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-一种有助于皮肤病痤疮发病机制的细菌，以开发用于治疗痤疮的有效的基于噬菌体的抗菌疗法。只有少量的痤疮丙酸杆菌被详细研究过，它们显示出许多有趣的特征。特别是，这些细菌能够与宿主细菌形成稳定的关系，称为溶原菌;然而，它们的基因组缺乏已知参与形成和维持溶原状态过程的所有蛋白质。因此，有人提出，通过对其他痤疮丙酸杆菌进行测序和表征，我们将能够更多地了解它们使宿主细菌溶原化的潜在新方式。彻底了解这一过程将是至关重要的，在最终选择的噬菌体用于治疗目的，促进溶原性的功能，必须避免在任何噬菌体为基础的治疗。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痤疮丙酸杆菌噬菌体，并根据溶解性与溶原性生长的倾向来表征其多样性，以及确定溶原性噬菌体赋予的免疫模式。该目标的最后一部分将涉及用于遗传工程痤疮丙酸杆菌的系统的开发。该系统将用于进行本提案第二部分中概述的功能性基因组研究，并最终用于最佳毒性治疗药物的工程设计。第二组实验将集中于阐明痤疮丙酸杆菌噬菌体能够使其宿主溶原化的潜在新机制。这将首先涉及对Aim 1中测序的p53进行仔细的比较基因组分析，重点是在裂解和溶原性p53中显示出不同免疫特征的变异性的基因。一旦鉴定出候选基因，将使用目标1中开发的遗传工具靶向这些基因进行诱变，并将确定突变体的表型，以确定溶原性和免疫性。似乎参与这些过程的基因将被克隆用于在痤疮丙酸杆菌中表达，以测定其体内功能，并在大肠杆菌中表达，用于纯化和用于生物化学测定，如测定DNA结合的那些，DNA结合是参与溶原性途径的许多蛋白质的特征。最终，这些研究将为皮肤来源的噬菌体与其宿主细菌痤疮丙酸杆菌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治疗有关。

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.