Genomic distribution of SINEs in Entamoeba histolytica strains: implication for genotyping
© Kumari et al.; licensee BioMed Central Ltd. 2013
Received: 26 November 2012
Accepted: 20 June 2013
Published: 1 July 2013
The major clinical manifestations of Entamoeba histolytica infection include amebic colitis and liver abscess. However the majority of infections remain asymptomatic. Earlier reports have shown that some E. histolytica isolates are more virulent than others, suggesting that virulence may be linked to genotype. Here we have looked at the genomic distribution of the retrotransposable short interspersed nuclear elements EhSINE1 and EhSINE2. Due to their mobile nature, some EhSINE copies may occupy different genomic locations among isolates of E. histolytica possibly affecting adjacent gene expression; this variability in location can be exploited to differentiate strains.
We have looked for EhSINE1- and EhSINE2-occupied loci in the genome sequence of Entamoeba histolytica HM-1:IMSS and searched for homologous loci in other strains to determine the insertion status of these elements. A total of 393 EhSINE1 and 119 EhSINE2 loci were analyzed in the available sequenced strains (Rahman, DS4-868, HM1:CA, KU48, KU50, KU27 and MS96-3382. Seventeen loci (13 EhSINE1 and 4 EhSINE2) were identified where a EhSINE1/EhSINE2 sequence was missing from the corresponding locus of other strains. Most of these loci were unoccupied in more than one strain. Some of the loci were analyzed experimentally for SINE occupancy using DNA from strain Rahman. These data helped to correctly assemble the nucleotide sequence at three loci in Rahman. SINE occupancy was also checked at these three loci in 7 other axenically cultivated E. histolytica strains and 16 clinical isolates. Each locus gave a single, specific amplicon with the primer sets used, making this a suitable method for strain typing. Based on presence/absence of SINE and amplification with locus-specific primers, the 23 strains could be divided into eleven genotypes. The results obtained by our method correlated with the data from other typing methods. We also report a bioinformatic analysis of EhSINE2 copies.
Our results reveal several loci with extensive polymorphism of SINE occupancy among different strains of E. histolytica and prove the principle that the genomic distribution of SINEs is a valid method for typing of E. histolytica strains.
Entamoeba histolytica, the etiological agent of amoebiasis, is a protistan parasite that lives in the human intestine. Amoebiasis is the third leading cause of death due to parasitic disease . According to the WHO, about 40–50 million people are infected annually causing approximately 100,000 deaths worldwide. About 90% of the infections with this parasite remain asymptomatic .What leads to the varied outcome of infection is not known, but it is possible that the genotype of the strain influences the outcome . The suggestion has been made that inherently avirulent strains exist that may be associated with unique genotypes . The E. histolytica strain Rahman is considered to be avirulent in axenic culture since it shows reduced cytopathic activity on epithelial cells and does not form liver abscesses in animal models [5, 6]. Data are, however, insufficient to assign virulence properties to specific genotypes of E. histolytica.
Retrotransposons without long terminal repeats are generally called long interspersed nuclear elements (LINEs) and their short non autonomous partners are called SINEs . LINEs are generally ~5 kb in length and encode the functions required for retrotransposition, while SINEs are short and do not code for proteins. They utilize the LINE-encoded proteins for their own retrotransposition. Both LINEs and SINEs are efficient genome invaders and are widespread in eukaryotes . In E. histolytica the EhLINEs (4.8 kb) and EhSINEs (0.5 to 0.7 kb) constitute 11.2% of the genome . They belong to three closely related families, of which EhLINE1/EhSINE1 are the most abundant. These elements are present mostly in the intergenic regions [10, 11], with a T- rich sequence within 50 bp upstream of the site of insertion [10, 12]. Due to their mobile nature they can occupy different genomic locations and may influence the phenotype of the organism by activating or silencing the genes in their vicinity. Previous work has shown that a number of SINE1 occupied sites in E. histolytica are unoccupied in the non pathogenic species Entamoeba dispar and vice versa [11, 13, 14] which may have important consequences for the pathogenicity of the parasite.
