Genetic Approaches to the Control of Mycobacterial Diseases 
ses are under way to track M. tuberculosis iso- 
lates that are multiply drug resistant. 
Luciferase Reporter Phages 
Accurate diagnosis of M. tuberculosis infec- 
tion routinely requires 4-6 weeks to allow culti- 
vation of the tubercle bacilli and time to perform 
tiie appropriate tests. Assessment of the drug- 
susceptibility patterns of clinical isolates can re- 
quire another 4-6 weeks of analysis. In light of 
the increasing numbers of drug-resistant speci- 
mens, we set out to devise a novel diagnostic test 
that would shorten the time required for both 
procedures. The test uses a mycobacteriophage, a 
virus that infects M. tuberculosis, into which we 
have cloned a gene that encodes a reporter en- 
zyme, the luciferase that makes fireflies glow. Lu- 
ciferase reporter mycobacteriophage (LRM) par- 
ticles, when mixed with bacterial cells, result in 
the production of light. 
The LRM test is extraordinarily sensitive, as 
photons can be detected at extremely low levels. 
In addition, it is exquisitely specific, as the phage 
only attaches to mycobacterial cells. Thus the pro- 
duction of light reveals the presence of M. tuber- 
culosis in the sample. We have undertaken stud- 
ies in clinical application. Since the luciferase 
reaction requires ATP for photon production, we 
also hope to use the test to assess the metabolic 
activity of a cell. And since drug treatment of my- 
cobacterial cells abrogates this activity, we are 
also exploring the use of the test to distinguish 
drug-resistant from drug-sensitive cells. 
Genetic Analysis of Mycobacterial 
Virulence Determinants 
We would like to know why pathogens such as 
M. tuberculosis or M. leprae cause severe disease 
when BCG can elicit an effective immune re- 
sponse against these organisms. To approach 
these questions, we have focused on developing 
systems for defining and characterizing the genes 
of mycobacteria. 
The first approach is to generate specific inser- 
tion mutations within the virulent M. tuberculo- 
sis genome and to screen for mutants that are no 
longer virulent in our animal models. Toward 
this goal, we are doing a number of studies de- 
signed to generate random insertions within the 
genome by taking advantage of natural mycobac- 
terial transposons or recombination systems. 
Complementary to this mutant isolation is the de- 
velopment of both extrachromosomal and inte- 
grating vectors that permit highly efficient in- 
troduction of libraries of genes from virulent 
mycobacteria into avirulent strains. 
The combination of these two strategies has al- 
ready allowed us to identify genes necessary for 
the biosynthesis of amino acids, purines, and 
complex polysaccharides found on the surface of 
the pathogenic mycobacteria. We are currently 
developing a variety of systems to screen these 
libraries for genes that confer virulence charac- 
teristics to the avirulent organisms. By identifying 
the genes and their products responsible for spe- 
cific virulence phenotypes, we hope to make pos- 
sible the design of strategies to prevent or control 
mycobacterial disease. In addition, the geneti- 
cally engineered avirulent mutants should pro- 
vide novel vaccine candidates. 
Recombinant BCG Vaccines and Novel 
Vaccine Strategies 
BCG is an attenuated mutant of bovine tuber- 
culosis bacillus. It has been used as a vaccine 
against tuberculosis in humans for over 50 years. 
The bacterium possesses several unique proper- 
ties that make it ideal for use as a live vector in 
generating multivalent vaccines. BCG is a safe 
vaccine, having been used in 2.5 billion individ- 
uals with a mortality rate significantly lower than 
that of the smallpox vaccine. It is the only live 
vaccine other than oral polio that WHO recom- 
mends for use in infants. Also, the mycobacterial 
cell wall has potent adjuvant properties that can 
engender excellent humoral responses; and since 
BCG is stored within macrophages, which are key 
antigen-presenting cells, it follows that it can also 
elicit cellular immune responses. 
In the last few years, our laboratory, in collabo- 
ration with Barry Bloom (HHMI, Albert Einstein 
College of Medicine), Medlmmune, and Graham 
Hatfull at the University of Pittsburgh, has devel- 
oped a series of expression vectors and transfor- 
mation systems whereby foreign genes encoding 
antigens from virtually any pathogen can be 
cloned and expressed in BCG. Mice immunized 
with these recombinant BCG cells have been 
shown to elicit both humoral and cellular im- 
mune responses to the expressed foreign pro- 
teins. We are currently cloning and expressing 
genes from pathogens that cause leishmaniasis, 
schistosomiasis, and toxoplasmosis in BCG. Im- 
munization of mice with recombinant BCG will 
provide useful models to test the types of im- 
mune responses that can be elicited with these 
live bacteria. Ultimately we hope to engineer a 
recombinant BCG that could protect humans 
from these dread diseases. 
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