Isolation, Identification And Functional Study Of Pathogenic Gene Of Mycobacterium

Tuberculosis （TB）, caused by Mycobacterium tuberculosis （M. tb）, continues to be a global health problem, causing approximately2million deaths and10million new infections annually. The situation has been further complicated with the advent of M. tb/HIV coinfection and the emergence of multidrug-resistant （MDR） and extensively drug-resistant （XDR） TB. In light of these developments, the search for new antibiotics against M. tb is of crucial importance and requires a comprehensive understanding of the pathogenicity and the intracellular lifestyle of this organism. Mycobacterium marinum, a pathogenic mycobacterium that causes disease in fish and amphibians, is genetically close to M. tb and is a useful model for studying the pathogenesis of TB and for uncovering potential therapeutic targets for drug development.In this study, a MycoMarT7mariner transposon insertion mutant library of approximately10,000mutants was constructed and visually inspected for mutants with altered colony morphology, as colony morphology was proved to be associated with the crucial virulence related cell wall components.66mutants were selected and insertion sites of53mutants were successfully identified and finally48mutated genes were obtained. Among the48hypothetical virulence related genes,15were reported as virulence genes in other papers （which is31.25%of the genes identified）. And this give high confidence the efficiency of the strategy of screening for virulence related mutants by observation of colony morghology. To efficiently obtain the virulence related genes involved in the pathogenesis of M. marinum, zebrafish infection model was applied. When we random picked up10strains to test the virulence changes in zebrafish model,3strains were attenuated in zebrafish infection model, which means we can directly select virulence related genes based on the morphological changes of mutants, and this is a new method of identifying potential genes involved in virulence. New virulence gene candidates obtained in this research are reserved for further study.The composition of cell surface in M. tuberculosis （Mtb） is intricate, while its role in surviving severe environment and manipulating the host immune system is conspicuous but rarely understood. Research on identifying virulence mechanism of infection with Mtb is currently focused on the context and function of cell surface lipid molecules. Cord is clarified as a characteristic phenotype of virulent strains of Mtb complex as they grow in rope-shape. We identified9mutants whose cord phenotype was missing and7of thses genes all belong to a synthesis pathway of the virulence factors phthiocerol dimycocerosate （PDIMs） in M. marinum. Further study showed that PDIMs of M. marinum are key virulence factors, and PDIMs play a significant role during bacterial surviving and escaping from the immune system of host zebra-fish. The other2gene associated with cord phenotype were PPE38and mmaA3, and futher test of virulence showed no significant change in virulence of M. marinum. And this differs from the common knowledge about the cord and virulence. Our finding will help to understand the function of important lipids of PDIMs in pathogenesis of Mtb and the relationship between PDIMs and cord phenotype and help to uncover the pathogenesis of this pathogen.Biotin （vitamin H） is essential for all living organisms and is a cofactor in a number of enzymatic carboxylation reactions involved in the biosynthesis of fatty acids and the metabolism of amino acids and carbohydrates. While most microbes, plants and fungi are cable of synthesizing biotin, humans and other mammals must obtain biotin from food or through bacteria in the large intestinal. Biotin is synthesized from pimeloyl-CoA through four enzymatic steps catalyzed by7-Keto-8-amino pelargonic acid （KAPA） synthase （bioF）,7,8-diaminopelargonic acid （DAPA） synthase （bioA）, dethiobiotin synthetase （bioD）, and biotin synthase （bioB）, respectively. This pathway is conserved in both Gram-positive and Gram-negative bacteria. Genes involved in this pathway are present in mycobacteria such as M. tb and M. marinum. A bio A mutant of M. smegmatis was found to be defective in stationary phase growth and enzymatic activities of KAPA synthase （Rv1569） and DAPA synthase （RV1568） of M. tb have been demonstrated and characterized biochemically. However, a mutant of pathogenic mycobacteria defective in biotin biosynthesis has not been isolated and the role of biotin biosynthesis in mycobacterial infection has not been demonstrated previously. In this study, we describe a mutant of M. marinum in which MMAR₂770, a predicted short-chain dehydrogenase/reductase, was disrupted. This mutant is a biotin auxotroph and is defective in growth in macrophages and zebrafish. Our results have provided direct evidence for the critical role of biotin synthesis for mycobacterial metabolisms in vitro and in vivo. Our studies have also identified MMAR₂770and its M. tb homolog Rv1882c as new enzymes involved in biotin biosynthesis and as potential targets for the development of antimycobacterial drugs.