Growth kinetics of <i>M</i>. <i>tuberculosis</i> Δ<i>pstA2</i> and Δ<i>pstA1</i>Δ<i>pstA2</i> mutants in aerosol-infected mice.

<p>Mice were aerosol-infected with ~100 CFU of <i>M</i>. <i>tuberculosis</i> WT (squares), Δ<i>pstA2</i> (circles), or Δ<i>pstA1</i>Δ<i>pstA2</i> (triangles). Groups of mice (n = 4) were sacrificed at the indicated time points. Bacterial CFU were enumerated by plating serially diluted lung homogenates on 7H10 agar and incubating 3–4 weeks at 37°C. All mouse strains were on the C57BL/6 background. Symbols represent means; error bars indicate standard error of the mean. Asterisks indicate statistically significant differences between WT and Δ<i>pstA1</i>Δ<i>pstA2</i>: *<i>P</i> < 0.05, **<i>P</i> < 0.005, ***<i>P</i> < 0.0005. No significant differences in pulmonary CFU were observed between the WT and Δ<i>pstA2</i> strains. (<b>A</b>) IFN-γ^-/-, (<b>B</b>) Irgm1^-/-, (<b>C</b>) NOS2^-/-, (<b>D</b>) C57BL/6.</p

Deletion of <i>pstA2</i> causes increased resistance to acidified medium.

<p><i>M</i>. <i>tuberculosis</i> WT, Δ<i>pstA1</i>, Δ<i>pstA2</i>, Δ<i>pstA1</i>Δ<i>pstA2</i>, Δ<i>pstA2</i> pMV<i>pstA2</i> and Δ<i>pstA1</i>Δ<i>pstA2</i> pMV<i>pstA2</i> strains were grown to mid-exponential phase in P_i-rich 7H9 medium and then subjected to stress conditions, as indicated. Bacterial CFU were enumerated by plating serially diluted cultures on 7H10 agar and incubating for 3 to 4 weeks at 37°C. Percent survival was calculated as (CFU poststress)/(CFU pre-stress)x100. Data shown are means ± standard deviations of three independent cultures from one experiment, and are representative of at least three independent experiments. Asterisks indicate statistically significant differences between the deletion mutant and WT control for panels A-C or between the deletion mutant and both the WT and complemented strains for panels D and E: *<i>P</i> < 0.05, **<i>P</i> < 0.005, ***<i>P</i> < 0.0005. <b>(A)</b> Cell wall stress (sodium dodecyl sulfate, SDS) and reactive oxygen stress (hydrogen peroxide, H₂O₂). Either 0.5% SDS or 5 mM H₂O₂ was added to each culture. CFU were enumerated at 0 and 24 hours and percent survival was calculated. <b>(B)</b> P_i starvation. Bacteria were shifted to P_i-free medium and CFU were enumerated at 0, 3, 5, 7, and 10 days. <b>(C-E)</b> Acidic pH stress. Bacteria were shifted to acidified 7H9 medium pH 4.5 containing 0.1% Tween-80 and CFU were enumerated at 0, 3, 7, and 10 days.</p

Deletion of <i>pstS1</i> causes increased resistance to acidified medium.

<p>(<b>A</b>) <i>M</i>. <i>tuberculosis</i> WT, Δ<i>pstS1</i>, and Δ<i>pstS1</i> pMV<i>pstS1</i> were grown to mid-exponential phase in P_i-rich 7H9 medium and then shifted to acidified 7H9 medium pH 4.5 containing 0.1% Tween-80. Bacterial CFU were enumerated on days 0, 3, 7, and 10 by plating serially diluted cultures on 7H10 agar and incubating for 3 to 4 weeks at 37°C. Data shown are means ± standard deviations of three independent cultures from one experiment and are representative of at least three independent experiments. Asterisks indicate statistically significant differences from the WT control: *<i>P</i> < 0.05, **<i>P</i> < 0.005, ***<i>P</i> < 0.0005. (<b>B</b>) PstS1 protein production. Whole cell lysate proteins (4 μg) of WT, Δ<i>pstS1</i>, Δ<i>pstS1</i> pMV<i>pstS1</i> were subjected to SDS-PAGE and Western blot analysis. Mouse monoclonal anti-PstS1 and anti-GroEL2 antibodies were used to detect PstS1 and GroEL2, a cell-associated protein that serves as a loading control. Results shown are representative of two independent experiments.</p

Virulence of <i>M</i>. <i>tuberculosis</i> Δ<i>pstA2</i> and Δ<i>pstA1</i>Δ<i>pstA2</i> mutants in immune-deficient mice.

<p>Groups of immune-deficient mice (n = 8), all on the C57BL/6 background, were aerosol-infected with ~100 CFU of wild-type <i>M</i>. <i>tuberculosis</i> (WT, squares), Δ<i>pstA2</i> (circles), or Δ<i>pstA1</i>Δ<i>pstA2</i> (triangles) and monitored for signs of illness. Moribund mice were euthanized and scored as dead. <b>(A</b>) IFN-γ^-/-, (<b>B</b>) Irgm1^-/-, (<b>C</b>) NOS2^-/-.</p

Confirmation of the Δ<i>pstA2</i> and Δ<i>pstA1</i>Δ<i>pstA2</i> deletions by Southern blotting.

<p><b>(A</b>) Genetic organization of the <i>M</i>. <i>tuberculosis pst</i> locus. Genes are indicated by arrows; probes used for Southern blotting are indicated by the gray rectangles. Locations of relevant restriction enzyme sites are shown. (<b>B</b>) Southern blot of wild-type Erdman (WT) and Δ<i>pstA2</i> genomic DNA digested with either SacI or XhoI, as indicated, and probed with probe2. Molecular weight standards are indicated at the right. The Δ<i>pstA2</i> deletion removes 0.9 kb. (<b>C</b>) Southern blot of WT and Δ<i>pstA1</i>Δ<i>pstA2</i> genomic DNA digested with PstI and probed with probe1 to detect the Δ<i>pstA1</i> deletion. Molecular weight standards are indicated at the right. The Δ<i>pstA1</i> deletion removes 0.89 kb. (<b>D, E</b>) Phthiocerol dimycocerosate (PDIM) production by the Δ<i>pstA2</i> (<b>D</b>) and Δ<i>pstA1</i>Δ<i>pstA2</i> (<b>E</b>) mutants. ¹⁴C propionate-labeled apolar lipid fractions were extracted and analyzed by thin-layer chromatography. Two spots that correspond to the phthiocerol (methoxy) and phthiodiolone (keto) forms of PDIM are indicated.</p