Limits...
Genomic distribution of SINEs in Entamoeba histolytica strains: implication for genotyping.

Kumari V, Iyer LR, Roy R, Bhargava V, Panda S, Paul J, Verweij JJ, Clark CG, Bhattacharya A, Bhattacharya S - BMC Genomics (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH

Related in: MedlinePlus

Strain identification in xenic cultures based on locus 13, 17 and 19: PCR was performed using genomic DNA of 16 different xenic cultures of E. histolytica for each locus, as described in Figure 6. PCR reactions were resolved on a 1 % agarose gel and subjected to Southern blotting with the locus specific probes 13 (Panel A), 17 (Panel B), or 19 (Panel C). Samples which did not give a product at locus 13 were amplified using an alternate reverse primer 13.1 R instead of 13.2 R followed by Southern blotting and hybridization with a locus specific probe. The expected size of the amplicon with each primer set is mentioned in the table below each locus panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3716655&req=5

Figure 7: Strain identification in xenic cultures based on locus 13, 17 and 19: PCR was performed using genomic DNA of 16 different xenic cultures of E. histolytica for each locus, as described in Figure 6. PCR reactions were resolved on a 1 % agarose gel and subjected to Southern blotting with the locus specific probes 13 (Panel A), 17 (Panel B), or 19 (Panel C). Samples which did not give a product at locus 13 were amplified using an alternate reverse primer 13.1 R instead of 13.2 R followed by Southern blotting and hybridization with a locus specific probe. The expected size of the amplicon with each primer set is mentioned in the table below each locus panel.

Mentions: We explored the possibility of using some of the polymorphic loci as markers for genotyping. For this we focused on loci 13, 17 and 19 and tested them using 23 axenic and xenic strains of E. histolytica. A genotyping method would need to be used for patient samples, where large amplicons may be difficult to obtain reproducibly due to impurities in DNA preparation and low E. histolytica DNA concentrations. We therefore designed primers as close to the EhSINE1 insertion site as possible to minimize amplicon size (Additional file 1: Figure S1). For each locus two primer sets were used; one set was designed from flanking sequences and the other set comprised one of the flanking primers combined with a primer from the EhSINE1 sequence (Figure 6A and Additional file 2: Table S1). Although care was taken to design primers for each locus that did not match the Entamoeba dispar genome, this was not possible in all cases due to extensive sequence conservation between the two species. However one primer from each pair for all three loci had no match in E. dispar (Additional file 2: Table S1). The amplicons obtained with each of the primer pairs for a given locus were combined and electrophoresed together in the same gel lane (Figure 6B shows the results for axenic strains). The identities of the bands were confirmed by Southern hybridization with a flanking region probe (middle panel, Figure 6B) or an EhSINE1 probe (bottom panel, Figure 6B). DNA from strains HM-1:IMSS and Rahman gave the expected amplicon with each primer pair, except for the 1.4 kb band with primers 13.1 F and 13.2 R expected from HM-1:IMSS, which could not be amplified efficiently. Hence HM-1:IMSS locus 13 was identified by the 0.2 kb 13.1 F/SINE R product. Results with the seven axenic strains showed that EhSINE1 was present at all three loci in strains MS84-1373 and MS27-5030. In this respect they behaved like HM-1:IMSS. However, primer set 17.2 F-17 .2 R could not amplify MS84 and primer set 17.2 R-SINE R could not amplify MS27, indicating that they were not identical to HM-1:IMSS at locus 17. Single nucleotide mutations in the flanking sequences could lead to sequence polymorphisms in these regions and give the observed result due to loss of primer recognition. Since the sequence of this region is not known in these other strains, an explanation for this result would have to await further sequence data. Similarly, strain HK-9 resembled Rahman at all three loci in terms of EhSINE1 occupancy but belonged to a third category since at locus 13 it repeatedly failed to give the expected amplicon size with primer pair 13.1 F-13.2R although the expected amplicon was obtained with primer pair 13.1 F-13.1R (Figure 7A). Strains PVBM08B and PVBM08F were like Rahman at locus 17 and like HM-1:IMSS at loci 13 and 19. Strain MS96-3382 was like Rahman at loci 13 and 17. However, genome sequence analysis (AmoebaDB) showed the presence of a 397 bp SINE sequence (truncated from both ends) at locus 17 in this strain. Since the PCR and Southern data for this locus were unambiguous we are inclined to believe that, as mentioned earlier (Figure 5), the discrepancy between our data and AmoebaDB may be due to sequence assembly problems. Strain 200:NIH was like Rahman at loci 17 and 19. Thus, based on the presence and absence of SINE1, and the amplicons obtained with each primer pair at these three loci, the axenic strains could be divided into five genotypes (Table 3).


