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Genetic engineering of Mycobacterium leprae to Glow and Grow

麻风分枝杆菌发光和生长的基因工程

基本信息

批准号：
10625474
负责人：
WILLIAM Robert JACOBS
金额：
$ 21万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-20 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10625474
关键词：
ATP-Binding Cassette Transporters Achievement Acid Fast Bacillae Staining Method Amino Acids Anabolism Animal Model Bacillus Bacteria Bacteriophages Binding Bioluminescence Carbon Cell Separation Cell Wall Cells Communities Complement Consumption Cosmids DNA DNA Sequence Data Detection Developing Countries Development Diagnosis Disease Electroporation Energy Transfer Engineering Enzymes Evolution Family Dasypodidae Fatty Acids Fireflies Gene Proteins Generations Genes Genetic Genetic Engineering Genome Genomics Glucose Goals Green Fluorescent Proteins Growth Harvest Homologous Gene Homoserine Human Immunologics In Vitro Infection Intravenous Iron Leprosy Libraries Light Lipids Luciferases Metabolic Methionine Methods Modeling Mus Mycobacterium leprae Mycobacterium tuberculosis NADH Nude Mice Organ Organism Oxidoreductase Pantothenic Acid Pathway interactions Persons Pharmaceutical Preparations Pharmacotherapy Plasmids Predisposition Process Proteins Pseudogenes Radioactivity Recording of previous events Regimen Regulatory Pathway Reporter Reporting Research Research Personnel Research Technics Siderophores Signal Transduction Source Stains Stigmatization System Techniques Testing Therapeutic Agents Time Tissue imaging Tissues Work acid fast bacteria cost drug development drug efficacy drug testing foot human disease improved in vivo imaging system intracellular parasitism monomer mycobacterial mycobactins nanoluciferase novel novel therapeutics oxidation pathogen promoter tool uptake vaccine development

项目摘要

Project Summary/ Abstract Leprosy has plagued mankind throughout human history and still afflicts 2 to 3 million people in Developing Countries. Although the leprosy bacillus was the first bacterium reported to be associated with human disease, it has yet never been cultivated on artificial media. It can be grown in the footpads of nude mice or systemically in nine-banded armadillos. With a generation time of 14 days, testing drug regimens are performed by counting acid-fast bacilli which is laborious and time-consuming. The comparison of the M. leprae DNA sequence to M. tuberculosis suggests the inability to culture M. leprae on artificial media might result from the inability to synthesize methionine, pantothenate and mycobactin. Using genetic tools, we have developed for M. tuberculosis, we hypothesize M. leprae can be stably transformed with phage- based integration proficient plasmids. We plan to incorporate a Bioluminescent Resonance Energy Transfer (BRET)-Nanoluc and Near infra-red proteins into the plasmid to detect transformants. Furthermore, we hypothesize that the transformation of M. leprae with the missing methionine and pantothenate biosynthetic genes and the mycobactin transporters will enable M. leprae to grow on artificial media. An M. leprae strain that glows would enhance drug development and the ability to assess the immunological mechanisms that control M. leprae infections. The genetic engineering of M. leprae to grow on artificial media would enhance our understanding of evolutionary processes that lead to obligate intracellular parasitism.

项目总结/摘要麻风病在整个人类历史上一直困扰着人类，发展中国家虽然麻风杆菌是第一个被报道的细菌，与人类疾病有关，但从未在人工培养基上培养过。可以生长在裸鼠的足垫中或系统地生长在九带犰狳中。持一代在14天的时间内，通过计数抗酸杆菌来进行测试药物方案，费力且耗时。比较了M.麻风杆菌DNA序列与M. 结核病提示不能培养M.人工媒介上的麻风病可能是由不能合成蛋氨酸、泛酸和分枝杆菌素。利用遗传学工具，为M.结核病，我们假设M。麻风可以稳定地转化噬菌体，基于整合精通质粒。我们计划将生物发光共振能量转移（BRET）-Nanoluc和近红外蛋白进入质粒进行检测转化体此外，我们假设M.麻风病人与失踪蛋氨酸和泛酸生物合成基因和分枝杆菌素转运蛋白将使M. 麻风病在人造培养基上生长个核磁会发光麻风菌株发展和评估控制M的免疫机制的能力。麻风感染.利用基因工程技术对M.在人造培养基上生长，理解导致专性细胞内寄生的进化过程。