Treatment of Clostridioides difficile infection (CDI) is expensive and complex, with a high proportion of patients suffering infection relapse (20-35%), and some having multiple relapses. A healthy, ...unperturbed gut microbiome provides colonisation resistance against CDI through competition for nutrients and space. However, antibiotic consumption can disturb the gut microbiota (dysbiosis) resulting in the loss of colonisation resistance allowing C. difficile to colonise and establish infection. A unique feature of C. difficile is the production of high concentrations of the antimicrobial compound para-cresol, which provides the bacterium with a competitive advantage over other bacteria found in the gut. p-cresol is produced by the conversion of para-Hydroxyphenylacetic acid (p-HPA) by the HpdBCA enzyme complex. In this study, we have identified several promising inhibitors of HpdBCA decarboxylase, which reduce p-cresol production and render C. difficile less able to compete with a gut dwelling Escherichia coli strain. We demonstrate that the lead compound, 4-Hydroxyphenylacetonitrile, reduced p-cresol production by 99.0 ± 0.4%, whereas 4-Hydroxyphenylacetamide, a previously identified inhibitor of HpdBCA decarboxylase, only reduced p-cresol production by 54.9 ± 13.5%. To interpret efficacy of these first-generation inhibitors, we undertook molecular docking studies that predict the binding mode for these compounds. Notably, the predicted binding energy correlated well with the experimentally determined level of inhibition, providing a molecular basis for the differences in efficacy between the compounds. This study has identified promising p-cresol production inhibitors whose development could lead to beneficial therapeutics that help to restore colonisation resistance and therefore reduce the likelihood of CDI relapse.
We describe an innovative system that exports diverse recombinant proteins in membrane-bound vesicles from E. coli. These recombinant vesicles compartmentalize proteins within a micro-environment ...that enables production of otherwise challenging insoluble, toxic, or disulfide-bond containing proteins from bacteria. The release of vesicle-packaged proteins supports isolation from the culture and allows long-term storage of active protein. This technology results in high yields of vesicle-packaged, functional proteins for efficient downstream processing for a wide range of applications from discovery science to applied biotechnology and medicine.
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•A simple peptide tag generates recombinant-protein-filled vesicles from E. coli•This approach allows production of recombinant protein at high yields•Enables production of disulfide-bond-containing and toxic proteins•Recombinant vesicles allow long-term storage of active soluble protein
The ability to reprogram a cell to direct the packaging of specific molecules into discrete membrane envelopes is one of the major challenges in the fields of synthetic biology and recombinant protein today. We thus set out to develop a system to allow the export of vesicle-packaged proteins from Escherichia coli. The resultant technology, involving a simple peptide tag, not only simplifies subsequent recombinant protein purification but the controlled packaging into membrane vesicles can be applied to the development of numerous technologies and commercializable products within the biotechnology and medical industries, including generation of recombinant bioreactors, environmental dispersion of biomolecules, and vehicles for drug delivery and vaccination, as well as providing a stable environment for isolation and storage of proteins.
Eastwood et al. describe a system for export of recombinant proteins in membrane-bound vesicles from E. coli. A simple peptide tag allows high-yield production of functional proteins within vesicle packages that simplify purification and enable long-term storage. This approach allows production of insoluble, toxic, and otherwise challenging proteins from bacteria.
