Engineering Temperate Bacteriophages for Induced Secretion of Proteins and Peptides by Oral Streptococcus Mitis

工程改造温带噬菌体以诱导口腔轻链球菌分泌蛋白质和肽

基本信息

批准号：
9258533
负责人：
TIMOTHY MICHAEL JACOBS
金额：
$ 5.51万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9258533
关键词：
Adopted Animals Antibiotic Resistance Antibiotic Therapy Antibodies Antibody Formation Antibody Response Antigens Bacteria Bacterial Genome Bacteriophages Binding Clinical Dental caries Development Disease Dose Engineering Excision Face Flow Cytometry Gene Transfer Genes Genetic Engineering Genetic Materials Genome Genomics Germ-Free Glycoproteins Gnotobiotic Goals Grant HIV Harvest Human In Situ Infection Life Life Cycle Stages Lytic Maintenance Measures Mediating Methods Modality Modeling Modification Mucous Membrane Mucous body substance Mus Oral Oral cavity Oral mucous membrane structure Organism Pathogenicity Patients Peptides Periodontal Diseases Play Population Prevention Production Prophages Protein Secretion Proteins Quality Control Reporter Genes Research Research Personnel Role Safety Saliva Sampling Serum Simplexvirus Species Specificity Specificity Streptococcus Streptococcus mitis Surface Testing Therapeutic Toxic effect Training Translations Tuberculosis Variant Virus Western Blotting Work antimicrobial base commensal microbes fecal transplantation gastrointestinal immunogenic insight interest killings link protein member microbiota oral bacteria oral streptococci oral vaccine particle pathogenic bacteria protein expression therapeutic protein vector

项目摘要

ABSTRACT Commensal bacterial populations in the oral cavity not only play a pivotal role in the maintenance of healthy oral mucosa, but also makes for attractive targets for induced secretion of therapeutic molecules, such as peptide/protein therapeutics or immunogens for oral vaccines. Studies have shown that genetically engineered commensals inoculated in germfree animals can produce therapeutically relevant proteins, such as immunogens that elicit mucosal antibody production. However, clinical translation of these these livebacterial therapies faces many hurdles, including (i) difficulty in displacing existing commensal populations with the engineered variants, (ii) safety concerns with the engineered variants, and (iii) characterization, storage and handling of live bacteria. With the exception of fecal transplants in patients who have received extensive antibiotics therapy, no live bacteria are currently used as a therapy in clinical setting. In this proposal, I seek to develop an alternative strategy that allows direct modification of existing commensal populations. Specifically, I will engineer bacterial viruses, or bacteriophages, to genetically modify commensal bacterial populations at mucosal surfaces in situ. Bacteriophages present no human toxicity or pathogenicity, and are efficient transduction vectors with high specificity. However, to date, virtually all bacteriophage development focuses on using lytic phages to kill specific pathogenic bacteria (i.e. bacteriophages as a new class of antimicrobial), and little work has been done on engineering temperate phages to modify the protein expression and secretion profiles of commensal bacteria. The primary research goal of this F32 training grant is to demonstrate the proof of concept that engineered phages can mediate efficient transfer of genetic material to commensal bacteria. In Aim 1, I will isolate three temperate bacteriophage from different S treptococcus Mitis strains, introduce a reporter gene (TagRFP) into its genome, and assess their potency in transducing S . Mitis isolated from human saliva via flow cytometry. Aim 2 extends this method to the display and secretion of model therapeutic molecules. Using the most potent bacteriophage vector, I will incorporate model proteins linked to bacterial secretion/display tags, and measure the extent that these proteins will be secreted by or displayed on the same human derived S. mitis. In Aim 3, using gnotobiotic mice inoculated with human S . mitis, I will quantify the amount of secreted protein in the oral and gastrointestinal mucosa induced by engineered phage particles, as well as antibody response to proteins presented on the surface of transduced S. mitis. The results will provide important insights into the potential use of temperate phages to modify commensal populations for delivery of specific protein therapies and immunogens.

抽象的口腔中的共生菌群不仅在维持健康方面发挥着关键作用口腔粘膜，而且还成为诱导治疗分子分泌的有吸引力的靶标，例如研究表明，口服疫苗的肽/蛋白质治疗剂或免疫原。接种在无菌动物中的共生体可以产生治疗相关的蛋白质，例如然而，这些活细菌的临床转化。疗法面临许多障碍，包括（i）难以用新疗法取代现有的共生群体工程变体，(ii) 工程变体的安全问题，以及 (iii) 表征、存储和活细菌的处理除外，接受过广泛治疗的患者的粪便移植除外。抗生素治疗，目前在临床环境中没有使用活细菌作为治疗方法。在本提案中，我寻求制定允许直接修改现有共生种群的替代策略。我将设计细菌病毒或噬菌体，以对共生细菌种群进行基因改造噬菌体原位粘膜表面，不存在对人体的毒性或致病性，并且是有效的。具有高特异性的转导载体。然而，迄今为止，几乎所有噬菌体开发都集中在使用裂解噬菌体杀死特定的病原菌（即噬菌体作为一类新型抗菌剂），并且在改造温带噬菌体以修改蛋白质表达和分泌方面所做的工作还很少 F32 培训资助的主要研究目标是展示共生细菌的概况。概念证明工程噬菌体可以介导遗传物质向共生体的有效转移在目标 1 中，我将从不同的 Mitis 链球菌菌株中分离出三种温带噬菌体，将报告基因 (TagRFP) 引入其基因组，并评估其转导分离的 Mitis 的效力。目的 2 将这种方法扩展到模型的展示和分泌。使用最有效的噬菌体载体，我将结合连接的模型蛋白。细菌分泌/展示标签，并测量这些蛋白质将被分泌或展示的程度在目标 3 中，使用接种了人类轻链球菌的无菌小鼠，我将使用相同的人源轻链球菌。量化工程噬菌体诱导的口腔和胃肠粘膜分泌蛋白的量颗粒，以及对转导的轻链球菌表面蛋白质的抗体反应。将为温带噬菌体的潜在用途提供重要见解，以改变共生群体提供特定的蛋白质疗法和免疫原。