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 Deltamce1 and Deltamce2 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 Deltamce3, Deltamce2 and Deltamce1 mutants. Ten weeks after infection, all mice infected with the Deltamce 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. Deltamce1-infected mice developed very few and small granulomas, while animals infected with Deltamce3 or Deltamce2 mutants showed delayed granuloma formation. Mice infected with Deltamce1 did not develop pneumonia, while animals infected with Deltamce3 and Deltamce2 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, Deltamce1 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.
Gioffré
Andrea
A
Institute of Biotechnology, CICVyA-INTA, Los Reseros y Las Cabañas, 1712 Castelar, Argentina.
Infante
Eduardo
E
Aguilar
Diana
D
Santangelo
María De la Paz
MP
Klepp
Laura
L
Amadio
Ariel
A
Meikle
Virginia
V
Etchechoury
Ignacio
I
Romano
María Isabel
MI
Cataldi
Angel
A
Hernández
Rogelio Pando
RP
Bigi
Fabiana
F
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics* / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D015870:Gene Expression; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000472:pathogenicity*; D009876:Operon / Q000235:genetics*; D013997:Time Factors; D014397:Tuberculosis, Pulmonary / Q000382:microbiology; D014774:Virulence / Q000235:genetics
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C473802:mammalian cell entry protein Mce1A, Mycobacterium tuberculosis; C443482:mce2 protein, Mycobacterium tuberculosis