Comparative genomic analysis of human infective Trypanosoma cruzi lineages with the bat-restricted subspecies T. cruzi marinkellei
© Franzén et al.; licensee BioMed Central Ltd. 2012
Received: 2 February 2012
Accepted: 1 October 2012
Published: 5 October 2012
Trypanosoma cruzi marinkellei is a bat-associated parasite of the subgenus Schizotrypanum and it is regarded as a T. cruzi subspecies. Here we report a draft genome sequence of T. c. marinkellei and comparison with T. c. cruzi. Our aims were to identify unique sequences and genomic features, which may relate to their distinct niches.
The T. c. marinkellei genome was found to be ~11% smaller than that of the human-derived parasite T. c. cruzi Sylvio X10. The genome size difference was attributed to copy number variation of coding and non-coding sequences. The sequence divergence in coding regions was ~7.5% between T. c. marinkellei and T. c. cruzi Sylvio X10. A unique acetyltransferase gene was identified in T. c. marinkellei, representing an example of a horizontal gene transfer from eukaryote to eukaryote. Six of eight examined gene families were expanded in T. c. cruzi Sylvio X10. The DGF gene family was expanded in T. c. marinkellei. T. c. cruzi Sylvio X10 contained ~1.5 fold more sequences related to VIPER and L1Tc elements. Experimental infections of mammalian cell lines indicated that T. c. marinkellei has the capacity to invade non-bat cells and undergo intracellular replication.
Several unique sequences were identified in the comparison, including a potential subspecies-specific gene acquisition in T. c. marinkellei. The identified differences reflect the distinct evolutionary trajectories of these parasites and represent targets for functional investigation.
The subgenus Schizotrypanum harbors the type species Trypanosoma cruzi, which is the causative agent of Chagas disease in humans. Other members of the Schizotrypanum subgenus are often referred to as T. cruzi-like species as they are morphologically similar or indistinguishable from T. cruzi. With the exception of the human infecting parasite, members of Schizotrypanum are restricted to bats (order Chiroptera) and occur in high prevalence among bats in Latin America and elsewhere in the world[1–4]. There is no evidence that T. cruzi-like parasites are harmful to bats, although this may reflect a paucity of data. Most infected bats are insectivorous and infection is thought to take place either through ingestion of infected arthropods or via stercorarian transmission from bat-feeding bugs[5, 6]. The genetic diversity of T. cruzi-like species and their evolutionary relationships are yet to be determined.
Trypanosoma cruzi marinkellei is a bat-associated subspecies of T. cruzi. The human infective parasite T. cruzi should accordingly be referred to as the nominate subspecies T. cruzi cruzi (T. c. cruzi). T. c. marinkellei is prevalent among bats in Central and South America, which are its only known mammalian hosts[1, 5]. It differs from T. c. cruzi in terms of isoenzyme electrophoresis patterns and buoyant DNA densities. T. c. marinkellei does not infect immunocompetent mice[1, 5], nor does it provide immunological protection against challenge with T. c. cruzi, suggesting that the infection is characterized by distinct antigenic profiles. Sequence-based phylogenies have confirmed a relatively close relationship with T. c. cruzi[5, 7–9] and estimated the divergence time at ~6.5-8.5 MYA[10–12]. Cavazzana et al. reported that T. c. marinkellei was associated with phyllostomid species (insectivorous, frugivorous, carnivorous and haematophagous bats) and transmission is thought to occur when triatomine bugs of the genus Cavernicola feed on bats. However, the natural transmission cycle among bats is not well characterized and there might be other vectors or direct transmission mechanisms. Some genetic substructure within the T. c. marinkellei population has been reported, but the strength of correlation between parasite lineage and host remains to be defined. Moreover, bat-restricted parasites are of evolutionary interest, since it has been proposed that T. c. cruzi may have originated from an ancestral bat-lineage that jumped into terrestrial mammals. The present day human lineage, T. c. cruzi, has been in contact with humans for no more than 10,000 to 30,000 years, which is the period of human presence in the Americas.
T. c. cruzi strains are currently sorted into six lineages or discrete typing units (DTUs), which illustrate the genetic diversity of this parasite. Several strains have to date been subjected to genome sequencing, among these are CL Brener and Sylvio X10. The CL Brener strain was selected for the original genome project and belongs to DTU VI. The size of the CL Brener genome was ~110 Mb and it was assembled mostly with Sanger paired-end reads. The CL Brener strain was shown to be a genetic hybrid of two diverged haplotypes named Esmeraldo-like and non-Esmeraldo-like. The hybrid and repetitive nature of this genome complicated sequence assembly and finishing, leaving the genome in many gaped scaffolds and contigs. Weatherly et al. later compiled scaffolds into more complete chromosome-wide sequences. Second-generation sequencing facilitates more cost-effective and rapid sequencing efforts. Recently, 454-sequencing was applied on the genome of the DTU I strain Sylvio X10, revealing a slightly smaller but still repeat-rich genome.
