Human serum phage induction in Aggregatibacter actinomycetemcomitans

放线聚集杆菌中人血清噬菌体的诱导

• 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
• 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

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.

项目摘要/摘要 牙周炎是一种由细菌生物膜引起的牙周炎症性疾病。 大约6500万美国人。牙周袋可以作为细菌的储存库，并创造潜力 发展成系统性疾病的风险。Aggregatibacter放线菌伴生菌，口腔定殖者占20% 是牙周炎和感染性心内膜炎的病原体。抗生素耐药性的增加 菌株和开发新抗生素的局限性需要替代疗法来治愈细菌 感染。噬菌体疗法被列为首选疗法，并已成功地治疗了几种细菌 感染。目前，还没有噬菌体疗法被用于治疗由口腔细菌引起的感染。 开发新的噬菌体疗法需要了解噬菌体在体内的感染和复制。这 人类口腔微生物区系缺乏相关知识。这项提议的最终目标是开发替代方案 治疗与细菌相关的口腔疾病，目前的目标是建立一种噬菌体 感染模型系统，以获取口腔病原体中噬菌体感染和复制的知识。 放线菌伴生菌开发出抵抗血清杀伤的机制并在炎性介质中存活 牙周袋和血迹。这两种环境都提供血清作为细菌的主要营养来源 成长。然而，我们的数据表明，这种微生物的选择性临床菌株清楚地显示出 只有在人血清存在的情况下才有“双峰”生长模式，光密度的二次增加 是由细菌裂解引起的，因此被归类为“血清敏感”菌株。这一新发现导致了 在血清敏感菌株中发现44kb的聚集素噬菌体S1249，占噬菌体基因的20% 在人血清中的表达水平是其他生长介质的10倍以上。我们建议人类 血清诱导这种噬菌体的复制，导致细菌被杀死，因此噬菌体S1249是一种 有可能用于研究噬菌体在该口腔病原体中的复制和调控。我们要开始了 我们的研究通过实现这一建议中的两个具体目标：首先，我们将发展一种噬菌体感染 通过从细菌染色体上删除原噬菌体DNA来治愈该噬菌体的模型系统，随后 用相同的噬菌体再次感染。将表征噬菌体诱导和细菌在人血清中的适合性 并在RNA和蛋白质水平上对野生型和其同基因治愈菌株进行了比较。其次，我们 将通过用此噬菌体感染其他抗血清来确定噬菌体S1249的感染特异性 以评估这些新感染的菌株在人血清中的生长适合性。这是生理上的 相关模型系统模拟牙周环境，为牙周环境模拟提供基础工具和知识 对于我们未来的研究，包括：鉴定噬菌体的细菌表面受体 对触发噬菌体诱导的人血清成分的吸收和测定。归根结底， 我们所获得的知识将揭开噬菌体感染和血清对这种口腔病原体的复制调控。