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。由溶组织埃希氏菌引起的疾病包括痢疾和肝脓肿，这种生物是全球范围内主要的寄生性死亡原因。在组织入侵过程中，基因表达调控是使寄生虫转变为囊肿期、传播疾病和适应宿主环境的关键因素。在溶组织性肠杆菌中，我们已经鉴定出一组大小为27个核苷酸的小RNA，它们(I)具有52-多聚磷酸末端，(Ii)将反义基因映射到基因，以及(Iii)与溶组织性肠杆菌ArgAerte蛋白相关。全基因组微阵列表达分析显示，基本上所有反义小RNA映射到的基因在滋养体条件下都不表达，滋养体是小RNA克隆的寄生阶段。然而，许多这些基因在其他溶组性肠杆菌菌株中表达，小RNA与基因表达水平呈负相关，表明这些小RNA介导了同源基因的沉默。总体而言，我们的结果表明，溶组织型乳杆菌具有丰富的27nt群体，具有与线虫次级siRNAs相似的特征，似乎调节基因表达。这些数据表明，由52-多聚磷酸siRNAs介导的沉默途径延伸到单细胞真核生物。我们的假设是，RNAi类沉默途径在溶组织埃希氏菌中具有功能，并可能调控寄生虫分化和毒力的关键基因。我们的目标是获得溶组织埃希氏菌中小RNA物种的全面图谱，重点是在强毒和非强毒寄生虫中以及在阶段转换过程中差异表达的sRNA。这项工作将提供内阿米巴小RNA的参考数据库，很容易与更广泛的科学界共享，并将成为该领域的重大进步。 公共卫生相关性：溶组织内阿米巴是一种在全球范围内对人类健康产生影响的重要病原体。尽管大多数疾病发生在发展中国家，但这种寄生虫可以在水净化系统受到不利影响的任何地方引起感染。我们对了解寄生虫致病的分子机制很感兴趣。

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.