Little is known about genomic variation among organisms within the Schizotrypanum genus. Genomic insights can provide information on evolutionary adaptation of these parasites, as well as being useful for advancing population genetics. Thus, exploring genomic diversity could reveal important genetic and biological characteristics, and potentially clues as to how these parasites relate to the human disease. Here we describe the genome of T. c. marinkellei B7, a bat-associated parasite originally isolated from a colony of the pale spear-nosed bat Phyllostomus discolor roosting in a hollow tree. The parasite was isolated in São Felipe, Bahia state, Brazil in 1974 and has since then been stored under cryogenic conditions with occasional short periods of in vitro cultivation. We combined Roche/454 and Illumina sequencing to generate a draft genome sequence of T. c. marinkellei. This is the first whole genome analysis of a T. c. cruzi-like species that is not associated with human infections. In addition, we also report re-assembly and re-annotation of the human infective strain T. c. cruzi Sylvio X10, a commonly used reference strain of T. c. cruzi I, using additional sequence data.
The comparative analyses with T. c. cruzi revealed that the genomes contain the same repertoire of housekeeping genes. Moreover, T. c. marinkellei contains an additional gene that appears to be an example of recent horizontal gene transfer. In addition, the genomes also exhibit copy number variation and diversification of gene families, which potentially give rise to a large number of strain-specific protein isoforms.
Results and discussion
Sequencing and Assembly of T. c. marinkellei and T. c. cruzi Sylvio X10
Raw sequence data
T. c. marinkellei
T. c. cruzi Sylvio X10
# reads (10 6 )
# nt (10 9 ) c
Average read length (nt) d
~ Coverage e
Genome assembly statistics and summary
Velvet (kmer 43)
Assembly of non-assembled Illumina reads
Velvet (kmer 53)
Velvet (kmer 43)
Assembly of non-assembled Illumina reads
Velvet (kmer 53)
The combined assembly lengths were 38.6 Mb and 43.4 Mb for Tcm and Tcc X10 respectively. The assembly size of Tcc X10 was very similar to our previous estimate from extrapolation of unassembled data and flow cytometry. Flow cytometry analysis estimated the haploid size of Tcm to ~39 Mb (Additional file1: Figure S1), which was close to the in silico assembly length. Thus, assembly sizes were consistent with experimental measurements. Moreover, this confirmed that the Tcm genome was ~4.8 Mb smaller than that of Tcc X10. The percentage of assembled bases in each assembly was very similar: Tcm 88.6% (34.2 Mb/38.6 Mb); Tcc X10 88.7% (38.5 Mb/43.4 Mb). We analyzed 29,422 unused 454 reads of Tcm with RepeatMasker, which identified 13,108 reads corresponding to kinetoplastid sequences. The remaining reads were analyzed with BLAST, showing them to correspond multicopy genes or other repeats.
868 (Tcm) and 987 (Tcc X10) scaffolds were longer than 5 kb, which corresponded to 25.7 and 26.8 Mb (including gaps). The longest scaffolds were 335 kb (Tcm) and 384 kb (Tcc X10). Some 200 gaps could be closed from the apparent overlap of adjacent contigs. Compared with 454 reads alone, addition of mated reads provided longer contigs and scaffolds, corrected 454 sequence errors and allowed accurate estimation of genome heterozygosity and copy number variation.
Comparison of heterozygosity and multicopy genes
Comparison of gene family content
T. c. marinkellei
T. c. cruzi Sylvio X10
Size in assemblyb
% Short readsc
Size in assemblyb
% Short readsc
2,129,983 (6.22 %)
1,265,650 (3.28 %)
2,109,163 (6.16 %)
2,953,602 (7.65 %)
540,360 (1.58 %)
727,537 (1.88 %)
521,665 (1.52 %)
1,314,589 (3.41 %)
452,732 (1.32 %)
514,422 (1.33 %)
273,890 (0.80 %)
334,544 (0.87 %)
37,490 (0.11 %)
42,072 (0.11 %)
25,946 (0.08 %)
26,732 (0.07 %)
Kinetoplastid DNA (maxicircle)
The mitochondrial genomes (maxicircles) of T. c. cruzi strains X10 (DTU I), Esmeraldo (DTU II) and CLBR (DTU VI) have been sequenced, and have provided insights into the structure and organization of kinetoplastid DNA of these strains. The T. c. marinkellei maxicircle was identified as a 20,037 bp contig from the 454 assembly. The length of this sequence was slightly longer (~ 5 kb) than those previously reported, and the difference was attributed to variability in the repetitive region. The coding region of the Tcm maxicircle was syntenic with the coding regions of the three complete T. c. cruzi maxicircle genomes, beginning with the 12S rRNA gene and ending with the ND5 gene. The lengths of the individual genes within the Tcm maxicircle coding region were comparable to those of the three T. c. cruzi strains (Additional file4: Table S1). The length of the complete maxicircle coding region (beginning at 12S rRNA and ending after ND5) for Tcm was 15,438 bp and began after 4,599 bp of non-coding sequence. With respect to coding sequences, the average maxicircle nucleotide identity between Tcm and Tcc X10 was (mean ± sd): 85.12% ± 6.1, between Tcm and Tcc CLBR was 85.4% ± 6.2 and between Tcm and Tcc Esmeraldo was 85.3% ± 6.1 (Additional file4: Table S1). Phylogenetic reconstruction of the maxicircles from Tcm, Tcc X10, Tcc CLBR and Tcc Esmeraldo confirmed that the Tcm maxicircle was slightly closer to Tcc Esmeraldo than Tcc X10/CLBR (Additional file5: Figure S4). The topology of the tree suggests that the Esmeraldo maxicircle might represent the ancestral maxicircle lineage of T. c. cruzi.
