2005 all data

This file contains all data used in the 2005 analyses for the paper. It lists pot and treatment details, and biomass, chemistry and microbial data

2011 all data

This file contains all data from the 2011 analyses including pot and treatment ID, biomass, chemistry and microbial data. See Readme file attached to the 2005 data for more details

Multiparametric analysis of mutants impaired for intracellular growth identified subsets with shared complex phenotypes.

<p>(A) Schematic diagram of the approach to analyzing the mutants. Bacterial attenuation was analyzed based on three imaging parameters (<i>Mtb</i> fluorescence intensity, percent infected, and macrophage count) and host response was analyzed based on multiplexed cytokine analysis for each mutant. The phenotypes for each mutant were then combined into a multiparametric analysis as described in B. (B) Unsupervised 2-D clustering of mutants based on a multiparametric representation of their phenotypes. This analysis combines the four host response clusters (top 4 rows) and three bacterial imaging clusters (bottom 3 rows). Each column represents one of the 113 mutants included in the analysis. Colors indicate low (blue) to high (red) normalized values for each phenotype. Mutants cluster into five groups (Clusters I-V). Mutants in the PDIM locus (labels in orange) and ESX-1 locus (labels in green) group together in Cluster V. (C) Contribution of each bacterial imaging phenotype (bacterial replication, host survival, and % infected: orange vectors) and clustered cytokine response phenotype (cyt-1, cyt-2, cyt-3, and cyt-4: black vectors) to principal component 1 (PC1) and principal component 2 (PC2) of the principal component analysis. PC1 is comprised almost entirely of cytokine phenotypes while PC2 is comprised almost entirely of bacterial imaging features, suggesting that the two sets of features represent orthogonal and complementary information. (D) The 113 mutants included in the multiparametric analysis separate when plotted by PC1 and PC2. Included mutants were those that induced macrophage responses most distinct from the response to wild-type <i>Mtb</i>. Coloring of mutant clusters reflects mapping of the analysis performed in 2D back to the PC1/PC2 graph.</p

Two additional genes that cluster with PDIM and ESX-1 are required for PDIM production and downstream induction of the type I IFN response.

<p>(A-B) The type I IFN response to infection was measured by quantitation of expression of the type I IFN response-representative gene IFIT2. Bone-marrow derived macrophages were infected with wild-type <i>Mtb</i>, the <i>Rv0712</i> transposon mutant and complemented mutant (A) or <i>hrp1</i> transposon mutant and complemented mutant (B). RNA was harvested at 24 hours for gene expression analysis by qRT-PCR. IFIT2 was quantitated relative to control expression of GAPDH. Loss of type I IFN response to infection seen with disruption of <i>Tn0712</i> or <i>hrp1</i> in each mutant is restored with expression of the respective gene in the complemented strains. (A-B) Mean +/- SD of biological duplicates. Two-tailed t-test p-value for the comparison of Rv with <i>Tn</i>::<i>hrp1</i> and <i>Tn</i>::<i>Rv0712</i> was < 0.001. (C) PDIM was quantitated using LC-MS analysis of total cell wall lipid extracts. The indicated strains were grown in tween-free medium; total cell wall lipids were extracted from the bacterial cells. Loss of PDIM production seen in <i>Rv0712</i> and <i>hrp1</i> transposon mutants was restored with expression of the respective genes in the complemented strains. Mean +/- SD of biological triplicates. Fragmentation patterns for each quantitated species is listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006363#ppat.1006363.s001" target="_blank">S1 Text</a>. (D) Western blot analysis of ESAT-6 and CFP-10 secretion in <i>Rv0712</i> and <i>hrp1</i> mutants. Supernatants from each indicated strain including H37Rv wild-type control and <i>Tn</i>::<i>ecca1</i> (ESX-1 mutant positive control) were harvested, concentrated, and probed for ESAT-6 or CFP-10, and GroEL (lysis control). Corresponding pellets were simultaneously lysed and probed for ESAT- or CFP-10 (production control) and GroEL (loading control). Disruption of <i>Rv0712</i> resulted in diminished secretion of both ESAT-6 and CFP10; loss of secretion was reversed with complementation. Disruption of <i>hrp1</i> resulted in diminished ESAT-6 secretion but no change in CFP-10 secretion. Diminished ESAT-6 secretion in the <i>hrp1</i> mutant was restored with complementation.</p

PDIM facilitates phagosome permeabilization and is required for coordinated ESX-1-mediated secretion.

