Identification of Entamoeba small RNAs by pyrosequencing

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DESCRIPTION (provided by applicant): Entamoeba histolytica, a protozoan parasite, is an important human pathogen. Diseases caused by E. histolytica include dysentery and liver abscesses and this organism is a leading parasitic cause of death on a global scale. Regulation of gene expression is a key factor that enables the parasite to convert to the cyst stage and propagate disease and adapt to the host environment during tissue invasion. In E. histolytica, we have identified a population of small RNAs of 27nt size that (i) have a 52-polyphosphate termini, (ii) map antisense to genes, and (iii) associate with an E. histolytica Argonaute protein. Whole genome microarray expression analysis revealed that essentially all genes to which antisense small RNAs map were not expressed under trophozoite conditions, the parasite stage from which the small RNAs were cloned. However, a number of these genes were expressed in other E. histolytica strains with an inverse correlation between small RNA and gene expression level suggesting that these small RNAs mediate silencing of the cognate gene. Overall, our results demonstrate that E. histolytica has an abundant 27nt population, with features similar to secondary siRNAs from C. elegans, and which appear to regulate gene expression. These data indicate that a silencing pathway mediated by 52-polyphosphate siRNAs extends to single celled eukaryotic organisms. Our hypothesis is that an RNAi-like silencing pathway is functional in E. histolytica and may regulate genes key to parasite differentiation and virulence. Our goal is to obtain a comprehensive profile of small RNA species in E. histolytica with an emphasis on sRNAs differentially expressed in virulent and non-virulent parasites and during stage conversion. This work will provide a reference database of Entamoeba small RNAs, be readily shared with the broader scientific community and be a significant advance for the field. PUBLIC HEALTH RELEVANCE: Entamoeba histolytica is an important pathogen with an impact on human health on a global scale. Although the majority of disease is in developing countries, this parasite can cause infections anywhere that water purification systems get adversely affected. We are interested in understanding the molecular mechanisms that the parasite uses to cause disease.