MOLECULAR SEARCH FOR BIOSYNTHETIC GENES OF MYCOBACTERIUM TUBERCULOSIS CORD FACTOR

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The principal focus of this work has been to search for biosynthetic genes of cord factor unique to Mycobacterium tuberculosis (Mtb). Since 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 genes encoding the synthesizing enzymes will increase our knowledge of mycobacterial cell-wall biosynthesis and function, hence permitting the design of a tailor-made drug(s) to inhibit key catalytic enzymes. Furthermore, since the cell wall mycolic glycoconjugates are large and complex molecules, 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. Though biosynthetic pathway(s) of Mtb wall lipid constituents are not known, experience in well-studied cell envelope features of gram-positive and gram-negative bacteria enables one to speculate on some principal catalytic steps involved in the formation of distinctive molecules such as TDM. The biosynthesis of Escherichia coli lipid A has been elucidated and several of the relevant genes are known. Among the known genes are lpxA and lpxD, which encode UDP-GlcNAc O-acyltransferases. Based on the primary sequence alignment of a number of lpxA/lpxD genes from various organisms (E. coli, Salmonella typhimurium, Yersinia enterocolitica, and Rickettsia rickettsii), degenerate oligonucleotide primers were designed to amplify the analogs in Mtb. A polymerase chain reaction (PCR) procedure amplified a lKb fragment from the chromosomal DNA of a virulent Mtb reference strain, H37Rv, while no comparable amplification product was observed from DNA controls of Bacillus subtilis or M. smegmatis. The length of the possible interdomain sequence amplified appeared to be about 2 times longer than those in the other species. A large open-reading frame in the sequence was deduced to contain hexad repeats, similar to those observed in LpxA/LpxD protein sequences. A Southern blot of chromosomal materials from H37Rv, H37Ra, M. smegmatis, and B. subtilis probed with the lKb fragment suggested that this PCR product was Mtb-specific - not present in any other species tested. The DNA sequence obtained from the lKb PCR product produced a perfect match to a Mtb gene proposed to encode a potential membrane-associated protein with an ATPase motif. Further investigations of the location and the biological function of this gene will be carried out in the next 12 months.