MOLECULAR GENETIC ANALYSIS OF MYCOBACTERIUM TUBERCULOSIS

Tuberculosis remains a devastating disease of mankind resulting in
3 million deaths per year. There are 10 million new cases of
tuberculosis worldwide, and for the first time after a 32 year
decline, the number of new cases of tuberculosis in the United
States has increased for the last 3 years. In New York city alone,
there was a 36% rise in the number of tuberculosis cases,
predominantly in the black and hispanic populations. This rise of
tuberculosis is most likely associated with the increase in AIDS
patients and so is still likely to worsen. M. avium infections are
a primary cause of fatality of AIDS patients. Although, M.
tuberculosis, the causative agent of tuberculosis, is one of
earliest described pathogens of man, analysis of this organism or
any other mycobacterium by genetic means has been previously
impossible. The goal of this proposal is to develop the systems
and methodology, using modern recombinant DNA technology, to permit
genetic analysis of the mycobacteria. By developing the first
efficient transfection system and a novel E. coli-mycobacteria
shuttle vector, we have introduced recombinant DNA molecules
into both M. smegmatis and BCG vaccine strains for the first time.
This novel vector, termed a shuttle plasmid, replicates in
mycobacteria as a phage and in E. coli as a plasmid. This vector
has been successfully used to introduce and stably express the
first selectable marker gene for mycobacteria genetic research.
We plan to use these genetic approaches to analyze the pathogenesis
of mycobacteria, which could lead to new avenues for drug design.
These methods will contribute to the development of BCG, currently
the most widely used vaccine in the world, into a recombinant
multivaccine vehicle, which would provide a novel approach to
development of vaccines where T cell memory and effector responses
are important.