Distinct genomic organization, mRNA expression and cellular localization of members of two amastin sub-families present in Trypanosoma cruzi
© Kangussu-Marcolino et al.; licensee BioMed Central Ltd. 2013
Received: 1 October 2012
Accepted: 14 January 2013
Published: 17 January 2013
Amastins are surface glycoproteins (approximately 180 residues long) initially described in Trypanosoma cruzi as particularly abundant during the amastigote stage of this protozoan parasite. Subsequently, they have been found to be encoded by large gene families also present in the genomes of several species of Leishmania and in other Trypanosomatids. Although most amastin genes are organized in clusters associated with tuzin genes and are up-regulated in the intracellular stage of T. cruzi and Leishmania spp, distinct genomic organizations and mRNA expression patterns have also been reported.
Based on the analysis of the complete genome sequences of two T. cruzi strains, we identified a total of 14 copies of amastin genes in T. cruzi and showed that they belong to two of the four previously described amastin subfamilies. Whereas δ-amastin genes are organized in two or more clusters with alternating copies of tuzin genes, the two copies of β-amastins are linked together in a distinct chromosome. Most T. cruzi amastins have similar surface localization as determined by confocal microscopy and western blot analyses. Transcript levels for δ-amastins were found to be up-regulated in amastigotes from several T. cruzi strains, except in the G strain, which is known to have low infection capacity. In contrast, in all strains analysed, β-amastin transcripts are more abundant in epimastigotes, the stage found in the insect vector.
Here we showed that not only the number and diversity of T. cruzi amastin genes is larger than what has been predicted, but also their mode of expression during the parasite life cycle is more complex. Although most T. cruzi amastins have a similar surface localization, only δ-amastin genes have their expression up-regulated in amastigotes. The results showing that a sub-group of this family is up-regulated in epimastigotes, suggest that, in addition of their role in intracellular amastigotes, T. cruzi amastins may also serve important functions during the insect stage of the parasite life cycle. Most importantly, evidence for their role as virulence factors was also unveiled from the data showing that δ-amastin expression is down regulated in a strain presenting low infection capacity.
Trypanosoma cruzi, the protozoan parasite that is the etiologic agent of Chagas disease , undergoes four developmental stages during its complex life cycle: epimastigotes and metacyclic trypomastigotes, present in the insect vector, and intracellular amastigotes and bloodstream trypomastigotes, present in the mammalian host. This parasite must rely on a broad set of genes that allow it to multiply in the insect gut, to differentiate into forms that are able to invade and multiply inside a large number of distinct mammalian cell types and to circumvent the host immune system. To meet the challenges it faces during its life cycle, complex regulatory mechanisms must control the expression of the T. cruzi repertoire of about 12,000 genes. Among them, there are several large gene families encoding surface proteins, which are key players directly involved in host-parasite interactions (reviewed by Epting et al. ).
The amastin gene family was initially reported as a group of T. cruzi genes encoding 174 amino acid transmembrane glycoproteins and whose mRNA are 60-fold more abundant in amastigotes than in epimastigotes or trypomastigotes . The differential expression of amastin mRNAs during the T. cruzi life cycle has been attributed to cis-acting elements present in the 3’UTR as well as to RNA binding proteins that may recognize this sequence [4, 5]. It is also known that amastin genes alternate with genes encoding a cytoplasmic protein named tuzin . After the completion of the genome sequences of several Trypanosomatids it was revealed that the amastin gene family is also present in various Leishmania species as well as in two related insect parasites, Leptomonas seymouri and Crithidia spp [7–9]. It has also been reported that this gene family is actually much larger in the genus Leishmania when compared to other Trypanosomatids. Predicted topology based on sequences found in the genomes of L. major, L. infantum and T. cruzi indicates that all amastins have four transmembrane regions, two extracellular domains and N- and C-terminal tails facing the cytosol . Moreover, comparative analyses of amastin genes belonging to six T. cruzi strains evidenced that sequences encoding the hydrophilic, extracellular domain, which is less conserved, have higher intragenomic variability in strains belonging to T. cruzi group II and hybrid strains compared to T. cruzi I strains . Based on phylogenetic analyses of amastin orthologs from various Trypanosomatids, it has been proposed that amastins can be classified into four subfamilies, named α-, β-, γ-, and δ- amastins. Importantly, in L. major and L. infantum, in which members of all four sub-families are found, amastin genes showed differences in genomic positions and expression patterns of their mRNAs [8, 9].
