Abstract
The mycobacterial cell envelope is a complex structure the components of which have been implicated in the survival, virulence, permeability and resistance to antibiotics of Mycobacterium. The elucidation of the synthesis of the cell envelope would improve the understanding of the structure and its influential properties and could lead to the identification of drug target sites. The isolation of genes encoding proteins involved in the biosynthesis of the cell envelope structures and their mutants would enable the biosynthetic pathways and functions of individual structures to be determined. The objective of this project was to isolate genes encoding cell envelope structures in Mycobacterium leprae, using Mycobacterium smegmatis as model organism. The aim was to isolate an M. smegmatis strain with a mutant cell envelope and to complement the mutation using a genomic library of M. leprae. M. smegmatis mc2155 was successfully mutagenised using /V-methyl-N-Nitro-fV- nitrosoguanidine (NTG), generating 0.1-0.2% auxotrophic mutants. A bank of 2,000 NTG- treated M. smegmatis strains were screened for alterations in phenotypes that may have reflected a change in the cell envelope i.e. mycobacteriophage resistance, temperature sensitivity and increased resistance and sensitivity to antibiotics. M. smegmatis strains with increased resistance to ofloxacin and ciprofloxacin (15) were isolated along with strains more sensitive to penicillin G(5) and pyrazinamide (1). The pyrazinamide sensitive mutant, Pyramidll was further characterised and found to be 20% more sensitive to pyrazinamide than the M. smegmatis mc2155 wild type strain. Pyramidll was found to be less hydrophobic than the wild type strain, variably more sensitive to penicillin G and to exhibit a smooth colony morphology. The cell wall components of Pyramid II were analysed but no gross differences were observed in comparison to the wild type M. smegmatis strain. Pyramidll is believed to contain a mutation effecting its permeability to pyrazinamide. Pyramidll was transformed with an M. leprae genomic pYUB18 shuttle vector library and complementing clones isolated (6%). A complementing cosmid, 57, found to map to cosmid B1308 in the ordered M. leprae library, was used to create a sub-library in pMV206 from which a 3.5kb fragment of complementing M. leprae DNA was isolated and found to contain three complete putative coding regions, possibly involved in osmoregulation.
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