Validation of the essential ClpP protease in Mycobacterium tuberculosis as a novel drug target

Abstract

Mycobacterium tuberculosis is a pathogen of major global importance. Validated drug targets are required in order to develop novel therapeutics for drug-resistant strains and to shorten therapy. The Clp protease complexes provide a means for quality control of cellular proteins; the proteolytic activity of ClpP in concert with the ATPase activity of the ClpX/ClpC subunits results in degradation of misfolded or damaged proteins. Thus, the Clp system plays a major role in basic metabolism, as well as in stress responses and pathogenic mechanisms. M. tuberculosis has two ClpP proteolytic subunits. Here we demonstrate that ClpP1 is essential for viability in this organism in culture, since the gene could only be deleted from the chromosome when a second functional copy was provided. Overexpression of clpP1 had no effect on growth in aerobic culture or viability under anaerobic conditions or during nutrient starvation. In contrast, clpP2 overexpression was toxic, suggesting different roles for the two homologs. We synthesized known activators of ClpP protease activity; these acyldepsipeptides (ADEPs) were active against M. tuberculosis. ADEP activity was enhanced by the addition of efflux pump inhibitors, demonstrating that ADEPs gain access to the cell but that export occurs. Taken together, the genetic and chemical validation of ClpP as a drug target leads to new avenues for drug discovery.

Fig 1

ClpP1 is essential for in vitro growth of M. tuberculosis. (A) Chromosomal organization of the probable clpP1-clpP2 operon. The region used in the complementation vector to generate the merodiploid and the region used as the probe for Southern blotting are indicated. (B) Schematics of the merodiploid and Del-Int (deletion, integration) strain showing the SphI restriction sites and expected sizes for Southern analysis. The attB site utilized by the integrating vector is marked. (C) Southern analysis of strains. Genomic DNA from wild-type (WT) H37Rv (lane 1), merodiploid (lane 2), Del-Int DCO (lane 3), and WT revertant (Rev) DCO (lane 4) strains was digested with SphI and hybridized with the clpP1 probe. The expected bands (and sizes) for WT, Del, WT Rev DCO, and Int alleles are indicated by arrows.

Fig 2

Structures of the ADEP compounds and derivatives. Three previously described ADEP compounds were synthesized (ADEP2, ADEP3, ADEP4); two additional new analogs (IDR-10001 and IDR-10011) were also synthesized.

Fig 3

Overexpression of clpP1 and clpP2 in recombinant strains of M. tuberculosis. clpP1 and clpP2 were independently overexpressed in the WT background, and levels of transcript were analyzed via qRT-PCR. POP-1, clpP1 overexpressor strain; POP-2, clpP2 overexpressor strain. Transcript levels were standardized to sigA expression and compared to those in WT and are represented as fold change. Solid bars, clpP1 mRNA; white bars, clpP2 mRNA. Results are average of duplicates; error bars are standard deviations.

Fig 4

Increased ADEP activity against M. tuberculosis using efflux pump inhibitors. M. tuberculosis was grown in the presence of two different concentrations of ADEP2 only (■) or in combination with reserpine (▴), verapamil (*), or both (×) at concentrations of 12 μg/ml and 40 μg/ml, respectively. (A) Growth in 10 μg/ml ADEP2. Aerobic growth in stirred culture tubes was monitored over 16 days. (B) Growth in 15 μg/ml ADEP2. Aerobic growth in stirred culture tubes was monitored over 16 days. (C) MICs against compound IDR-10011 were determined against M. tuberculosis in liquid medium (in 96-well plates) in the presence/absence of 12 μg/ml reserpine and/or 40 μg/ml verapamil. The experiment was performed in triplicate, and results are presented as average with standard deviations.