Listeria monocytogenes utilizes the ClpP1/2 proteolytic machinery for fine-tuned substrate degradation at elevated temperatures.

Listeria monocytogenes exhibits two ClpP isoforms (ClpP1/ClpP2) which assemble into a heterooligomeric complex with enhanced proteolytic activity. Herein, we demonstrate that the formation of this complex depends on temperature and reaches a maximum ratio of about 1 : 1 at 30 °C, while almost no complex formation occurred below 4 °C. In order to decipher the role of the two isoforms at elevated temperatures, we constructed L. monocytogenes ClpP1, ClpP2 and ClpP1/2 knockout strains and analyzed their protein regulation in comparison to the wild type (WT) strain via whole proteome mass-spectrometry (MS) at 37 °C and 42 °C. While the ΔclpP1 strain only altered the expression of very few proteins, the ΔclpP2 and ΔclpP1/2 strains revealed the dysregulation of many proteins at both temperatures. These effects were corroborated by crosslinking co-immunoprecipitation MS analysis. Thus, while ClpP1 serves as a mere enhancer of protein degradation in the heterocomplex, ClpP2 is essential for ClpX binding and functions as a gatekeeper for substrate entry. Applying an integrated proteomic approach combining whole proteome and co-immunoprecipitation datasets, several putative ClpP2 substrates were identified in the context of different temperatures and discussed with regards to their function in cellular pathways such as the SOS response.

Total synthesis and mechanism of action of the antibiotic armeniaspirol A.

Emerging antimicrobial resistance urges the discovery of antibiotics with unexplored, resistance-breaking mechanisms. Armeniaspirols represent a novel class of antibiotics with a unique spiro[4.4]non-8-ene scaffold and potent activities against Gram-positive pathogens. We report a concise total synthesis of (±) armeniaspirol A in six steps with a yield of 20.3% that includes the formation of the spirocycle through a copper-catalyzed radical cross-coupling reaction. In mechanistic biological experiments, armeniaspirol A exerted potent membrane depolarization, accounting for the pH-dependent antibiotic activity. Armeniaspirol A also disrupted the membrane potential and decreased oxygen consumption in mitochondria. In planar lipid bilayers and in unilamellar vesicles, armeniaspirol A transported protons across membranes in a protein-independent manner, demonstrating that armeniaspirol A acted as a protonophore. We provide evidence that this mechanism might account for the antibiotic activity of multiple chloropyrrole-containing natural products isolated from various origins that share a 4-acylphenol moiety coupled to chloropyrrole as a joint pharmacophore. We additionally describe an efflux-mediated mechanism of resistance against armeniaspirols.