), gp85/trans-sialidases (84 copies), retroelements (81 copies that include SIRE, VIPER, DIRE, L1Tc and NARTc), dispersed gene family 1 (46 copies), and hypothetical proteins (42 copies). The only four masp genes found in those regions include three pseudogenes and a MASP/SAP chimeric sequence. Although the T. cruzi chromosome structure has not been completely elucidated and only about half of the T. cruzi telomeres are represented in the assembled genome, the available data suggests that the large clusters of surface protein-encoding genes reside internally in the chromosomes. More specifically, those clusters are located in the T. cruzi-specific regions of the genome which fall outside of the regions of synteny between the three trypanosomatids (Supplementary Figure S1).

To identify the chromosomal localization of masp genes, we performed Southern blot experiments on filters kindly provided by Dr Björn Andersson (Karolinska Institute). T. cruzi chromosomes were separated by pulsed-field gel electrophoresis under three distinct conditions, each optimized to separate small (1.1–0.3 Mb), medium (2.2–0.6 Mb) or large (3.5–2.0 Mb) chromosomes from three T. cruzi strains: CL-Brener, Sylvio X10/7 and CAI/72 (20). Two probes derived from the C-terminal domain and 3′ UTR region of masp were used since they represent the longest conserved regions among all members of this family (see below). Similar hybridization patterns were obtained with both probes and with the three T. cruzi strains. Masp genes appear to locate predominantly in large chromosomes (Figure 3).

Figure 3.
PFGE mapping of the masp genes onto T. cruzi karyotype. Three panels represent distinct pulsed-field gel conditions optimized to separate small (left); medium (center) and large chromosomes (right). The blots were hybridized with a probe derived from ...

MASP protein features

An alignment of the amino-acid sequences of 771 full-length MASP proteins revealed highly conserved N- and C-terminal domains that were predicted to encode a signal peptide and a GPI-anchor addition site, respectively (Figure 4A), suggesting a surface location in the parasite. The central region varies both in sequence (Figure 4A) and in length (ranging from 176 to 645 amino-acid residues) (Supplementary Figure S2) and often contains repetitive motifs.

Figure 4.
MASP protein features. (A) MASP protein sequence. All 771 MASP proteins containing both N- and C-terminal conserved regions were aligned using ClustalW (6). A consensus sequence was generated and a percentage identity score was computed for each position ...

To further investigate the repertoire of repetitive motifs in the family, all 944 MASP proteins encoded by intact genes were analyzed. The sequences corresponding to the signal peptide and the GPI-anchor addition site were removed, so that only the central region of each protein, which is likely to be exposed at the parasite surface, was analyzed. The following repetitions were searched: (i) single amino acids repeated at least five times; (ii) di-peptides repeated at least four times; (iii) tri-peptides repeated at least three times and (iv) tetra- to dodeca-peptides repeated at least twice. From the total of 944 sequences, 406 proteins were found to contain at least one repetitive motif. A total of 679 repetitions were identified in the dataset (Supplementary Table S1). Single residue repetitions are the most common, and those consisting of glutamic acid are the most frequent corresponding to ~27% of the total of the identified repetitive motifs.

A large number of T. cruzi surface proteins are heavily glycosylated. Analyses of full-length MASP proteins revealed at least four potential O-glycosylation sites per sequence, 70% of which correspond to threonines. Also, a total of 2000 predicted N-glycosylation sites were predicted in 710 out of 771 MASP intact proteins. MASP may also be subjected to phosphorylation, since 11–59 potential phosphorylation sites were predicted in the same MASP family subset analyzed.

A search of all MASP proteins against the Pfam database revealed only a few weak hits, 18 in total, to a Mucin-like glycoprotein Pfam domain (PF01456) found in mucin proteins (Supplementary Table S2). A close inspection of these 18 members revealed that in all but two cases the predicted proteins correspond to chimeras containing the N-terminal domain characteristic of TcMUC family and the MASP C-terminal conserved domain. Alignments of the PF01456 domain matches (data not shown) indicate this domain is degenerated in the MASP family. This is reflected by less significant P-values when compared to matches from the TcMUC family (Supplementary Table S2). It is also worth noting that the consensus N- and C-terminal regions of TcMUC and MASP gene families show some level of identity at the nucleotide level, with the N-terminal domains sharing 57% identity and the C-terminal domain 38% (data not shown). In addition, threonine residues located near the C-terminal of TcMUC proteins are also found in the consensus MASP sequence (Figure 4A). Despite these similarities, sequence comparison of the N- and C-terminal conserved regions from all MASP and TcMUC members against each other confirms the unique identity of each family. A multidimensional scaling plot (Figure 4B) which represents the visual schema of the distance matrix reveals two main clusters, one representing the MASP and the other one the TcMUC sequences. The amino-acid composition of these families is also quite distinct (Supplementary Table S3).

