Luciferase expression in M. leprae infected with phAE129

Using a combination of freshly harvested, nude mouse-propagated Mycobacterium leprae which have been enriched for viable bacilli by Percol-gradient centrifugation and a modified D29 mycobacteriophage (phAE129) carrying the luciferase gene we have obtained the first evidence of both successful phage infection and foreign gene expression in the leprosy bacillus. M. leprae was incubated at 33°C in a Middle-brook-based medium in the presence of phAE129 at MOI ranging from approximately 1-800. Luciferase expression was monitored at time intervals by addition of luciferin and detection of light in a microplate luminometer. In four independent experiments, expression of luciferase in phAE129-infected M. leprae was detected at greater than 10 times background levels, reaching peak levels at 24-48 hr post-infection. At 1 × 10⁷ and 1 × 10⁸ M. leprae per assay, net luciferase production was in excess of 2500 and 4000 relative light units (rlu), respectively. Significant luciferase expression (2-3 × background) could be detected with as few as 1 × 10⁶ M. leprae per assay. Luciferase expression was reduced by greater than 95% in the presence of 1 μg/ml rifampin or clarithromycin and was unaffected by 50 μg/ml ampicillin. Preliminary data indicate that exclusion of palmitic acid may enhance luciferase expression. Following peak light production the decay of light was very gradual and no increases in phage titer (determined by plaque assay on M. smegmatis) were observed, suggesting the absences of a phage-mediated lytic burst. The ultimate goal of this line of investigation is the construction of shuttle cosmid vectors capable of stable expression of foreign DNA in M. leprae for the purpose of establishing in vitro growth competence. Such a vector could conceivably carry DNA libraries from slow-growing, cultivable mycobacteria. Alternatively, pending the completion of the M. leprae genome sequence and its comparison with that of M. tuberculosis, it may be possible to choose selected genes for introduction. The results of the current investigation suggest that these approaches will indeed be possible by demonstrating the ability to successfully introduce and express foreign DNA in the leprosy bacillus.