Transcriptome analysis of Leishmania infantum PNA+ and PNA- promastigotes by complete shotgun DNA microarrays. Leishmania infantum

Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis in Mediterranean areas and also acts as an opportunistic parasite in HIV patients. Metacyclic promastigotes are transmitted during bloodmeals of the sand-fly host after development. Metacyclogenesis can be micmiked in axenic cultures and peanut lectin (PNA) agglutination followed by two-step centrifugation allows the separation of procyclic and metacyclic promastigotes in L. major. The purpose of this study is to isolate both fractions simultaneously from the same population of L. infantum in stationary phase of axenic culture and compare their expression profiles through DNA microarrays, specially focusing on metacyclic promastigotes. Whole-genome shotgun DNA microarrays were constructed and used to analyse the stationary-phase procyclic and metacyclic expression profiles. Four biological replicates of the experiment were performed and analysed, so that 322 clones with meaningful values of stage-specific regulation were selected. We found several genes dealing with primary metabolism, differentiation in procyclic promastigotes and with development of infectivity in metacyclic promastigotes. The differences we have found between the procyclic (PNA+) and metacyclic (PNA-) transcriptomes demonstrate that negative selection of metacyclic promastigotes through PNA agglutination is suitable in L. infantum and both fractions can be isolated. In addition, up-regulation of genes implied in lipophosphoglycan (LPG), proteophosphoglycan (PPG) and glycoprotein biosynthesis indicate that metacyclic promastigotes are related with infectivity. Keywords: comparative hybridization between cDNAs from procyclic PNA+ and metacyclic PNA- promastigotes of L.infantum Overall design: -CONSTRUCTION OF SHOTGUN DNA GENOME LIBRARY IN PUC18 VECTOR -PCR AMPLIFICATION WITH EXPAND LONG TEMPLATE. 29952 AMPLIFICATIONS -CONTROL ITEMS: positive control genes from Leishmania sp., genomic DNA from L.infantum, herring sperm DNA; negative control genes (from the nif regulon) from Leptospirillum ferrooxidans (BACTERIA), buffer 1xSSC -SPOTTING OF 30576 PCR PRODUCTS/CONTROL ITEMS: ARRAY CONSTRUCTION -LEISHMANIA GROWTH CURVE AND PNA+/- SEPARATION BY CENTRIFUGATION AFTER AGGLUTINATION -TOTAL RNA EXTRACTION, PURIFICATION, AMPLIFICATION AND CDNA SYNTHESIS/INDIRECT LABELLING -HYBRIDIZATION AND SCANNING (DUAL CHANNEL) -ANALYSIS: RAW DATA (GENEPIX 6.0) -NORMALIZATION: LOWESS PER PIN (ALMAZEN SOFTWARE) -COMPARATIVE EXPERIMENT OF THREE BIOLOGICAL REPLICATES BY T-TEST -FILTERING OF INTERESTING SPOTS (THAT MAY CONTAIN DIFFERENTIALLY REGULATED GENES) -CULTURES OF THE SELECTED CLONES, MINIPREPS AND SEQUENCING REACTIONS. -ASSEMBLY OF SEQUENCES, BLAST, PROCESSING WITH A CUSTOM PERL SCRIPT AND MAPPING AGAINST THE GENOME OF L.INFANTUM SEQUENCED BY THE SANGER CENTER WITH A GBROWSE APPLICATION. -CLUSTERING: GENE ONTOLOGY MOLECULAR FUNCTIONS

利什曼原虫（Leishmania infantum）是地中海地区人畜共患内脏利什曼病的致病菌，同时也是艾滋病患者的机会性寄生虫。后循环前鞭毛体（metacyclic promastigotes）在白蛉宿主吸血时，经发育成熟后完成传播。无菌培养体系可模拟后循环分化过程；在利什曼原虫（L. major）中，花生凝集素（peanut lectin, PNA）凝集结合两步离心法，可实现前循环（procyclic）与后循环前鞭毛体的分离。本研究旨在从静止期无菌培养的婴儿利什曼原虫同一群体中同时分离这两个组分，并通过DNA微阵列（DNA microarray）比较其表达谱，重点聚焦于后循环前鞭毛体。本研究构建了全基因组鸟枪法DNA微阵列，用于分析静止期前循环与后循环前鞭毛体的表达谱。实验共开展4次生物学重复，最终筛选出322个具有显著阶段特异性调控特征的克隆。研究发现多个参与初级代谢、前循环前鞭毛体分化以及后循环前鞭毛体感染性发育的基因。前循环（PNA+）与后循环（PNA-）转录组之间的差异表明，通过PNA凝集筛选后循环前鞭毛体的方法适用于婴儿利什曼原虫，且可成功分离这两个组分。此外，参与脂磷聚糖（lipophosphoglycan, LPG）、蛋白磷酸聚糖（proteophosphoglycan, PPG）以及糖蛋白生物合成的基因呈现上调表达，提示后循环前鞭毛体与感染性密切相关。
关键词：婴儿利什曼原虫前循环PNA+与后循环PNA-前鞭毛体cDNA的比较杂交
整体实验设计：
1. 基于PUC18载体的鸟枪法DNA基因组文库构建
2. 采用Expand Long Template聚合酶进行PCR扩增，共计完成29952次扩增反应
3. 对照样本设置：利什曼原虫属阳性对照基因、婴儿利什曼原虫基因组DNA、鲱鱼精DNA；以氧化亚铁钩端螺旋菌（Leptospirillum ferrooxidans，细菌）的固氮调控子（nif regulon）序列作为阴性对照基因，辅以1×SSC缓冲液
4. 点样30576个PCR产物及对照样本，完成微阵列构建
5. 利什曼原虫生长曲线绘制，以及凝集后通过离心分离PNA+与PNA-组分
6. 总RNA提取、纯化、扩增及cDNA合成与间接标记
7. 杂交与双通道扫描
8. 数据分析：原始数据采集基于GENEPIX 6.0平台
9. 归一化处理：采用ALMAZEN软件进行基于每个点针的LOWESS归一化
10. 采用t检验完成3次生物学重复的比较实验
11. 筛选候选差异表达基因斑点
12. 候选克隆的培养、质粒小量提取及测序反应
13. 序列组装、BLAST比对、自定义Perl脚本处理，并映射至由桑格中心（Sanger Center）测序的婴儿利什曼原虫基因组，辅以GBROWSE工具进行可视化
14. 聚类分析：基于基因本体论（Gene Ontology）分子功能注释