Characterization of the Lysogenic Pathway in P. acnes Bacteriophages

痤疮丙酸杆菌噬菌体溶原途径的表征

• 批准号: 8526192
• 负责人: Laura Jane Marinelli
• 金额: $ 2.06万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8526192
• 关键词: Acne; Antibiotic Resistance; Area; Bacteria; Bacteriophages; Basic Science; Biochemical; Biological; Biological Assay; Biology; Bypass; Candidate Disease Gene; Cells; Clinical Research; Comparative Genomic Analysis; DNA Binding; Development; Effectiveness; Engineering; Escherichia coli; Genes; Genetic; Genetic Engineering; Genetic Variation; Genome; Goals; Growth; Human; Immune; Immunity; Infection; Integrase; Knowledge; Learning; Life; Lysogeny; Lytic; Maintenance; Modality; Mutagenesis; Pathogenesis; Pathway interactions; Patients; Pattern; Phage Display; Phenotype; Process; Property; Propionibacterium acnes; Proteins; Research; Skin; System; Testing; Therapeutic; Therapeutic Agents; Training; Tropism; Virulent; Virus; antimicrobial; base; career development; expression cloning; functional genomics; gene function; genome sequencing; in vivo; insight; interest; killings; mutant; novel; novel therapeutics; research study; resistant strain; site-specific integration; skin disorder; superinfection; tool

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.

描述（由申请人提供）：该提案的长期目标是增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。仅对少数痤疮丙酸杆菌噬菌体进行了详细研究，并且它们表现出许多有趣的特征。特别是，这些噬菌体能够与宿主细菌（称为溶原菌）建立稳定的关系。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，建议通过对其他痤疮丙酸杆菌噬菌体进行测序和表征，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。该项目的首要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，并表征其在溶解与溶原生长倾向方面的多样性，以及确定溶原噬菌体赋予的免疫模式。该目标的最后一部分将涉及开发用于基因工程痤疮丙酸杆菌噬菌体的系统。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，并将最终用于最佳毒力治疗噬菌体的工程设计。第二组实验将重点阐明痤疮丙酸杆菌噬菌体能够溶原其宿主的潜在新机制。这首先涉及对目标 1 中测序的噬菌体进行仔细的比较基因组分析，重点关注显示不同免疫特征的裂解噬菌体和溶原噬菌体中表现出变异的基因。一旦确定了候选基因，将使用目标 1 中开发的遗传工具对这些基因进行诱变，并确定突变噬菌体的溶源性和免疫性表型。似乎参与这些过程的基因将被克隆，以便在痤疮丙酸杆菌中表达，以测定其体内功能，并在大肠杆菌中进行纯化和用于生化测定，例如测定 DNA 结合（溶原途径中涉及的许多蛋白质的一个特征）。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相关。