In vivo selection systems for the development of recombinant BCG vaccines and analysis of mycobacterial pathogenesis

Abstract

The goal of this dissertation was the development of selection systems that would allow stable maintenance of recombinant DNA in mycobacteria for the construction of recombinant vaccines in BCG, study of mycobacterial pathogenesis, and development of new antimycobacterial drugs. A molecular genetic approach to the study of mycobacterial biosynthetic pathways was undertaken to develop appropriate selection systems for in vivo studies. Mycobacterial genes involved in glutamate, aromatic amino acid, and diaminopimelic acid (DAP) biosynthetic pathways were isolated by complementation of defined Escherichia coli mutants. The DAP biosynthetic pathway was of particular interest due to its potential use for in vivo selection systems as well as for design of new antibiotics for the treatment of mycobacterial diseases. The asd, dapA, dapB, dapD, and dapE genes of BCG were isolated by complementing E. coli mutations. Further characterization of these genes allowed the development of a model for biosynthesis of DAP in mycobacteria.;The asd gene, encoding aspartate semialdehyde dehydrogenase, was chosen as a candidate for the development of an in vivo selection system for mycobacteria. The nucleotide sequences of the M. smegmatis and BCG asd genes were determined, demonstrating that the mycobacterial aspartokinase gene was upstream of and possibly within an operon with asd. Vectors were constructed to allow isolation of mycobacterial asd mutants using aminoglycoside phosphotransferase (aph) as a positive selection for recombination and {dollar}\beta{dollar}-galactosidase as a screen for allelic exchange. Methods were developed for the use of generalized transduction and intrachromosomal recombination to construct these mutants as well. These experiments resulted in: (1) the conclusion that the asd mutation in mycobacteria may be lethal, (2) the isolation of an asd mutation in a strain of M. smegmatis diploid for the asd gene, (3) the first demonstration of generalized transduction of linked chromosomal markers and plasmids in mycobacteria, and, (4) the serendipitous isolation of a novel insertion element, IS1096, from M. smegmatis. This insertion element promises to be a useful tool for genetic analysis of mycobacteria and the study of mechanisms of pathogenesis.