Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal.

Decades of previous efforts to develop renal-sparing polyene antifungals were misguided by the classic membrane permeabilization model1. Recently, the clinically vital but also highly renal-toxic small-molecule natural product amphotericin B was instead found to kill fungi primarily by forming extramembraneous sponge-like aggregates that extract ergosterol from lipid bilayers2-6. Here we show that rapid and selective extraction of fungal ergosterol can yield potent and renal-sparing polyene antifungals. Cholesterol extraction was found to drive the toxicity of amphotericin B to human renal cells. Our examination of high-resolution structures of amphotericin B sponges in sterol-free and sterol-bound states guided us to a promising structural derivative that does not bind cholesterol and is thus renal sparing. This derivative was also less potent because it extracts ergosterol more slowly. Selective acceleration of ergosterol extraction with a second structural modification yielded a new polyene, AM-2-19, that is renal sparing in mice and primary human renal cells, potent against hundreds of pathogenic fungal strains, resistance evasive following serial passage in vitro and highly efficacious in animal models of invasive fungal infections. Thus, rational tuning of the dynamics of interactions between small molecules may lead to better treatments for fungal infections that still kill millions of people annually7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids9.

A Versatile Strategy for Characterization and Imaging of Drip Flow Microbial Biofilms.

The inherent architectural and chemical complexities of microbial biofilms mask our understanding of how these communities form, survive, propagate, and influence their surrounding environment. Here we describe a simple and versatile workflow for the cultivation and characterization of model flow-cell-based microbial ecosystems. A customized low-shear drip flow reactor was designed and employed to cultivate single and coculture flow-cell biofilms at the air-liquid interface of several metal surfaces. Pseudomonas putida F1 and Shewanella oneidensis MR-1 were selected as model organisms for this study. The utility and versatility of this platform was demonstrated via the application of several chemical and morphological imaging techniques-including matrix-assisted laser desorption/ionization mass spectrometry imaging, secondary ion mass spectrometry imaging, and scanning electron microscopy-and through the examination of model systems grown on iron substrates of varying compositions. Implementation of these techniques in combination with tandem mass spectrometry and a two-step imaging principal component analysis strategy resulted in the identification and characterization of 23 lipids and 3 oligosaccharides in P. putida F1 biofilms, the discovery of interaction-specific analytes, and the observation of several variations in cell and substrate morphology present during microbially influenced corrosion. The presented workflow is well-suited for examination of both single and multispecies drip flow biofilms and offers a platform for fundamental inquiries into biofilm formation, microbe-microbe interactions, and microbially influenced corrosion.

Sterol Sponge Mechanism Is Conserved for Glycosylated Polyene Macrolides.

Amphotericin-like glycosylated polyene macrolides (GPMs) are a clinically and industrially important family of natural products, but the mechanisms by which they exert their extraordinary biological activities have remained unclear for more than half a century. Amphotericin B exerts fungicidal action primarily via self-assembly into an extramembranous sponge that rapidly extracts ergosterol from fungal membranes, but it has remained unclear whether this mechanism is applicable to other GPMs. Using a highly conserved polyene-hemiketal region of GPMs that we hypothesized to represent a conserved ergosterol-binding domain, we bioinformatically mapped the entirety of the GPM sequence-function space and expanded the number of GPM biosynthetic gene clusters (BGCs) by 10-fold. We further leveraged bioinformatic predictions and tetrazine-based reactivity screening targeting the electron-rich polyene region of GPMs to discover a first-in-class methyltetraene- and diepoxide-containing GPM, kineosporicin, and to assign BGCs to many new producers of previously reported members. Leveraging a range of structurally diverse known and newly discovered GPMs, we found that the sterol sponge mechanism of fungicidal action is conserved.