Microarray Analysis of Infective and Noninfective Larvae of Strongyloides stercoralis. 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. Overall design: For the present study, four technical replicates using pooled samples of L1 stage larvae versus L3i stage larvae were performed, including one dye swap.

研究背景：寄生线虫粪类圆线虫（Strongyloides stercoralis）的无感染性第一期（L1）幼虫与感染性第三期（L3i）幼虫之间的分子层面差异，目前尚未得到充分阐明。本研究采用DNA微阵列（DNA microarray）技术对此展开研究。
研究方法与结果：本微阵列所用的寡核苷酸杂交探针（oligonucleotide hybridization probe），靶向结合由线虫EST计划产生的11335条表达序列标签（expressed sequence tags, EST）分析预测得到的3571个推定mRNA转录本。分别用不同荧光标签标记粪类圆线虫L3i与L1期幼虫的RNA后，将两种RNA共同杂交至每张微阵列芯片。采用新型cDNA注释系统（cDNA Annotation System）软件开展基因功能的生物信息学预测。本研究共鉴定得到967个差异表达基因（differentially expressed genes），其中457个为L3i偏向性基因，510个为L1偏向性基因，其表达差异倍数大于2且P值小于0.01。功能分析结果显示，L1期幼虫中参与转录过程的推定基因数量更多（P=0.0158），而L3i期幼虫的应激相关基因（如推定热休克蛋白（heat shock protein）dnj-12、daf-21、dnj-10）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编码基因（SsIR与NIE）同样被发现为L3i偏向性基因。值得关注的特异性高丰度L3i期重叠群（contig）包括粪类圆线虫的细胞色素氧化酶ucr 2.1、daf-12及daf-21直系同源基因（ortholog），上述基因可能成为潜在化疗靶点。成功用于锡兰钩口线虫（Ancylostoma ceylanicum）疫苗的脂肪酸与视黄醇结合蛋白-1的粪类圆线虫直系同源基因，也位列L3i偏向性排名前25的基因之中。用于指状库柏线虫（Cooperia punctata）疫苗的含精子糖蛋白结构域，仅在L3i期幼虫组中被检出，可作为粪类圆线虫极具研究价值的潜在靶点。
研究结论：本研究开发了一种用于粪类圆线虫生物学研究的新型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极具价值的视角。本研究鉴定得到了潜在的治疗与疫苗靶点，可供后续研究开展。
实验整体设计：本研究采用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次染料互换实验。