Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS Negl Trop Dis. 2011 May 3;5(5):e1039. doi: 10.1371/journal.pntd.0001039.
Differences between noninfective first-stage (L1) and infective third-stage (L3i) larvae of parasitic nematode Strongyloides stercoralis at the molecular level are relatively uncharacterized. DNA microarrays were developed and utilized for this purpose.
Oligonucleotide hybridization probes for the array were designed to bind 3,571 putative mRNA transcripts predicted by analysis of 11,335 expressed sequence tags (ESTs) obtained as part of the Nematode EST project. RNA obtained from S. stercoralis L3i and L1 was co-hybridized to each array after labeling the individual samples with different fluorescent tags. Bioinformatic predictions of gene function were developed using a novel cDNA Annotation System software. We identified 935 differentially expressed genes (469 L3i-biased; 466 L1-biased) having two-fold expression differences or greater and microarray signals with a p value<0.01. Based on a functional analysis, L1 larvae have a larger number of genes putatively involved in transcription (p = 0.004), and L3i larvae have biased expression of putative heat shock proteins (such as hsp-90). Genes with products known to be immunoreactive in S. stercoralis-infected humans (such as SsIR and NIE) had L3i biased expression. Abundantly expressed L3i contigs of interest included S. stercoralis orthologs of cytochrome oxidase ucr 2.1 and hsp-90, which may be potential chemotherapeutic targets. The S. stercoralis ortholog of fatty acid and retinol binding protein-1, successfully used in a vaccine against Ancylostoma ceylanicum, was identified among the 25 most highly expressed L3i genes. The sperm-containing glycoprotein domain, utilized in a vaccine against the nematode Cooperia punctata, was exclusively found in L3i biased genes and may be a valuable S. stercoralis target of interest.
A new DNA microarray tool for the examination of S. stercoralis biology has been developed and provides new and valuable insights regarding differences between infective and noninfective S. stercoralis larvae. Potential therapeutic and vaccine targets were identified for further study.
寄生虫旋毛虫的非感染性第一期（L1）和感染性第三期（L3i）幼虫在分子水平上的差异相对不明显。为此，我们开发并利用 DNA 微阵列进行了研究。
为该阵列设计了寡核苷酸杂交探针，以与作为线虫 EST 项目的一部分获得的 11335 个表达序列标签（EST）的分析所预测的 3571 个假定 mRNA 转录物结合。将来自 S. stercoralis L3i 和 L1 的 RNA 分别用不同的荧光标记物标记后，共同杂交到每个阵列上。使用新的 cDNA 注释系统软件对基因功能的生物信息学预测进行了开发。我们鉴定了 935 个差异表达基因（469 个 L3i 偏向表达；466 个 L1 偏向表达），这些基因的表达差异倍数为 2 倍或更高，且微阵列信号的 p 值<0.01。基于功能分析，L1 幼虫具有更多的假定参与转录的基因（p = 0.004），而 L3i 幼虫则偏向表达假定的热休克蛋白（如 hsp-90）。在感染旋毛虫的人类中具有免疫反应性的已知产物的基因（如 SsIR 和 NIE）具有 L3i 偏向表达。大量表达的 L3i 感兴趣的连续体包括 S. stercoralis 细胞色素氧化酶 ucr 2.1 和 hsp-90 的同源物，这些可能是潜在的化学治疗靶点。在针对Ancylostoma ceylanicum 的疫苗中成功使用的脂肪酸和视黄醇结合蛋白-1 的 S. stercoralis 同源物被鉴定为 25 个表达最丰富的 L3i 基因之一。在针对线虫 Cooperia punctata 的疫苗中使用的精子包含糖蛋白结构域仅在 L3i 偏向基因中发现，可能是旋毛虫的一个有价值的目标。
我们开发了一种新的旋毛虫生物学检查的 DNA 微阵列工具，为感染性和非感染性旋毛虫幼虫之间的差异提供了新的有价值的见解。鉴定了潜在的治疗和疫苗靶点，以供进一步研究。