More than fifteen years after their discovery, the function of amastins remains unknown. Because of the predicted structure and surface localization in the intracellular stage of T. cruzi and Leishmania spp, it has been proposed that amastins may play a role in host-parasite interactions within the mammalian cell: they could be involved in transport of ions, nutrients, across the membrane, or involved with cell signaling events that trigger parasite differentiation . Its preferential expression in the intracellular stage also suggest that it may constitute a relevant antigen during parasite infection, a prediction that was confirmed by studies showing that amastins peptides elicit strong immune response during Leishmanial infection . Amastin antigens are considered a relevant immune biomarker of cutaneous and visceral Leishmaniasis as well as protective antigens in mice .
Although complete genome sequences of two strains of T. cruzi (CL Brener and SylvioX-10) have been reported, their assemblies were only partially achieved because of their unusually high repeat content [13, 14]. Therefore, for several multi-gene families, such as the amastin gene family, their exact number of copies is not yet known. According to the current assembly , only four δ-amastins and two β-amastins were identified in the CL Brener genome. Herein, we used the entire data set of sequencing reads from the CL Brener  and Sylvio X-10  genomes, to analyzed all sequences encoding amastin orthologues present in the genomes of these two T. cruzi strains and determine their copy number as well as their genome organization. Expression of distinct amastin genes in fusion with the green fluorescent protein, allowed us to examine the cellular localization of different members of both amastin sub-families. By determining the levels of transcripts corresponding to each sub-family in all three parasite stages of various strains we showed that, whereas the levels of δ-amastins are up-regulated in amastigotes, β-amastin transcripts are significantly increased in the epimastigote insect stage. Most importantly, evidence indicating that amastins may constitute T. cruzi virulence factors was suggested by the analyses showing reduced expression of δ-amastins in amastigotes from strains known to have lower infection capacity.
Results and discussion
The amastin gene repertoire of Trypanosoma cruzi
In spite of the sequence divergence, an alignment of polypeptide sequences belonging to all amastin sub-families shows increased amino acid conservation within the putative hydrophobic transmembrane domains. Within the predicted extracellular domains, two highly conserved cysteine and one tryptophan residues, that are part of the 10 amino acid “amastin signature” , may be critical for amastin function (Additional file 1: Figure S1B). On the other hand, the more variable sequences present in the two predicted extracellular, hydrophilic domains suggest that this portion of the protein, which, in amastigotes, are in contact with the host cell cytoplasm, may interact with distinct host cell proteins.
Because the assembly of CL Brener genome does not include its complete sequence, we conducted a read-based analysis to estimate the total number of amastin genes in this strain of the parasite. It is well known that the assembly of the CL Brener genome is only accurate for non-repetitive regions, and for tandemly repeated genes, misassembles frequently occurred since most repetitive copies usually collapse into one or two copies. Therefore, we used the entire dataset of reads generated by the Tri-Tryp consortium to select reads containing sequences homologous to amastin and, based on a 13 × genome coverage , we estimated a total number of 14 copies of amastin genes, 2 β-amastins and 12 δ-amastins in the CL Brener genome. Similar analyses performed with sequencing reads generated by Franzen et al. (2011)  from the genome of Sylvio X-10 indicated a comparable number of copies in the genome of this T. cruzi I strain.
Distinct patterns of amastin gene expression
Also, to investigate the mechanisms controlling the expression of the different sub-classes of amastins, sequence alignment of the 3’UTR sequences from β- and δ-amastins were done. Previous work has identified regulatory elements in the 3’ UTR of δ-amastins as well as in other T. cruzi genes controlling mRNA stability [4–6, 21, 22] and mRNA translation . Since we observed that the two groups of amastin genes have highly divergent sequences in their 3’UTR (not shown), we are preparing luciferase reporter constructs to identify regulatory elements that might be present in the β-amastin transcripts as well as to identify the factors responsible for the differences observed in the amastin gene expression in distinct T. cruzi strains.
