The existence of steroid antibacterials has been studied since the golden age of antibiotics, although the literature on this topic is fragmented. To address this the current review seeks to bring these fragmented lines of enquiry together to assess the utility of steroidal compounds as antibacterials. In doing so this review will document the structural features, biosynthetic/synthetic origins, antibacterial toxicities, potential for fungal biotransformations, and catabolic pathways in the human host. Herein, we identify steroid antibacterials from fungal origins, synthetic steroids, and approved drugs that have the potential for future repurposing as antibacterials. Of particular note are the fusidane steroids of fungal origin, displaying low MIC values and being effective against a variety of Gram-positive and Gram-negative species. At present, fusidic acid is the only steroid that is prescribed as an antibacterial, despite many publications describing antibacterial activity in a range of well-known steroids. The impact of bacterial exposure to steroids naturally differs depending on the nature of the cell membranes, cell wall, and the ability of bacterial to import steroids for use as a carbon source, which is discussed herein. Also, this study explores the biotechnological potential of fungi for the production of antibacterial steroids, and how steroid drugs are metabolised in the human host. Together, this provides an assessment of the potential for steroids as future antibacterial drugs.

Natural products are valuable starting points for drug discovery, although individual modes of action are often difficult to pin down. Ursanes such as madecassic acid have been shown to have antibacterial properties, but a variety of mechanisms have been proposed. In this paper, we report previously uninvestigated activity against cytochrome bd oxidases which are only found in prokaryotes and are therefore promising new targets, using madecassic acid and a set of synthetically modified derivatives. Our work shows that madecassic acid and its derivatives can block activity of these enzymes, while phenotypic effects in membrane and whole organism assays are more complex, consistent with modulation of multiple pathways depending upon molecular structure. This provides a new route to ursane-based antibacterial action while highlighting the importance of chemical modifications in fine-tuning biological activity of natural products.

BACKGROUND:Cytochrome bd complexes are respiratory oxidases found exclusively in prokaryotes that are important during infection for numerous bacterial pathogens. METHODS:In silico docking was employed to screen approved drugs for their ability to bind to the quinol site of Escherichia coli cytochrome bd-I. Respiratory inhibition was assessed with oxygen electrodes using membranes isolated from E. coli and methicillin-resistant Staphylococcus aureus strains expressing single respiratory oxidases (ie, cytochromes bd, bo', or aa3). Growth/viability assays were used to measure bacteriostatic and bactericidal effects. RESULTS:The steroid drugs ethinylestradiol and quinestrol inhibited E. coli bd-I activity with median inhibitory concentration (IC50) values of 47 ± 28.9 µg/mL (158 ± 97.2 µM) and 0.2 ± 0.04 µg/mL (0.5 ± 0.1 µM), respectively. Quinestrol inhibited growth of an E. coli "bd-I only" strain with an IC50 of 0.06 ± 0.02 µg/mL (0.2 ± 0.07 µM). Growth of an S. aureus "bd only" strain was inhibited by quinestrol with an IC50 of 2.2 ± 0.43 µg/mL (6.0 ± 1.2 µM). Quinestrol exhibited potent bactericidal effects against S. aureus but not E. coli. CONCLUSIONS:Quinestrol inhibits cytochrome bd in E. coli and S. aureus membranes and inhibits the growth of both species, yet is only bactericidal toward S. aureus.

It is well-known that antibiotics target energy-consuming processes and a significant body of research now supports the conclusion that the metabolic state of bacteria can have a profound impact upon the efficacy of antibiotics. Several articles implicate bacterial energetics and the respiratory inhibitor nitric oxide (NO) in this process, although pinpointing the precise mechanism for how NO can diminish the potency of a range of antibiotics through modulating bacterial energy metabolism has proved challenging. Herein, we introduce the role of NO during infection, consider known links between NO and antibiotic efficacy, and discuss potential mechanisms via which NO present at the site of infection could mediate these effects through controlling bacterial energetics. This perspective article highlights an important relationship between NO and antibiotic action that has largely been overlooked and outlines future considerations for the development of new drugs and therapies that target bacterial energy metabolism.

