Epigenetic Control of Bacterial Virulence

描述（由申请人提供）：在动物和植物中，基因表达的表观遗传控制使基因相同的细胞追求不同的命运，这种细胞分化对它们的发育至关重要。表观遗传修饰和信息传递也可以在细菌感染周期中使用，从而在时间和空间上改变宿主内毒力功能的表达。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，我们提出验证基于甲基化的表观遗传系统控制沙门氏菌发病机制的假设。我们的建议是基于改变细菌甲基转移酶的表达，称为DNA腺嘌呤甲基化酶（Dam），它参与DNA甲基化模式的形成，并且已知是许多病原体毒性所必需的。我们打算确定鼠伤寒沙门氏菌中短暂过表达Dam活性是否会导致基因典型野生型、表观遗传修饰细胞谱系的建立，这些细胞表现出DNA甲基化模式的改变和毒力状态的改变，并持续数代细胞。如果是这样，一个细菌细胞中毒力基因表达的现状可能依赖于其过去的历史（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，我们建议使用鼠伤寒沙门氏菌pef操纵子作为模型系统来指定细胞记忆控制在单个基因水平上发生的机制。鼠伤寒沙门氏菌表达的质粒编码菌毛（Pef）介导肠上皮的粘附，是产生毒力所必需的。pef操纵子受甲基化依赖性转录调控，类似于大肠杆菌pap操纵子，其编码尿路感染中重要的毒力决定因素。我们建议测试大坝过度生产介导的pef表达缺陷是否是DNA甲基化模式改变的直接结果，这些模式可以映射到特定的上游pef大坝靶点（GATC序列）。我们进一步提出确定短暂暴露于坝过表达是否会导致Pef合成的持续变化，以及这种变化是否通过上游Per GATC位点DNA甲基化模式的改变而遗传地维持。在细菌中发现基于甲基化的记忆系统将为毒力基因表达控制的基本分子机制以及由此产生的病原体行为变化提供深刻的见解，这些变化对感染至关重要。这些信息对于开发针对生物战剂和新出现的传染病的新型抗菌剂和疫苗至关重要，这些传染病目前威胁着全世界的公共卫生。

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.