Analysis of conserved flanking regions and masp mRNA levels during the T. cruzi life cycle

An alignment of the 5′ and 3′ flanking regions from all 771 masp intact genes revealed that both regions are highly conserved (Figure 5A). Based on sequence similarity with two expressed sequence tags (ESTs) derived from an amastigote cDNA library (accession number CB923887 and S.M.R. Teixeira, unpublished data) we were able to map the polyadenylation addition site (Figure 5A) and to verify that the 3′ flanking conserved region is part of the masp 3′ UTR. This conserved region has been previously described as the repetitive TcIRE element (24), which in fact corresponds to the reverse complement of the MASP C-terminal region and the 3′ UTR conserved sequences. Consistent with our masp chromosomal location results, hybridization of PFGE blots with the TcIRE probe showed that this sequence is located in high molecular weight chromosome bands (24).

Figure 5.
MASP conserved domains and mRNA expression during the T. cruzi life cycle. (A) The coding and flanking regions of the 771 MASP members containing both the N- and C-terminal conserved domains were aligned as in Figure 4. The mRNA structure is depicted ...

Because the 3′ UTR is the largest and the most conserved region of masp transcripts, we selected this sequence to use as a probe in northern blots. As shown in Figure 5B, masp is preferentially expressed in trypomastigotes with low levels of expression also detected in amastigotes and epimastigotes. A similar pattern of hybridization was obtained using a probe derived from the C-terminal conserved region (data not shown). This differential expression is consistent with the results of a T. cruzi proteome study in which nine MASP peptide fragments (out of 5792 total unique peptides) were detected exclusively in trypomastigotes (25). Interestingly, despite the fact that the length of masp coding regions is quite variable (Supplementary Figure S2), a predominant hybridization band of ~1.4 kb was detected.

MASP expression profile and mRNA structure

To investigate the repertoire of masp expression in trypomastigotes, we constructed and probed a trypomastigote cDNA library with the masp 3′ UTR conserved region. A total of ~15 000 phages were screened yielding a total of ~600 positive clones (~4%). A total of 52 independent cDNA clones were sequenced from both ends and searched against the T. cruzi coding sequence database. The T. cruzi genes corresponding to the best hit of each cDNA clone are shown in the Supplementary Table S4. The corresponding gene could be identified for 36 of the cDNA clones. A total of 12 cDNAs could not be unambiguously associated to a specific masp gene, since their sequences covered only the C-terminal conserved domain and the downstream conserved 3′UTR. Four clone sequences were not related to masp.

The analysis of the cDNA sequences revealed that 24 clones correspond to full length masp genes or masp pseudogenes whose N- and C-terminal domains could be reconstituted. Another 13 clones could not be matched to a specific masp gene because the cDNA sequence did not cover the central variable region of the corresponding gene or because the 5′ region is partial due to contig ends. Taken together, this suggests that 46–71% of the 52 sequenced cDNA match full length masp genes or pseudogenes whose N- and C-terminal conserved regions could be reconstituted. Since ~4% of the trypomastigote cDNA clones hybridized with the masp probe and assuming that 46–71% of the positives clones are in fact full-length masp sequences, we estimate that masp non-chimeric genes corresponds to around 1.8–2.8% of the trypomastigote transcripts.

We have previously subjected all MASP predicted proteins containing both N- and C-terminal conserved domains to the MEME algorithm to identify conserved motifs in the family (2). MASP members sharing the same combination of motifs were then clustered, yielding a total of 135 MASP subgroups (2) and Supplementary File 1). As shown in Supplementary Table S4, 7 out of 20 cDNAs (35%) that match full-length masp genes belong to a single MASP subgroup (S008). The remaining 13 cDNAs are derived from genes that belong to distinct subgroups. The cDNA sequences derived from S008 MASP subgroup are however not identical and are derived from distinct genes. In summary, our data indicates an expression bias towards MASP subgroup S008 in the trypomastigote population used to construct the cDNA library.

