Generation of Lasso Peptide-Based ClpP Binders

The Clp protease system fulfills a plethora of important functions in bacteria. It consists of a tetradecameric ClpP barrel holding the proteolytic centers and two hexameric Clp-ATPase rings, which recognize, unfold, and then feed substrate proteins into the ClpP barrel for proteolytic degradation. Flexible loops carrying conserved tripeptide motifs protrude from the Clp-ATPases and bind into hydrophobic pockets (H-pockets) on ClpP. Here, we set out to engineer microcin J25 (MccJ25), a ribosomally synthesized and post-translationally modified peptide (RiPP) of the lasso peptide subfamily, by introducing the conserved tripeptide motifs into the lasso peptide loop region to mimic the Clp-ATPase loops. We studied the capacity of the resulting lasso peptide variants to bind to ClpP and affect its activity. From the nine variants generated, one in particular (12IGF) was able to activate ClpP from Staphylococcus aureus and Bacillus subtilis. While 12IGF conferred stability to ClpP tetradecamers and stimulated peptide degradation, it did not trigger unregulated protein degradation, in contrast to the H-pocket-binding acyldepsipeptide antibiotics (ADEPs). Interestingly, synergistic interactions between 12IGF and ADEP were observed

Synergetic Antimicrobial Activity and Mechanism of Clotrimazole-Linked CO-Releasing Molecules

This work was financially supported by Fundação para a Ciência e Tecnologia (Portugal) through fellowship PD/BD/ 148006/2019 (SSM), PTDC/SAU-INF/29313/2017 grant, and R&D unit LISBOA-01-0145-FEDER007660 (MostMicro) cofounded by FCT/MCTES and FEDER funds under the PT2020 Partnership Agreement. The NMR data was acquired at CERMAX, Instituto de Tecnologia Quıḿ ica e Bioloǵ ica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal, with equipment funded by FCT, project AAC 01/ SAICT/2016. This work was partially supported by the PPBIPortuguese Platform of BioImaging (PPBI-POCI-01- 0145-FEDER-022122) cofunded by national funds from OE “Orçamento de Estado” and by European funds from FEDER“Fundo Europeu de Desenvolvimento Regional”. LMS and SSM acknowledge funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 810856. H.B.-O., T.S., C.M., F.O., J.B., and M.A. gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), project ID 398967434 (TRR 261, projects A01, A06, A10, and Z02). A.B. appreciates funding by the German Federal Ministry for Education and Research (project Gramneg. Design).Several metal-based carbon monoxide-releasing molecules (CORMs) are active CO donors with established antibacterial activity. Among them, CORM conjugates with azole antibiotics of type [Mn(CO)3(2,2′-bipyridyl)(azole)]+ display important synergies against several microbes. We carried out a structure-activity relationship study based upon the lead structure of [Mn(CO)3(Bpy)(Ctz)]+ by producing clotrimazole (Ctz) conjugates with varying metal and ligands. We concluded that the nature of the bidentate ligand strongly influences the bactericidal activity, with the substitution of bipyridyl by small bicyclic ligands leading to highly active clotrimazole conjugates. On the contrary, the metal did not influence the activity. We found that conjugate [Re(CO)3(Bpy)(Ctz)]+ is more than the sum of its parts: while precursor [Re(CO)3(Bpy)Br] has no antibacterial activity and clotrimazole shows only moderate minimal inhibitory concentrations, the potency of [Re(CO)3(Bpy)(Ctz)]+ is one order of magnitude higher than that of clotrimazole, and the spectrum of bacterial target species includes Gram-positive and Gram-negative bacteria. The addition of [Re(CO)3(Bpy)(Ctz)]+ to Staphylococcus aureus causes a general impact on the membrane topology, has inhibitory effects on peptidoglycan biosynthesis, and affects energy functions. The mechanism of action of this kind of CORM conjugates involves a sequence of events initiated by membrane insertion, followed by membrane disorganization, inhibition of peptidoglycan synthesis, CO release, and break down of the membrane potential. These results suggest that conjugation of CORMs to known antibiotics may produce useful structures with synergistic effects that increase the conjugate's activity relative to that of the antibiotic alone.publishersversionpublishe

Reprogramming of the Caseinolytic Protease by ADEP Antibiotics: Molecular Mechanism, Cellular Consequences, Therapeutic Potential

Rising antibiotic resistance urgently calls for the discovery and evaluation of novel antibiotic classes and unique antibiotic targets. The caseinolytic protease Clp emerged as an unprecedented target for antibiotic therapy 15 years ago when it was observed that natural product-derived acyldepsipeptide antibiotics (ADEP) dysregulated its proteolytic core ClpP towards destructive proteolysis in bacterial cells. A substantial database has accumulated since on the interaction of ADEP with ClpP, which is comprehensively compiled in this review. On the molecular level, we describe the conformational control that ADEP exerts over ClpP, the nature of the protein substrates degraded, and the emerging structure-activity-relationship of the ADEP compound class. On the physiological level, we review the multi-faceted antibacterial mechanism, species-dependent killing modes, the activity against carcinogenic cells, and the therapeutic potential of the compound class

Purification of the Caseinolytic protease ClpP.

The caseinolytic protease P (ClpP) is an essential protein for bacteria and bacterial derived organelles. It belongs to the AAA+ family of proteases and has proteolytic activity, thereby, it plays an important role in protein digestion. This protease is involved in essential processes such as, cellular regulatory mechanisms, protein homeostasis, responses to environmental stimuli and host infections.The ClpP complex is formed by two principal components. In one hand, two heptameric rings of ClpP forms the proteolytic core, with catalytic activity. The second major component is the Clp-ATPases, which forms an hexameric ring in both edges of the complex. The function of this ATPase is selecting the substrate for degradation. There are different Clp-ATPases depending on the organism, and each of them recognise a different substrate. For a further specificity, the Clp-ATPase interacts with adaptor proteins.In humans, the only Clp-ATPase interacting with ClpP is ClpX. The human Clp system plays its role inside the mitochondrial matrix, therefore, a transport of both ClpP and ClpX to the mitochondrial matrix is required for the function of the protease. For this transport to happen, both proteins are first translated as premature proteins, with a Mitochondrial Target Sequence (MTS). This sequence will be then recognised by the cell machinery to transport the proteins inside the mitochondrial matrix. Afterwards, the MTS sequence will be cleaved when the proteins pass into the matrix.In this study, the proteins of the human Clp system, as well as the Clp protease of S. aureus, were purified through the Äkta Column, using the Strep-tag purification technique. Prior to the purification, E. coli cells were transformed with a pET plasmid containing the gen of interest, and then the expression of the protein was induced with IPTG. Later, the purification through the column was performed.Finally, after the purification, the protein concentration was measured, and the proteins were stored at -80°C for further in vitro assays. Afterwards, the purification results were also confirmed by doing an SDS-PAGE Electrophoresis.As a conclusion, this study fathom in the understanding of Clp protease system expression in the cells, in order to use this knowledge in future studies with this complex.<br /