Submitted to: Frontiers in Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/16/2011
Publication Date: 7/26/2011
Citation: Bannantine, J.P., Stabel, J.R., Lamont, E.A., Briggs, R.E., Sreevatsan, S. 2011. Monoclonal antibodies bind a SNP-sensitive epitope that is present uniquely in Mycobacterium avium subspecies paratuberculosis. Frontiers in Microbiology. 2(163):1-13. Interpretive Summary: This manuscript is the second part of a companion manuscript. In the initial manuscript, a specific monoclonal antibody was produced and characterized. In the present manuscript, the protein that the antibody binds to is characterized through start site determination and expression under different conditions. It turns out that this protein is full of surprises. First, it was not annotated when the genome was sequenced. This is because the gene that codes for the protein is on the opposite strand and overlaps with a gene that was annotated. Overlapping genes in this arrangement are rarely seen in bacteria. The most surprising observation is that the gene is also present in other closely related mycobacteria, yet none of those mycobacteria express the protein! Only M. avium subsp paratuberculosis produces this protein. This novel observation has huge implications.
Technical Abstract: Although there are an ever-increasing number of sequenced and annotated bacterial genomes, there still exist genes present in those genomes that are missed by manual or automated annotation. One such “hidden” gene, termed up1, is expressed only by M. avium subsp paratuberculosis and was discovered in the bovine strain K-10 genome using a monoclonal antibody (Bannantine & Sreevatsan, J. Bacteriol, 2010). In the current study, we characterize this novel gene’s structure and demonstrate extensive overlap with the annotated MAP3422c gene on the opposite strand. Surprisingly, similarity searches reveal that the DNA sequence encoding UP1 is present in other MAC complex species, yet mRNA transcripts are apparently made from this strand only in M. avium subsp paratuberculosis. RT-PCR analysis shows that M. avium subsp paratuberculosis makes a up1 transcript and expresses the protein in macrophages as well as when cultured in Middlebrook 7H9 media. The protein is not a strong antigen, but it is detected in mice and rabbits immunized with M. avium subsp paratuberculosis and furthermore, is detected by sera from cattle with Johne’s disease. These findings have new implications for comparative genomics and gene regulation differences among mycobacteria.