Construction and evaluation of next-generation reporter mycobacteriophages

下一代报告分枝杆菌噬菌体的构建和评估

• 批准号: 8475398
• 负责人: Graham F. Hatfull
• 金额: $ 4.91万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8475398
• 关键词: Affinity; Anabolism; Antibiotics; Antitubercular Agents; Argentina; Bacteria; Bacteriophages; Biological Assay; Biological Models; Cells; Cessation of life; Clinical; Collaborations; Complex; Cytolysis; DNA biosynthesis; Detection; Developing Countries; Development; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostic tests; Drug Tolerance; Drug resistance; Ensure; Evaluation; Flow Cytometry; Fluorescence; Gene Expression; Genes; Genus Mycobacterium; Goals; Grant; Growth; Hour; Human; Immune response; Immunocompetent; Incidence; Infection; Infectious Agent; Laboratory Research; Metabolic; Methods; Microbial Biofilms; Microscopy; Mus; Mycobacteriophages; Mycobacterium Infections; Mycobacterium smegmatis; Mycobacterium tuberculosis; Mycolic Acid; Organism; Pathogenesis; Patients; Pattern; Performance; Pharmaceutical Preparations; Physiological; Predisposition; Process; Protocols documentation; Recovery; Regulation; Reporter; Reporter Genes; Reporting; Research; Resistance; Role; Sampling; Sensitivity and Specificity; Signal Transduction; Solutions; Specimen; Speed; Sputum; System; Temperature; Testing; Time; Transportation; Tuberculosis; United States National Institutes of Health; Universities; Virus; base; clinical Diagnosis; cost; density; human mortality; macrophage; mouse model; mutant; mycobacterial; mycolate; next generation; novel diagnostics; novel strategies; parent grant; particle; resistant strain; response; sample fixation; tool; tuberculosis drugs

DESCRIPTION (provided by applicant): Tuberculosis (TB) is a major cause of human mortality with 9 million new cases and nearly two million deaths annually; approximately two billion people are infected with the causative agent, Mycobacterium tuberculosis. In Argentina there are about 12,000 cases and one thousand deaths per year. While M. tuberculosis infections can be effectively resolved with a standard 6-9 month course of antibiotics with at least three drugs, the emergence of drug resistant strains severely complicates treatment. There is a need for new diagnostic approaches that combine speed (time-to-detection), sensitivity, specificity, biosafety, rapid and accurate determination of resistance to the commonly used anti-tuberculosis drugs at a low cost to be applied in developing countries where the incidence of TB is high. Mycobacteriophages are excellent candidates for the development of diagnostic tools since they efficiently and specifically infect and replicate in Mycobacteria. We recently described the development of Fluoromycobacteriophages - reporter phages containing a fluorescent reporter gene - that provide a simple means of revealing the metabolic state of M. tuberculosis cells, and therefore their response to antibiotics. Fluorescence can be detected easily by fluorescent microscopy or by flow cytometry. The assay is responsive to antibiotics, and fluorescence is maintained for at least two weeks following fixation, increasing biosafety and facilitating storage or transportation of samples. Fluoromycobacteriophages have promising attributes in the research laboratory, and our goal is to develop the next generation of fluoromycobacteriophages that can be used for direct analysis of clinical samples, with a readout within one hour. We propose to modify the current phages such DNA replication contributes to signal amplification, which can be accomplished by the construction of lysis- defective phage mutants; this is a particularly desirable feature for use in developing countries, since they can be used at any infection temperature. Incorporation of optimized versions of fluorescent genes with an enhanced mycobacterial expression will also enhance the signal and shorten the time-to-detection. We also propose to develop a system for addition of affinity tags to phage particles to ensure efficient capture of mycobacterial cells, and thus optimize the sensitivity of detection. Finally, the construction of these optimized versions of Fluoromycobacteriophages will facilitate the testing of specific protocols for sputum processing to achieve efficient phage infection of mycobacterial cells directly in these samples. Together, these developments will result in a simple, rapid, and specific diagnostic test for tuberculosis. This research will be primarily done in Argentina at University of Buenos Aires in collaboration with Dr. Mariana Piuri as an extension of NIH Grant AI064494 (7/1/06 - 6/30/11).

