Article Figures & Data
Figures
- Fig 1
Reversal of the ΔsecA2 mutant apoptosis phenotype does not rescue growth in macrophages or mice. (A) Nonactivated BMDM were infected with the H37Rv, ΔsecA2, ΔsecA2 plus psecA2 (complemented strain), or ΔsecA2 plus α-sodA strain of M. tuberculosis, and intracellular replication was monitored as described in the text. The data shown are plotted on a linear scale and are representative of two experiments; the points represent means of triplicate wells, and the error bars represent standard deviations (SD). *, P < 0.05 by Student's t test. (B) Lung and spleen burdens in mice infected through the aerosol route at an initial dose of ∼200 CFU/lung with H37Rv or the ΔsecA2 or ΔsecA2-plus-α-sodA strain. We determined CFU counts by plating lung or spleen homogenates at various time points for viable bacteria. The data shown are from a single experiment; the bars represent the mean organ burdens from four mice, and the error bars represent SD. *, P < 0.05 by Student's t test. d, day(s); p.i., postinfection.
- Fig 2
M. tuberculosis autofluorescence can be used to identify bacilli by microscopy. The M. tuberculosis strains used in this study carrying a GFP expression plasmid were grown to mid-log phase and fixed in 4% paraformaldehyde in PBS. The fixed bacteria were loaded into the well of a chambered coverslip and visualized in the CFP and GFP channels on a wide-field fluorescence microscope. Autofluorescence in the CFP channel is compared to GFP fluorescence in strains expressing GFP from a plasmid. The overlap is demonstrated in the merged images, where yellow indicates a positive correlation.
- Fig 3
Compared to H37Rv, the ΔsecA2 mutant is enriched in LysoTracker-positive phagosomes. Nonactivated BMDM were infected with H37Rv, the ΔsecA2, mutant or the complemented strain (A); H37Rv, ΔsecA2, or ΔeccD1 (B); or BCG Pasteur or a ΔsecA2 mutant on the BCG Pasteur background (C). At the indicated times, the slides were stained with LysoTracker and scored for LysoTracker-positive phagosomes, as described in the text. Also shown in panel A are representative images from at least three independent experiments (LT, LysoTracker). The bars represent mean percentages of bacterium-containing phagosomes that stain positive for LysoTracker; The error bars represent SD of three replicate wells, with each well having >100 phagosomes scored. *, P ≤ 0.05 by Student's t test compared to the WT.
- Fig 4
Not all M. tuberculosis mutants with intracellular growth defects are enriched in LysoTracker-positive phagosomes. Nonactivated BMDM were infected with the H37Rv, ΔsecA2, or ΔleuD (A) or H37Rv, ΔsecA2, mce1A::tn, mce2F::tn, or rv0199::tn (B) strain. At the indicated times, the slides were stained with LysoTracker and scored for LysoTracker-positive phagosomes as described in the text. Shown are representative data from at least three independent experiments. The bars represent mean percentages of bacterium-containing phagosomes that stain positive for LysoTracker; the error bars represent SD of three replicate wells. *, P ≤ 0.05 by Student's t test compared to H37Rv.
- Fig 5
Compared to H37Rv, the ΔsecA2 mutant is enriched in phagosomes positive for late endocytic/lysosomal markers. Nonactivated BMDM were infected with H37Rv or the ΔsecA2, complemented ΔsecA2, or ΔeccD1 mutant for 24 h and immunofluorescently stained for markers of phagosome maturation as described in the text. (A) V-ATPase. (B) CD63. (C) Rab7. A representative of three independent experiments is shown. The bars represent mean percentages of bacterium-containing phagosomes that stain positive for marker; the error bars represent SD of three replicate wells, with each well having >100 phagosomes scored. *, P ≤ 0.05 by Student's t test compared to H37Rv. Representative microscopy images for each set of markers are shown.
- Fig 6
MyD88 has no effect on ΔsecA2 mutant phagosome trafficking or intracellular growth. (A) Nonactivated BMDM derived from C57BL/6 or MyD88−/− mice were infected with H37Rv or the ΔsecA2 mutant. At 24 h postinfection, the slides were stained with LysoTracker or antibodies to CD63, Rab7, or V-ATPase and scored for marker-positive bacterium-containing phagosomes as described in the text. The bars represent mean percentages of bacterium-containing phagosomes that stain positive for marker; the error bars represent SD of three replicate wells. n.s., not significant. There are no significant differences between C57BL/6 and MyD88−/− macrophages infected with the ΔsecA2 mutant. (B) Nonactivated BMDM from C57BL/6 (open symbols) or MyD88−/− (closed symbols) mice were infected with H37Rv or the ΔsecA2 mutant, and intracellular replication was monitored as described. The data shown are plotted on a linear scale and are from a representative experiment out of three; the points are means of three replicate wells, and the error bars represent SD. *, P ≤ 0.05 by student's t test compared to H37Rv.
- Fig 7
Growth of the ΔsecA2 mutant is inhibited by phagosome acidification. (A) LysoTracker-positive phagosomes in BMDM infected with H37Rv or the ΔsecA2 mutant and treated with bafilomycin A1 or concanamycin A. Shown are the lowest inhibitor concentrations that bring ΔsecA2 LysoTracker-positive phagosomes to H37Rv levels. ND, not determined. (B) Nonactivated BMDM treated with bafilomycin A1 or vehicle control (DMSO) were infected with H37Rv or the ΔsecA2 or ΔleuD mutant, and intracellular replication was monitored as described. Shown is a representative of four independent experiments. The bars represent the mean fold growth over 5 days of three replicate wells plus SD. *, P < 0.05 by Student's t test. (C) Data from the experiment described in panel B combined with three independent experiments to show the fold effect of bafilomycin A1 on intracellular growth compared to untreated macrophages. The bars represent the mean fold effect of bafilomycin A1 in four individual experiments plus the standard errors of the mean (SEM). *, P < 0.05 by Student's t test compared to H37Rv. (D and E) As in panels B and C, with concanamycin A or DMSO. (D) Shown is a representative of three experiments. (E) The bars represent the mean fold effect of concanamycin in three experiments plus SEM. *, P < 0.05 by Student's t test compared to H37Rv.
Tables
- Table 1
Mycobacterium strains used
Strain Description Reference or source M. tuberculosis H37Rv Wild type Trudeau Institute mc23112 H37Rv ΔsecA2 6 MBTB74 H37Rv ΔsecA2 attB::secA2 This study MBTB109 H37Rv ΔsecA2 attB::αsodA 25, this study mc23032 H37Rv ΔleuD 26 MBTB3 H37Rv ΔeccD1 This study PM638 H37Rv ΔblaC 18 MBTB204 PM638; mce1A::tn 40 MBTB156 PM638; mce2F::tn 40 MBTB183 PM638; rv0199::tn 40 MBTB157 H37Rv; gfp This study MBTB158 mc23112; gfp This study MBTB159 MBTB74; gfp This study MBTB390 mc23032; gfp This study M. bovis BCG BCG Pasteur Wild type Staten Serum Institut MB544 BCG Pasteur ΔsecA2 This study
