Mtb ClpP1 and ClpP2 interact, forming a multi-component protease, and share substantial similarity with ClpP1 and ClpP2 homologs in Msm.

<p>(A) C-terminally myc-tagged Mtb ClpP1 and 6×His-tagged Mtb ClpP2 were expressed in Msm. Lysate (lane 1) was prepared and loaded onto a Ni-column. After washing with PBS (lanes 2,3), Ni-bound material was eluted with 50 mM (lane 4), 100 mM (lane 5), 250 mM (lane 6, 7) of imidazole in PBS, and analyzed by immunoblotting using anti α-myc and α-6×His antibodies. (B) Fraction 6 from (A) was applied to an anti-myc column (lane 1). The flow through (lane 2), and bound material (lane 3) were analyzed by immunoblot with α-myc and α-His antibodies. Bound material was released from the anti-myc agarose beads by boiling in Laemmli buffer after washing with PBS. (C) Bands representing ClpP1 and ClpP2 from (B) were sequenced by MS/MS revealing the presence of both Mtb and Msm homologs. Msm specific peptides are indicated by black lines, those specific to Mtb are indicated by red lines. (D) Cleavage of fluorescent peptide Z-Gly-Gly-Leu-AMC was measured in the presence of 1 µg ClpP1, 1 µg Clp2, and the activating peptide Z-Leu-Leu (see accompanying paper). Addition of 5 µg of catalytically inactive mutants of either ClpP1 (ClpP1S) or ClpP2 (ClpP2S) markedly inhibited cleavage by the ClpP1P2 protease. Results graphed are a representative sample of results obtained.</p

Inducible protein degradation demonstrates requirement of ClpP2 for normal growth.

<p>(A) Schematic representation of the inducible degradation system used to inducibly deplete ClpP2 (Msm strain clpP2_ID). Induction of HIV-2 protease with ATc leads to cleavage of the HIV-2 protease cutting site and exposure of a SsrA tag on the tagged protein. Cleavage by HIV-2 protease and subsequent degradation can be tracked via the FLAG (square) and c-myc (circle) epitope tags, respectively, included on the inducible degradation tag. (B) Degradation of ClpP2 in clpP2_ID was tracked by Western in the absence or presence of inducer ATc. Blots were probed α-FLAG (loss indicates HIV-2 protease cleavage), α-myc (loss indicates target degradation), and α-RpoB (loading control). (C) Growth curves of Msm clpP2_ID in the absence or presence (50 ng/mL) of inducer ATc. Msm clpP2_ID was also complemented with clpP2 in the presence of ATc. Data are represented as mean CFU/mL +/− standard deviation.</p

A catalytically inactive ClpP allele inhibit Mtb growth <i>in vitro and during infection</i>.

<p>(A) Growth curves for Mtb overexpressing wild type ClpP1 or ClpP1 S98A via an ATc-inducible expression vector. Data are represented as mean OD₆₀₀ +/− standard deviation. Dashed lines represent assumed growth rates until first measured growth point. (B) Growth of Mtb containing a doxycycline-inducible plasmid expressing the mutant allele ClpP1 S98A in lungs of C57BL/6 mice 30 days post aerosol infection. Mice were infected via aerosol with a 3∶1 mixture of mutant and wild type bacteria. Mice were fed either with chow containing (dark squares, N = 5 mice) or lacking (gray triangles, N = 5 mice) the inducer doxycycline. As a control, wild type Mtb was co-infected, and representative CFU/organs for the control are represented (right). Each point represents calculated total CFU/organ for each mouse. Not all mice received enough wild type bacteria to quantitate.</p

Both ClpP1 and ClpP2 are essential for normal growth in mycobacteria.

<p>(A) Schematic representation of mycobacterial recombineering, employed to replace the endogenous promoter of the clpP1P2 operon with a ATc-inducible promoter (Msm strain ptet_clpP1P2). (B) Growth curves of Msm ptet_clpP1P2 in the presence (50 ng/mL) or absence of inducer ATc. Data are represented as mean CFU/mL +/− standard deviation. (C) Growth curves of Msm ptet_clpP1P2 complemented with clpP1, clpP2 or both clpP1 and clpP2 in the absence of inducer ATc. Data are represented as mean CFU/mL +/− standard deviation. (D) Schematic representation of genetic strategy used to create a tetracycline inducible conditional Msm ClpP2 mutant (Msm strain ptet_ClpP2) (E) Growth curves of Msm ptet_clpP2 in the presence (50 ng/mL) or absence of inducer ATc. Msm ptet_clpP2 was also complemented with clpP2 in the absence of ATc. Data are represented as mean OD₆₀₀ +/− standard deviation. Dashed lines represent assumed growth rates until first measured growth point.</p

Clp protease is required for degradation of abnormal proteins and SsrA-tagged proteins in mycobacteria.

