Characterization of ethanol extracted cell wall components of Mycobacterium avium subsp. paratuberculosis

Authors: Bannantine, John; WADHWA, ASHUTOSH - University Of Tennessee; Stabel, Judith; EDA, SHIGETOSHI - University Of Tennessee

Submitted to: Veterinary Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/28/2019
Publication Date: 10/31/2019
Citation: Bannantine, J.P., Wadhwa, A., Stabel, J.R., Eda, S. 2019. Characterization of ethanol extracted cell wall components of Mycobacterium avium subsp. paratuberculosis. Veterinary Sciences. 6(4):88. https://doi.org/10.3390/vetsci6040088.
DOI: https://doi.org/10.3390/vetsci6040088

Interpretive Summary: In the past, our group has discovered and developed a surface antigen for Mycobacterium avium subspecies paratuberculosis, the bacterium that causes Johne's disease. This antigen has shown strong potential for use in a diagnostic test for Johne's disease. The problem is that the antigen has a complex make up, consisting of lipid, carbohydrate and proteins and thus it would be beneficial to determine more precisely what these components are and if they are necessary for the antigenicity of the diagnostic test. We discovered that it is the carbohydrate component and not the proteins that are antigenic. We further defined exactly what those components are. This new knowledge should enable researchers to construct this antigen using only the more immunogenic components to develop the best possible diagnostic test.

Technical Abstract: The antigens extracted using ethanol (EtOH) and incorporated in the EVELISA test have previously shown high specificity and sensitivity for detecting Mycobacterium avium subspecies paratuberculosis (Map) and M. bovis infections in cattle. The objective of this study is to define the components present in the EtOH extract of Map. We show that this extract is composed of lipid, carbohydrate and protein antigens on the surface of the bacilli and that EtOH removes the outer layer structure of Map which comprise these elements. The lipid and carbohydrate components of the extract were analyzed by thin layer chromatography and lectin binding, respectively. To identify proteins, polyclonal antibodies to the EtOH prep were produced and used to screen a Map genomic expression library. Seven overlapping clones were identified with a single open reading frame, MAP_0585, common to all. MAP_0585, which encodes a hypothetical protein, was recombinantly produced and used to demonstrate strong reactivity in sera from hyperimmunized rabbits, but this protein is not strongly immunogenic in cattle with Johne’s disease. A panel of well-characterized mAbs were used to determine the presence of additional proteins in the EtOH extract. These antibodies demonstrated that MBP83 is present in M. bovis EtOH extracts and a fatty acid desaturase (MAP_2698c) is present in Map EtOH extracts, while lipoarabinomannan (LAM) was common to both. Lectin biding and protease treatment of the EtOH extract suggest the antigenic component is carbohydrate and not protein. Collectively, analysis shows that this preparation is composed predominantly of carbohydrate and lipid along with a non-antigenic protein component. These results give further insight into this important antigen prep for detecting mycobacterial diseases of cattle.