The consensus maxicircle genome sequence is derived from the predominant nucleotide present across multiple read alignments at each position. However, this criterion disregards low abundance single nucleotide polymorphisms (SNPs) and therefore masks minor maxicircle haplotypes (heteroplasmy), which has previously been reported from Tcc X10. Illumina reads were used to assess the presence/absence of minor Tcm maxicircle haplotypes. In total, this identified 19,821 reads that aligned to the Tcm maxicircle. Low levels of heteroplasmy were observed in the Tcm maxicircle protein-coding region. Twenty SNPs were identified among four genes (ND8, MURF1, COI and ND3) and one intergenic region (between CR4 and ND4). Average read depth for each SNP site was 47. At heterozygous sites, the minor nucleotide was present among an average of 9.5% (± 3.3%) of reads. All SNPs were bi-variable except for at two intergenic positions, where two minor nucleotides were present. These observations imply the occurrence of at least two minor mitochondrial haplotypes.
Gene content analysis and comparison
In order to identify isolate-specific genes, we compared the gene complements of the three genomes (Tcm, Tcc X10 and Tcc CLBR) using BLAST. Initially, the predicted proteomes of Tcm and Tcc X10 were queried with BLASTp against the predicted proteome of Tcc CLBR using the E-value threshold 1e-20. This resulted in 237 (Tcm) and 290 (Tcc X10) proteins longer than 250 amino acids that were not found in the Tcc CLBR proteome. These protein sequences were queried using tBLASTn against the Tcc CLBR genome to exclude the possibility that these putative genes were present as non-annotated open reading frames, using the same E-value threshold. This decreased the number of hits to 22 (Tcm) and 3 (Tcc X10). The composition of the 22 putative Tcm-specific genes were as follows: 11 TcMUCII mucin genes; 1 acetyltransferase (MOQ_006101); 5 putative genes with weak hits to microbial sequences (MOQ_006053, MOQ_007485, MOQ_009774, MOQ_006631, MOQ_003304); and 5 putative genes with no hits in public databases (MOQ_003636, MOQ_009528, MOQ_006983, MOQ_009799, MOQ_005225). For Tcc X10, one of the specific genes corresponded to a diverged mucin-associated surface protein (TCSYLVIO_008353). The remaining two putative genes did not show any significant hits in public databases (TCSYLVIO_011068 and TCSYLVIO_008789). Thus, the improved Tcc X10 genome sequence facilitated the detection of two putative Tcc X10-specific protein-coding genes not apparent in the earlier version. The two unknown genes were found to also be present in the previously reported draft genome sequence of Tcc X10.
We used the same strategy to perform the reversed search, i.e. searching for genes specific for Tcc CLBR. This resulted in 344 and 206 protein sequences that were not found in Tcm and Tcc X10. Searches using tBLASTn towards Tcm and Tcc X10 further decreased this number to 70 and 100, and of these 52 and 21 were mucin-associated surface proteins or TcMUCII mucin. 8 (Tcm) and 26 (Tcc X10) contained low complexity repeats. The remaining 10 (Tcm) and 53 (Tcc X10) genes were queried against the raw 454 reads of Tcm and Tcc X10, which further decreased the number of Tcc CLBR specific genes to 3 that were not present in Tcm (Tc00.1047053511585.110, Tc00.1047053509525.260, Tc00.1047053510073.24). The 3 genes were uncharacterized (hypothetical). The Tcc CLBR-specific genes, compared with Tcc X10, were identical to those previously reported. In conclusion, the total number of specific genes was remarkably low in relation to the number of coding sequences in these genomes. As a perspective, comparative genomics of T. brucei brucei and T. brucei gambiense did not identify any gene that could explain the ability to infect different species, despite interspecific pathological variation.
A specific acetyltransferase gene in T. c. marinkellei
List of hits obtained from PSI-BLAST after 4 iterations querying MOQ _006101 against GenBank non-redundant database
CAS1 domain-containing protein 1-like
Arabidopsis lyrata subsp. Lyrata
O-acetyltransferase family protein
Arabidopsis lyrata subsp. Lyrata
CAS1 domain-containing protein 1-like
Codon Adaptation Index (CAI) is a measure of synonymous codon usage bias and can be used to evaluate the extent of which codon usage of a supposed foreign gene is similar to highly expressed genes in the host genome. CAI can range between 0 and 1 and values closer to 0 imply equal use of synonymous codons whereas values closer to 1 imply strong codon usage bias. MOQ_006101 displayed a CAI value of 0.518 (Figure5C). In contrast, the median CAI across all genes was 0.545 ± 0.05 (median ± median absolute deviation). Thus, CAI was lower than the mean but still within the expected range, suggesting that the gene has conformed to the host genome.
