ClpC1-targeting peptide natural products differentially dysregulate the proteome of Mycobacterium tuberculosis.
Targeting the protein quality control system in Mycobacterium tuberculosis offers a powerful and largely untapped opportunity for antibiotic development. The ClpC1:ClpP1P2 protease is an essential component of the system and is involved in both regulatory and stress-related protein degradation. Several non-ribosomal peptide natural product families have been discovered, including ecumicin, ilamycins (also called rufomycins) and cyclomarins, which bind to the ClpC1 chaperone component of the system and possess potent antimycobacterial activity. This has led to significant interest in these molecules, and the ClpC1:ClpP1P2 system more generally, as a bona fide target for the development of new tuberculosis drugs. In this study, we apply a systematic and multi-layered approach combining quantitative proteomics, bioinformatics, transcriptomic profiling, CRISPRi gene knockdown, and targeted biochemical and biophysical assays to dissect the mechanisms of ecumicin, ilamycin, and cyclomarin in clinically relevant M. tuberculosis. Strikingly, despite exhibiting similar modes to ClpC1, each natural product elicits distinct substrate-specific effects on protein-degradation. We further show that derivatives of ilamycin and ecumicin engage the stress-response chaperones ClpC2 and Hsp20 respectively, albeit with varied sequestration capacity. Together our data reveal that these natural products dysregulate ClpC1 substrate recognition and perturb stress response chaperones in distinct ways, highlighting opportunities to target multiple nodes of protein quality control for the development of next generation antimycobacterials. RNA isolation, library preparation, and sequencing Exponentially growing Mtb H37Rv cultures were treated with MIC90 Ecu* (1) and incubated for a further 48 h at 37 C. The Mtb cells were then centrifuged at 3725 g for 10 min to pellet bacteria. Pellets were resuspended with 1 mL TRIzol and transferred to a 2 mL O-ring tube with acid-washed glass beads. To disrupt the mycobacterial cell envelope, cells were subjected to six cycles of bead beating (20 s at 4600 rpm and 40 s on ice) with a (Biopec products) mini beadbeater. Samples were then clarified at 18000 g for 1 min. After a short room temp incubation chloroform was added to each sample and samples were inverted by hand for 15 seconds and incubated at room temperature for 3 min. Samples were then centrifuged at 12000 g for 15 min at 4 C. The upper aqueous phase containing RNA was collected. RNA was purified from the aqueous phase with the Isolate II RNA micro kit. Libraries were prepared using the (Illumina) Total RNA library kit with Ribo-Zero Plus chemistry for rRNA depletion and sequenced on an (Illumina) NovaSeq 6000 platform to generate 150 bp paired-end reads (>65 million pairs per sample).