<p>(A) Bone marrow-derived macrophages were infected with wild-type <i>Mtb</i>, a <i>ppsD</i> clean deletion strain (<i>ΔppsD</i>), <i>ΔppsD</i> complemented with <i>ppsD</i>, or the <i>ΔppsD</i> expressing listeriolysin O (<i>hly</i>) at an MOI of 2:1. RNA was harvested at 24 hours for gene expression analysis by qRT-PCR. Hly-mediated mediated phagosomal permeabilization restores the type I IFN response in the PDIM mutant. Mean +/- SD of biological duplicates. Two-tailed t-test p-value for the comparison of Rv with <i>ΔppsD</i> was < 0.001 and for the comparison of Rv with <i>ΔppsD</i>::<i>hly</i> was non-significant. (B) Western blot analysis of ESAT-6 and CFP-10 secretion in PDIM mutants. Supernatants from wild-type, ESX-1 mutant (<i>Tn</i>::<i>eccCa1</i>), or PDIM mutants and complements were harvested, concentrated, and probed for ESAT-6, CFP-10, antigen 85 (Sec-secreted control), and GroEL (lysis control). Corresponding pellets were simultaneously lysed and probed for ESAT- or CFP-10 (production control) and GroEL (loading control). Disruption of <i>drrC</i> results in loss of ESAT-6 and CFP-10 secretion; inducibly restoring <i>drrC</i> expression with 50ng/ml aTc restores ESAT-6 and CFP-10 secretion. Loss of <i>ppsD</i> function due to a point mutation in the active site (<i>ppsD</i>(G44C)) similarly results in loss of ESAT-6 secretion, but results in enhanced CFP-10 secretion. Reversion of the mutation back to wild-type <i>ppsD</i>(G44C)rev restores the wild-type phenotype. (C) Western blot analysis of ESX-5 secretion of EsxN and PPE41 in the <i>ppsD</i> point mutant and <i>drrC</i> transposon mutant. Supernatants from phosphate-starved wild-type or PDIM mutant and complement were harvested, concentrated, and probed for ESX-5 substrates EsxN or PPE41 and GroEL (lysis control). Supernatant from an ESX-5 hypersecreter strain (<i>ΔpstA1</i>) was used for definitive band identification [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006363#ppat.1006363.ref040" target="_blank">40</a>]. Loss of <i>ppsD</i> function due to the G44C active site point mutation or loss of <i>drrC</i> function in the transposon mutant leads to significant loss of secretion of ESX-5 substrates PPE41 and EsxN. In both cases, secretion of EsxN and PPE41 is restored with complementation. Pellets below demonstrate that the <i>ppsD</i> mutant and <i>drrC</i> transposon mutant do not have a defect in EsxN or PPE41 production.</p

Identification of <i>M</i>. <i>tuberculosis</i> mutants defective for intracellular growth using a high-content imaging screen.

<p>(A) Protocol for the high-content imaging assay for the transposon mutant library growth in macrophages. Wild-type <i>Mtb</i> at MOIs varying two-fold from 2:1 to 0.25:1 was included in each plate to allow both accurate normalization between plates and validation of a cutoff for hits based on control population behavior. Because the transposon mutant library does not express a fluorescent protein, <i>Mtb</i> was externally stained with auramine-rhodamine after fixation to permit visualization. (B) Correlation of PC1 score of technical replicates in the full screen. (C) Density function of PC1 values for mutants and controls. While hits were selected based on comparison with OD-matched mutants, the distribution of hit mutants falls almost entirely below the wild-type control distribution as well, confirming that hit mutants also behave differently than wild-type.</p

PDIM production and export is required for induction of the macrophage type I IFN response.

