The principal focus of this work has been to search for biosynthetic genes of cord factor unique to Mycobacterium tuberculosis (Mtb). Because the virulence factor trehalose dimycolate (TDM), the molecular element responsible for cord formation in virulent Mtb (cord factor), is found exclusively in the lipid constituents of the cell walls of pathogenic Mtb, study of the synthetic enzyme encoding genes will increase our knowledge of mycobacterial cell-wall bio-synthesis and function, hence permitting the design of a tailor-made drug to inhibit key catalytic enzyme. Furthermore, because the cell wall mycolic glycoconjugates are large and complex mole-cules, the biosynthetic enzymes are believed to be membrane-bound proteins that carry out some of the major condensation steps outside of the cytoplasm to avoid employing complex transport systems. Therefore, these membrane-bound proteins could, in theory, be used for vaccine targets. The current chemical knowledge of the mycobacterial cellular envelope greatly outweighs our understanding of its genetic and molecular synthetic processes. Nevertheless, experience in well-studied cell envelope features of gram-positive and gram-negative bacteria makes it possible to speculate about principal catalytic steps involved in formation of distinctive molecules such as TDM. The biosynthesis of gram-negative outer membrane component lipid A has been elucidated, and several of the relevant genes are known. By homology search in Sanger MycDB genome database, I have found lpx-analogous genes in the Mtb genome that have not been given definitive functional assignment. One of the corresponding Mtb sequencez79701/e264134exhibited profound hexapeptid tandem-repeats resembling the E. coli and B. subtilis counterparts. The unique hexapeptide repeat feature of the UDP-GlcNAc O-acyltransferases in bacteria was further confirmed by searching and excluding its counterpart in two eukaryotic genome databases: the Saccharomyces cerevisiae and the human genome databases. In conclusion, a possible sugar acylation enzyme-encoding gene (z79701/e264134) has been found in the genome of the Mtb H37Rv strain. Characterization of the gene will be carried out in three steps. First, the gene is going to be cloned and in-frame fused to a fluorescent reporter gene. Therefore, the cell- surface location of the gene product will be determined by fluorescence micro-scopy. The second step will involve over-expression and crystallization of the protein to compare it to the crystallographic model of the E. coli LpxA. The final step will be the collaborative tests performed with experts in computational biology to design drugs to inhibit the UDP-GlcNAc O-acyltransferases-like catalytic activities, and with immunologists to identify unique antigenic epitops for vaccine development. This project has been terminated with the departure of Dr. Qin.
