Characterization of the Lysogenic Pathway in P. acnes Bacteriophages

描述(由申请人提供)：这项建议的长期目标是加强我们对痤疮的了解，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，以开发一种有效的基于噬菌体的抗菌疗法来治疗痤疮。只有一小部分痤疮假单胞菌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进入一种稳定的关系，称为溶原菌；然而，它们的基因组缺乏所有已知的与形成和维持溶原性状态过程有关的蛋白质。因此，有人建议，通过对更多的痤疮噬菌体进行测序和鉴定，我们将能够更多地了解它们溶原化宿主细菌的潜在的新颖方式。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性的功能。该项目的第一个目标将是从痤疮患者和正常捐赠者的皮肤中分离痤疮假单胞菌噬菌体，并根据溶源性与溶原性生长倾向来表征它们的多样性，以及确定溶原性噬菌体所产生的免疫模式。这一目标的最后部分将涉及开发一种用于基因工程痤疮噬菌体的系统。该系统将被用来进行本提案第二部分概述的功能基因组研究，并最终将用于设计最具毒力的治疗性噬菌体。第二组实验将集中于阐明潜在的新机制(S)，通过这种机制，痤疮杆菌噬菌体能够溶原化它们的宿主。这将首先涉及对目标1中测序的噬菌体进行仔细的比较基因组分析，重点放在显示不同免疫谱的裂解噬菌体和溶源噬菌体中显示可变性的基因。一旦确定了候选基因，将利用目标1中开发的遗传工具对这些基因进行定向诱变，并将从溶原性和免疫性方面确定突变噬菌体的表型。似乎参与这些过程的基因将被克隆在痤疮假单胞菌中表达，在体内测试它们的功能，在大肠杆菌中进行纯化，并用于生化分析，如确定DNA结合的那些，这是许多参与溶源途径的蛋白质的特征。最终，这些研究将为从皮肤获得的噬菌体与其宿主细菌痤疮假单胞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的治疗方法有关。 与公共卫生相关：导致痤疮发病的细菌被称为痤疮丙酸杆菌，它可以被生活在人类皮肤上的病毒攻击和杀死，这种病毒被称为噬菌体。我们建议了解这些病毒与痤疮细菌相互作用的机制，以指导基于噬菌体的新疗法的发展，以克服抗生素耐药细菌的出现。

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. PUBLIC HEALTH RELEVANCE: The bacterium that contributes to the pathogenesis of acne is known as Propionibacterium acnes, and it can be attacked and killed by viruses, known as bacteriophages, that live on the human skin. We propose to learn about the mechanisms by which these viruses interact with the acne bacteria in order to guide the development of new bacteriophage-based therapies to overcome the emergence of antibiotic resistant bacteria.