<p>(A) Measurement of macrophage CCL5 production after infection with <i>Mtb</i> strains. J774.A1 cells were infected with wild-type <i>Mtb</i> strains or transposon mutants at an MOI of 1:1. Supernatants were harvested at 48 or 72 hours after infection for cytokine quantitation using Luminex. Mean +/- SD of three biological replicates. (B-D) The type I IFN response was measured by quantitation of expression of the type I IFN-responsive gene <i>ifit2</i> expression after macrophage infection with <i>Mtb</i>. Bone marrow-derived macrophages were infected with the indicated <i>Mtb</i> strains at an MOI of 2:1 and RNA was harvested at 24 hours for gene expression analysis by qRT-PCR. (B) Transposon mutants across the PDIM locus, including synthetic function mutants (<i>ppsC</i>, <i>ppsD</i>, <i>mas</i>), transport mutants (<i>drrA</i>, <i>drrB</i>, <i>drrC</i>, <i>mmpL7</i>), and acyl activating mutants (<i>fadD28</i>) failed to induce expression of type I IFN-responsive gene <i>ifit2</i>. P-value by two-tailed t-test for comparisons of Rv with Tn mutants in <i>eccCb1</i>, <i>eccCa1</i>, <i>ppsC</i>, <i>ppsD</i>, <i>drrA</i>, <i>drrB</i>, <i>drrC</i>, <i>papA5</i>, <i>mas</i>, <i>fadD28</i>, and <i>fadD22</i> were < 0.05, for the comparison of Rv and <i>mmpL7</i> was <i>0</i>.<i>05</i>, and for the comparison of Rv with <i>lppX</i>, <i>pks15</i>, <i>Rv2949c</i>, and <i>mutT4</i> were non-significant. (C) A <i>drrC</i> transposon mutant was complemented with tetracycline-inducible, chromosomally-integrated <i>drrC</i>; <i>drrC</i> expression was induced with 50ng/ml or 100ng/ml aTc. While in the absence of inducer <i>ifit2</i> expression was substantially decreased relative to wild-type-infected macrophages, induction of <i>drrC</i> expression with aTc restored <i>ifit2</i> expression. Two-tailed t-test p-value for the comparison of Rv with <i>Tn</i>::<i>drrC</i> and <i>Tn</i>::<i>drrC</i>::<i>ptet-drrC</i> 0 ng/ml aTc was < 0.01. (D) Infection with wild-type H37Rv, <i>ppsD</i> point mutant <i>ppsD</i>(G44C) that fails to produce PDIM, or complemented mutants <i>ppsD</i>(G44C)rev and <i>ppsD</i>(G44C)::<i>pMVppsD</i>. While loss of functional <i>ppsD</i> in the mutant resulted in loss of <i>ifit2</i> expression, restoring <i>ppsD</i> function restored <i>ifit2</i> production. Two-tailed t-test p-value for the comparison of Rv with <i>ppsD</i>mut was < 0.05. (B-D) Mean +/- SD of biological duplicates. (E) Microscopy of macrophages infected with PDIM mutant and complemented strains demonstrates that complementation restores wild-type image analysis phenotypes. J774A.1 macrophages were infected with wild-type, PDIM mutant, or complemented strains. After 3 days of infection, cells were washed, fixed, and stained with DAPI to visualize macrophage nuclei and auramine-rhodamine to visualize <i>Mtb</i>. Cells were imaged and CellProfiler automated image analysis was used [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006363#ppat.1006363.ref029" target="_blank">29</a>] to quantitate bacterial fluorescent intensity, percent macrophages infected, and macrophage cell count. Mean +/- SD of six biological replicates.</p