Mycobacterium avium subsp. paratuberculosis Candidate Vaccine Strains Are Pro-apoptotic in RAW 264.7 Murine Macrophages

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.

Keywords: Mycobacterium avium subsp. paratuberculosis; apoptosis; bovine; macrophages; necrosis; vaccine; virulence.

Figure 1

Determination of growth curves for MAP strains. Bacilli were inoculated into MOADC media at an initial OD₆₀₀ of about 0.05. Cultures were incubated standing at 37 °C for 28 days: (A) OD₆₀₀ readings were carried out at various time points, and (B) the corresponding CFU counts were determined; both are in the log₁₀ scale. The slopes for each strain on Days 0 to 14, as well as the statistical significance of pairwise determinations, are indicated as * p ≤ 0.05 and ** p ≤ 0.01. Results represent means (n = 3) ± standard errors of the mean. A legend displays the correlation between each strain and the color used.

Figure 2

Determination of cell morphology using transmission electron microscopy: (A) micrographs of MAP strains with a reference bar of 0.380 µm; (B) strain 4H2 (orange tick mark) was significantly longer than NADC K-10. DMAP52 (purple tick marks) and DMAP56 (blue tick marks) showed significantly increased length compared with both wild types and 4H2; (C) the deletion mutants also had significantly increased width compared with these strains. The asterisk color represents the strain to which pairwise comparisons were statistically significant: * p ≤ 0.05 and *** p ≤ 0.001. Results represent means (n = 3) ± standard errors of the mean.

Figure 3

RAW 264.7 uninfected control (gray) and MAP-infected cells were assayed for real-time apoptosis and necrosis. Measurements for the various time points from 1 to 48 h are indicated by a light-to-dark color gradient: (A) apoptosis (RLU) was determined as a luminescence output. Pairwise comparisons are shown for DMAP56 (top) and DMAP52 (bottom) vs. the control, wild types, and 4H2 for 24/48 h (upper line) and 6/12 h (middle line). The lower line displays pairwise comparisons between 4H2 (top), UNL K-10 (inline), and NADC K-10 (bottom) vs. the uninfected cells for 48 h. The asterisk color represents the strain to which pairwise comparisons were statistically significant: * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001; (B) secondary necrosis (RFU) was recorded as a fluorescence output. The upper line has pairwise comparisons with the p-values listed that are close to being significant for DMAP56 at 12, 24, and 48 h. Pairwise comparisons are shown for DMAP56 (top) and DMAP52 (bottom) vs. the control cells for 24 h (lower line): * p ≤ 0.05. Results represent means (n = 3) ± standard errors of the mean.

Figure 4

Determination of apoptosis using a rapid staining assay. RAW 264.7 uninfected control (gray) and MAP-infected cells were stained with DAPI at 6 h post-infection: (A) the percent of apoptotic nuclei is displayed. The asterisk color represents the strain to which pairwise comparisons were statistically significant: *** p ≤ 0.001. Pairwise comparisons are presented for DMAP56 (top) and DMAP52 (bottom) vs. the control, wild types, and 4H2 (upper line). The lower line shows pairwise comparisons between 4H2 (top), UNL K-10 (inline), and NADC K-10 (bottom) vs. the uninfected cells. Results represent means (n = 3) ± standard errors of the mean; (B) phase contrast (left) and fluorescent (right) micrographs for representative samples for the uninfected cells and each strain. These images were merged using Adobe Photoshop Version 24.4.1 (San Jose, CA, USA) and modified for uniformity and clarity.

Figure 5

RAW 264.7 uninfected control (gray) and MAP-infected cells were assayed using flow cytometry 6 h post-infection: (A) dot plots (FlowJo™ software) for representative samples for the control and each strain are displayed with cell percentiles for each quadrant (see results in Section 3.4 for details); (B) the percent of early (diagonal lines), late (solid), and total (early plus late) apoptosis are presented. Pairwise comparisons are shown for DMAP56 (top) and DMAP52 (bottom) for early, late, and total apoptosis vs. the control, wild types, and the transposon mutant. The asterisk color represents the strain to which pairwise comparisons were statistically significant: * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001. Results represent means (n = 3) ± standard errors of the mean, with varying technical replicates (r) as follows: Control r = 13, NADC K-10 r = 4, UNL K-10 r = 6, 4H2 r = 5, DMAP52 r = 13, and DMAP56 r = 11.