Enzyme-based lysis of mycobacteria

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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.