The knockout of the lprG-Rv1410 operon produces strong attenuation of Mycobacterium tuberculosis.
P27 lipoprotein was previously described as an antigen in the Mycobacterium tuberculosis complex, encoded by the lprG gene, also named Rv1411 in the TubercuList (http://genolist.pasteur.fr/TubercuList) gene bank. It forms an operon with Rv1410 that encodes for an efflux pump, P55. A mutant of the H37Rv strain of M. tuberculosis not producing P27 (strain DeltaP27) was obtained by two-step mutagenesis using the counterselectable marker sacB and a thermosensitive origin of replication in the shuttle plasmid pPR27. By RT-PCR, we observed no lprG or Rv1410 mRNA in the DeltaP27 mutant strain compared with the wild type and complemented strains. Western blot experiments using anti-P27 polyclonal sera showed that the P27 protein was present both in the parental and in a complemented strain, in which the entire lprG-Rv1410 operon was reintroduced, but absent in the mutant strain. The three strains showed similar growth kinetics and characteristics in culture broth. To study the effect of the lprG mutation on M. tuberculosis virulence, BALB/c mice were inoculated to determine bacterial loads in spleens. At days 15 and 35 after infection, decreases of 1.5 and 2.5 logs in the bacterial load were found, respectively, in animals inoculated with the DeltaP27 mutant strain or with the wild type. This attenuation was reverted in the complemented strain. These results demonstrated that lprG gene is required for growth of M. tuberculosis in immunocompetent mice. The reversion of attenuation in the complemented strain indicates that the attenuated phenotype resulted from disruption of the lprG-Rv1410 operon.
Institute of Biotechnology, CICVyA-INTA, Los Reseros y Las Cabañas, 1712 Castelar, Argentina.
Fabiana
F
Bigi
Andrea
A
Gioffré
Laura
L
Klepp
María de la Paz
MP
Santangelo
Alicia
A
Alito
Karina
K
Caimi
Virginia
V
Meikle
Martín
M
Zumárraga
Oscar
O
Taboga
María I
MI
Romano
Angel
A
Cataldi
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D005816:Genetic Complementation Test; D008074:Lipoproteins / Q000172:deficiency* / Q000235:genetics / Q000502:physiology; D026901:Membrane Transport Proteins / Q000172:deficiency* / Q000235:genetics / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity*; D009876:Operon / Q000235:genetics*; D014376:Tuberculosis / Q000382:microbiology; D014774:Virulence / Q000235:genetics*
D001426:Bacterial Proteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; C110121:P27 protein, Mycobacterium; C425143:P55 protein, Mycobacterium