Epigenetic Control of Bacterial Virulence

描述(申请人提供)：在动物和植物中采用表观遗传控制基因表达，以使遗传相同的细胞追求不同的命运，这种细胞分化是它们发育的中心。在细菌感染周期中，也可以采用表观遗传修饰和信息传递，从而在时间和空间上改变毒力功能在宿主内的表达。这些基因表达的改变可能伴随着细菌在宿主部位的传播，以及对宿主炎症、组织破坏和免疫反应的反应。在目标1中，我们建议检验基于甲基化的表观遗传系统控制沙门氏菌发病的假设。我们的建议是基于改变细菌甲基转移酶的表达，称为DNA腺嘌呤甲基酶(DAM)，它参与DNA甲基化模式的形成，并被认为是许多病原体毒力所必需的。我们建议确定在鼠伤寒沙门氏菌中瞬时过表达Dam活性是否会导致建立一个基因典型的野生型、表观遗传修饰的细胞谱系，这些细胞表现出改变的DNA甲基化模式和改变的毒力状态，并持续几代细胞。如果是这样的话，给定细菌细胞中毒力基因表达的当前状态可能取决于其过去的历史(即细胞记忆)，类似于在高等生物的细胞分化和发育程序中观察到的情况。在目标2中，我们建议以鼠伤寒沙门氏菌PEF操纵子为模型系统，在单基因水平上阐明细胞记忆控制的机制。鼠伤寒沙门氏菌表达的菌毛编码子(PEF)介导与肠道上皮细胞的黏附，是致病所必需的。PEF操纵子处于甲基化依赖的转录调控之下，类似于特征明确的大肠杆菌纸质操纵子，后者编码尿路感染的重要毒力决定因素。我们建议测试Dam过度生产介导的PEF表达缺陷是否是映射到特定上游PEF Dam靶点(GATC序列)的DNA甲基化模式改变的直接结果。我们进一步建议确定短暂暴露于DAM过度表达是否导致PEF合成的持续变化，以及这些变化是否通过改变上游每个GATC位点的DNA甲基化模式而遗传地保持。细菌中基于甲基化的记忆系统的发现，将为控制毒力基因表达的基本分子机制(S)以及由此导致的对感染至关重要的病原体行为的变化提供深刻的见解。这些信息对于开发针对生物制剂和目前威胁全球公共健康的新出现的传染病的新型抗菌剂和疫苗至关重要。

DESCRIPTION (provided by applicant): Epigenetic control of gene expression is employed in animals and plants to enable genetically identical cells to pursue different fates, and such cell differentiation is central to their development. Epigenetic modification and information transfer may also be employed during the bacterial infection cycle so as to change the expression of virulence functions temporally and spatially within the host. These alterations in gene expression may accompany bacterial dissemination among host sites, and in response to host inflammation, tissue breakdown, and immune reactions. In Aim 1, we propose to test the hypothesis that a methylation-based epigenetic system controls Salmonella pathogenesis. Our proposal is based on altering the expression of the bacterial methyltransferase, termed DNA adenine methylase (Dam), which is involved in the formation of DNA methylation patterns and is known to be required for virulence in a number of pathogens. We propose to determine whether transient overexpression of Dam activity in S. typhimurium leads to the establishment of a lineage of genotypically wild-type, epigenetically modified cells that exhibit altered DNA methylation patterns and altered virulence states that persist for a number of cell generations. If so, the present state of virulence gene expression in a given bacterial cell may be dependent on its past history (i.e., cellular memory), similar to that observed in cell-differentiation and developmental programs in higher organisms. In Aim 2, we propose to specify the mechanism by which cellular memory control occurs at the level of a single gene using the S. typhimurium pef operon as a model system. Plasmid encoded fimbriae (Pef) expressed by S. typhimurium mediate adherence to intestinal epithelium and are required for virulence. The pef operon is under methylation-dependent transcriptional regulation similar to the well characterized E. coli pap operon, which encodes important virulence determinants in urinary tract infections. We propose to test whether Dam overproduction-mediated defects in pef expression are a direct consequence of altered DNA methylation patterns that map to specific upstream pef Dam-target sites (GATC sequences). We further propose to determine whether transient exposure to dam overexpression leads to persistent changes in Pef synthesis and whether such changes are heritably maintained via altered DNA methylation patterns at upstream Per GATC sites. The finding of a methylation-based memory system in bacteria would provide profound insights into the fundamental molecular mechanism(s) underlying control of virulence gene expression and the resultant changes in pathogen behavior that are critical to infection. Such information is vital toward developing novel antimicrobials and vaccines against biowarfare agents and emerging infectious diseases that currently threaten public health worldwide.