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

Epifadin leads to rapid cell lysis of <i>S. aureus</i>

Epifadin leads to rapid cell lysis of S. aureus. S. aureus USA300 JE2 cells were applied to an agarose pad, onto which 2 µL of extracts (50 mg/mL) of the epifadin producer IVK83 ((E9.czi) ) or ∆efiTP ((D1_4h_time.czi) had been previously spotted. Image acquisition was started in the surrounding of the respective extract spot 15 min after S. aureus application. The agarose contained FM4-64 (red, 0.25 µg/mL, membrane dye) and Sytox Green (green, 0.25 µM, only visible upon membrane barrier malfunction).</p

Antibacterial marinopyrroles and pseudilins act as protonophores

Elucidating the mechanism of action (MoA) of antibacterial natural products is crucial to evaluating their potential as novel antibiotics. The marinopyrroles, pentachloropseudilin, and pentabromopseudilin are densely halogenated, hybrid pyrrole-phenol natural products with potent activity against Gram-positive bacterial pathogens like Staphylococcus aureus. However, the exact way in which they exert this antibacterial activity has not been established. In this study, we explore their structure-activity relationship, determine their spatial location in bacterial cells, and investigate their MoA. We show that the natural products share a common MoA based on membrane depolarization and dissipation of the proton motive force (PMF) that is essential for cell viability. The compounds show potent protonophore activity, but do not appear to destroy the integrity of the cytoplasmic membrane via the formation of larger pores or interfere with the stability of the peptidoglycan sacculus. Thus, our current model for the antibacterial MoA of marinopyrrole, pentachloropseudilin, and pentabromopseudilin stipulates that the acidic compounds insert into the membrane and transport protons inside the cell. This MoA may explain many of the deleterious biological effects in mammalian cells, plants, phytoplankton, viruses, and protozoans that have been reported for these compounds

Commensal production of a broad-spectrum and short-lived antimicrobial peptide polyene eliminates nasal Staphylococcus aureus.

Antagonistic bacterial interactions often rely on antimicrobial bacteriocins, which attack only a narrow range of target bacteria. However, antimicrobials with broader activity may be advantageous. Here we identify an antimicrobial called epifadin, which is produced by nasal Staphylococcus epidermidis IVK83. It has an unprecedented architecture consisting of a non-ribosomally synthesized peptide, a polyketide component and a terminal modified amino acid moiety. Epifadin combines a wide antimicrobial target spectrum with a short life span of only a few hours. It is highly unstable under in vivo-like conditions, potentially as a means to limit collateral damage of bacterial mutualists. However, Staphylococcus aureus is eliminated by epifadin-producing S. epidermidis during co-cultivation in vitro and in vivo, indicating that epifadin-producing commensals could help prevent nasal S. aureus carriage. These insights into a microbiome-derived, previously unknown antimicrobial compound class suggest that limiting the half-life of an antimicrobial may help to balance its beneficial and detrimental activities

Commensal production of a broad-spectrum and short-lived antimicrobial peptide polyene eliminates nasal Staphylococcus aureus

Antagonistic bacterial interactions often rely on antimicrobial bacteriocins, which attack only a narrow range of target bacteria. However, antimicrobials with broader activity may be advantageous. Here we identify an antimicrobial called epifadin, which is produced by nasal Staphylococcus epidermidis IVK83. It has an unprecedented architecture consisting of a non-ribosomally synthesized peptide, a polyketide component and a terminal modified amino acid moiety. Epifadin combines a wide antimicrobial target spectrum with a short life span of only a few hours. It is highly unstable under in vivo-like conditions, potentially as a means to limit collateral damage of bacterial mutualists. However, Staphylococcus aureus is eliminated by epifadin-producing S. epidermidis during co-cultivation in vitro and in vivo, indicating that epifadin-producing commensals could help prevent nasal S. aureus carriage. These insights into a microbiome-derived, previously unknown antimicrobial compound class suggest that limiting the half-life of an antimicrobial may help to balance its beneficial and detrimental activities.</p