Enzyme-based lysis of mycobacteria

基于酶的分枝杆菌裂解

• 批准号: 8698490
• 负责人: Anil Kumar Ojha
• 金额: $ 22.88万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8698490
• 关键词: Acid Fast Bacillae Staining Method; Acquired Immunodeficiency Syndrome; Affect; Algorithms; Bacillus (bacterium); Biochemical; Biological Assay; Biological Markers; Cause of Death; Cell Wall; Cells; Centers for Disease Control and Prevention (U.S.); Chemical Agents; Chemicals; Clinical; Clinical Research; Colony-forming units; Complex; Cord Factors; Cytolysis; DNA; Data; Detection; Development; Diagnosis; Disease; Early Diagnosis; Environment; Enzymes; Epidemiology; Exposure to; Fluorescence; Glycolipids; Growth; HIV; HIV-2; Hour; Human; Hydrolase; Immune system; Immunity; Immunocompromised Host; In Vitro; Individual; Infection; Laboratories; Liquid substance; Manuscripts; Molecular; Mycobacteriophages; Mycobacterium bovis; Mycobacterium marinum; Mycobacterium tuberculosis; Nucleic Acids; Patients; Peptidoglycan; Polymerase Chain Reaction; Population; Preparation; Recombinants; Rest; Rifampicin resistance; Risk; Sampling; Severities; Sonication; Specimen; Speed; Sputum; Suspension substance; Suspensions; Symptoms; Techniques; Testing; Thick; Time; Tissues; Trehalose; Tuberculosis; Virus Diseases; World Health Organization; animal tissue; arabinogalactan; base; clinically relevant; cutinase; improved; killings; lysin; mortality; mycobacterial; mycolate; novel; pathogen; public health relevance; serine esterase; synergism; tool

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.

描述（由申请人提供）：结核分枝杆菌是人类结核病（TB）的致病病原体，估计世界上三分之一的人口都携带结核分枝杆菌。其中，约 5-10% 出现活动性疾病，其余人则处于潜伏状态，无症状感染；在损害免疫力的情况下重新激活的风险成比例地增加。毫不奇怪，结核病的流行病学状况对免疫功能低下的个体（例如感染人类免疫缺陷病毒（HIV）的个体）具有严重的破坏性。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，特别是在杆菌数量较少的结核病-艾滋病毒合并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) 最近已将基于 PCR 的核酸扩增检测纳入结核病诊断算法中。该策略的一个重要限制是从嵌入痰或组织中的病原体中有效提取核酸。核酸提取的效率对于分枝杆菌物种来说是一个更重要的挑战，因为它们厚而蜡质的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，超声作为裂解技术已被纳入基于 PCR 的结核病标本诊断的样品制备中。我们最近发现，体外暴露于重组角质酶样丝氨酸酯酶（TDM）可水解分枝杆菌糖脂，会导致液体悬浮液中多种分枝杆菌的广泛裂解。这一发现为开发基于酶的分枝杆菌裂解方法以改进基于 PCR 的结核病诊断开辟了巨大的可能性。此外，裂解技术也可用于实验室环境中分枝杆菌的基础生化研究。在该项目中，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效率。接下来，我们将测试基于 RT-PCR 检测受感染动物组织和痰中结核分枝杆菌的酶裂解效率。