Enzyme-based lysis of mycobacteria

描述（由申请人提供）：结核分枝杆菌是人类结核病（TB）的病原体，估计世界上三分之一的人口携带结核分枝杆菌。其中，约5-10%发展为活动性疾病，而其余的则在潜伏状态下无症状地携带感染;在损害免疫状态的情况下，再激活的风险成比例地增加。毫不奇怪，结核病的流行病学景观在免疫功能低下的个体中具有严重的破坏性，例如感染人类免疫缺陷病毒（HIV）的个体。可以说，更好地控制TB的关键瓶颈是有效诊断临床标本中的病原体相关标志物，特别是在具有较低数量的杆菌的TB-HIV共感染患者的痰样品中。基于PCR的核酸扩增测定最近已被世界卫生组织（WHO）和美国疾病控制中心（CDC）纳入TB诊断算法中。这种策略的一个重要限制是从痰或组织中包埋的病原体中有效提取核酸。核酸提取的效率成为分枝杆菌物种的更大意义的挑战，因为它们的厚和蜡状包膜可以抵抗暴露于用于细菌裂解的大多数化学试剂。因此，超声处理作为裂解技术已被纳入基于PCR的TB诊断标本的样品制备。我们最近发现，外源性暴露于重组角质酶样丝氨酸酯酶水解分枝杆菌糖脂，TDM，在体外引起广泛的溶解多种分枝杆菌物种的液体悬浮液。这一发现为开发基于酶的分枝杆菌裂解以改进基于PCR的TB诊断提供了巨大的可能性。此外，裂解技术也将是有用的分支杆菌在实验室设置的基础生化研究。在这个项目中，我们将首先开发TDMH和分枝杆菌噬菌体溶素的酶混合物，以最大限度地提高分枝杆菌裂解的效率。接下来，我们将测试基于酶的裂解在基于RT-PCR的M检测中的效率。受感染动物组织和痰中的结核病。

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.