Characterization of the Lysogenic Pathway in P. acnes Bacteriophages

描述（由申请人提供）：本提案的长期目标是增强我们对痤疮的理解，特别是感染和杀死痤疮丙酸杆菌的噬菌体-一种有助于皮肤疾病痤疮发病机制的细菌，以开发有效的基于噬菌体的痤疮治疗抗菌疗法。只有少量的痤疮噬菌体被详细研究过，它们显示出许多有趣的特征。特别是，这些噬菌体能够与宿主细菌（称为溶菌原）建立稳定的关系；然而，它们的基因组缺乏所有已知参与形成和维持溶原状态过程的蛋白质。因此，建议通过测序和表征额外的P. acnes噬菌体，我们将能够更多地了解它们溶原宿主细菌的潜在新方式。彻底了解这一过程对于最终选择用于治疗目的的噬菌体至关重要，因为在任何基于噬菌体的治疗中都必须避免促进溶原的功能。该项目的第一个目标是从痤疮患者和正常供体的皮肤中分离出痤疮假单胞杆菌噬菌体，并根据其溶解性与溶原性生长的倾向来表征其多样性，同时确定溶原性噬菌体赋予的免疫模式。这一目标的最后一部分将涉及到一个系统的遗传工程痤疮噬菌体的发展。该系统将用于执行本提案第二部分中概述的功能基因组研究，并最终用于最佳毒性治疗噬菌体的工程设计。第二组实验将集中于阐明痤疮假单胞杆菌噬菌体能够溶原宿主的潜在新机制。这将首先涉及对Aim 1中测序的噬菌体进行仔细的比较基因组分析，重点关注在裂解性和溶原性噬菌体中表现出不同免疫特征的变异基因。一旦确定了候选基因，这些基因将使用Aim 1中开发的遗传工具进行靶向诱变，并且突变噬菌体的表型将根据溶原性和免疫力来确定。似乎参与这些过程的基因将被克隆并在痤疮假单胞菌中表达，以测定其在体内的功能，并在大肠杆菌中纯化并用于生化分析，例如确定DNA结合的生物化学分析，这是许多参与溶原途径的蛋白质的特征。最终，这些研究将为来自皮肤的噬菌体与其宿主细菌（痤疮P.）相互作用的机制提供新的见解，这与我们对痤疮的理解和潜在的治疗方法有关。

DESCRIPTION (provided by applicant): The long-term goal of this proposal it to enhance our understanding of acne, in particular the bacteriophages that infect and kill Propionibacterium acnes - a bacterium that contributes to the pathogenesis of the skin disease acne towards the development an effective phage-based antimicrobial therapy for the treatment of acne. Only a small number of P. acnes phages have been studied in any detail, and they display a number of interesting features. In particular, these phages are able to enter into a stable relationship with the host bacterium, known as a lysogen; however, their genomes lack all proteins known to be involved in the process of forming and maintaining a lysogenic state. It is proposed, therefore, that by sequencing and characterizing additional P. acnes phages, we will be able to learn more about the potentially novel way in which they lysogenize their host bacteria. A thorough understanding of this process will be critically important in the eventual choice of phages to be employed for therapeutic purposes, as functions that promote lysogeny must be avoided in any phage-based treatments. The first aim of this project will be to isolate P. acnes bacteriophages from the skin of acne patients and normal donors and to characterize their diversity in terms of propensity for lytic vs. lysogenic growth, as well as to determine the patterns of immunity conferred by the lysogenic phages. The last part of this aim will involve the development of a system for the genetic engineering P. acnes phages. This system will be utilized to perform the functional genomic studies outlined in the second part of this proposal and will ultimately be useful for the engineering of optimally virulent therapeutic phages. The second set of experiments will focus on elucidating the potentially novel mechanism(s) by which P. acnes bacteriophages are able to lysogenize their hosts. This will first involve performing a careful comparative genomic analysis of the phages sequenced in Aim 1, with a focus on genes that show variability in lytic and in lysogenic phages displaying differing immunity profiles. Once candidate genes are identified, these will be targeted for mutagenesis using the genetic tools developed in Aim 1, and the phenotypes of the mutant phages will be determined with regards to lysogeny and immunity. Genes that appear to be involved in these processes will be cloned for expression in P. acnes, to assay their function in vivo, and in Escherichia coli, for purification and use in biochemical assays, such as those to determine DNA binding, a feature of many proteins involved in the lysogenic pathway. Ultimately, these studies will provide new insight into the mechanisms by which bacteriophages derived from skin interact with their host bacteria, P. acnes, relevant to our understanding of, and potential therapy for acne.