The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring

Mycobacterium tuberculosis (Mtb) contains two clpP genes, both of which are essential for viability. We expressed and purified Mtb ClpP1 and ClpP2 separately. Although each formed a tetradecameric structure and was processed, they lacked proteolytic activity. We could, however, reconstitute an active, mixed ClpP1P2 complex after identifying N-blocked dipeptides that stimulate dramatically (>1000-fold) ClpP1P2 activity against certain peptides and proteins. These activators function cooperatively to induce the dissociation of ClpP1 and ClpP2 tetradecamers into heptameric rings, which then re-associate to form the active ClpP1P2 2-ring mixed complex. No analogous small molecule-induced enzyme activation mechanism involving dissociation and re-association of multimeric rings has been described. ClpP1P2 possesses chymotrypsin and caspase-like activities, and ClpP1 and ClpP2 differ in cleavage preferences. The regulatory ATPase ClpC1 was purified and shown to increase hydrolysis of proteins by ClpP1P2, but not peptides. ClpC1 did not activate ClpP1 or ClpP2 homotetradecamers and stimulated ClpP1P2 only when both ATP and a dipeptide activator were present. ClpP1P2 activity, its unusual activation mechanism and ClpC1 ATPase represent attractive drug targets to combat tuberculosis

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

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

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

The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo

Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development

Mycobacterium tuberculosis ClpP1 and ClpP2 function together in protein degradation and are required for viability in vitro and during infection

In most bacteria, Clp protease is a conserved, non-essential serine protease that regulates the response to various stresses. Mycobacteria, including Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis, unlike most well studied prokaryotes, encode two ClpP homologs, ClpP1 and ClpP2, in a single operon. Here we demonstrate that the two proteins form a mixed complex (ClpP1P2) in mycobacteria. Using two different approaches, promoter replacement, and a novel system of inducible protein degradation, leading to inducible expression of clpP1 and clpP2, we demonstrate that both genes are essential for growth and that a marked depletion of either one results in rapid bacterial death. ClpP1P2 protease appears important in degrading missense and prematurely terminated peptides, as partial depletion of ClpP2 reduced growth specifically in the presence of antibiotics that increase errors in translation. We further show that the ClpP1P2 protease is required for the degradation of proteins tagged with the SsrA motif, a tag co-translationally added to incomplete protein products. Using active site mutants of ClpP1 and ClpP2, we show that the activity of each subunit is required for proteolysis, for normal growth of Mtb in vitro and during infection of mice. These observations suggest that the Clp protease plays an unusual and essential role in Mtb and may serve as an ideal target for antimycobacterial therapy