Overall, these findings point to that MOQ_006101 was acquired by the lineage leading to Tcm rather than lost in T. c. cruzi and demonstrates an example of horizontal gene transfer between a photosynthesizing organism and a protozoan parasite. Interestingly, a genome comparison of two strains of the protozoan Giardia intestinalis also identified a strain-specific acetyltransferase. Finally, the biological function of MOQ_006101, if any, remains to be determined.
Comparison of synteny reveals putative rearrangements
T. c. cruzi has previously been reported to exhibit extensive DNA content and karyotype variability[24, 41–43]. We investigated sequence co-linearity of the assembled data and compared with the current chromosome-level assembly of Tcc CLBR. Scaffolds >25 kb were extracted from the assemblies, which resulted in 307 and 229 scaffolds for Tcm and Tcc X10 respectively, amounting to 50.7% (19.6 Mb/38.6 Mb) and 44.7% (19.4 Mb/43.4 Mb) of the genomes. In order to identify putative inter- and intra-chromosome rearrangements, scaffolds were queried against Tcc CLBR (non-Esm) using the alignment program promer. The number of chromosome hits per scaffold was plotted and the results were inspected. A total of 73 (Tcm) and 114 (Tcc X10) scaffolds contained hits to more than one chromosome from Tcc CLBR. However, manual examination showed that the vast majority of these hits were to gene family members (e. g. DGF, trans-sialidase, TcMUCII mucin) or other repeats. Hence, these were not likely to be rearrangements between chromosomes. 4 scaffolds were identified from Tcm (244, 732, 1101 and 2169) and 6 from Tcc X10 (94, 737, 1353, 2784, 2065 and 2359) that were involved in inter-chromosome rearrangements (Additional file7: Figure S5). Moreover, Tcm and Tcc X10 both contained rearrangement in a region on chromosome 34, containing a repeat-like composition in Tcc CLBR. Scaffold 1101 from Tcm aligned with chromosomes 34 and 27. Scaffold 94 from Tcc X10 aligned with chromosomes 34 and 12. Also, scaffold 732 from Tcm aligned with the distal parts of Tcc CLBR chromosomes 22 and 42. In Tcm, VIPER elements were frequently found in regions where synteny was discontinued. Regions where rearrangements had occurred were frequently found inside unidirectional gene clusters.
Intra-chromosome rearrangements were searched for using the same strategy. This identified 23 and 13 scaffolds in Tcm and Tcc X10 respectively, where intra-chromosome rearrangements were identified. Frequently, one or several genes were found to have shifted location and were found to be located distally on the same chromosome. In a few cases, a certain structural variant was present in Tcm and Tcc X10 but not in Tcc CLBR, suggesting that it was introduced in the lineage leading to Tcc CLBR. Tcm scaffold 836 contained a large inverted region, flanked by VIPER elements. This inversion causes disruption of a head-to-head strand switch region. The larger number of structural rearrangements in Tcm likely reflects its phylogenetic distance from T. c. cruzi.
PCR validation was performed in order to validate the accuracy of the assembly and some of the identified rearrangements. Representative regions were selected from Tcm, Tcc X10 and Tcc CLBR and targeted for PCR amplification. The size of the PCR product was compared with the in silico expected size and confirm assembly consistency. In total, 3 of 4 genomic regions were successfully amplified from Tcm, 2 of 2 from Tcc X10 and 1 of 2 from Tcc CLBR (Additional file8: Figure S6). Of which Tcm yielded the following PCR product sizes: 4, 0.5 and 0.8 kb. The first and second PCR products spanned across assembly gaps and therefore did not allow estimation of the expected sizes, but confirmed contigs to be accurately linked together. The third PCR reaction from Tcm resulted in the expected product size of 0.8 kb. Tcc X10 resulted in PCR products of sizes 0.8 kb and 1 kb, which were expected. The Tcc CLBR reaction resulted in the expected product size of 3 kb. Two PCR reactions did not work, which could either be due to non-optimal PCR conditions, formation of primer-secondary structures/duplexes or misassembly.
It is important to note that the present analyses are limited by the sequence continuity of the particular scaffold and therefore the presented numbers of observed rearrangements are likely to be underestimates. In conclusion, the majority of analyzed genomic regions from Tcm and Tcc X10 exhibited conserved local synteny with Tcc CLBR. However, insertions, deletions or other types of structural alterations occasionally interrupted synteny. These observations suggest that different T. c. cruzi lineages contain distinct karyotypes and other types of structural features, which have been fixed in a certain lineage. The cause of these rearrangements could either be due to random processes, i.e. oxidative stress or mistakes introduced by spontaneous cellular processes or perhaps less likely, physiological processes. Clearly, the presence of genetic variation other than SNPs provides an additional layer of complexity to studies of T. c. cruzi genetic variability.
