Construction and evaluation of next-generation reporter mycobacteriophages

描述(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20亿人感染了病原体--结核分枝杆菌。在阿根廷，每年约有12000例病例和1000例死亡。虽然结核分枝杆菌感染可以通过标准的6-9个月疗程的抗生素和至少三种药物有效地解决，但耐药菌株的出现使治疗严重复杂化。需要一种新的诊断方法，将快速(检测时间)、敏感性、特异性、生物安全性、快速和准确地低成本地确定常用抗结核药物的耐药性结合起来，应用于结核病发病率较高的发展中国家。分支杆菌噬菌体是开发诊断工具的极佳候选者，因为它们有效和特异地感染和复制分支杆菌。我们最近描述了氟分支杆菌噬菌体-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。荧光显微镜或流式细胞仪可以很容易地检测到荧光。该检测对抗生素敏感，固定后荧光至少保持两周，提高了生物安全性，方便了样品的储存或运输。在研究实验室中，氟分支杆菌噬菌体具有很有前途的特性，我们的目标是开发下一代氟分支杆菌噬菌体，可以用于临床样本的直接分析，并在一小时内读出。我们建议修改目前的噬菌体，这种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；这是发展中国家使用的一个特别理想的特征，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在一起，也将增强信号并缩短检测时间。我们还建议开发一种在噬菌体颗粒上添加亲和标签的系统，以确保有效地捕获分枝杆菌细胞，从而优化检测的灵敏度。最后，这些优化版本的氟分支杆菌噬菌体的构建将有助于测试特殊的痰处理方案，以实现在这些样本中直接对分枝杆菌细胞进行高效的噬菌体感染。总而言之，这些发展将导致一种简单、快速和特异的结核病诊断测试。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补助金AI064494的延伸(7/1/06-6/30/11)。

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).