Another interesting observation was the significant number of cDNA clones containing chimeric MASPs. A total of 11 clones encoded 5′ segments of hypothetical proteins, TcMUCII or TS-like protein and 3′ segments of MASP C-terminal domain and 3′ UTR. One of the chimeric sequences (Supplementary Figure S3) corresponds to the TcMUCII-MASP gene Tc00.1047053508219.50 present in contig 7643 (Genbank acc. #AAHK01000303). This chimera contains the first 126 nt of TcMUCII that encode a signal peptide and 20 amino acids of the putative mature protein (the best non-chimeric TcMUCII hit is Tc00.1047053508873.240). The remaining sequence corresponds to MASP coding sequence (the best non-chimeric MASP hit is Tc00.1047053504239.30) followed by the conserved MASP 3′ UTR. This chimeric cDNA is colinear with the genome sequence, ruling out an artifact of the genome assembly or cDNA library construction. In addition, two individual whole genome shotgun reads, TCHKU21TF and TCJTB48TR, match the cDNA perfectly (Supplementary Figure S3). Also, this expressed chimera sequence codes for potential signal peptide and GPI-anchor addition site and therefore contains a priori the required signals for a surface localization.

Analysis of cDNA sequences containing the spliced leader and/or poly-A tail allowed us to map the masp untranslated regions. As previously predicted from the alignment of all flanking sequences of the masp coding regions (Figure 5), both 5′ and 3′ UTRs are very well conserved among the cDNA clones. The 5′ UTRs are short (50 nt on average) while the 3′ UTRs range from 361 to 498 nt in size.

MASP protein expression and cellular localization

To analyze the expression of MASP throughout the parasite life cycle, protein extracts from the epimastigote and trypomastigote stages were separated on SDS–PAGE followed by immunoblotting using anti-MASP antibodies. The antibodies were raised against a MASP peptide (peptide 7) which, according to the MEME analysis (2), is found in 109 members. As shown in Figure 6A, anti-MASP antibodies reacted with trypomastigote proteins of around 45 kDa. Light additional bands are likely due to a non-specific recognition as they do not appear consistently and reproducibly in independent experiments. Pre-immune sera do not react with the parasite protein extracts (data not shown).

Figure 6.
MASP protein expression and cellular localization. Western blot analysis of total protein extracts (10 μg) from the epimastigote (E) and trypomastigote (T) forms using anti-MASP peptide 7 rabbit antisera (A). (B) PI-PLC treatment of parasites. ...

To determine the cellular localization of MASP proteins, non-permeabilized trypomastigotes were analyzed by immunofluorescence using anti-MASP antibodies. As shown in Figure 6C the entire cell surface of trypomastigotes expressing MASPs containing the peptide 7 was labeled. Only a small proportion (~5%) of the parasites, however, reacted with this antibody, indicating that the expression of peptide 7-containing MASP is limited to a subset of the population.

To confirm the in silico prediction that suggested MASP is GPI-anchored, trypomastigotes were treated with phosphatidylinositol-specific phospholipase C (PI-PLC) and the supernatant and the pellet fractions were analyzed by western blot. Proteins in the expected range (~45 kDa) were detected by anti-MASP antibodies in the supernatant of the PI-PLC-treated trypomastigotes (Figure 6B, lane 4) suggesting that MASP is a GPI-anchored surface protein. The supernatant of non-treated parasites also reacted with the anti-MASP antibodies (Figure 6C, lane 2), suggesting that at least some of the MASP family members are shed into the culture by trypomastigotes.