描述（由申请人提供）：结核病 (TB) 是人类死亡的主要原因，每年新增病例 900 万，死亡人数近 200 万人；大约二十亿人感染了病原体结核分枝杆菌。阿根廷每年约有 12,000 例病例和 1,000 人死亡。虽然结核分枝杆菌感染可以通过至少三种药物的标准 6-9 个月抗生素疗程有效解决，但耐药菌株的出现使治疗严重复杂化。结核病发病率高的发展中国家需要新的诊断方法，将速度（检测时间）、灵敏度、特异性、生物安全性、快速、准确地确定常用抗结核药物的耐药性结合起来，并且成本低廉。分枝杆菌噬菌体是开发诊断工具的绝佳候选者，因为它们能够高效且特异性地在分枝杆菌中感染和复制。我们最近描述了氟分枝杆菌噬菌体（含有荧光报告基因的报告噬菌体）的开发，它提供了一种简单的方法来揭示结核分枝杆菌细胞的代谢状态，从而揭示它们对抗生素的反应。通过荧光显微镜或流式细胞术可以轻松检测荧光。该测定对抗生素有反应，并且荧光在固定后保持至少两周，提高了生物安全性并便于样品的储存或运输。氟分枝杆菌噬菌体在研究实验室中具有广阔的前景，我们的目标是开发下一代氟分枝杆菌噬菌体，可用于直接分析临床样本，并在一小时内读出结果。我们建议修改现有的噬菌体，例如 DNA 复制有助于信号放大，这可以通过构建裂解缺陷噬菌体突变体来实现；对于发展中国家来说，这是一个特别理想的功能，因为它们可以在任何感染温度下使用。将优化版本的荧光基因与增强的分枝杆菌表达结合起来也将增强信号并缩短检测时间。我们还建议开发一种向噬菌体颗粒添加亲和标签的系统，以确保有效捕获分枝杆菌细胞，从而优化检测的灵敏度。最后，这些优化版本的氟分枝杆菌噬菌体的构建将有助于测试痰处理的特定方案，以直接在这些样品中实现分枝杆菌细胞的高效噬菌体感染。总之，这些进展将带来一种简单、快速、特异性的结核病诊断测试。这项研究将主要在阿根廷布宜诺斯艾利斯大学与 Mariana Piuri 博士合作完成，作为 NIH 拨款 AI064494（2006 年 7 月 1 日 - 2011 年 6 月 30 日）的延伸。

项目成果

Cluster K mycobacteriophages: insights into the evolutionary origins of mycobacteriophage TM4.

• DOI: 10.1371/journal.pone.0026750
• 发表时间: 2011
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Pope WH;Ferreira CM;Jacobs-Sera D;Benjamin RC;Davis AJ;DeJong RJ;Elgin SC;Guilfoile FR;Forsyth MH;Harris AD;Harvey SE;Hughes LE;Hynes PM;Jackson AS;Jalal MD;MacMurray EA;Manley CM;McDonough MJ;Mosier JL;Osterbann LJ;Rabinowitz HS;Rhyan CN;Russell DA;Saha MS;Shaffer CD;Simon SE;Sims EF;Tovar IG;Weisser EG;Wertz JT;Weston-Hafer KA;Williamson KE;Zhang B;Cresawn SG;Jain P;Piuri M;Jacobs WR Jr;Hendrix RW;Hatfull GF
• 通讯作者: Hatfull GF

Recombineering: A powerful tool for modification of bacteriophage genomes.

• DOI: 10.4161/bact.18778
• 发表时间: 2012-01-01
• 期刊: Bacteriophage
• 作者: Marinelli LJ;Hatfull GF;Piuri M
• 通讯作者: Piuri M

Generation of affinity-tagged fluoromycobacteriophages by mixed assembly of phage capsids.

通过噬菌体衣壳的混合组装生成亲和标记的荧光分枝杆菌噬菌体。

• DOI: 10.1128/aem.01016-13
• 发表时间: 2013
• 期刊: Applied and environmental microbiology
• 影响因子: 4.4
• 作者: Piuri,Mariana;Rondón,Liliana;Urdániz,Estefanía;Hatfull,GrahamF
• 通讯作者: Hatfull,GrahamF