Mycobacterium avium subsp. paratuberculosis Candidate Vaccine Strains Are Pro-apoptotic in RAW264.7MurineMacrophages

Authors

Raul G. Barletta, University of Nebraska - LincolnFollow
John P. Bannantine, USDA, Agricultural Research Service
Judith R. Stabel, USDA, Agricultural Research Service
Ezhumalai Muthukrishnan, University of Nebraska-Lincoln
Dirk Anderson, University of Nebraska - LincolnFollow
Enakshy Dutta, University of Nebraska-LincolnFollow
Vamsi Manthena, University of Nebraska-Lincoln
Mostafa Hanafy, Cairo UniversityFollow
Denise K. Zinniel, University of Nebraska - LincolnFollow

Date of this Version

6-10-2023

Citation

Barletta, R.G.; Bannantine, J.P.; Stabel, J.R.; Muthukrishnan, E.; Anderson, D.K.; Dutta, E.; Manthena, V.; Hanafy, M.; Zinniel, D.K. Mycobacterium avium subsp. paratuberculosis Candidate Vaccine Strains Are Pro-apoptotic in RAW 264.7 Murine Macrophages. Vaccines 2023, 11, 1085. https://doi.org/ 10.3390/vaccines11061085

Comments

Open access.

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease, a severe gastroenteritis of ruminants. This study developed a model cell culture system to rapidly screen MAP mutants with vaccine potential for apoptosis. Two wild-type strains, a transposon mutant, and two deletion mutant MAP strains (MOI of 10 with 1.2 × 10⁶ CFU) were tested in murine RAW 264.7 macrophages to determine if they induce apoptosis and/or necrosis. Both deletion mutants were previously shown to be attenuated and immunogenic in primary bovine macrophages. All strains had similar growth rates, but cell morphology indicated that both deletion mutants were elongated with cell wall bulging. Cell death kinetics were followed by a real-time cellular assay to measure luminescence (apoptosis) and fluorescence (necrosis). A 6 h infection period was the appropriate time to assess apoptosis that was followed by secondary necrosis. Apoptosis was also quantified via DAPI-stained nuclear morphology and validated via flow cytometry. The combined analysis confirmed the hypothesis that candidate vaccine deletion mutants are pro-apoptotic in RAW 264.7 cells. In conclusion, the increased apoptosis seen in the deletion mutants correlates with the attenuated phenotype and immunogenicity observed in bovine macrophages, a property associated with good vaccine candidates.