<p>(A) Growth curves of Msm ptet_clpP2 in growth medium containing low (1 ng/mL) or high (100 ng/mL) concentrations of inducer ATc, in the presence of either no drug, or amikacin (top left, 0.03 µg/mL), streptomycin (top right, 0.125 µg/mL), and chloramphenicol (bottom, 7.5 µg/mL). Data are represented as mean OD₆₀₀ +/− standard deviation. Dashed lines represent assumed growth rates until first measured growth point. (B) Increase in fluorescence (RFU, 485/520) and initial growth curve (OD₆₀₀) of Msm clpP2_ID expressing the fusion construct GFP-SsrA on a constitutively expressing plasmid, in the presence and absence of inducer, ATc. Data are represented as mean RFU or OD₆₀₀ +/− standard deviation. (C) Depletion of ClpP2 and increase in GFP-SsrA in Msm clpP2_ID expressing the fusion construct GFP-SsrA on a constitutively expressing plasmid was tracked by immunoblot. Blots were probed α-GFP, α-myc, α-FLAG, and α-RpoB (loading control).</p

Proteomic profiling of P750-clpP1P2DAS in the presence and absence of ATc reveals a wide array of potential Clp protease substrates.

<p>(A) In triplicate, P750-clpP1P2DAS was grown for 48 hours in the absence, denoted “wt”, or presence of ATc (1.5 µg/mL), denoted “mut”, from a starting OD₆₀₀ of 0.02. Immunoblotting of protein lysates with α-ClpP2 and α-RpoB (loading control) demonstrates degree of ClpP2-DAS depletion in mut cells. Samples were then used for TMT₆ MS3-based quantitative proteomics. The specific TMT reagent used for each condition is listed under the immunoblot. (B) Normalized, summed intensities for all quantified proteins was used to perform Pearson correlational hierarchical clustering of biological replicates. (C) The Log2 ratio of average mutant protein intensity to average wildtype protein intensity plotted against the p-value determined by t-test, grouping the three biological replicates. The threshold for over-representation was set at an average ratio of greater than equal to 2, while the cut-off for under-representation was 0.5. In both instances, p-values below 0.01 were deemed significant. Proteins considered for further analysis are denoted in red. (D) The relative quantity of specific proteins plotted across the six TMT channels, for highly (left) and moderately (center) over-represented, and under-represented (right) proteins in the mutant versus wildtype conditions.</p

Validation of proteomic hits reveals that WhiB1 and CarD are likely Clp protease substrates.

<p>(A) Quantitative PCR to determine transcript levels of over-represented proteins upon depletion of Clp protease using RNA generated from Mtb P750-clpP1P2DAS after growth for 48 h in the presence or absence of ATc (1.5 µg/mL). Relative standard curves were generated for each probe set, and sigA transcript was used as an endogenous control. Data are represented as mean fold change, normalized to transcript in (−) ATc cultures +/− SEM of technical replicates. Protein ratios are derived from TMT experiments described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003994#ppat-1003994-g002" target="_blank">Figure 2</a>, and represented as average ratios +/− standard deviation of biological replicates (B) Fluorescence (485/538) was measured for N- and C-terminal GFP fusions constructed for WhiB1 (left) and CarD (right), and induced for 8 hours in wildtype or clpP2-ID Msm with ATc (100 ng/mL). In clpP2-ID, ATc simultaneously induced fusion protein production and degradation of ClpP2. (C) Fluorescence (485/538) was measured for GFP fusions bearing a variable number of C-terminal residues from WhiB1 in clpP2-ID Msm, grown in the absence or presence of ATc (100 ng/mL) for 8 hours. In (B) and (C), data are represented as mean RFU +/− standard deviation of biological replicates. Asterisks denote a p-value <0.05 determined by t-test.</p

Blocking Clp-dependent degradation of WhiB1 is toxic in mycobacteria.

<p>(A) Inducible production of GFP-WhiB1, WhiB1-GFP, and WhiB1wt in Msm demonstrates growth inhibition of WhiB1-GFP producing bacteria. Over-production was induced with ATc (100 ng/mL). (B) Growth curves of strains producing WhiB1 constructs in the presence of ATc (100 ng/mL). As a control, strains producing WhiB1wt were grown in the absence of inducer. Data are represented as mean OD₆₀₀ +/− standard deviation of biological replicates. (C) Quantitative PCR, using probe sets that hybridize to the whiB1 5′utr, to determine transcriptional repression at the endogenous <i>whiB1</i> locus in wildtype Msm, or Msm inducibly expressing <i>gfp-whiB1</i> or <i>whiB1-gfp</i>. RNA was isolated from cultures grown for 6 hours from a starting OD₆₀₀ of 0.06 in the presence of the inducer ATc (100 ng/mL). Relative standard curves were generated for each probe set, and sigA transcript was used as an endogenous control. Data are represented as mean fold change, normalized to transcript in wildtype cultures +/− SEM of technical replicates. (D) Promoter reporters were constructed fusing the 500 bp upstream of whiB1 to luciferase. Luminescence (RLU, 100 ms exposure, grey bars) was measured in wildtype Msm, or Msm inducibly producing GFP-WhiB1 or WhiB1-GFP. Amounts of the fusion proteins were monitored by fluorescence (RFU, 485/538, black bars). Data are represented as RFU or RLU +/− standard deviation of biological replicates. (E) GFP whiB1 fusions and wildtype whiB1 were cloned into integrative plasmids, in which expression was driven by the native <i>whiB1</i> promoter. Constructs were transformed into Msm, and transformation efficiency was determined by calculating colony forming units (CFU) obtained per ng DNA. In (C), (D), and (E), asterisks denote a p-value <0.05, determined by t-test.</p