In this study, we identify a novel two-component system in Acinetobacter baumannii (herein named AmsSR for regulator of alternative metabolic systems) only present in select gammaproteobacterial and betaproteobacterial species. Bioinformatic analysis revealed that the histidine kinase, AmsS, contains 14 predicted N-terminal transmembrane domains and harbors a hybrid histidine kinase arrangement in its C-terminus. Transcriptional analysis revealed the proton ionophore CCCP selectively induces PamsSR expression. Disruption of amsSR resulted in decreased intracellular pH and increased depolarization of cytoplasmic membranes. Transcriptome profiling revealed a major reordering of metabolic circuits upon amsR disruption, with energy generation pathways typically used by bacteria growing in limited oxygen being favored. Interestingly, we observed enhanced growth rates for mutant strains in the presence of glucose, which led to overproduction of pyruvate. To mitigate the toxic effects of carbon overflow, we noted acetate overproduction in amsSR-null strains, resulting from a hyperactive Pta-AckA pathway. Additionally, due to altered expression of key metabolic genes, amsSR mutants favor an incomplete TCA cycle, relying heavily on an overactive glyoxylate shunt. This metabolic reordering overproduces NADH, which is not oxidized by the ETC; components of which were significantly downregulated upon amsSR disruption. As a result, the mutants almost exclusively rely on substrate phosphorylation for ATP production, and consequently display reduced oxygen consumption in the presence of glucose. Collectively, our data suggests that disruption of amsSR affects the function of the aerobic respiratory chain, impacting the energy status of the cell, which in turn upregulates alternative metabolic and energy generation pathways.

Iron-bound cyclic tetrapyrroles (hemes) are redox-active cofactors in bioenergetic enzymes. However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here, we used cellular, biochemical, structural and computational methods to characterize the structure and function of the heterodimeric bacterial ABC transporter CydDC. We provide multi-level evidence that CydDC is a heme transporter required for functional maturation of cytochrome bd , a pharmaceutically relevant drug target. Our systematic single-particle cryogenic-electron microscopy approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and occlusion. Our simulations reveal that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to rotate 180°.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Antimicrobial resistance is one of the greatest threats to human health. Gram-positive methicillin resistant Staphylococcus aureus (MRSA), in both its planktonic and biofilm form, is of particular concern. Herein we identify the hydrogelation properties for a series of intrinsically fluorescent, structurally related supramolecular self-associating amphiphiles and determine their efficacy against both planktonic and biofilm forms of MRSA. To further explore the potential translation of this hydrogel technology for real-world applications, the toxicity of the amphiphiles was determined against the eukaryotic multicellular model organism, Caenorhabditis elegans. Due to the intrinsic fluorescent nature of these supramolecular amphiphiles, material characterisation of their molecular self-associating properties included; comparative optical density plate reader assays, rheometry and widefield fluorescence microscopy. This enabled determination of amphiphile structure and hydrogel sol dependence on resultant fibre formation.

It is well known that loss of aerobic respiration in Gram-negative bacteria can diminish the efficacy of a variety of bactericidal antibiotics, which has lead to subsequent demonstrations that the formation of reactive oxygen species (ROS) and the proton motive force (PMF) can both play a role in antibiotic toxicity. The susceptibility of Gram-negative bacteria to aminoglycoside antibiotics, particularly gentamicin, has previously been linked to both the production of ROS and the rate of antibiotic uptake that is mediated by the PMF, although the relative contributions of ROS and PMF to aminoglycoside toxicity has remained poorly understood. Herein, gentamicin was shown to elicit a very modest increase in ROS levels in an aerobically grown Escherichia coli clinical isolate. The well-characterised uncoupler 2,4-dinitrophenol (DNP) was used to disrupt the PMF, which resulted in a significant decrease in gentamicin lethality towards E. coli . DNP did not significantly alter respiratory oxygen consumption, supporting the hypothesis that this uncoupler does not increase ROS production via elevated respiratory oxidase activity. These observations support the hypothesis that maintenance of PMF rather than induction of ROS production underpins the mechanism for how the respiratory chain potentiates the toxicity of aminoglycosides. This was further supported by the demonstration that the uncoupler DNP elicits a dramatic decrease in gentamicin lethality under anaerobic conditions. Together, these data strongly suggest that maintenance of the PMF is the dominant mechanism for the respiratory chain in potentiating the toxic effects of aminoglycosides.