DISCUSSION

The T. cruzi surface protein-encoding genes are often clustered into large haploid and heterogeneous arrays that can be as large as 600 kb (2). Here we show that the occurrence of genes in the vicinity of masps is not random, with a strong bias towards the occurrence of TcMUCII genes upstream of masp genes and a frequent occurrence of members of the gp85/TS-like superfamily downstream of masp intact genes as well as pseudogenes (Figure 2). Interestingly relatively few head-to-tail arrays of masp genes were observed in the genome (Figure 2). We speculate this genomic organization with masp interspersed with other surface protein gene families may play a contributing factor in maintaining diversity within the masp family by avoiding sequence homogenization. We also show that clusters of surface protein genes containing members of the masp gene family are preferentially associated with large T. cruzi chromosomes (Figure 3). The same pattern of chromosome distribution has been reported for TcMUC (26), the T. cruzi specific SAP family (27) and the satellite SAT 195 (28,29), which are all associated with the same arrays (2,27,30). Although not all subtelomeric regions could be assembled with confidence in the CL-Brener genome (2), the available data clearly demonstrates that the clusters of surface proteins containing masp genes and other large gene families such as TcMUC and SAP are internal in the chromosomes at regions of synteny breaks with T. brucei and L. major. This is in contrast to other protozoans such as T. brucei and P. falciparum where variable surface protein genes (vsg and var, respectively) are often subtelomeric in the megachromosomes (3,31). This is also the case for all chromosomes of T. brucei, including mini- (50–150 kb) and intermediate (200–900 kb) chromosomes, which contain telomere-linked vsgs (3,32). The clustering of T. cruzi surface protein genes internally within the chromosomes may correlate with the absence of telomere-associated classical antigenic variation in this parasite. Instead, it has been demonstrated that different members of T. cruzi surface protein families such as mucin TcMUC and gp85/TS family are co-expressed (25,33). Our survey of expressed masp genes in the trypomastigote revealed that many are co-expressed in a parasite population. The expression profile is not random, however, since one MASP subgroup is over-represented (Supplementary Table S4). Our IFA results (Figure 6C) also reveal that MASP expression is heterogeneous in the trypomastigote culture analyzed since only ~5% of the parasites express MASP members containing the peptide 7, which is present in 14% of all possible MASP proteins encoded by intact genes. Whether an individual trypomastigote expresses a single masp gene, co-expresses different members of the masp family or none at all remains to be investigated.

In addition to the large repertoire of polypeptides derived from the various polymorphic surface protein families, T. cruzi combines sequences from different gene families to increase diversity. T. cruzi mosaic genes formed by group II and III members of the gp85/TS superfamily (34) and containing the N- or C-terminal conserved domain of MASP combined with the N- or C-terminal domain of TcMUC, SAP or gp85/TS superfamily have been identified (2). As part of this study, we identified two cDNAs that contain masp sequences associated with segments of TcMUCII or TS-like genes (Supplementary Table S4 and Supplementary Figure S3), indicating that at least some of the T. cruzi predicted mosaic genes are expressed. It is possible that these mosaic sequences have originated by means of segmental gene conversion, favored by the clustering of surface protein genes. Recombination events within and between genes families can generate an unlimited repertoire of parasite protein exposed to the host. In fact, several lines of evidence suggest that those areas containing arrays of surface proteins are subject to intense rearrangements: (i) the average length of the directional gene clusters in these T. cruzi specific regions is much smaller than for syntenic regions with T. brucei and L. major genomes; (ii) the presence of large number of pseudogenes (~42% of all predicted gp85/ts, masp, mucins, retrotransposon hot spot, dispersed gene family-1 and metalloprotease gp63 genes), (iii) the abundance of retroelements and (iv) syntenic break between the two CL-Brener haplotypes.

It has been proposed that sequence polymorphism of T. cruzi surface proteins might represent an evasion mechanism to drive the immune system into a series of spurious and inefficient activations of naïve CD8+ T cells, resulting in their delayed generation of protective immune responses in the initial phase of the infection (35). Also, Pitcovsky et al. (36) proposed that trans-sialidase family variability might contribute to simultaneous presence of B-cell-related epitopes during the infection resulting in a series of non-neutralizing antibody response.

The MASP protein resembles other T. cruzi surface proteins with N- and C-terminal conserved regions that encode putative signal peptide and GPI-anchor addition site, respectively (Figure 4A). We showed in this study that MASP may be GPI-anchored at the surface of the trypomastigote and shed (at least some of the members) into the culture medium (Figure 6B). We have also shown that MASP central region is highly variable in length and in sequence, with frequent repeated motifs (Figure 4, Supplementary Figure S2 and Supplementary Table S1). It has been recognized that the expression of repetitive proteins is a common phenomenon among parasites that persistently evade immune destruction in their hosts (37). In fact, T. cruzi infection induces a strong antibody response against many proteins containing tandem repeats (38–43). No experimental evidence, however, supports the concept that repetitive antigens are involved in a specific mechanism to subvert host immune responses. Other functions have also been attributed to T. cruzi tandem amino-acid repeats. In one study, repetitive motifs derived from trans-sialidase and SAPA (shed-acute phase antigen) were shown to delay trans-sialidase clearance in the blood (44).

