Human serum phage induction in Aggregatibacter actinomycetemcomitans

放线聚集杆菌中人血清噬菌体的诱导

基本信息

批准号：
10056404
负责人：
Gaoyan Grace Tang-Siegel
金额：
$ 15.6万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10056404
关键词：
Actinobacillus actinomycetemcomitans Adopted Affect American Antibiotic Resistance Antibiotics Area Bacteria Bacterial Chromosomes Bacterial DNA Bacterial Infections Bacteriophages Biological Models Blood Blood Vessels Cells Clinical Culture Media Cytolysis DNA DNA sequencing Data Development Disease Enhancers Environment Foundations Future Genes Genetic Genomic DNA Goals Gram-Negative Bacteria Growth Human Immune Sera Immune system Infection Infective endocarditis Inflammation Inflammatory Insertion Mutation Investigation Knowledge Laboratories Lead Libraries Lytic Lytic Phase Mammals Membrane Microbial Biofilms Modeling Mouth Diseases Nutrient Optics Oral Parents Pattern Periodontal Diseases Periodontal Pocket Periodontitis Permeability Phage Receptors Phenotype Physiological Population Process Prophages Proteins RNA Regulation Research Research Proposals Resistance Risk Serotyping Serum Source Specificity Stream Surface Systemic infection Therapeutic Tissues Work absorption bacterial fitness bacterial metabolism combat density fitness in vivo microorganism mutant novel novel therapeutic intervention novel therapeutics oral bacteria oral microbial community oral pathogen pathogenic bacteria prevent protein aggregation receptor resistant strain tool transcriptomics

项目摘要

Project Summary/Abstract Periodontitis is an inflammatory disease of the periodontium caused by bacterial biofilms and affects approximately 65 million Americans. Periodontal pockets serve as bacterial reservoirs and create potential risks to develop systemic illness. Aggregatibacter actinomycetemcomitans, an oral colonizer in 20% of the population, is a causative agent of periodontitis and infective endocarditis. The increasing antibiotic-resistant strains and the limitation of developing new antibiotics demand alternative therapeutics to cure bacterial infections. Bacteriophage therapy is listed as the top alternative and has successfully treated several bacterial infections. Currently, no phage therapeutics has been pursued to treat infections caused by oral bacteria. Development of new phage therapeutics requires understanding phage infection and replication in vivo. This knowledge is lacking in the human oral microbiota. The ultimate goal of this proposal is to develop alternative therapeutics to combat bacteria-associated oral diseases and, the immediate goal is to establish a phage infection model system to acquire the knowledge of phage infection and replication in oral pathogens. A. actinomycetemcomitans develops mechanisms to resist serum killing and survives in the inflammatory periodontal pockets and the blood. Both environments provide serum as the main nutrient source for bacterial growth. However, our data indicated that selective clinical strains of this microorganism clearly demonstrated a “bimodal” growth pattern only in the presence of human serum, and the secondary increase of optical density was due to bacterial lysis, therefore were grouped as “serum-sensitive” strains. This novel finding leaded to the discovery of a 44kb Aggregatibacter phage S1249 in the serum-sensitive strain and, 20% of the phage genes were up regulated over 10-fold in human serum comparing to other growth media. We propose that human serum induces the replication of this phage resulting in bacterial killing and, therefore phage S1249 is a potential candidate to be used for studying phage replication and regulation in this oral pathogen. We will start our investigation by accomplishing two specific aims in this proposal: Firstly, we will develop a phage infection model system by deletion of the prophage DNA from the bacterial chromosome to cure this phage, following by re-infection with the same phage. Phage induction and bacterial fitness in human serum will be characterized and compared between the wild type and its isogenic cured strain at the RNA and protein levels. Secondly, we will determinate the infection specificity of phage S1249 by using this phage to infect other serum-resistant strains to evaluate the growth fitness of those newly infected strains in human serum. This physiologically relevant model system simulates periodontal environment and will provide fundamental tools and knowledge for our future investigations, which includes: identification of bacterial surface receptors for the phage absorption and, determination of human serum components that trigger phage induction. Ultimately, the knowledge we gain will unravel phage infection and replication regulation by serum in this oral pathogen.

项目概要/摘要牙周炎是由细菌生物膜引起的牙周组织炎症性疾病，影响牙齿健康。大约 6500 万美国人的牙周袋是细菌储存库并创造了潜在的细菌。伴随放线菌聚集菌（Aggregatibacter actinomycetemcomitans）有发生全身性疾病的风险，这是 20% 的人的口腔定植菌。人群中，是牙周炎和感染性心内膜炎的病原体。菌株和开发新抗生素的限制需要治疗替代品来治愈细菌噬菌体疗法被列为最佳替代疗法，并已成功治疗多种细菌。目前，尚未采用噬菌体疗法来治疗口腔细菌引起的感染。新噬菌体疗法的开发需要了解噬菌体在体内的感染和复制。人类口腔微生物群缺乏知识该提案的最终目标是开发替代品。对抗细菌相关的口腔治疗疾病，近期目标是建立噬菌体感染模型系统，以获得口腔病原体中噬菌体感染和复制的知识。伴放线放线菌发展出抵抗血清杀伤的机制并在炎症中存活牙周袋和血液这两种环境都提供血清作为细菌的主要营养来源。然而，我们的数据表明，这种微生物的选择性临床菌株明显表现出生长。仅在人血清存在下的“双峰”生长模式，以及光密度的二次增加由于细菌裂解，因此被归类为“血清敏感”菌株。这一新发现导致了这一结果。在血清敏感菌株中发现 44kb 聚合杆菌噬菌体 S1249，以及 20% 的噬菌体基因与其他生长培养基相比，人血清中的表达上调超过 10 倍。血清诱导该噬菌体的复制，从而杀死细菌，因此噬菌体 S1249 是一种用于研究噬菌体在这种口腔病原体中的复制和调节的潜在候选者我们将开始。我们的研究通过实现该提案中的两个具体目标：首先，我们将开发噬菌体感染模型系统通过从细菌染色体中删除原噬菌体 DNA 来治愈该噬菌体，然后将表征相同噬菌体的再次感染和人血清中的细菌适应性。并在RNA和蛋白质水平上比较野生型及其同基因治愈株。将通过使用该噬菌体感染其他血清抗性来确定噬菌体S1249的感染特异性菌株来评估这些新感染菌株在人血清中的生长适应性。相关模型系统模拟牙周环境，提供基础工具和知识用于我们未来的研究，其中包括：噬菌体细菌表面受体的鉴定吸收和测定触发噬菌体诱导的人血清成分。我们获得的知识将揭示这种口腔病原体中噬菌体感染和血清复制调节。