Mutation in mce operons attenuates Mycobacterium tuberculosis virulence

On the Mycobacterium tuberculosis genome there are four mce operons, all of which are similar in sequence and organization, and code for putatively exported proteins. To investigate whether Mce proteins are essential for virulence, we generated knock-out mutants in mce1, mce2 and mce3 operons of M. tuberculosis and evaluated their ability to multiply in a mammalian host. The allelic replacement was confirmed in each mutant strain by Southern blotting. RT-PCR experiments demonstrated the lack of in vitro expression of mutated genes in Δmce1 and Δmce2 mutants. On the other hand, no expression of mce3 was detected in either the wild-type or mutant strains. Similar doubling time and growth characteristics in in vitro culture were observed for mutants and parental strains. The intratracheal route was used to infect BALB/c mice with the Δmce3, Δmce2 and Δmce1 mutants. Ten weeks after infection, all mice infected with the Δmce mutants survived, while those infected with the wild-type strain died. This long survival correlated with very low counts of colony-forming units (CFU) in the lungs. Δmce1-infected mice developed very few and small granulomas, while animals infected with Δmce3 or Δmce2 mutants showed delayed granuloma formation. Mice infected with Δmce1 did not develop pneumonia, while animals infected with Δmce3 and Δmce2 mutants showed small pneumonic patches. In spleens, bacterial counts of mutant strains were less reduced than in lungs, compared with those of wild-type. In contrast, no such attenuation was observed when the intraperitoneal route was used for infection. Moreover, Δmce1 mutants appear to be more virulent in lungs after intraperitoneal inoculation. In conclusion, mce operons seem to affect the virulence of M. tuberculosis in mice, depending on the route of infection. Hypotheses are discussed to explain this last issue. Thus, mutants in these genes seem to be good candidates for vaccine testing.