Disruption of the gene encoding for secretion antigen SA5K affects growth of Mycobacterium bovis bacillus Calmette-Guerin in human macrophages and in mice.
An 8.3-kDa secretory antigen of Mycobacterium bovis bacillus Calmette-Guerin (BCG), called SA5K, was previously identified and characterized in our laboratory. Sequence analysis of the BCG sa5k gene, including the corresponding promoter region, showed that it is identical to the homologous gene in Mycobacterium tuberculosis (Rv1174c). No significant homology with other proteins was found and the physiologic role of SA5K for mycobacteria remains unknown. In the present study, a BCG mutant strain (BCGsa5k::aph) was constructed by allelic exchange involving the replacement of the sa5k gene with a kanamycin-inactivated copy. Mutant and parental strains showed similar growth rates in liquid medium, suggesting that the loss of the sa5k gene does not affect the in vitro growth of BCG. Nevertheless, BCGsa5k::aph showed a reduced ability to grow in human macrophages compared with the wild-type BCG, suggesting that SA5K is involved in intracellular survival/multiplication mechanisms. The mutant strain was also attenuated in vivo in a mouse infection model, showing impaired growth/survival in spleen and liver and fewer and smaller granulomatous lesions compared to the parental strain. Complementation of the mutation restored the parental phenotype. Taken together, results presented in this study suggest a role for SA5K in the growth capacity of BCG both in an intracellular milieu and in infected mice.
Bottai
Daria
D
Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, University of Pisa, Italy.
Esin
Semih
S
Batoni
Giovanna
G
Pardini
Manuela
M
Maisetta
Giuseppantonio
G
Donati
Valentina
V
Favilli
Flavia
F
Florio
Walter
W
Campa
Mario
M
D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D015139:Blotting, Southern; D015153:Blotting, Western; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005260:Female; D005798:Genes, Bacterial; D006651:Histocytochemistry; D006801:Humans; D008099:Liver / Q000382:microbiology / Q000473:pathology; D008264:Macrophages / Q000276:immunology / Q000382:microbiology* / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D016254:Mutagenesis, Insertional; D009163:Mycobacterium bovis / Q000235:genetics / Q000254:growth & development* / Q000276:immunology; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013154:Spleen / Q000382:microbiology / Q000473:pathology
D000942:Antigens, Bacterial; D004269:DNA, Bacterial