Enzyme-based lysis of mycobacteria

描述(申请人提供)：结核分枝杆菌是人类结核病(TB)的病原体，据估计，世界上三分之一的人口都携带结核杆菌。其中，大约5%-10%的人发展为活动性疾病，其余的人在潜伏状态下无症状地携带感染；在免疫功能受损的状态下，重新激活的风险按比例增加。不足为奇的是，结核病的流行病学格局对免疫受损的人，如感染人类免疫缺陷病毒(HIV)的人来说是严重破坏性的。可以说，更好地控制结核病的一个关键瓶颈是对临床标本中病原体相关标记物的有效诊断，特别是在细菌数量较少的结核病-艾滋病毒混合感染患者的痰标本中。世界卫生组织(WHO)和美国疾病控制中心(CDC)最近将基于聚合酶链式反应的核酸扩增分析纳入结核病诊断算法。这一策略的一个重要限制是无法有效地从痰或组织中嵌入的病原体中提取核酸。核酸提取的效率对分枝杆菌物种来说是一个更重要的挑战，因为它们厚厚的蜡状包膜可以抵抗大多数用于细菌裂解的化学试剂的暴露。因此，超声波作为一种裂解技术已被纳入到从标本中进行基于PCR的结核病诊断的样品制备中。我们最近发现，外源暴露于体外降解分枝杆菌糖脂TDM的重组角质酶样丝氨酸酯酶，会导致液体悬浮液中多种分枝杆菌的广泛裂解。这一发现为开发一种基于酶的分枝杆菌裂解方法以改进基于聚合酶链式反应的结核病诊断提供了巨大的可能性。此外，裂解技术还将用于实验室环境中分枝杆菌的基本生化研究。在这个项目中，我们将首先开发一种tdmh和分枝杆菌噬菌体裂解蛋白的酶鸡尾酒，以最大限度地提高分枝杆菌裂解的效率。接下来，我们将测试基于酶裂解的效率，在基于RT-PCR的检测感染动物组织和痰中的结核分枝杆菌。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis, the causative pathogen of human tuberculosis (TB), is harbored by an estimated one third of the world's population. Of these, about 5-10% develop active disease while the rest carry the infection asymptomatically in latent state; with a proportionately increasing risk of reactivation in compromising state of immunity. Not surprisingly, the epidemiological landscape of TB is severely devastating in immunocompromised individuals, such as those infected with Human Immunodeficiency virus (HIV). Arguably a key bottleneck for a better control of TB is an efficient diagnosis of pathogen-associated markers in clinical specimens, particularly in sputum samples of TB-HIV co-infected patients with lower number of bacilli. A PCR-based nucleic acid amplification assay has been recently incorporated in the TB diagnosis algorithm by the World Health Organization (WHO) and the Center for Disease Control (CDC), USA. An important limitation in this strategy is the efficient extraction of nucleic acids from the pathogen embedded in sputum or tissues. The efficiency of nucleic acid extraction becomes a challenge of greater significance for mycobacterial species, because of their thick and waxy envelope that can resist exposure to most chemical agents used for bacterial lysis. As a result, sonication as a lysis technique has been incorporated in sample preparation of PCR-based TB diagnosis from specimens. We recently discovered that exogenous exposure to a recombinant cutinase-like serine esterase hydrolyzing a mycobacterial glycolipid, TDM, in vitro causes extensive lysis of multiple mycobacterial species in liquid suspension. This discovery opens up enormous possibility to develop an enzyme-based mycobacterial lysis towards an improved PCR-based diagnosis of TB. Furthermore, the lysis technique will also be useful in basic biochemical studies of mycobacteria in laboratory settings. In this project we will first develop an enzyme cocktail of TDMH and lysins of mycobacteriophages to maximize the efficiency of mycobacterial lysis. Next we will test the efficiency of enzyme-based lysis in RT-PCR based detection of M. tuberculosis in infected animal tissues and sputum.