Genomic distribution of SINEs in Entamoeba histolytica strains: implication for genotyping.

Kumari V, Iyer LR, Roy R, Bhargava V, Panda S, Paul J, Verweij JJ, Clark CG, Bhattacharya A, Bhattacharya S - BMC Genomics (2013)

Strain identification in xenic cultures based on locus 13, 17 and 19: PCR was performed using genomic DNA of 16 different xenic cultures of E. histolytica for each locus, as described in Figure 6. PCR reactions were resolved on a 1 % agarose gel and subjected to Southern blotting with the locus specific probes 13 (Panel A), 17 (Panel B), or 19 (Panel C). Samples which did not give a product at locus 13 were amplified using an alternate reverse primer 13.1 R instead of 13.2 R followed by Southern blotting and hybridization with a locus specific probe. The expected size of the amplicon with each primer set is mentioned in the table below each locus panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716655&req=5

Figure 7: Strain identification in xenic cultures based on locus 13, 17 and 19: PCR was performed using genomic DNA of 16 different xenic cultures of E. histolytica for each locus, as described in Figure 6. PCR reactions were resolved on a 1 % agarose gel and subjected to Southern blotting with the locus specific probes 13 (Panel A), 17 (Panel B), or 19 (Panel C). Samples which did not give a product at locus 13 were amplified using an alternate reverse primer 13.1 R instead of 13.2 R followed by Southern blotting and hybridization with a locus specific probe. The expected size of the amplicon with each primer set is mentioned in the table below each locus panel.
Mentions: We explored the possibility of using some of the polymorphic loci as markers for genotyping. For this we focused on loci 13, 17 and 19 and tested them using 23 axenic and xenic strains of E. histolytica. A genotyping method would need to be used for patient samples, where large amplicons may be difficult to obtain reproducibly due to impurities in DNA preparation and low E. histolytica DNA concentrations. We therefore designed primers as close to the EhSINE1 insertion site as possible to minimize amplicon size (Additional file 1: Figure S1). For each locus two primer sets were used; one set was designed from flanking sequences and the other set comprised one of the flanking primers combined with a primer from the EhSINE1 sequence (Figure 6A and Additional file 2: Table S1). Although care was taken to design primers for each locus that did not match the Entamoeba dispar genome, this was not possible in all cases due to extensive sequence conservation between the two species. However one primer from each pair for all three loci had no match in E. dispar (Additional file 2: Table S1). The amplicons obtained with each of the primer pairs for a given locus were combined and electrophoresed together in the same gel lane (Figure 6B shows the results for axenic strains). The identities of the bands were confirmed by Southern hybridization with a flanking region probe (middle panel, Figure 6B) or an EhSINE1 probe (bottom panel, Figure 6B). DNA from strains HM-1:IMSS and Rahman gave the expected amplicon with each primer pair, except for the 1.4 kb band with primers 13.1 F and 13.2 R expected from HM-1:IMSS, which could not be amplified efficiently. Hence HM-1:IMSS locus 13 was identified by the 0.2 kb 13.1 F/SINE R product. Results with the seven axenic strains showed that EhSINE1 was present at all three loci in strains MS84-1373 and MS27-5030. In this respect they behaved like HM-1:IMSS. However, primer set 17.2 F-17 .2 R could not amplify MS84 and primer set 17.2 R-SINE R could not amplify MS27, indicating that they were not identical to HM-1:IMSS at locus 17. Single nucleotide mutations in the flanking sequences could lead to sequence polymorphisms in these regions and give the observed result due to loss of primer recognition. Since the sequence of this region is not known in these other strains, an explanation for this result would have to await further sequence data. Similarly, strain HK-9 resembled Rahman at all three loci in terms of EhSINE1 occupancy but belonged to a third category since at locus 13 it repeatedly failed to give the expected amplicon size with primer pair 13.1 F-13.2R although the expected amplicon was obtained with primer pair 13.1 F-13.1R (Figure 7A). Strains PVBM08B and PVBM08F were like Rahman at locus 17 and like HM-1:IMSS at loci 13 and 19. Strain MS96-3382 was like Rahman at loci 13 and 17. However, genome sequence analysis (AmoebaDB) showed the presence of a 397 bp SINE sequence (truncated from both ends) at locus 17 in this strain. Since the PCR and Southern data for this locus were unambiguous we are inclined to believe that, as mentioned earlier (Figure 5), the discrepancy between our data and AmoebaDB may be due to sequence assembly problems. Strain 200:NIH was like Rahman at loci 17 and 19. Thus, based on the presence and absence of SINE1, and the amplicons obtained with each primer pair at these three loci, the axenic strains could be divided into five genotypes (Table 3).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus