Pyrrolomycins Are Potent Natural Protonophores

Abstract

The escalating burden of antibiotic drug resistance necessitates research into novel classes of antibiotics and their mechanism of action. Pyrrolomycins are a family of potent natural product antibiotics with nanomolar activity against Gram-positive bacteria, yet with an elusive mechanism of action. In this work, we dissect the apparent Gram-positive specific activity of pyrrolomycins and show that Gram-negative bacteria are equally sensitive to pyrrolomycins when drug efflux transporters are removed and that albumin in medium plays a large role in pyrrolomycin activity. The selection of resistant mutants allowed for the characterization and validation of a number of mechanisms of resistance to pyrrolomycins in both Staphylococcus aureus and an Escherichia coli ΔtolC mutant, all of which appear to affect compound penetration rather than being target associated. Imaging of the impact of pyrrolomycin on the E. coli ΔtolC mutant using scanning electron microscopy showed blebbing of the bacterial cell wall often at the site of bacterial division. Using potentiometric probes and an electrophysiological technique with an artificial bilayer lipid membrane, it was demonstrated that pyrrolomycins C and D are very potent membrane-depolarizing agents, an order of magnitude more active than conventional carbonyl cyanide m-chlorophenylhydrazone (CCCP), specifically disturbing the proton gradient and uncoupling oxidative phosphorylation via protonophoric action. This work clearly unveils the until-now-elusive mechanism of action of pyrrolomycins and explains their antibiotic activity as well as mechanisms of innate and acquired drug resistance in bacteria.

BrevicidineB, a New Member of the Brevicidine Family, Displays an Extended Target Specificity

“…The dye DiSC 3 (5) (3,3’-dipropylthiadicarbocyanine iodide) is widely used in the mode of action studies of antibiotics ( Rink et al, 2007 ; Jangra et al, 2019 ; Valderrama et al, 2019 ; Stokes et al, 2020 ). DiSC 3 (5) accumulates in the cytoplasmic membrane in response to the Δψ component of the proton motive force and self-quenches its fluorescence ( Wu et al, 1999 ; Stokes et al, 2020 ).…”

Fluorescein Derivatives as Antibacterial Agents Acting via Membrane Depolarization

“…The main arguments in favor of Mitchell's chemiosmotic theory were based on the ability of uncouplers to cause the collapse of the membrane potential by carrying protons across membranes. At present, there is a revival of interest in uncouplers because of the growing evidence of their broad-spectrum therapeutic efficacy [1][2][3][4][5][6][7][8][9], including antibacterial potency [10][11][12][13][14][15][16][17][18][19], which was actually found very early [20]. To this end, a series of esters of fluorescein were obtained and studied at our laboratory, manifesting themselves as rather effective mitochondrial uncouplers [21][22][23].…”

New Synthetic Nitro-Pyrrolomycins as Promising Antibacterial and Anticancer Agents

“…PM-C can be defined as a tetrachlorine compound since it features two chlorine atoms on both the pyrrolic and the phenolic moieties; differently, the synthetic PM 1 contains five bromines on its scaffold, and this represents the most active pyrrolomycin against Staphylococcus aureus synthesized so far [5]. Pyrrolomycins are recognized as molecules with diverse biological activities; indeed, they show potent antimicrobial and antibiofilm properties mainly against Gram-positive bacteria [5][6][7][8][9][10][11]. Besides, some of them display anthelmintic activity [12], as well as neuro-modulatory [13], immunemodulatory [14], insecticidal [15] and anticancer properties.…”

“…PMs are thought to explicate their function by binding sortase A in the Gram-positive strain S. aureus [9,20,21]. Recently, PMs, such as PM-C and PM-D, were shown to be powerful depolarizing membrane agents capable of specifically disturbing the proton gradient and decoupling oxidative phosphorylation by protonophoric action [11]. Although PM-C resulted more effective than PM-D in depolarizing the membrane potential, the biological activity of PM-D on both bacterial and cancer cells was greater, likely for its increased lipophilicity and therefore ability to permeate the membrane [11].…”