Biology subjects

Winterhalter, M.

Publications and source records attributed to Winterhalter, M..

4 recordsLinked to original sources

bioRxiv2025

Aetokthonotoxin, the causative agent of vacuolar myelinopathy, uncouples oxidative phosphorylation due to protonophore activity

Aetokthonotoxin (AETX) is an emerging environmental toxin produced by the freshwater cyanobacterium Aetokthonos hydrillicola. Accumulating in the food chain, it causes vacuolar myelinopathy, a neurological disease affecting a wide range of wildlife characterized by the development of large intra-myelinic vacuoles in the white matter of the brain. So far, the mode of action of AETX is unknown. After discovering that AETX is cytostatic and arrests cancer cell lines in G1-phase, metabolomic profiling of AETX-treated cells as well as an assessment of the physico-chemical properties of the compound suggested that AETX is a weakly acidic uncoupler of mitochondrial respiration. We confirmed this hypothesis by in vitro assays on mammalian cells, finding that AETX has the expected effects on the mitochondrial network morphology, mitochondrial membrane potential, and oxygen consumption rates, resulting in affected ATP generation. We confirmed that AETX is capable of transporting protons across lipid bilayers. In summary, we demonstrate that AETX is a protonophore that uncouples oxidative phosphorylation in mitochondria, the primary event of AETX intoxication. Significance statementAetokthonotoxin (AETX) is an emerging cyanotoxin. Produced by the cyanobacterium Aetokthonos hydrillicola, it is transferred through the food chain, affects the nervous system, and eventually causes mortality in animals of various taxa. Our finding that AETX is an unspecific uncoupler of mitochondrial respiration implies that it might also be harmful for human health upon ingestion and trophic accumulation. First steps towards a full risk assessment are needed. An important aspect in this regard is the elucidation of the toxins mode of action. We anticipate our findings to be a starting point for the development of an adverse outcome pathway addressing the formation of vacuolar myelinopathy, expanding the significance of our results to the future risk assessment of other environmental neurotoxins.

pharmacology and toxicology↗

bioRxiv2022

Uptake of aminoglycosides through outer membrane porins in Escherichia coli

Aminoglycosides are important clinical antibiotics but their molecular uptake mechanism is still not completely understood. Here we quantify and compare the passive transport of three aminoglycosides (kanamycin, gentamicin, and amikacin) across general or sugar specific porins of Escherichia coli (OmpF, OmpC, LamB and ChiP). Our analysis revealed that permeation of aminoglycosides (Kanamycin/Gentamycin/Amikacin) is about the same through ChiP ({approx}5/3/2 molecules/s), OmpF ({approx}10/15/<1 molecules/s) and OmpC ({approx}11/8/<1 molecules/s). In contrast, LamB of smaller pore diameter has no significant permeation ([≤]1/1/1 molecules/s, all values recalculated for a gradient of 10 {micro}M). Biological assays confirmed the relevance of these translocations for antibiotic potency.

biophysics↗

bioRxiv2022

Single TolC-AcrA complex formation monitoredby time dependent single-channel electrophysiology

Characterizing protein-protein interaction on a single molecular level is a challenge, experimentally as well as the interpretation of the data. For example, Gram-negative bacteria contain protein complexes spanning the outer and inner cell wall being able to efflux effectively cell toxic substances. Within the search for antibiotics with novel modes of action, inhibition of the efflux pump assembly is an interesting possible target. Recent seminal work revealed the high-resolution structure of the tripartic composition TolC-AcrA-AcrB. Here, we reconstitute a single TolC homotrimer into a planar lipid membrane and follow the assembly of AcrA using the modulation of the ion current through TolC during binding. In particular, the presence of AcrA increases the average ionic current through TolC and, moreover, reduces the ion-current fluctuations caused by flickering of TolC. Here, we demonstrate that statistical properties of ion-current fluctuations (the power spectral density) provide a complementary measure of the interaction of the TolC-AcrA complex with potential inhibitors. Both characteristics, the average current and the current noise, taken into consideration together, provide more robust information.

biophysics↗

bioRxiv2018

A multidisciplinary approach towards identification of novel antibiotic scaffolds for Acinetobacter baumannii

Research efforts to discover potential new antibiotics for Gram-negative bacteria suffer from high attrition rates due to the synergistic action of efflux systems and the limited permeability of the outer membrane (OM). One potential strategy to overcome the OM permeability barrier is to identify small molecules that are natural substrates for abundant OM channels, and to use such compounds as scaffolds for the design of efficiently-permeating antibacterials. Here we present a multidisciplinary approach to identify such potential small-molecule scaffolds. Focusing on the pathogenic bacterium Acinetobacter baumannii, we use OM proteomics to identify DcaP as the most abundant channel under various conditions that are relevant for infection. High-resolution X-ray structure determination of DcaP surprisingly reveals a trimeric, porin-like structure and suggests that dicarboxylic acids are potential transport substrates. Electrophysiological experiments and allatom molecular dynamics simulations confirm this notion and provide atomistic information on likely permeation pathways and energy barriers for several small molecules, including a clinically-relevant {beta}-lactamase inhibitor. Our study provides a general blueprint for the identification of molecular scaffolds that will inform the rational design of future antibacterials.

microbiology↗