Widespread occurrence of copy number variation in Tcm and Tcc X10
In order to identify chromosomal aneuploidies, we calculated the baseline coverage for each scaffold. Scaffolds with a median <3.2 and median absolute deviation <0.7 were extracted (empirically determined thresholds). This identified 7 scaffolds in Tcm with a lower average coverage: 950, 938, 79, 78, 70, 2392 and 2744. These genomic regions were homologous with chromosomes 25, 25, 12, 12, 12 and 7 in Tcc CLBR. No scaffolds with low overall coverage were identified in Tcc X10. This suggested the existence of monosomic chromosomes in Tcm but not in Tcc X10. As expected, heterozygosity was absent in these putatively monosomic Tcm regions. However, the homologous region in Tcc X10 displayed heterozygosity. This further supported the likely monosomic state of these regions in Tcm. Genomic qPCR with a primer pair targeting scaffold 78 in Tcm resulted in Ct=17.81 for the putative monosomic scaffold, whereas for putative disomic regions on scaffold 1093 and 1101 Ct was 15.08 and 15.30. Moreover, we searched for evidence of higher ploidy levels. Scaffolds with median > 3.5 were extracted. This identified 14 and 5 scaffolds in Tcm and Tcc X10 respectively, with an increased overall coverage. These scaffolds showed homology with large mega-base chromosomes from Tcc CLBR, suggesting that higher ploidy levels may be more common in larger chromosomes.
The presented analysis confirms that copy number variation is a common feature of the Tcm and Tcc X10 genomes. In theory, copy number variation would not be beneficial for the parasite as it increases the amount of DNA that needs to be replicated and the energy cost of the cell. The evolutionary benefit of having such an excessive amount of genes would seem to be limited. It is possible that copy variation does not infer any evolutionary advantage for the parasite, but is only a consequence of sloppy or non-perfect DNA replication mechanisms of these parasites.
Retrotransposons, repetitive elements and low complexity repeats
Comparison of repetitive elements
T. c. marinkellei
T. c. cruzi Sylvio X10
% Short readsb
% Short readsb
574,697 (1.679 %)
1,116,378 (2.892 %)
433,619 (1.267 %)
655,064 (1.697 %)
432,474 (1.263 %)
805,885 (2.088 %)
382,416 (1.117 %)
481,685 (1.248 %)
223,679 (0.653 %)
281,491 (0.729 %)
176,724 (0.516 %)
238,914 (0.619 %)
94,765 (0.277 %)
151,879 (0.393 %)
18,338 (0.054 %)
102,810 (0.266 %)
4,705 (0.014 %)
10,936 (0.028 %)
2,944 (0.009 %)
167 (0.000 %)
621 (0.002 %)
7,573 (0.020 %)
The Long Terminal Repeat (LTR)-like retroelement VIPER belongs to the superfamily tyrosine recombinase retrotransposons and was the most abundant element in Tcm and Tcc X10 respectively, representing 24.5% (574,697 Mb/2,344,982 Mb) and 28.9% (1,116,378 Mb/3,852,782 Mb) of the repetitive elements (Table5). The large amount of sequence related to these elements suggested that large-scale proliferation occurred before the split of T. c. cruzi and T. c. marinkellei. Furthermore, phylogenetic reconstruction based on a multiple sequence alignment (MSA) indicated some substructure between Tcc X10 and Tcc CLBR, whereas Tcm in large formed a more distant clade (Additional file9: Figure S7). 69% (3,450/4,968) of the MSA sites were too diverged to be included in the multiple-alignment, suggesting that these elements have been inactive for a substantial time. The human infecting lineage does contain a larger amount of these elements, possibly due to a loss of VIPER-related sequences in Tcm. In conclusion, repetitive elements explain in part the smaller genome size of Tcm. 3 repeat categories were on the contrary expanded in Tcm; the 2 low-abundance repeats NARTc and C6 and the abundant DIRE (degenerate Ingi/L1Tc-related retroelement) element.
De novo repeat discovery was performed in order to identify unique sequence repeats, using the program RepeatScout and RepeatMasker. RepeatScout identified 2,225 (Tcm) and 2,263 (Tcc X10) repeats of variable lengths. These repeats were then filtered using these criteria; i) removal of repeats shorter than 50 bp, ii) removal of repeats containing more than 50% low complexity sequence, iii) removal of repeats with fewer than 10 genomic copies, iv) removal of known repeats (i.e. present in Tcc CLBR). This decreased the number of hits to 20 (Tcm) and 3 (Tcc X10) using the outlined criteria. Manual examination of the Tcm repeats revealed that 12 corresponded to diverged Tcc CLBR sequences, including a spliced leader sequence and sequences related to MASP and TcMUCII mucin. We searched for these 8 repeats in the genome assemblies of Tcc X10 and Tcc CLBR as well as in raw reads, which decreased the number of Tcm-specific repeats to 7. The length of the identified Tcm-specific repeats varied between 60 to 896 bp, and BLAST searches resulted in non-significant hits to sequences of metazoan origin. These repeats were found exclusively on short contigs (0.5-1 kb), corroborating the idea that the repetitive components of these genomes have evolved faster. We estimated the copy number of the two longest repeats, Tcm-Rep1 (825 bp) and Tcm-Rep2 (896 bp) from the depth of 454 read coverage mapped on these sequences. The average 454 read coverage (12x) was then used to estimate copy number. The average read coverage was 1974 reads/position for Tcm-Rep1 and 1,494 reads/position for Tcm-Rep2. Hence, the estimated copy number became 164 and 124 for Tcm-Rep1 and Tcm-Rep2 respectively. Taken together, these two repetitive elements contribute ~250 kb of sequences to the Tcm genome and also represent a large set of putative Tcm-specific sequences. Since the repeats were not found in Tcc X10 and Tcc CLBR, it is possible that a loss has occurred in the lineage leading to the human infective T. c. cruzi.
