Microarray Analysis of Infective and Noninfective Larvae of Strongyloides stercoralis

BACKGROUND: Differences between noninfective first-stage (L1) and infective third-stage (L3i) larvae of parasitic nematode Strongyloides stercoralisat the molecular level are relatively uncharacterized. DNA microarrays were developed and utilized for this purpose. METHODS AND FINDINGS: Oligonucleotide hybridization probes for the array were designed to bind 3571 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 967 differentially expressed genes (457 L3i-biased; 510 L1-biased) having greater than two-fold expression differences and p < 0.01. Based on functional analysis, L1s have a larger number of genes putatively involved in transcription (p = 0.0158), and L3is have higher expression of stress-related genes (such as putative heat shock proteins dnj-12, daf-21, dnj-10). Genes with products that have been shown to be immunoreactive in S. sterocoralis-infected humans (SsIR and NIE) were additionally found to be L3i biased. Unique and abundant L3i contigs of interest included S. stercoralis orthologs of cytochrome oxidase ucr 2.1, daf-12, and daf-21, 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 top 25 most L3i-biased genes. The sperm-containing glycoprotein domain, utilized in a vaccine against the nematode Cooperia punctata, was exclusively found in the L3i group and may be a valuable S. stercoralis target of interest. Conclusions 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. For the present study, four technical replicates using pooled samples of L1 stage larvae versus L3i stage larvae were performed, including one dye swap.

背景：寄生线虫粪类圆线虫（Strongyloides stercoralis）的非感染性第一期（L1）幼虫与感染性第三期（L3i）幼虫之间的分子水平差异迄今尚未得到充分阐释。为此，本研究开发并应用了DNA微阵列（DNA microarray）技术。
方法与结果：本微阵列的寡核苷酸杂交探针（oligonucleotide hybridization probe）设计用于结合3571个推定mRNA转录本，这些转录本通过对11335条表达序列标签（expressed sequence tags, ESTs）的分析预测得到，而这些ESTs是线虫EST项目的一部分。将取自粪类圆线虫L3i与L1幼虫的RNA分别用不同荧光标签标记后，与同一微阵列进行共杂交。本研究采用新型cDNA注释系统（cDNA Annotation System）软件进行基因功能的生物信息学预测。最终鉴定出967个差异表达基因（其中457个在L3i中表达上调，510个在L1中表达上调），这些基因的表达差异倍数大于2，且P值小于0.01。功能分析结果显示，L1幼虫中参与转录过程的推定基因数量更多（P=0.0158），而L3i幼虫中应激相关基因（如推定热休克蛋白dnj-12、daf-21、dnj-10）的表达水平更高。此外，在粪类圆线虫感染人体中被证实具有免疫反应性的基因产物（SsIR与NIE）对应的基因，也被发现为L3i表达上调。值得关注的独特且高丰度的L3i重叠群（contig）包括粪类圆线虫的细胞色素氧化酶ucr 2.1、daf-12与daf-21的同源基因，这些可能成为潜在的化疗靶点。成功用于防治锡兰钩口线虫（Ancylostoma ceylanicum）疫苗的脂肪酸视黄醇结合蛋白-1的粪类圆线虫同源基因，被鉴定为前25个L3i表达上调最显著的基因之一。含精子糖蛋白结构域（该结构域曾被用于制备类库柏线虫（Cooperia punctata）疫苗）仅在L3i组中被发现，可能成为粪类圆线虫的潜在研究靶点。
结论：本研究开发了一种用于研究粪类圆线虫生物学特性的新型DNA微阵列工具，为阐明感染性与非感染性粪类圆线虫幼虫之间的差异提供了全新且有价值的见解。本研究还鉴定出潜在的治疗靶点与疫苗靶点，以供后续研究。本研究针对L1期幼虫与L3i期幼虫的混合样本开展了4次技术重复实验，其中包含1次荧光染料交换实验。