Bacteriomimetic Liposomes Improve Antibiotic Activity of a Novel Energy-Coupling Factor Transporter Inhibitor

Liposomes have been studied for decades as nanoparticulate drug delivery systems for cytostatics, and more recently, for antibiotics. Such nanoantibiotics show improved antibacterial efficacy compared to the free drug and can be effective despite bacterial recalcitrance. In this work, we present a loading method of bacteriomimetic liposomes for a novel, hydrophobic compound (HIPS5031) inhibiting energy-coupling factor transporters (ECF transporters), an underexplored antimicrobial target. The liposomes were composed of DOPG (18:1 (Δ9-cis) phosphatidylglycerol) and CL (cardiolipin), resembling the cell membrane of Gram-positive Staphylococcus aureus and Streptococcus pneumoniae, and enriched with cholesterol (Chol). The size and polydispersity of the DOPG/CL/± Chol liposomes remained stable over 8 weeks when stored at 4 °C. Loading of the ECF transporter inhibitor was achieved by thin film hydration and led to a high encapsulation efficiency of 33.19% ± 9.5% into the DOPG/CL/Chol liposomes compared to the phosphatidylcholine liposomes (DMPC/DPPC). Bacterial growth inhibition assays on the model organism Bacillus subtilis revealed liposomal HIPS5031 as superior to the free drug, showing a 3.5-fold reduction in CFU/mL at a concentration of 9.64 µM. Liposomal HIPS5031 was also shown to reduce B. subtilis biofilm. Our findings present an explorative basis for bacteriomimetic liposomes as a strategy against drug-resistant pathogens by surpassing the drug-formulation barriers of innovative, yet unfavorably hydrophobic, antibiotics.

Structure-Guided Optimization of Small-Molecule Folate Uptake Inhibitors Targeting the Energy-Coupling Factor Transporters

Here, we report on a potent class of substituted ureidothiophenes targeting energy-coupling factor (ECF) transporters, an unexplored target, which is not addressed by any antibiotic on the market. Since the ECF module is crucial for the vitamin transport mechanism, prevention of substrate uptake should ultimately lead to cell death. By utilizing a combination of virtual and functional whole-cell screening of our in-house library, the membrane-bound protein mediated uptake of folate could be effectively inhibited. Structure-based optimization of our hit compound yielded low-micromolar inhibitors, whereby the most active compounds showed in addition potent antimicrobial activities against a panel of clinically relevant Gram-positive pathogens without significant cytotoxic effects.

An efficient way to screen inhibitors of energy-coupling factor (ECF) transporters in bacteria uptake assay

Herein, we report a novel whole-cell screening assay using Lactobacillus casei as model microorganism to identify inhibitors of energy-coupling factor (ECF) transporters. This promising and underexplored target may have important pharmacological potential through modulation of vitamin homeostasis in bacteria and, importantly, it is absent in humans. The assay represents an alternative, cost-effective and fast solution to demonstrate the direct involvement of these membrane transporters in a native biological environment rather than using a low-throughput in vitro assay employing reconstituted proteins in a membrane bilayer system. Based on this new whole-cell screening approach, we demonstrated the optimization of a weak hit compound (2) into a small molecule (3) with improved in vitro and whole-cell activities. This study opens the possibility to quickly identify novel inhibitors of ECF transporters and optimize them based on structure–activity relationships.

Targeting the energy-coupling factor (ECF) transporters: identification of new tool compounds

The energy-coupling factor (ECF) transporters are a family of transmembrane proteins involved in the uptake of vitamins in a wide range of bacteria. Inhibition of the activity of these proteins could reduce the viability of pathogens that depend on vitamin uptake. Their central role in the metabolism of bacteria and absence in humans make the ECF transporters a potential antibacterial target, which can be further investigated making use of a selective chemical probe. Here, we report on the virtual screening, design, synthesis, structure–activity relationships (SARs) and coarse-grained molecular dynamics simulations of the first class of inhibitors of the ECF transporters. We investigated the mechanism of action of this chemical class and profiled the best hit compounds regarding their pharmaceutical properties. The optimized hit has a minimum inhibitory concentration (MIC) value of 2 µg/mL against Streptococcus pneumoniae, which opens up the possibility to use this chemical class to investigate the role of the ECF transporters in health and disease.

