Construction and evaluation of next-generation reporter mycobacteriophages

描述（由申请人提供）：结核病（TB）是人类死亡的主要原因，每年有900万新病例和近200万人死亡；大约有20亿人感染了病原体结核分枝杆菌。在阿根廷，每年大约有12000个病例和1000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，但耐药菌株的出现严重复杂化了治疗。需要一种新的诊断方法，将速度（检测时间）、灵敏度、特异性、生物安全性、快速和准确地以低成本测定对常用抗结核药物的耐药性结合起来，应用于结核病发病率高的发展中国家。分枝杆菌噬菌体是开发诊断工具的极好候选者，因为它们在分枝杆菌中有效地和特异性地感染和复制。我们最近描述了含荧光报告基因的报告噬菌体——含荧光报告基因的报告噬菌体——的发展，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。荧光可以很容易地通过荧光显微镜或流式细胞术检测到。该方法对抗生素有反应，固定后荧光至少可维持两周，提高了生物安全性，便于样品的储存或运输。荧光菌噬菌体在研究实验室中具有很好的特性，我们的目标是开发下一代可用于直接分析临床样品的荧光菌噬菌体，并在一小时内读取结果。我们建议对现有的噬菌体进行修饰，如DNA复制有助于信号扩增，这可以通过构建裂解缺陷噬菌体突变体来实现；这是在发展中国家使用的一个特别理想的特点，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也将增强信号并缩短检测时间。我们还建议开发一种系统，将亲和力标签添加到噬菌体颗粒中，以确保有效捕获分枝杆菌细胞，从而优化检测灵敏度。最后，构建这些优化版本的氟菌噬菌体将有助于测试痰液处理的特定方案，从而在这些样品中直接实现分枝杆菌细胞的高效噬菌体感染。总之，这些发展将产生一种简单、快速和特定的结核病诊断检测方法。这项研究将主要在阿根廷布宜诺斯艾利斯大学与Mariana Piuri博士合作进行，作为NIH资助AI064494（7/1/06 - 6/30/11）的延伸。

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). PUBLIC HEALTH RELEVANCE: 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. Diagnosis of TB is complicated by the slow growth of the organism and the need for rapid drug susceptibility testing. The development of a simple, rapid, cheap, and accurate test for determining drug susceptibilities of clinical samples would dramatically enhance the control of tuberculosis across the world.