Screening And Characterization Of Mycobacterium Tuberculosis Gene Related To Surfactant Stress Tolerance

Tuberculosis is the world's greatest infectious killer of humans. In 1998 and 2001, the genome sequence of strain H37Rv and CDC1551 have been completed and represented a fundamental resource for Mycobacterium tuberculosis research projects. But the sequence gives no clue to the function of 16% of the genes. Investigating the functions of anonymous genes will give clues to understand why Mycobacterium tuberculosis is able to survive in human body and find new candidates of pharmaceutical target.The main stresses faced during Mycobacterium tuberculosis infection involve oxidizing agents, low pH and surfactant. Alveolar surfactant is a mild detergent with antibacterial activity and could damage the structure of its fatty acid-rich cell envelope. In addition, some researches indicate that alveolar surfactant can mediate Mycobacterium tuberculosis attachment to alveolar macrophages, and suggest possible underlying mechanisms for that.In the present study, an Mycobacterium tuberculosis genomic library was constructed in pUC18 vector under the controlling of the inducible T7 promoter and transformed into Escherichia coli. Mycobacterium tuberculosis sequences were expressed and a function-driver screening was used to selected the recombinant clones in 0.4% SDS stress. We insolated an anonymous protein. Overexpression of this proteins in Escherichia coli cells afforded significantly increase in SDS tolerance. Sequence analysising found the gene encode this protein was partial Rv0621. Product of Rv0621 is a putative membrane protein. Membrane proteins fulfil a wide range of central functions in the cell.We have prepared genomic expression library of Mycobacterium tuberculosis in Escherichia coli. Taking advantage of the genetic simplicity of Escherichia coli and of the functional conservation of some prokaryote protein, by the "cross-phylogenetic" screen, Rv0621 gene was isolated encoding a 37kD putative membrane protein. The colony with partial Rv0621 gene insert, named S1, was able to grow in the medium containing 0.4% SDS, while the strain carried empty vector was unable to grow. Plasmid pET32a (+) was used to express the Rv0621 gene in Escherichia coli BL21 (DE3). Using gas chromatographic - mass spectrometric (GC-MS), we compared the fatty acid composition of the Escherichia coli BL21 (DE3) carrying Rv0621-pET32a (+) and the Escherichia coli BL21 (DE3) carrying empty pET32a (+), we found Escherichia coli BL21 (DE3) carrying Rv0621 -pET32a (+) contained more oleic acid. And we found the recombinant protein changed the shape of the A549 cells. This suggests the gene may be involved in fatty acid synthesis regulation and help pathogens to resist the surfactant defense of the host.