T. c. marinkellei invades non-bat epithelial cells in small numbers and divides intracellularly
We also investigated the ability of Tcm to replicate intracellularly using the same cell lines. The human infective Tcc was included as a positive control. Infected cells were incubated over a 5 day time course, the development of intracellular amastigotes during this period indicated that Tcm is capable of intracellular proliferation (Figure7). Amastigogenesis and amastigote proliferation of Tcm following metacyclic invasion appeared to be analogous to Tcc controls. These data suggested that Tcm is capable of infecting other cells than strictly those from bats, and hence, that the infection is not blocked by species-specific host cell tropism mediated at the level parasite entry. In conclusion, the infection barrier must therefore arise in a different context, though whether this relates to different aspects of Tcm specific biology or as physiological or immunological differences between hosts, or as a combination of both, remains to be elucidated.
This study is the first genome analysis of a non-human associated member of Schizotrypanum. Our aim was to identify genome sequence differences that may relate to host specificity or other phenotypical differences, as well as to further understand the evolution of these parasite lineages. We found a slightly smaller genome of T. c. marinkellei compared with the human infective strains, although it remains an open question if this is a general tendency among bat-associated trypanosomes. T. c. marinkellei and T. c. cruzi shared the same set of core genes, i.e. there were no missing coding sequences in terms of housekeeping genes. On the contrary, several gene families were expanded in T. c. cruzi Sylvio X10, contributing to the larger genome size. This suggested that T. c. cruzi Sylvio X10 have a more versatile toolbox of surface antigens, which may reflect an adaptation to its host. Interestingly, one subspecies specific acetyltransferase gene was identified in T. c. marinkellei, containing detectable homology with genes from photosynthesizing organisms. It appears likely that this gene was acquired after the split of T. c. cruzi and T. c. marinkellei, since the gene was missing from T. c. cruzi strains Sylvio X10 and CL Brener. The gene represents a rare example of gene transfer between distantly related eukaryotes and may provide additional functionality to T. c. marinkellei. Future efforts will be required to understand its function. Considering the divergence time between T. c. marinkellei and T. c. cruzi (~6.5-8.5 MYA[10–12]), remarkably few absolute gene differences were present. This suggests that the core gene content of T. cruzi lineages is relatively stable, whereas the repetitive component is allowed to undergo more rapid changes. The low number of subspecies specific genes suggests that phenotypic variation, like host specificity, might be encoded by more discrete variation, e.g. via non-synonymous nucleotide variants leading to specific protein isoforms. The difficulty to explain how the genome encodes phenotypes like host-specificity is further illustrated by our finding that T. c. marinkellei invades non-bat cells, which indicates that the machinery to invade host cells is functionally conserved. The two subspecies T. c. marinkellei and T. c. cruzi were on average ~7.5% diverged in coding sequences with respect to single nucleotide differences. The large number of small nucleotide differences may have implications on phenotypic variation via the formation of new alleles. The present study has provided many new candidate genes, including putative antigens that can provide starting points for functional investigation of phenotypic variation of these parasite lineages.
Extensive copy number variation of various genes was identified. Copy number variation has been suggested as means for the parasite to increase gene expression in the absence of transcriptional regulation. These findings are not surprising and are corroborated by the long-standing knowledge of genomic variability in T. c. cruzi[24, 33, 41–43]. It is possible that phenotypes may be encoded at the transcriptional level. Interestingly, the T. c. marinkellei and T. c. cruzi genomes also contained variation in the amounts of non-coding repeats, related to retroelements and other previously uncharacterized repeats. As these differences were substantial, it remains plausible that whole chromosomes or chromosomal chunks have been lost in T. c. marinkellei. None of the larger chromosomes were missing, suggesting that smaller chromosomes harboring surface antigens or other repeats have been lost. The plasticity of the T. cruzi karyotype is further demonstrated by the fact that certain chromosomes appear to be monosomic in one subspecies but not in the other. The existence of such monosomic chromosomes reduces allelic redundancy and might have implications on transcript abundance. Karyotype variability therefore stands as another possible source of phenotypic variation. Finally, the amount of intraspecific genetic variation identified in this study is likely to represent only the tip of the iceberg in terms of the actual genetic variation present in natural reservoirs.
Taken together, the presented draft genomes raise further questions about genome evolution and diversity in this group of protozoa, and the putative functional implications of this variation. Further exploration of the genetic diversity within Schizotrypanum should therefore be a future priority as this may help to resolve complex relationships between parasites, vectors and hosts. The decreasing cost and time for whole genome sequencing should therefore pave the way for further large-scale efforts to understand the underlying genetic basis of these parasites.
Sequence data and annotations have been deposited in NCBI GenBank under the accession numbers AHKC01000000 (T. c. marinkellei B7) and ADWP02000000 (T. c. cruzi Sylvio X10). The data can also be downloaded fromhttp://www.ki.se/chagasepinet/genomes.html.
