Bruchhaus Iris, Roeder Thomas, Lotter Hannelore, Schwerdtfeger Maike, Tannich Egbert
Bernhard Nocht Institute for Tropical Medicine, Bernhard Nocht Str. 74, 20359 Hamburg, Germany.
Mol Microbiol. 2002 May;44(4):1063-72. doi: 10.1046/j.1365-2958.2002.02941.x.
The majority of human infections with the intestinal protozoan parasite Entamoeba histolytica remain asymptomatic. In a small proportion of infections, however, E. histolytica trophozoites penetrate the intestinal mucosa and disseminate to other organs, most commonly to the liver, where they induce abscess formation. It is believed that the ability of E. histolytica trophozoites to destroy host tissues and to survive within the liver is accomplished by a strong adaptive response, which requires the specific regulation of a number of amoeba proteins. Using differential display polymerase chain reaction (DD-PCR), we compared RNA expression between E. histolytica trophozoites isolated from liver abscesses of infected gerbils and those grown under normal culture conditions. A total of 3000 cDNA-derived amplicons were compared between the two groups of amoebae, which were calculated to represent about one-third of all E. histolytica mRNA species (transcriptome). Among these, 55 were found to be specifically present or absent in abscess-derived amoebae, of which 42 were successfully cloned and sequenced. Database searches and Northern blot analyses revealed that the 42 amplicons correspond to 29 independent E. histolytica genes, of which at least seven are specifically upregulated and five are downregulated in abscess-derived amoebae. Specific expression of most of these genes was not simply the result of a heat shock response, which might be expected during abscess formation, as only five of the genes revealed an expression profile similar to that found in amoebae cultured under elevated temperatures. The two genes specifically downregulated in abscess-derived amoebae encode members of a family of so far unknown proteins, which contain repetitive stretches of sequences that are rich in lysine and glutamic acid residues. In contrast, a diverse set of genes is specifically upregulated, encoding ribosomal proteins (S30, L37A), cyclophilin, ferredoxin 2 and GTP-binding protein RAB7D, supporting the notion that liver abscess formation requires the regulation and concerted action of a variety of amoeba proteins. These proteins are associated with stress response, signal transduction, regulation of transcription and vesicular trafficking. However, transcriptome analysis will not be sufficient to identify all proteins specifically upregulated during abscess formation, as at least an increase in the expression of actin was found to be regulated at the post-transcriptional level.
大多数人体感染肠道原生动物寄生虫溶组织内阿米巴后并无症状。然而，在一小部分感染病例中，溶组织内阿米巴滋养体穿透肠黏膜并扩散至其他器官，最常见的是肝脏，在那里它们会引发脓肿形成。据信，溶组织内阿米巴滋养体破坏宿主组织并在肝脏内存活的能力是通过强烈的适应性反应实现的，这需要对多种阿米巴蛋白进行特定调控。我们使用差异显示聚合酶链反应（DD-PCR），比较了从感染沙鼠的肝脓肿中分离出的溶组织内阿米巴滋养体与在正常培养条件下生长的滋养体之间的RNA表达。在两组阿米巴中比较了总共3000个cDNA衍生的扩增子，据计算这些扩增子约占所有溶组织内阿米巴mRNA种类（转录组）的三分之一。其中，发现55个在脓肿来源的阿米巴中特异性存在或缺失，其中42个成功克隆并测序。数据库搜索和Northern印迹分析表明，这42个扩增子对应于29个独立的溶组织内阿米巴基因，其中至少7个在脓肿来源的阿米巴中特异性上调，5个下调。这些基因中的大多数的特异性表达并非仅仅是脓肿形成过程中可能预期的热休克反应的结果，因为只有5个基因显示出与在高温下培养的阿米巴中发现的表达谱相似。在脓肿来源的阿米巴中特异性下调的两个基因编码一个迄今未知的蛋白质家族的成员，这些蛋白质含有富含赖氨酸和谷氨酸残基的重复序列片段。相反，一组不同的基因特异性上调，编码核糖体蛋白（S30、L37A）、亲环蛋白、铁氧化还原蛋白2和GTP结合蛋白RAB7D，支持肝脓肿形成需要多种阿米巴蛋白的调控和协同作用这一观点。这些蛋白质与应激反应、信号转导、转录调控和囊泡运输有关。然而，转录组分析不足以鉴定脓肿形成过程中所有特异性上调的蛋白质，因为至少发现肌动蛋白表达的增加是在转录后水平受到调控的。