In recent years, much attention has been focused on the biogenesis, engineering and utilisation of outer membrane vesicles (OMVs) in Gram-negative bacteria in a range of environments and niches. While the precise mechanism of biogenesis is unknown, it is focused on the modification of the Gram-negative cell wall to facilitate blebbing at sites of weakness in and around the characteristically thin peptidoglycan layer within the periplasm. Here, we investigate the biogenesis of membrane vesicles (MVs) in the Gram-positive organism Streptomyces albus S4 (Seipke et al. J Bacteriol 193:4270–4271, 2011 and Fazal et al. Antonie Van Leeuwenhoek 113:511–520, 2020). The S. albus S4 strain is an antifungal (candicidin and antimycin) producing organism that was isolated from attine ants (Barke et al. BMC Biol 8:109, 2010). The biogenesis and characterisation of S. albus S4 MVs is demonstrated using the wild-type (WT) and mutant strains ΔantC (no antimycin production) ΔfscC (no candicidin production) and ΔantC ΔfscC (produces neither antimycin nor candicidin). Here, we have shown that the S. albus S4 strain produces MVs and that these are comprised of both specific protein profiles and secondary metabolites, with a clear demonstration of the ability to selectively package one antifungal (candicidin) but not the other (antimycin).

Nitric oxide was once considered to be of marginal interest to the biological sciences and medicine; however, there is now wide recognition, but not yet a comprehensive understanding, of its functions and effects. NO is a reactive, toxic free radical with numerous biological targets, especially metal ions. However, NO and its reaction products also play key roles as reductant and oxidant in biological redox processes, in signal transduction, immunity and infection, as well as other roles. Consequently, it can be sensed, metabolized and modified in biological systems. Here, we present a brief overview of the chemistry and biology of NO—in particular, its origins in geological time and in contemporary biology, its toxic consequences and its critical biological functions. Given that NO, with its intrinsic reactivity, appeared in the early Earth’s atmosphere before the evolution of complex lifeforms, we speculate that the potential for toxicity preceded biological function. To examine this hypothesis, we consider the nature of non-biological and biological targets of NO, the evolution of biological mechanisms for NO detoxification, and how living organisms generate this multifunctional gas.

Following the discovery of streptomycin from Streptomyces griseus in the 1940s by Selman Waksman and colleagues, aminoglycosides were first used to treat tuberculosis and then numerous derivatives have since been used to combat a wide variety of bacterial infections. These bactericidal antibiotics were used as first-line treatments for several decades but were largely replaced by ß-lactams and fluoroquinolones in the 1980s, although widespread emergence of antibiotic-resistance has led to renewed interest in aminoglycosides. The primary site of action for aminoglycosides is the 30 S ribosomal subunit where they disrupt protein translation, which contributes to widespread cellular damage through a number of secondary effects including rapid uptake of aminoglycosides via elevated proton-motive force (PMF), membrane damage and breakdown, oxidative stress, and hyperpolarisation of the membrane. Several factors associated with aminoglycoside entry have been shown to impact upon bacterial killing, and more recent work has revealed a complex relationship between metabolic states and the efficacy of different aminoglycosides. Hence, it is imperative to consider the environmental conditions and bacterial physiology and how this can impact upon aminoglycoside entry and potency. This mini-review seeks to discuss recent advances in this area and how this might affect the future use of aminoglycosides.

