Mycobacterium bovis BCG recA deletion mutant shows increased susceptibility to DNA-damaging agents but wild-type survival in a mouse infection model

Abstract

Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxifies reactive oxygen species, and DNA repair systems, which repair damage resulting from oxidative stress. To (i) determine the relative importance of the DNA repair system when oxidative stress is encountered by the Mycobacterium tuberculosis complex during infection of the host and to (ii) provide improved mycobacterial hosts as live carriers to express foreign antigens, the recA locus was inactivated by allelic exchange in Mycobacterium bovis BCG. The recA mutants are sensitive to DNA-damaging agents and show increased susceptibility to metronidazole, the first lead compound active against the dormant M. tuberculosis complex. Surprisingly, the recA genotype does not affect the in vitro dormancy response, nor does the defect in the DNA repair system lead to attenuation as determined in a mouse infection model. The recA mutants will be a valuable tool for further development of BCG as an antigen delivery system to express foreign antigens and as a source of a genetically stable vaccine against tuberculosis.

FIG. 1

(A) Southern blot analysis of the recA locus. Lane 1, parental M. bovis BCG; lane 2, single-crossover transformant obtained by transformation with plasmid precA::aph-rpsL; lanes 3 to 5, recA (recA::aph) knockout mutants obtained after counterselection of a single-crossover transformant on medium containing kanamycin plus streptomycin. Approximately 200 ng of genomic DNA was digested with SmaI and hybridized to a recA probe. (B) Schematic drawing of the BCG recA locus: the wild-type locus is shown along with the vector used for inactivation, a 5′ single-crossover transformant, and a knockout mutant. S, SmaI recognition site.

FIG. 2

Western blot analysis of RecA. Lane 1, parental M. bovis BCG; lane 2, single-crossover transformant; lane 3, recA knockout mutant. Approximately 30 μg of protein was separated on a polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and probed with an antibody raised against M. tuberculosis RecA.

FIG. 3

Survival after UV irradiation. Parental M. bovis BCG (●), a recA single-crossover transformant (▪), and a recA knockout mutant (▴) were irradiated with UV light for the indicated times. Following irradiation, the numbers of viable cells were determined by plating.

FIG. 4

Growth under dormancy conditions. Parental M. bovis BCG (●) and the recA knockout mutant (▴) were grown under dormancy culture conditions. Growth was determined by measuring the OD. F and D, fading and complete decolorization of the methylene blue indicator, respectively.

FIG. 5

Survival under dormancy conditions. Survival of parental M. bovis BCG and the recA knockout mutant under dormancy culture conditions was investigated by determining the numbers of CFU after 20 days of incubation in the presence or absence of metronidazole (MTZ; 10 μg/ml).

FIG. 6

Course of infection in BALB/c mice. Parental M. bovis BCG (●), a recA single-crossover transformant (▪), and a recA knockout mutant (▴) were injected into the tail vein (approximately 10⁶ CFU/animal). The numbers of bacteria in spleens (A) and lungs (B) were determined at different time points.

FIG. 7

Course of infection in nude mice. Parental M. bovis BCG (●), a recA single-crossover transformant (▪), and a recA knockout mutant (▴) were injected into the tail vein (approximately 10⁶ CFU/animal). The numbers of bacteria in spleens (A) and lungs (B) were determined at different time points.