A number of studies in different organisms have utilized SINEs as useful markers for phylogeny . It has been argued that SINE insertion analysis is one of the best methods for determining relationships of closely related species since SINEs are widely dispersed in the genome and, unlike DNA transposons, there is no evidence of any process that removes SINEs from the genome once they are inserted. Nonspecific SINE deletions due to unequal crossing over are relatively rare. Thus the absence of a SINE at a particular locus signifies the ancestral state. The probability of independent insertions at the same locus is exceedingly low, which links SINE-containing loci as related by descent [16, 17]. For these reasons population genetic analysis can be performed more accurately with SINEs than with RFLPs and microsatellite loci (where the same allele may be shared by two individuals by chance). Here we have explored the possibility of using EhSINE insertions as strain-specific markers.
Several methods have been developed for the genotyping of this parasite [18–24], which have their individual limitations. Polymorphisms are observed in short tandem repeat numbers, and repeat sequences present in the genes encoding chitinase and the surface antigen SREHP, as well as in the arrays of tRNA genes of E. histolytica. These have been utilized successfully for strain identification [25, 26]. However the size variation in most of these loci is small, sometimes making it difficult to detect polymorphism by agarose gel electrophoresis, so DNA sequencing is normally used for confirmation. A transposon display technique was also devised for strain identification based on the genomic distribution of EhSINE1 . However, this method is not suitable for use with clinical isolates.
Here we analysed 393 EhSINE1 and 119 EhSINE2 loci present in the HM-1:IMSS strain of E. histolytica for insertion polymorphism in other sequenced strains (http://www.Amoebadb.org) [28, 29]. Seventeen loci were found (13 for EhSINE1 and 4 for EhSINE2) that showed insertion polymorphism. Of these, six loci were validated experimentally in strain Rahman. Three of these loci were tested in 7 other axenically grown strains and 16 clinical isolates. Each of the loci gave a single specific amplicon with the primer sets used, making this a suitable method for genotyping. We also report a bioinformatic analysis of EhSINE2 elements.
Analysis of polymorphic loci
The E. histolytica HM-1:IMSS genome sequence is available in 1529 scaffolds as the full genome could not be assembled into chromosomes. The sequences were downloaded from NCBI [accession number AAFB00000000]. Different strains of E. histolytica, namely HM1:CA, DS4-868, KU27, KU48, KU50, MS96-3382 and Rahman were downloaded from AmoebaDB (http://www.amoebadb.org) . These are partially assembled sequences obtained using next generation sequencing technologies.
Genome sequence data of different strains of E. histolytica used in this study
Number of scaffolds
Entamoeba histolytica HM-1:IMSS (REFERENCE)
Entamoeba histolytica DS4-868
Entamoeba histolytica KU27
Entamoeba histolytica KU48
Entamoeba histolytica KU50
Entamoeba histolytica MS96-3382
Entamoeba histolytica Rahman
Entamoeba histolytica HM1:CA
Axenic and xenic cultivation of E. histolytica- Axenic strains HM-1:IMSS and Rahman were maintained by continuous subculturing in TYI-S-33 medium , and the rest of the axenic strains were maintained in LYI-S-2 medium . Xenic strains were maintained by continuous subculturing in Robinson′s medium .
Genomic DNA isolation- Genomic DNA of axenic and xenic E. histolytica strains was isolated using a genomic DNA isolation kit (Promega, USA) and the QIAamp® DNA Mini Kit (Qiagen, Germany), respectively, according to the manufacturer’s instructions.
Polymerase chain reaction (PCR) - Primers were designed from the flanking sequences of different EhSINE1 copies obtained from the E. histolytica HM-1:IMSS database (Additional file 1: Figure S1). All PCR reactions were performed with Biotools DNA polymerase (Biotools, B&M Labs, Spain); the PCR programme consisted of initial denaturation for 5 min at 94°C followed by 30 cycles of 30 sec at 94°C, annealing for 30 sec at a temperature dependent on the Tm of the primers used, and an extension time at 72°C dependent on the size of amplicon. Products were resolved on a 1% agarose gel (USB, Spain) containing 0.5 μg/ ml of ethidium bromide using 0.5X TBE (Tris borate EDTA pH8) buffer.