Amastin cellular localization
Taken together, the results present here provided further information on the amastin sequence diversity, mRNA expression and cellular localization, which may help elucidating the function of this highly regulated family of T. cruzi surface proteins. Our analyses showed that the number of members of this gene family is larger than what has been predicted from the analysis of the T. cruzi genome and actually includes members of two distinct amastin sub-families. Although most T. cruzi amastins have a similar surface localization, as initially described, not all amastins genes have their expression up-regulated in amastigotes: although we confirmed that transcript levels of δ-amastins are up-regulated in amastigotes from different T. cruzi strains, β-amastin transcripts are more abundant in epimastigotes than in amastigotes or trypomastigotes. Together with the results showing that, in the G strain, which is known to have lower infection capacity, expression of δ-amastin is down-regulated, the additional data on amastin gene expression presented here indicated that, besides a role in the intracellular, amastigote stage, T. cruzi amastins may also serve important functions in the insect stage of this parasite. Hence, based on this more detailed study on T. cruzi amastins, we should be able to test several hypotheses regarding their functions using a combination of protein interaction assays and parasite genetic manipulation.
Amastin sequences were obtained from the genome databases of T. cruzi CL Brener, Esmeraldo and Sylvio X-10 strains [25, 26]. The sequences, listed in Additional file 4: Table S1, were named according to the genome annotation of CL Brener or the contig or scaffold ID for the Sylvio X10/1 and. All coding sequences were translated and aligned using ClustalW . Amino acid sequences from CL Brener, Esmeraldo, Sylvio X-10, and Crithidia sp (ATCC 30255) were subjected to maximum-likelihood tree building using the SeaView version 4.4  and the phylogenetic tree was built using an α-amastin from Crithidia sp as root. Weblogo 3.2 was used to display the levels of sequence conservation throughout the protein . Amino acid sequences from one amastin from each sub-family were used to predict trans membrane domains, using SOSUI  as well as signal peptide, using SignalP 3.0 . For copy number estimations, individual reads from the genome sequence of T. cruzi CL Brener  were aligned by reciprocal BLAST against each amastin coding sequences. Unique reads showing at least 99.7% of identity were mapped on the CDS and the coverage for each nucleotide was determined. Coverage values were normalized through z-score and the copy numbers were determined after determining the ratios between z-score and the whole genome coverage.
T. cruzi strains or clones, obtained from different sources, were classified according to the nomenclature and genotyping protocols described by . Epimastigote forms of T. cruzi strains or clones Colombiana, G, Sylvio X-10, Dm28c, Y and CL Brener were maintained at 28°C in liver infusion tryptose (LIT) medium supplemented with 10% fetal calf serum (FCS) as previously described . Tissue culture derived trypomastigotes and amastigotes were obtained after infection of LLC-MK2 or L6 cells with metacyclic trypomastigotes generated in LIT medium as previously described .
Pulse-field gel electrophoresis and Southern blot analyses
Genomic DNA, extracted from 107epimastigotes and included in agarose blocks were separated as chromosomal bands by pulse-field gel electrophoresis (PFGE) using the Gene Navigator System (Pharmacia) as described by Cano et al. (1995) , with the following modifications: separation was done in 0.8% agarose gels using a program with 5 phases of homogeneous pulses (north/south, east/west) with interpolation for 135 h at 83 V. Phase 1 had pulse time of 90 s (run time 30 h); phase 2 120 s (30 h); phase 3200 s (24 h); phase 4 350 s (25 h); phase 5 800 s (26 h). Chromosomes from Saccharomyces cerevisiae (Bio-Rad) were used as molecular mass standards. Separated chromosomes were transferred to nylon filters and hybridized with 32P labelled probes prepared as described in the following section.
RNA purification and Northern blot assays
Sequence of primers used to amplify amastin isoforms ORFs.