Iron-bound cyclic tetrapyrroles (hemes) are key redox-active cofactors in membrane-integrated oxygen reductases and other bioenergetic enzymes. However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here, we used a combination of cellular, biochemical, structural and computational methods to resolve ongoing controversies around the function of the heterodimeric bacterial ABC transporter CydDC. We provide multi-level evidence that CydDC is a heme transporter required for assembly and functional maturation of cytochrome bd, a pharmaceutically relevant drug target. Our systematic single-particle cryo-EM approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and occlusion. Our simulations reveal that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to flip 180 degrees. The membrane-accessible heme entry site of CydDC is primarily controlled by the conformational plasticity of CydD transmembrane helix 4, the extended cytoplasmic segment of which also couples heme confinement to a rotational movement of the CydC nucleotide-binding domain. Our cryo-EM data highlight that this signal transduction mechanism is necessary to drive conformational transitions toward occluded and outward-facing states. One Sentence Summary The heterodimeric bacterial ABC transporter CydDC is a heme flippase essential for the functional maturation of cytochrome bd.

The spread of multidrug-resistance in Gram-negative bacterial pathogens presents a major clinical challenge, and new approaches are required to combat these organisms. Nitric oxide (NO) is a well-known antimicrobial that is produced by the immune system in response to infection, and numerous studies have demonstrated that NO is a respiratory inhibitor with both bacteriostatic and bactericidal properties. However, given that loss of aerobic respiratory complexes is known to diminish antibiotic efficacy, it was hypothesised that the potent respiratory inhibitor NO would elicit similar effects. Indeed, the current work demonstrates that pre-exposure to NO-releasers elicits a > tenfold increase in IC50 for gentamicin against pathogenic E. coli (i.e. a huge decrease in lethality). It was therefore hypothesised that hyper-sensitivity to NO may have arisen in bacterial pathogens and that this trait could promote the acquisition of antibiotic-resistance mechanisms through enabling cells to persist in the presence of toxic levels of antibiotic. To test this hypothesis, genomics and microbiological approaches were used to screen a collection of E. coli clinical isolates for antibiotic susceptibility and NO tolerance, although the data did not support a correlation between increased carriage of antibiotic resistance genes and NO tolerance. However, the current work has important implications for how antibiotic susceptibility might be measured in future (i.e. ± NO) and underlines the evolutionary advantage for bacterial pathogens to maintain tolerance to toxic levels of NO.

Herein, we present the synthesis of two novel adamantane appended supramolecular self-associating amphiphiles. The antimicrobial efficacy of these compounds is determined against both clinically relevant Gram-positive methicillin-resistant Staphylococcus aureus and Gram-negative Escherichia coli. We also explore the self-associative properties of these amphiphiles in both polar organic DMSO-d(6) 0.5% H2O and H2O (D2O)/EtOH 19:1 solutions, confirming aggregate stability through the determination of zeta potential values, aggregate size through a combination of H-1 NMR DOSY and dynamic light scattering studies as appropriate, and critical aggregate concentration through the derivation of concentration-dependent surface tension values. We also perform a variety of H-1 NMR dilution studies and in-silico modelling to further explore the roles of intermolecular hydrogen bonding and lipophilicity within aggregate formation and antimicrobial efficacy. Finally, we perform haemolysis and Galleria mellonella toxicity assays to establish the potential of these compounds to undergo further development as antibiotic agents.

When working with anaerobic bacteria it is important to have the capability to perform parallel bioreactor growth experiments that are both controllable and reproducible, although capital and consumables costs for commercially available systems are often prohibitively high. Hence, a three-vessel parallel bioreactor system was designed and constructed that has the capabilities for batch and fed batch processes and can also be set up for continuous culture at a fraction of the cost of commercial systems. This system carries over many of the same functionalities of those systems with a higher price point of entry, including in-line monitoring of temperature, pH, and redox poise. To validate the performance of this system Clostridium saccharoperbutylacetonicum was grown under conditions that promote ABE fermentation, an established industrial process used to produce the solvents acetone, butanol and ethanol. Measurements of cell density, pH, and redox poise all confirmed reproducible culture conditions for these parallel vessels, and solvent quantitation via GCMS verified consistent metabolic activities for the separate cultures. In future, this system will be of interest to researchers that require high performance parallel fermentation platforms but where commercial systems are not accessible.