Southern blotting and hybridization- DNA was transferred to HYbond™-N + Nylon membrane (GE Healthcare) using standard methods . Labeled probes were prepared using α-32P–dATP by the random priming method using the NEBlot(R) kit (NEB, USA) according to the manufacturer’s instructions. Blots were hybridized overnight with probe at 65°C in a solution of 1% SDS, 1 M NaCl and 100 μg/ml of salmon sperm DNA, washed to remove nonspecific probe, exposed (Fujifilm) and scanned by phosphorimager.
DNA sequencing- Amplicons were extracted from agarose gels using a gel extraction kit (Qiagen) and cloned into the pGEM-T vector (Promega, USA). Sequences were generated commercially (TCGA, India) and compared using ClustalW software (Bioedit).
Analysis of Target Site duplication (TSD) and internal repeats (IRs) using MEME- The online tool MEME  was used for the analysis of TSDs and IRs of SINE2. 50 bp of sequence upstream and downstream of the EhSINE2 were extracted from the E. histolytica HM-1:IMSS genome and these were analysed for TSD. Since the longest TSDs found were in the range of 16–20 bp, and some of the shorter TSDs may result from accumulation of mutations in older SINE insertions, TSDs having size < 8 bp were excluded. The input consisted of 79 FASTA formatted sequences of TSDs with the default settings of width (Minimum 6 and Maximum 50) and the search was optimized for identifying zero or one motif per sequence. For IR analysis 150 sequences were subjected to MEME analysis in a similar way.
Results and discussion
Identification of genomic loci with differential EhSINE1/EhSINE2 occupancy in the sequenced E. histolyticastrains
The availability of genome sequences of a number of E. histolytica strains is likely to help define the level of polymorphism in SINE distribution in E. histolytica. EhSINE1 (445 copies) and EhSINE2 (256 copies) constitute the majority of the SINE population of E. histolytica. There are only 49 copies of EhSINE3 , therefore we focused only on EhSINE1 and EhSINE2 for this study. Out of 445 copies of EhSINE1, 393 are full-length (>450 bp) , and only full length copies were used for analysis. We performed a similar analysis with EhSINE2 and found 119 full length copies (length >400 bp and similarity >70% with the EhSINE2 consensus) in strain HM-1:IMSS.
Similarly, out of the 119 full-length copies of EhSINE2 it was possible to use only 69 copies for our analysis (Figure 2), and only 2 unoccupied sites were identified in Rahman following the criteria described for EhSINE1. Since the total number of unoccupied sites obtained was rather small (4 out of 270 for EhSINE1, and 2 out of 119 for EhSINE2), we checked to see if we were missing some polymorphic loci in the copies that could not be computationally analyzed. PCR primers were designed using the genes flanking a number of EhSINE1 loci in HM-1:IMSS and were used to amplify the same loci from genomic DNA of strain Rahman. A total of 159 loci were tested from the various categories listed in Figure 1. Of these, the amplicon size in Rahman was identical with HM-1:IMSS at 157 loci, showing that these loci were all occupied, while at the remaining two loci (17 and 19) the EhSINE1 was absent from Rahman. Locus 17 was missed in the computational analysis because the sequence of the SINE, and some sequence upstream of it, contained undefined nucleotides in Rahman. In the case of locus 19 the corresponding sequence was located in three different contigs in Rahman. Therefore the combined experimental and computational analysis allowed us to identify 6 EhSINE1 loci that are polymorphic between strains HM-1:IMSS and Rahman.
SINE polymorphic loci in sequenced strains (AmoebaDB)
Scaffold ID in HM1:IMSS
Position of Sine in HM1
Scaffold ID in strain
DS571157 (locus 13) ★
DS571247 (locus 17) ●
90 bp of SINE present *
DS571226 (locus 19) ▲
DS571158 (locus 42)
40 bp of SINE present
50 bp of SINE from 5’ end present
Truncated from both side (397 bp present) *
150 bp upstream flank also missing
60 bp downstream flank also missing
Only 80 bp of SINE present*
312 bp upstream and 275 bp downstream flank missing
60 bp of SINE from 5’ end present and 500 bp downstream flank missing
DS571418 (locus 18) ↷
DS571150 (locus 50) ♦
These loci were also found to be polymorphic among different strains and isolates of E. histolytica as deduced from analysis of NGS data (compiled in Additional file 4: Table S2). In some strains, although the SINE was present at the locus, the sequence showed some truncations or short deletions. If these changes are not due to assembly errors in the database one could envision various factors that may contribute to this. Most of the truncations were at the 5′-end of the SINE, which could result from the well known phenomenon of incomplete reverse transcription of the SINE RNA template during retrotransposition . Short deletions may appear due to recombination between genomic SINE copies, or due to replication slippage at the short internal repeats in the EhSINEs (described later). However, some of these changes are, indeed, due to sequence assembly errors in the database, which we document below for locus 17 in strains Rahman and MS96-3382.