Primer name / gene ID
Primer Sequence (5’-3’)
pδ1-amastin (F) Tc00.1047053511071.40
pδ1-amastin (R) Tc00.1047053511071.40
β1-amastin (F) Tc00.1047053509965.390
β1-amastin (R) Tc00.1047053509965.390
β2-amastin (F) Tc00.1047053509965.394
β2-amastin (R) Tc00.1047053509965.394
To express different amastin genes in fusion with GFP we initially constructed a plasmid named pTREXAmastinGFP. The coding sequence of the TcA21 cDNA clone  (accession number U04339) was PCR-amplified using a forward primer (5’-CATCTAGAAAGCAATGAGCAAAC-3’) and a reverse primer (5’-CTGGATCCCTAGCATACGCAGAAGCAC-3’) containing the XbaI and BamHI restriction sites (underlined in the primers), respectively. After digesting the PCR product with XbaI and BamHI, the fragment was ligated with the vector fragment of pTREX-GFP  that was previously cleaved with BamHI and XhoI. To generate the GFP constructions with other amastin genes, their corresponding ORFs were PCR-amplified using the primers listed in Table 1 and total genomic DNA that was purified from epimastigote cultures of T. cruzi CL Brener according to previously described protocols . The PCR products were cloned initially into pTZ (Qiagen) and the amastin sequences, digested with the indicated enzymes, were purified from agarose gels with Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). The fragment corresponding to the TcA21 amastin cDNA was removed from pTREXAmastinGFP after digestion with XbaI/BamHI and the fragments corresponding to the other amastin sequences were ligated in the same vector, generating pTREXAma40GFP, pTREXAma390GFPand pTREXAma394GFP. All plasmids were purified using QIAGEN plasmid purification kits and sequenced to confirm that the amastin sequences were properly inserted, in frame with the GFP sequence.
Parasite transfections and fluorescence microscopy analyses
Epimastigotes of T. cruzi CL Brener, growing to a density of 1 to 2 × 107 parasites/mL, were transfected as described by DaRocha et al., 2004 . After electroporation, cells were recovered in 5 ml LIT plus 10% FCS 28°C for 24 h and analysed by confocal microscopy using the ConfocalRadiance2100 (BioRad) system with a 63/100x NA 1.4 oil immersion objective. To perform co-localization analyses, transfected parasites expressing amastin-GFP fusions were prepared for immunofluorescence assays by fixing the cells for 20 minutes in 4% PFA-PBS at room temperature. Parasites adhered to poly-L-lysine coverslips (Sigma) were permeabilized with 0.1% Triton X-100-PBS for 2 minutes, blocked with 4% BSA-PBS for 1 hour and incubated with primary antibodies (rabbit polyclonal antibody anti-phosphoenolpyruvate carboxykinase (anti-PEPCK, kindly provided by Stenio Fragoso, Instituto Carlos Chagas, Curitiba, Brazil) in blocking solution (5.0% non-fat dry milk) for 1 hour followed by incubation with secondary anti-rabbit IgG conjugated with Alexa546. Samples were also stained with 0.1 μg / mL 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI, from Sigma) at room temperature for 5 min before confocal microscopy.
Parasite membrane fractionation and western blot analyses
Aproximately 109 epimastigotes growing at a cell density of 2 × 107 parasites/mL were harvest, washed with saline buffer (PBS) and ressuspended in lysis buffer (Hepes 20mM; KCl 10 mM; MgCl2 1,5 mM; sacarose 250 mM; DTT 1 mM; PMSF 0,1 mM). After lysing cells with five cycles of freezing in liquid nitrogen and thawing at 37°C, an aliquot corresponding to total protein (T) extract was collected. Total cell lysate was centrifuged at a low speed (2,000 × g) for 10 min and the supernatant was subjected to ultracentrifugation (100,000 × g) for one hour. The resulting supernatant was collected and analysed as soluble, cytoplasmic fraction (C) whereas the pellet, corresponding to the membrane fraction (M) was ressuspended in lysis buffer. Volumes corresponding to 10 μg of total parasite protein extract (T), cytoplasmic (C) and membrane (M) fractions, mixed with Laemmli’s sample buffer, were loaded onto a 12% SDS–PAGE gel, transferred to Hybond-ECL membranes (GE HealthCare), blocked with 5.0% non-fat dry milk and incubated with anti-GFP antibody (Santa Cruz Biotechnology) or anti-PEPCK antibody, followed by incubation with peroxidase conjugated anti-rabbit IgG and the ECL Plus reagent (GE HealthCare).
This study was supported by funds from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil), Fundação de Amparo a pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil) and the Instituto Nacional de Ciência e Tecnologia de Vacinas (INCTV, Brazil). DCB, RAM and SMRT are recipients of CNPq fellowships; The work of WDDR, MMKM and LL is supported by Fundação Araucária, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), PPSUS/MS and CNPq.
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