Figure 1.
Mtb ClpP1 and ClpP2 interact, forming a multi-component protease, and share substantial similarity with ClpP1 and ClpP2 homologs in Msm.
(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.
Figure 2.
Both ClpP1 and ClpP2 are essential for normal growth in mycobacteria.
(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.
Figure 3.
Inducible protein degradation demonstrates requirement of ClpP2 for normal growth.
(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.
Figure 4.
Clp protease is required for degradation of abnormal proteins and SsrA-tagged proteins in mycobacteria.
(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).
Figure 5.
A catalytically inactive ClpP allele inhibit Mtb growth in vitro and during infection.
(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.