Sequence analysis of some of the polymorphic loci in strains HM-1:IMSS and Rahman
Sequence data available for the two genomes in AmoebaDB shows that the assembled genome data of Rahman has many more undefined regions and gaps. There are 1529 scaffolds defining the HM-1:IMSS genome (in the size range of 0.9 kb-500 kb) compared to 1145 of Rahman (in the size range of 2 kb-170 kb) and 17378 unassembled contigs. We examined the sequences at loci 13, 17, 19 and 42 more closely and found that the locus 13 sequence was located in a single scaffold in both strains and the sequence was identical except for the loss of EhSINE1 in Rahman. However, the sequences at the other loci were either found in multiple scaffolds/contigs in Rahman, or contained undefined regions, as described below.
Locus 19 was present in the scaffolds DS571126 (HM-1:IMSS) and EhRmscaffold_00536 (Rahman). The sequence upstream of the EhSINE1 location in HM-1:IMSS was undefined in Rahman. However we found three unassembled contigs (EhRmcontig_00303, EhRmcontig_00523 and EhRm_contig21711) in the Rahman database that matched the HM-1:IMSS sequence (Additional file 6: Figure S5). An amplicon from Rahman generated by PCR amplification using a primer each designed from EhRmcontig_00523 and EhRcontig_21011 displayed the expected size (Figure 3B), showing that these contigs likely belong to this locus. Sequence analysis of the amplicon confirmed that the two strains were identical except for the loss of EhSINE1 in Rahman (Figure 5).
Locus 42 in HM-1:IMSS was in one scaffold (DS571158), while in Rahman the syntenic sequence was present across three different scaffolds/contigs (Additional file 7: Figure S3). One contig spanned the downstream gene sequence with which primer 42.1 R was an exact match. However, in primer 42.1 F (Additional file 7: Figure S3) the 3′ nucleotide was a mismatch. Sequence comparison of this region revealed single nucleotide differences at several positions, which may explain our failure to amplify this locus from Rahman using HM-1:IMSS primers.
These results suggest that some of the sequence data currently available in the database needs reanalysis and the predictions need to be validated by experimentation. Our analysis has helped to correctly assemble the sequences at loci 17, 19 and 42 in Rahman.
Genotyping using SINE sequences
Categorization of E. histolytica strains
The same primer pairs were used for analysis of 16 clinical isolates of E. histolytica (Figure 7, Additional file 8: Table S3). The results are summarized in Table 3. The amplicons were clearly visible only after Southern hybridization for most clinical isolates. The results clearly show mosaic patterns in the three loci, displaying characters of both HM-1:IMSS and Rahman in many strains.
To sum up the above data, a total of 25 E. histolytica strains were used in this study, of which HM-1:IMSS contains EhSINE1 at all three loci (HHH), while Rahman lacks the element at all three loci (RRR). In the remaining 23 E. histolytica strains (including axenic and xenic clinical isolates), EhSINE1 was absent at loci 13, 17 and 19 in 7, 10 and 8 strains respectively. Based on the presence/absence of EhSINE1, and amplicons obtained with the primer pairs at these three loci, the 23 strains were categorized into eleven genotypes (Table 3). Based on SINE occupancy there can only be eight combinations at the three loci (i.e. 23). Additional variations (designated N, which are neither H nor R) have come about due to alterations in flanking sequences leading to loss of primer recognition sites. In the 23 strains tested the most frequent combination was HHH (5 strains) followed by HRR and HRH (3 strains each) and HNH, NRR, HNR and NNH (2 strains each). The use of multiple loci for strain identification is preferred [23, 25] as a single locus cannot differentiate all the strains. The results obtained by our method corroborated with the data from tRNA-STRs. Both methods distinguished the strains HM-1:IMSS, Rahman, 200:NIH and HK-9 from one another [20, 25, 26] and gave the same pattern for strains PVB and PVF (Clark C.G., unpublished observation). Thus our results suggest that in principle genomic distribution of SINEs can be used as a valid method for typing of E. histolytica strains.