SummaryThe development and advent of mutagenesis tools for solventogenic clostridial species in recent years has allowed for the increased refinement of industrially relevant strains. In this study we have utilised CLEAVE™, a CRISPR/Cas genome editing system developed by Green Biologics Ltd., to engineer a strain of Clostridium saccharoperbutylacetonicum N1‐4(HMT) with potentially useful solvents titres and energy metabolism. As one of two enzymes responsible for the conversion of glyceraldehyde‐3‐phosphate (GAP) to 3‐phosphoglyceric acid in glycolysis, it was hypothesised that deletion of gapN would increase ATP and NADH production that could in turn improve solvent production. Herein, whole genome sequencing has been used to evaluate CLEAVE™ and the successful knockout of gapN, demonstrating a clean knockout with no other detectable variations from the wild type sequence. Elevated solvent levels were detected during the first 24 h of batch fermentation, indicating an earlier shift to solventogenesis. A 2.4‐fold increase in ATP concentration was observed, and quantitation of NAD(P)H derivatives revealed a more reducing cytoplasm for the gapN strain. These findings expand our understanding of clostridium carbon metabolism and report a new approach to optimising biofuel production.

Fundamental aspects of outer membrane vesicle (OMV) biogenesis and the engineering of producer strains have been major research foci for many in recent years. The focus of this study was OMV production in a variety of Escherichia coli strains including wild type (WT) (K12 and BW25113), mutants (from the Keio collection) and proprietary [BL21 and BL21 (DE3)] strains. The present study investigated the proteome and prospective mechanism that underpinned the key finding that the dominant protein present in E. coli K-12 WT OMVs was fimbrial protein monomer (FimA) (a polymerizable protein which is the key structural monomer from which Type 1 fimbriae are made). However, mutations in genes involved in fimbriae biosynthesis (Δ fimA , B , C , and F ) resulted in the packaging of flagella protein monomer (FliC) (the major structural protein of flagella) into OMVs instead of FimA. Other mutations (Δ fimE, G, H, I , and Δ lrhA –a transcriptional regulator of fimbriation and flagella biosynthesis) lead to the packaging of both FimA and Flagellin into the OMVs. In the majority of instances shown within this research, the production of OMVs is considered in K-12 WT strains where structural appendages including fimbriae or flagella are temporally co-expressed throughout the growth curve as shown previously in the literature. The hypothesis, proposed and supported within the present paper, is that the vesicular packaging of the major FimA is reciprocally regulated with the major FliC in E. coli K-12 OMVs but this is abrogated in a range of mutated, non-WT E. coli strains. We also demonstrate, that a protein of interest (GFP) can be targeted to OMVs in an E. coli K-12 strain by protein fusion with FimA and that this causes normal packaging to be disrupted. The findings and underlying implications for host interactions and use in biotechnology are discussed.

The scsABCD (suppressor of copper sensitivity) locus of Salmonella encodes four proteins that resemble the disulfide folding machinery of other bacteria. Previous work has shown that Salmonella encounters toxic levels of copper during infection and the Scs system provides protection against this copper‐mediated toxicity. The current work reports that expression of the soluble periplasmic protein StScsC is induced by copper and that intramacrophage survival in the presence of copper is diminished by the loss of StScsC. Using a combination of genetic and proteomic approaches, the abundance of various cysteine‐containing periplasmic proteins was found to be elevated by StScsC in the Salmonella periplasm, implicating StScsC in the disulfide folding of superoxide dismutases and proteins involved in amino acid sensing and import. Co‐purification and mass spectrometry approaches confirmed that the arginine‐sensing periplasmic protein ArtI associates with StScsC via a disulfide interaction, and purified ArtI was shown to alter the thiol redox state of purified StScsC. This work reports the first demonstration of a redox partner for the Scs system of Salmonella and provides insights into how this bacterial pathogen responds to copper stress during infection.

The CydDC family of ABC transporters export the low molecular weight thiols glutathione and cysteine to the periplasm of a variety of bacterial species. The CydDC complex has previously been shown to be important for disulfide folding, motility, respiration, and tolerance to nitric oxide and antibiotics. In addition, CydDC is thus far unique amongst ABC transporters in that it binds a haem cofactor that appears to modulate ATPase activity. CydDC has a diverse impact upon bacterial metabolism, growth, and virulence, and is of interest to those working on membrane transport mechanisms, redox biology, aerobic respiration, and stress sensing/tolerance during infection.