|JOHNSTON, C - Cork Institute Of Technology|
|GOVENDER, RODNEY - Cork Institute Of Technology|
|ENDERSEN, LORRAINE - Cork Institute Of Technology|
|PLETZER, DANIEL - Jacobs University Bremen|
|WEINGART, HELGE - Jacobs University Bremen|
|COFFEY, A - Cork Institute Of Technology|
|O'MAHONY, J - Cork Institute Of Technology|
|SLEATOR, R - Cork Institute Of Technology|
Submitted to: Frontiers in Cellular and Infection Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/15/2014
Publication Date: 9/4/2014
Publication URL: http://handle.nal.usda.gov/10113/59667
Citation: Johnston, C., Bannantine, J.P., Govender, R., Endersen, L., Pletzer, D., Weingart, H., Coffey, A., O'Mahony, J., Sleator, R.D. 2014. Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius. Frontiers in Cellular and Infection Microbiology. 4:120. doi: 10.3389/fcimb.2014.00120.
Interpretive Summary: Johne’s disease in livestock such as dairy cattle and sheep is caused by the bacterium Mycobacterium avium subspecies paratuberculosis (MAP). A protein vaccine would be an excellent tool for controlling Johne’s disease; however, there are difficulties in getting MAP proteins expressed in a vaccine delivery vehicle. In this study, we examined Lactobacillus salivarius as a host for cloning MAP vaccine candidate proteins. Through a combination of molecular approaches, we were able to achieve high expression of MAP proteins in this host. The use of antibodies specific to the recombinant proteins also demonstrated faithful production of each protein in the Lactobacillus host. This work underscores the potential of Lactobacillus salivarius as a viable antigen delivery vehicle, which may provide a realistic route to an effective mucosal vaccine against Johne’s disease.
Technical Abstract: We have previously identified the mycobacterial high G+C codon usage bias as a limiting factor in heterologous expression of MAP proteins from Lb.salivarius, and demonstrated that codon optimisation of a synthetic coding gene greatly enhances MAP protein production. Here, we effectively demonstrate that codon-usage manipulation of two synthetic MAP genes (MAP2121synth and MAP3733synth) can facilitate the synthesis of conformationally folded antigens (MMP and MptD respectively), analogous to the form to which they are presented by virulent MAP bacilli. When heterologously over-expressed, antigenic determinants were detected from synthetic MMP proteins by monoclonal antibody (mAb8G2 and mAb13EI) mediated ELISA, including a discontinuous epitope associated with native protein folding. Moreover, MMP was present on the cellular surface of recombinant Lb.salivarius comparable to levels detected for MAP K10. Additionally, codon optimised MptD displayed the autochthonous tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adhered with the MptD-specific bacteriophage fMptD. This work substantiates the potential of Lb. salivarius as a viable delivery vehicle for MAP antigens.