Pushing the Envelope: The Mysterious Journey Through the Bacterial Secretory Machinery, and Beyond

Gram-negative bacteria are contained by an envelope composed of inner and outer-membranes with the peptidoglycan (PG) layer between them. Protein translocation across the inner membrane for secretion, or insertion into the inner membrane is primarily conducted using the highly conserved, hourglass-shaped channel, SecYEG: the core-complex of the Sec translocon. This transport process is facilitated by interactions with ancillary subcomplex SecDF-YajC (secretion) and YidC (insertion) forming the holo-translocon (HTL). This review recaps the transport process across the inner-membrane and then further explores how delivery and folding into the periplasm or outer-membrane is achieved. It seems very unlikely that proteins are jettisoned into the periplasm and left to their own devices. Indeed, chaperones such as SurA, Skp, DegP are known to play a part in protein folding, quality control and, if necessary degradation. YfgM and PpiD, by their association at the periplasmic surface of the Sec machinery, most probably are also involved in some way. Yet, it is not entirely clear how outer-membrane proteins are smuggled past the proteases and across the PG to the barrel-assembly machinery (BAM) and their final destination. Moreover, how can this be achieved, as is thought, without the input of energy? Recently, we proposed that the Sec and BAM translocons interact with one another, and most likely other factors, to provide a conduit to the periplasm and the outer-membrane. As it happens, numerous other specialized proteins secretion systems also form trans-envelope structures for this very purpose. The direct interaction between components across the envelope raises the prospect of energy coupling from the inner membrane for active transport to the outer-membrane. Indeed, this kind of long-range energy coupling through large inter-membrane assemblies occurs for small molecule import (e.g., nutrient import by the Ton complex) and export (e.g., drug efflux by the AcrAB-TolC complex). This review will consider this hypothetical prospect in the context of outer-membrane protein biogenesis.

Coupling mechanism of materials and screen surface motion in multi-stage variable inclination equal thickness screening process

Multi-stage variable inclination equal-thickness screen (MSVIETS) is widely used in separating coal and mineral particles because of its large production capacity and good screening performance. In this study, the kinematic characteristics of infeed and outfeed ends surface under the conditions of load and no load was investigated by using a high-speed camera analysis system. The motion speed of the screen surface at the infeed end was approximately 5 times higher under load than under no-load conditions, and motion speed of the screen surface at the outfeed end was approximately 4 times higher than under no-load conditions. The mechanism of coupled motion of material and screen surface in the process of multi-stage variable inclination equal thickness screening was elucidated, and the energy coupling transfer law was “strong in and weak out”.

Stormtime Energetics: Energy Transport Across the Magnetopause in a Global MHD Simulation

Coupling between the solar wind and magnetosphere can be expressed in terms of energy transfer through the separating boundary known as the magnetopause. Geospace simulation is performed using the Space Weather Modeling Framework (SWMF) of a multi-ICME impact event on February 18–20, 2014 in order to study the energy transfer through the magnetopause during storm conditions. The magnetopause boundary is identified using a modified plasma β and fully closed field line criteria to a downstream distance of −20Re. Observations from Geotail, Themis, and Cluster are used as well as the Shue 1998 model to verify the simulation field data results and magnetopause boundary location. Once the boundary is identified, energy transfer is calculated in terms of total energy flux K, Poynting flux S, and hydrodynamic flux H. Surface motion effects are considered and the regional distribution of energy transfer on the magnetopause surface is explored in terms of dayside X>0, flank X<0, and tail cross section X=Xmin regions. It is found that total integrated energy flux over the boundary is nearly balanced between injection and escape, and flank contributions dominate the Poynting flux injection. Poynting flux dominates net energy input, while hydrodynamic flux dominates energy output. Surface fluctuations contribute significantly to net energy transfer and comparison with the Shue model reveals varying levels of cylindrical asymmetry in the magnetopause flank throughout the event. Finally existing energy coupling proxies such as the Akasofu ϵ parameter and Newell coupling function are compared with the energy transfer results.