Although SINEs are mobile genetic elements, their mobilization in present-day E. histolytica is probably a very infrequent event. This can be inferred from the fact that most genomic copies of the EhLINE1 retrotransposon (which provides the machinery for EhSINE1 mobilization through retrotransposition) are inactive. We have shown experimentally that the retrotransposition activity in these cells is very low or absent . Therefore the genomic location of SINEs in a given strain is stable enough to be used as a strain-specific signature.
Bioinformatic analysis of EhSINE2 copies
As already mentioned, retrotransposition is accompanied by generation of TSDs. Newly retrotransposed copies are expected to be flanked by identical TSDs, while over time these accumulate mutations, become shorter in length and are finally unrecognizable. Therefore length of TSDs may be a marker of age of SINEs . We analyzed the TSDs of all 119 EhSINE2 copies, and could find TSD in 97 cases. The longest TSDs (ranging in sizes from 16–20 bp) were found in elements with IRs, while copies lacking intact IRs displayed smaller TSDs, in the range of 8–9 bp (Figure 8). This suggests that copies lacking IR may be older and may have suffered loss of IR sequences subsequent to retrotransposition. In the case of EhSINE1, the 2 IR-containing copies were reported to be the most recently transposed elements as they had longer TSDs than the other copies . The TSDs of 81 EhSINE2 sites (excluding those below 8 bp in length) were analyzed by MEME. All 81 TSDs showed the consensus motif T(T/C)T(T/C)TN(A/T)T, suggesting a high percentage of pyrimidines is needed at the insertion point.
SINE elements are useful genomic markers due to their wide occurrence and property of irreversible re-integration in the host genome . The loss of SINEs from genomic loci is a rare event and is generally accompanied by changes in flanking sequences as well . Therefore, as stated earlier, SINEs are better suited to establish genealogies below the species level with minimal assumptions compared with other standard markers, such as microsatellites, RFLPs, and SNPs, which can result from independent mutations at different times that are not inherited from a common ancestor [16, 42–46]. For this reason the analysis of SINE occupancy in E. histolytica strains reported here will be significant to establish intraspecific relationships.
Retrotransposons are known to influence the expression of genes in their vicinity by various mechanisms, including silencing by heterochromatinization, up-regulation by providing alternate promoters, and novel expression patterns through alternative splicing and polyadenylation [47–50]. Thus the gain or loss of EhSINE1 element from a genomic locus could potentially influence the phenotype of the organism in a profound manner. For this reason the strain typing method used here has a potential to reveal loci that may be associated with different phenotypes, including the virulence properties of the parasite. However more samples need to be tested to provide a correlation between virulence and genotype. A combination of rapid genome sequencing and expression analysis from a variety of clinical isolates of E. histolytica by NGS will reveal whether retrotransposons in E. histolytica have the ability to influence neighboring gene expression. This method of strain typing based on retrotransposon occupancy could then have physiological relevance.
This work was supported by a grant to SB from Indian Council of Medical Research and Department of Biotechnology, to JP from Department of Biotechnology and to LRI from Department of Science and Technology, under the Women Scientists Scheme (WOS-A), Government of India. VK received a fellowship from Council of Scientific and Industrial Research, India. Grateful thanks are extended to Dr. Rashidul Haque for providing strain MS96-3382. We acknowledge Gareth Weedall and Neil Hall, Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK for Genome Assembly of E. histolytica strain Rahman; J. Craig Venter Institute and the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, USA for E. histolytica strains KU50, MS96, KU27, KU48, HM-1:CA and DS4, and Lis Caler at the J. Craig Venter Institute for E. histolytica HM-1:IMSS sequence and annotation.
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