Reduced expression of antigenic proteins MPB70 and MPB83 in Mycobacterium bovis BCG strains due to a start codon mutation in sigK.
Mycobacterium bovis Bacille Calmette-Guerin (BCG) strains are genetically and phenotypically heterogeneous. Expression of the antigenic proteins MPB70 and MPB83 is known to vary considerably across BCG strains; however, the reason for this phenotypic difference has remained unknown. By immunoblot, we separated BCG into high- and low-producing strains. By quantitative reverse transcription polymerase chain reaction (RT-PCR), we determined that transcription of the antigen-encoding genes, mpb70 and mpb83, follows the same strain pattern with mRNA levels reduced over 50-fold in low-producing strains. Transcriptome comparison of the same BCG strains by DNA microarray revealed two gene regions consistently downregulated in low-producing strains compared with high-producing strains, one including mpb70 (Rv2875) and mpb83 (Rv2873) and a second that includes the predicted sigma factor, sigK. DNA sequence analysis revealed a point mutation in the start codon of sigK in all low-producing BCG strains. Complementation of a low-producing strain, BCG Pasteur, with wild-type sigK fully restored MPB70 and MPB83 production. Microarray-based analysis and confirmatory RT-PCR of the complemented strains revealed an upregulation in gene transcription limited to the sigK and the mpb83/mpb70 gene regions. These data demonstrate that a mutation of sigK is responsible for decreased expression of MPB70 and MPB83 in low-producing BCG strains and provide clues into the role of Mycobacterium tuberculosis SigK.
Charlet
Danielle
D
Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, Canada.
Mostowy
Serge
S
Alexander
David
D
Sit
Louis
L
Wiker
Harald G
HG
Behr
Marcel A
MA
D000942:Antigens, Bacterial / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000096:biosynthesis*; D018387:Codon, Initiator; D015870:Gene Expression; D005816:Genetic Complementation Test; D015151:Immunoblotting; D008565:Membrane Proteins / Q000096:biosynthesis*; D009163:Mycobacterium bovis / Q000235:genetics* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D017354:Point Mutation; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D018387:Codon, Initiator; C094904:MPB83 protein, Mycobacterium; D008565:Membrane Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D014157:Transcription Factors; C084190:sigma K; C060216:MPB70 protein, Mycobacterium bovis