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. 2007 Jan 5:8:7.
doi: 10.1186/1471-2164-8-7.

Growth of the protozoan parasite Entamoeba histolytica in 5-azacytidine has limited effects on parasite gene expression

Ibne Karim M Ali  1 , Gretchen M Ehrenkaufer, Jason A Hackney, Upinder Singh
Affiliations

Growth of the protozoan parasite Entamoeba histolytica in 5-azacytidine has limited effects on parasite gene expression

Ibne Karim M Ali et al. BMC Genomics. .

Abstract

Background: In higher eukaryotes DNA methylation regulates important biological functions including silencing of gene expression and protection from adverse effects of retrotransposons. In the protozoan parasite Entamoeba histolytica, a DNA methyltransferase has been identified and treatment with 5-azacytidine (5-AzaC), a potent inhibitor of DNA methyltransferase, has been reported to attenuate parasite virulence. However, the overall extent of DNA methylation and its subsequent effects on global gene expression in this parasite are currently unknown.

Results: In order to identify the genome-wide effects of DNA methylation in E. histolytica, we used a short oligonucleotide microarray representing 9,435 genes (approximately 95% of all annotated amebic genes) and compared the expression profile of E. histolytica HM-1:IMSS parasites with those treated with 23 microM 5-AzaC for up to one week. Overall, 2.1% of genes tested were transcriptionally modulated under these conditions. 68 genes were upregulated and 131 genes down regulated (2-fold change; p-value < 0.05). Sodium-bisulfite treatment and sequencing of genes indicated that there were at least two subsets of genes with genomic DNA methylation in E. histolytica: (i) genes that were endogenously silenced by genomic DNA methylation and for which 5-AzaC treatment induced transcriptional de-repression, and (ii) genes that have genomic DNA methylation, but which were not endogenously silenced by the methylation. We identified among the genes down regulated by 5-AzaC treatment a cysteine proteinase (2.m00545) and lysozyme (52.m00148) both of which have known roles in amebic pathogenesis. Decreased expression of these genes in the 5-AzaC treated E. histolytica may account in part for the parasites reduced cytolytic abilities.

Conclusion: This work represents the first genome-wide analysis of DNA-methylation in Entamoeba histolytica and indicates that DNA methylation has relatively limited effects on gene expression in this parasite.

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Figures

Figure 1
Figure 1
Effects of 5-AzaC on E. histolytica growth and in vitro virulence. (A) and (B) Growth curves for untreated and 5-AzaC treated E. histolytica HM-1:IMSS and E. histolytica 200:NIH strains respectively. On day zero 50,000 trophozoites were inoculated in 15 ml culture tubes and grown with or without 23 μM 5-AzaC. On days two and four (marked by a downward arrow), 50,000 trophozoites were subcultured into fresh media and 5-AzaC added to the appropriate tubes. (C) Monolayer destruction of CHO cells by untreated, three-day, and seven-day 5-AzaC treated trophozoites of E. histolytica strains HM-1:IMSS and 200:NIH. In both parasite strains the 3-day and 7-day 5-AzaC treated parasites showed significantly decreased virulence compared to that with corresponding untreated parasites (p-value < 0.05) (shown by *).
Figure 2
Figure 2
Verification of array data by RT-PCR. Array data were confirmed for a subset of genes by semi-quantitative RT-PCR. Total RNA was isolated from untreated and 5-AzaC treated parasites (7 days) and subjected to RT-PCR. Sequential 1:10 dilutions of cDNA were used as template for the PCR and a genomic DNA and minus RT control (-RT) were included. The microarray expression fold-change for each gene is shown based on average array data from 3 day and 7 day 5-AzaC treated parasites. Two genes (115.m00143 and 141.m00082) that were predicted to be upregulated (based on array data) after 5-AzaC exposure were confirmed by RT-PCR. Two genes (2.m00545 and 226.m00092) that were predicted to be down-regulated (based on array data) after 5-AzaC exposure were confirmed by RT-PCR. A gene whose expression did not change (based on array data) with 5-AzaC exposure (147.m00095) was found to be unchanged in the two conditions by RT-PCR. Primers used are given in Table 1.
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