Cell culture, library preparation and sequencing
T. c. marinkellei B7 clone 11 and T. c. cruzi Sylvio X10 clone 1 were cultured using standard conditions (supplemented RPMI + 10% FBS). Parasite DNA was extracted using the Puregene kit. Genome size estimation of T. c. marinkellei was performed using flow cytometry as described by Lewis et al.. Illumina sequencing: The Tcm and Tcc X10 mate pair libraries were prepared according to Van Nieuwerburgh et al.. Initially, the paired-end protocol for 3 kb libraries from Roche/454 was used until circularized. After circularization, the libraries were prepared following the Illumina mate-pair protocol: 5 μg of genomic DNA was sheared to approximately 3 kb and end-polished. Fragments were then ligated to the Roche/454 circularization adapters and size selected using AMPure beads (Beckman Coulter). The ligated DNA was circularized using Cre-recombinase and then exonuclease treated. All enzymes were obtained from New England Biolabs. The circular DNA was fragmented using Covaris S2, end-repaired and purified using streptavidin coated magnetic beads. The DNA was then A-tailed and ligated with sequencing adapters and PCR amplified. The post-circularization steps were performed using the reagents either supplied or referenced by the Illumina mate-pair protocol. The clustering was performed on a cBot cluster generation system using a HiSeq paired-end read cluster generation kit. The samples were sequenced on an Illumina HiSeq2000 as 2x100-nt reads (one lane for each genome). Base conversion was done using Illumina OLB v1.9. 454 sequencing: Prepared according to the manufacturer’s instructions and sequenced on a 454 instrument with Titanium chemistry.
Illumina reads were quality filtered and trimmed using the fastq_quality_filter program of the fastx toolkit (parameters: -q 20 -p 95). Illumina reads were assembled with Velvet v1.1.04, using the empirically determined kmer length of 43 and a minimum contig length of 500 bp. velveth and velvetg were called with the following commands, respectively: ‘<name> 43 -fastq -shortPaired1 input.fastq’ and ‘<name> −min_contig_lgth 500 -exp_cov auto -ins_length 2000 -ins_length_sd 2000 -amos_file no -scaffolding no -unused_reads yes’. Unused reads were extracted and subjected to a second round of Velvet assembly using a kmer length of 53 (empirically determined) and a minimum contig length of 400 bp (velvetg: ‘-min_contig_lgth 400 -exp_cov auto -ins_length 10 -ins_length_sd 5000 -scaffolding no’). 454 reads were assembled with CELERA v6.1 (default settings). 454-related insertion-deletion errors in the assembly were corrected using the Illumina reads: Illumina reads were aligned with bwa, and the resulting SAM file was then scanned in order to identify indels. In order to correct a position, at least 10 Illumina reads were required to support the change. The three assemblies (i. Illumina Velvet 1; ii. Illumina Velvet 2; iii. 454 CELERA) were pooled and merged into a non-redundant assembly. Assembly merging was performed using the Zorro pipeline, relying on minimus2 and mummer to split and merge contigs. The merged assembly was filtered to include only contigs longer than 500 bp. Maxicircle (mitochondrial) sequences were identified using megablast and removed. Minicircle (mitochondrial) sequences were identified and removed by searching for the TCKIN2 signature sequence. Distance information from mate-pairs were used to order and orient contigs into scaffolds. The software SSPACE was used for scaffolding. A small number of intra-scaffold gaps (~200/genome) could be closed using the overlap between adjacent contigs. The final assembly was subjected to gap closure using the IMAGE pipeline.
Annotation: Gene models were transferred from Tcc CLBR using Perl scripts, and additional genes were called using GeneMarkS. Annotations were manually curated using the Artemis Comparison Tool. Orthologous genes were identified using the best reciprocal BLASTp hit (E-value 1e-10). Unique genes were identified using BLASTp and tBLASTn searches. Genes in synteny were determined using homology of surrounding genes. At least one adjacent homologous gene was required to be present in order to call a gene syntenic. dN and dS values were calculated using the yn00 program of the PAML package. Rearrangements: Chromosomal re-arrangements were identified from alignments generated using promer. Repeats: Sequence repeats were identified with RepeatMasker, Repbase and Tandem Repeat Finder. Phylogenetic analysis of the candidate horizontal gene transfer: A dataset was extracted with Blast Explorer (E-value <1e-40). Multiple sequence alignment was done with ClustalW v2.1 and filtered with Gblocks to remove ambiguous positions. Alignments were manually inspected in Jalview. A maximum likelihood tree was generated with RAxML v7.0.4, using the PROTGAMMAJTT model and 100 bootstrap replicates. Multicopy genes: Genome sequence reads (Illumina) were mapped back to the assembly using bwa (default settings) and the coverage was calculated. Sequence entropy was calculated using bio3d. Copy number variation: Genome sequence reads (Illumina) were mapped back to the assembly and the reference sequence was divided into 100 bp windows with 50 bp overlap. The sum of coverage for each position in the window was computed, log10-scaled and plotted. Heterozygosity: Reads were aligned with the assembly and samtools and awk were used to extract polymorphic positions. Maxicircle analysis: Manual annotation of maxicircle coding regions was performed by comparison to the published CLBR (GenBank: DQ343645), Esmeraldo (GenBank: DQ343646) and Sylvio X10/1 (GenBank: FJ203996) maxicircle coding sequences. Sequence identity was calculated using BioEdit v188.8.131.52. Heteroplasmy was called with samtools mpileup. A SNP was defined as a nucleotide variant present in at least 5 independent reads (with parameters: 20X coverage and mapping quality, 30). All scripts are available from the authors on request.
Normal PCR and quantitative real-time PCR
Normal PCRs: Primers were selected with Primer3 and synthesized by Sigma-aldrich. Amplification was performed using the Phire Hot Start II DNA polymerase kit (Finnzymes). The targets were amplified in a mixture containing 1X Phire Reaction Buffer, 0.2 mM of dNTPs (Fermentas), 0.4 μM of each primer, 2% DMSO, 50 ng of genomic DNA, 0.4 μl of Phire Hot Start II DNA polymerase and water to a final volume of 20 μl. The cycling conditions were as follows: Initial denaturation at 98°C for 2 minutes, 35 cycles of 98°C for 10 seconds, 60–68°C for 10 seconds and 72°C for 10 seconds and a final extension step at 72°C for 2 minutes. The Tc_CLB1 amplicon of 3 kb size uses a 45 seconds extension step, in comparison with the other amplicons that uses just 10 seconds. Amplicons were visualized using a 1.3% agarose gel stained with ethidium bromide. Quantitative Real-time PCR (RT-qPCR) on the specific acetyltransferase gene in Tcm: RNA extraction was performed using the RNeasy Mini kit (Qiagen). RNA was converted to cDNA with reverse transcriptase and random hexamer primers. Reactions were performed using Power SYBR Green MasterMix (Life Technologies) under standard conditions. Template concentration was 50 ng/μl and 1 μl of template was used in each reaction. Primer concentration was 0.2 μM in 20 μl of final volume. Each experiment was performed in triplicate and the average cycle threshold (Ct) value was used as a measurement of initial template abundance. All reactions were performed on an ABI 7300 Real-time PCR system. The following primer pairs were selected for the experiment (5′ to 3′); unique gene: TTGCAGCATATGTGTGGATG (F), ACGTTAAAGAAACGGCTGCT (R), hypoxanthine-guanine phosphoribosyltransferase: GCCTTCATGTCAACCCTCTT (F), AAGACGTGACACCTTCACCA (R), 18S rRNA: TTACGTCCCTGCCATTTGTA (F), TTCGGTCAAGTGAAGCACTC (R). RT-qPCR to validate copy number variation: Experimental conditions were similar as for the previous experiment, except that genomic DNA was used (20 ng/μl, 1 μl loaded).
Cell invasion assay
Vero cells were maintained in DMEM + glutaMAX (Gibco, Invitrogen, UK) supplemented with 10% fetal calf serum (PAA laboratories, UK), 5mM l-glutamine, 50 μg/ml streptomycin and 50 units/ml penicillin. Tb1 lu cells (HPACC, UK) were maintained in MEM (ATCC, UK) and supplemented as described above. OK cells (HPACC, UK) were maintained in MEM (Sigma-Aldrich, UK) with 10% fetal calf serum, 5 mM l-glutamine, 50 μg/ml streptomycin and 50 units/ml penicillin and 5% non-essential amino acids. T. c. marinkellei epimastigotes from lineage B7 cl11 were grown in Liver Infusion Tryptose (LIT) and T. c. cruzi strain M6241 was grown in RPMI for 2 weeks prior to experiments. Each cell line was seeded at a density of 105 per ml onto 13 mm diameter coverslips and allowed to grow overnight. Cells were then washed and the growth media replaced with media containing 105 metacyclic T. c. marinkellei and incubated at 37°C for either overnight or 5 days. The media was removed and cells were washed twice with PBS and either fixed with 4% paraformaldehyde for 20 min or ice cold methanol. Methanol fixed cells were stained with Giemsa for 10 min and imaged with a Zeiss Axioplan 2 microscope and a Zeiss AxioCam Hrc camera. Paraformaldehyde fixed cells were blocked in 10% goat serum and labelled with T. c. marinkellei positive serum for 1 h before incubating with AffiniPure Fab fragments (Stratech Scientific Ltd., UK) for 10 min, these epitopes were then recognised by anti-rabbit Alexa Fluor 568. The cells were blocked again in 10% goat serum and permeabilised in 1% NP40 for 3 min before labelling with anti-whole cell body (WCB) antibody (kindly provided by Prof. K. Gull) for 1 h recognised by anti-mouse Alexa Fluor 488 and finally DAPI stained before mounting in Fluoromount (Sigma-Aldrich, UK). Antibody labelled cells were visualised by a Zeiss Axioplan 2 microscope and Zeiss AxioCam MRm camera all image processing was done with Axiovision 4.7 software. Two hundred cells were assayed in the overnight experiments and the results are expressed as an average of three independent experiments.
We would like to acknowledge Rickard Sandberg and UPPMAX for providing computational resources and the Genomics Platform at SciLifeLab Stockholm for library preparation and sequencing. We also would like to thank the anonymous reviewers for their suggestions on how to improve the manuscript. This work was supported by the European Union Seventh Framework Program (http://cordis.europa.eu/fp7) grant 223034 (ChagasEpiNet). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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