Identification of Entamoeba small RNAs by pyrosequencing

描述（申请人提供）：溶组织内阿米巴是一种原生动物寄生虫，是一种重要的人类病原体。由溶组织芽胞杆菌引起的疾病包括痢疾和肝脓肿，这种有机体是全球范围内导致死亡的主要寄生虫。基因表达的调控是寄生虫在组织侵袭过程中向包囊期转化、传播疾病和适应宿主环境的关键因素。在溶组织芽孢杆菌中，我们发现了一个27nt大小的小rna群体，它们(i)具有52-多磷酸末端，（ii）与基因反义，（iii）与溶组织芽孢杆菌Argonaute蛋白相关。全基因组微阵列表达分析显示，在滋养体条件下，基本上所有反义小rna所映射的基因都不表达，这是克隆小rna的寄生虫阶段。然而，许多这些基因在其他溶组织芽胞杆菌菌株中表达，小RNA与基因表达水平呈负相关，表明这些小RNA介导同源基因的沉默。总体而言，我们的研究结果表明，溶组织芽胞杆菌具有丰富的27nt群体，其特征与秀丽隐杆线虫的次级sirna相似，并且似乎调节基因表达。这些数据表明，52-多磷酸sirna介导的沉默途径延伸到单细胞真核生物。我们的假设是一种rnai样沉默途径在溶组织芽胞杆菌中起作用，并可能调节寄生虫分化和毒力的关键基因。我们的目标是获得溶组织芽胞杆菌中小RNA物种的全面概况，重点是在毒力和非毒力寄生虫以及阶段转化过程中差异表达的sRNAs。这项工作将提供内阿米巴小rna的参考数据库，易于与更广泛的科学界共享，是该领域的重大进展。

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.