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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Infectious Bacterial Diseases Research » Research » Publications at this Location » Publication #404247

Research Project: Identification of Antigens and Host Innate Immune Responses for Control of Johne's Disease

Location: Infectious Bacterial Diseases Research

Title: Mycobacterium avium subsp. paratuberculosis candidate vaccine strains are pro-apoptotic in RAW 264.7 murine macrophageS

item BARLETTA, RAUL - University Of Nebraska
item Bannantine, John
item STABEL, JUDITH - Retired ARS Employee
item MUTHUKRISHNAN, EZHUMALAI - University Of Nebraska
item ANDERSON, DIRK - University Of Nebraska
item DUTTA, ENAKSHY - University Of Nebraska
item MANTHENA, VAMSI - University Of Nebraska
item HANAFY, MOSTAFA - University Of Nebraska
item ZINNIEL, DENISE - University Of Nebraska

Submitted to: Vaccines
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/7/2023
Publication Date: 6/10/2023
Citation: Barletta, R.G., Bannantine, J.P., Stabel, J.R., Muthukrishnan, E., Anderson, D., Dutta, E., Manthena, V., Hanafy, M., Zinniel, D.K. 2023. Mycobacterium avium subsp. paratuberculosis candidate vaccine strains are pro-apoptotic in RAW 264.7 murine macrophageS. Vaccines. 11(6).

Interpretive Summary: This work represents a result from my USDA-NIFA subcontract, which examines two Mycobacterium avium subsp. paratuberculosis mutants in a variety of ways. In this study we specifically looked at their lowered virulence using an apoptosis assay. The host cell that we used for this assay is mouse macrophages. Because Mycobacterium avium subsp. paratuberculosis is an intracellular pathogen, it does NOT want to cause apoptosis (programmed cell death) in macrophages. Therefore, virulent strains prevent apoptosis while attenuated strains actually allow apoptosis of macrophages to occur. We showed exactly that - macrophages infected with the mutants showed apoptosis while those infected with the wild-type strain did not show apoptosis. So we concluded that the mutant strains are not virulent and hence are good vaccine candidates.

Technical Abstract: Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease, a severe gastroenteritis of ruminants. In this study, the goal was to develop a model cell culture system to rapidly screen MAP mutants for apoptosis that could then be tested for vaccine potential. We tested whether MAP wild type (NADC K-10 and UNL K-10) and transposon (4H2) and deletion (MAP'MAP_1152 and MAP'_MAP1156 deletions) mutants induce apoptosis and/or necrosis in murine RAW 264.7 macrophage cells. Growth rates were similar for all strains, but cell morphology indicated that DMAP52 and DMAP56 were elongated and displayed bulging of the cell wall. Cells were infected at a MOI of 10 with 1.2 x 106 CFU of MAP grown to mid-exponential phase in complete Middlebrook media. To follow the kinetics of cell death, a novel real-time cellular assay was used and the corresponding luminescence (apoptosis) and fluorescence (necrosis) outputs were measured. Based on these assays, a 6 h infection period was determined to be appropriate to assess apoptosis in MAP deletion mutant strains. Apoptosis was also quantified by DAPI stained nuclear morphology and confirmed by flow cytometry. The combined analysis of all methods confirmed the hypothesis that candidate vaccine strains DMAP52 and DMAP56 are pro-apoptotic in RAW 264.7 cells while wild type strains did not induce apoptotic changes. The real-time assay indicated early induction of apoptosis for the deletion mutants followed by secondary necrosis. In conclusion, the methods developed in this study provide a rapid assessment to clearly show that deletion mutants have a significant increase in apoptosis and apoptotic nuclei compared to both wild type strains and uninfected cells. These results indicate that the increased apoptosis observed in the deletion mutants suggest attenuation and are thus associated with good vaccine candidates.