As for other T. cruzi surface proteins, MASP may also be subjected to post-translational modifications since a large number of N- and O-glycosylation sites as well as phosphorylation sites were predicted in MASP proteins. Proteomic analyses have confirmed that at least some MASP members are subject to N-glycosylation (45). The glycosylation status of MASP proteins may, in part, explain why this large family has escaped detection for so long. Studies using library screening approaches with antisera from chagasic patients have typically relied on bacterial expression systems that lack the glycosylation machinery.

We have demonstrated that MASP conserved coding domains display limited, yet detectable, nuclei-acid sequence identity with the TcMUC gene family. In addition, a few MASP members contain a degenerate version of Pfam domain PF01456, characteristic of the mucin proteins, including those of T. cruzi (TcMUC) (Supplementary Table S2). No other Pfam domain was identified in the MASP proteins. MASP may have originated from an ancestral TcMUC member, followed by divergence, extensive gene duplications and sequence diversification to generate the actual MASP repertoire.

In addition to the conservation of sequences coding for the signal peptide and the GPI-anchor addition site, striking identity levels were observed in the 5′ and 3′ UTRs of the MASP transcripts (Figure 5A). Those UTRs may contribute to the differential abundance of MASPs throughout the T. cruzi life cycle. Northern blots reveal that MASP transcripts are preferentially expressed during the trypomastigote stage (Figure 5B). It is well established that trypanosomatids rely primarily on post-transcriptional mechanisms for control of gene expression. One of the most well known mechanisms responsible for developmental regulation of gene expression in trypanosomatids involves the presence of regulatory sequences within untranslated regions that modulate transcript stability and/or translation efficiency (19,46–50). Another possible explanation for the conservation of MASP untranslated regions may be related to the generation of sequence diversity within the family. An alternative model of recombination was recently proposed as a way to generate diversity in the surface protein MSP2 involved in antigenic variation in Anaplasma marginale, a bacterial pathogen (51). According to this model, one recombination site occurs in the conserved flanking domains and the other within the hypervariable region. There is no apparent minimum region of sequence identity required for recombination in the hypervariable region; however, this is only possible due to the strong anchoring of the conserved ends holding the two recombining molecules in close proximity (51). It is noteworthy that MASP proteins display a mosaic structure with different members having a distinct combination of similar motifs (2).

The massive expansion of masp genes in the T. cruzi genome suggests this family may be critical for parasite survival. Its pattern of expression in the surface of the circulating trypomastigote forms, shedding properties, extensive sequence variability and repetitive nature suggests MASP may be involved in important parasite-host cell interactions. The ability of T. cruzi to infect and replicate within a variety of cell types is an essential feature of the parasite's survival strategy. Genetic polymorphism of T. cruzi surface glycoproteins has been hypothesized to be an important factor that may contribute to this phenomenon (52,53), although no clear association between a specific profile of parasite surface molecules and the ability of the parasite to infect a given host cell has been established. We speculate that the highly variable MASP central region may contribute to a large repertoire of peptides that may interact with different receptors from a variety of host cell types. Alternatively, the strong pressure for diversification may be imposed by the host immune system. In this regards, it will be interestingly to investigate whether MASP elicit the immune system especially during the acute phase of the infection when a large number of trypomastigotes are circulating.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

FUNDING

National Institute of Allergy and Infectious Diseases (grant number AI45038 to N.E.S.); UNICEF/UNDP/World Bank WHO for Research and Training in Tropical Diseases (grant number A50993 to D.C.B.); Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) (to D.C.B.); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (to D.C.B.); Howard Hughes Medical Institute (to S.M.R.T.). D.C.B. and S.M.R.T. are CNPq research fellows. Funding for open access charge: University of Maryland, Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and WHO.

Conflict of interest statement. None declared.

Supplementary Material

[Supplementary Data]

ACKNOWLEDGEMENTS

We greatly appreciate the gift of the pulsed field gel blots from Dr Björn Andersson (Karolinska Institute). We would like to thank João Luis Reis Cunha and Gabriela Caldas for technical assistance.

Footnotes

Present address: Wanderson D. daRocha, Department of Biochemistry and Molecular Biology, Federal University of Paraná, Brazil.

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