The cell membrane, made up of a complex arrangement of proteins and lipids, is an integral component of cells and functions as a protective barrier around cells. Interactions with cell membranes can ...impact the membrane dynamics, integrity and morphology and therefore have biological consequences. The study of these interactions allows a deeper understanding of fundamental biological processes, particularly the vast number of membrane related proteins whose physiological function is still currently unknown. This information may also allow us to determine the pathogenesis of diseases associated with these membrane related proteins. It can be utilised in the development of effective therapeutic agents against these diseases, such as bacterial infections, due to the significance of membranes in cell survival.Thus, this project is focused on understanding the interactions between biotechnological and therapeutic molecules with cell membranes through the use of synthetic liposomes, and a range of membrane binding assays. In the first part of this project, the antimicrobial activity of a series of membrane binding supramolecular self-associating amphiphiles (SSAs) was determined. Stepwise modifications were made to these molecules to ascertain their structure-activity relationships which were then utilised to produce second generation antimicrobials with improved E. coli activity. The anionic geometry of SSAs heavily influenced the acidity of the molecules and subsequent intermolecular interactions, which in turn impacted the antimicrobial activity observed. Lipid binding of SSAs was confirmed through the use of a competitive binding microscopy assay and fluorescence anisotropy experiments. The second-generation antimicrobials were found to gelate in salt solutions; thus, fluorescence microscopy was employed to characterise the gelation properties of these SSAs.In the second part of this study, the lipid binding properties of the membrane related protein alpha synuclein (α-Syn), which has been implicated in Parkinson's Disease (PD) was investigated. In addition, the impact of the post-translational modifications acetylation and phosphorylation was also explored. Unacetylated and Nt-acetylated α-Syn exhibited high affinities to CL and PE lipids in the thermal shift assays conducted. Further, fluorescence anisotropy binding experiments revealed binding of acetylated and unacetylated α-Syn to POPA, a key signalling lipid. Additionally, unacetylated α-Syn exhibited increased lipid binding activities in comparison to acetylated α-Syn, suggesting negative regulation of α-Syn by Nt-acetylation. The final part of this project was concerned with the lipid binding interactions of the C-elegans myosin 1 homolog, Hum-1, specifically its TH1 domain. Hum-1 TH1 was found to predominantly interact with anionic lipids, and phosphorylation of Hum-1 TH1 increased binding to PA lipids. This project demonstrated the value of investigating membrane lipid interactions in order to further understand the native roles and regulation of membrane proteins and improve the activity of membrane binding therapeutic molecules.
Herein, we identify supramolecular self-associating amphiphiles (SSAs) as a novel class of antibacterials with activity towards methicillin-resistant Staphylococcus aureus. Structure-activity ...relationships have been identified in the solid, solution and gas phases. Finally, we show that when supplied in combination, SSAs exhibit increased antibacterial efficacy against these clinically relevant microbes.
Organophosphorus (OP) chemical warfare agents (CWAs) represent an ongoing threat but the understandable widespread prohibition of their use places limitations on the development of technologies to ...counter the effects of any OP CWA release. Herein, we describe new, accessible methods for the identification of appropriate molecular simulants to mimic the hydrogen bond accepting capacity of the P&z.dbd;O moiety, common to every member of this class of CWAs. Using the predictive methodologies developed herein, we have identified OP CWA hydrogen bond acceptor simulants for soman and sarin. It is hoped that the effective use of these physical property specific simulants will aid future countermeasure developments.
Using low-level computational modelling to predict solution state association constants and binding modes for the identification of appropriate CWA simulants.
Herein, we identify supramolecular self-associating amphiphiles (SSAs) as a novel class of antibacterials with activity towards methicillin-resistant
Staphylococcus aureus
. Structure-activity ...relationships have been identified in the solid, solution and gas phases. Finally, we show that when supplied in combination, SSAs exhibit increased antibacterial efficacy against these clinically relevant microbes.
The co-formulation of supramolecular self-associating amphiphiles (SSAs) enhances solution state physicochemical properties and increases efficacy against methicillin-resistant
Staphylococcus aureus
.
The co-formulation of supramolecular self-associating amphiphiles (SSAs) enhances solution state physicochemical properties and increases efficacy against methicillin-resistant
Staphylococcus aureus
....
Herein, we identify supramolecular self-associating amphiphiles (SSAs) as a novel class of antibacterials with activity towards methicillin-resistant
Staphylococcus aureus
. Structure–activity relationships have been identified in the solid, solution and gas phases. Finally, we show that when supplied in combination, SSAs exhibit increased antibacterial efficacy against these clinically relevant microbes.
Herein we report 50 structurally related supramolecular self‐associating amphiphilic (SSA) salts and related compounds. These SSAs are shown to act as antimicrobial agents, active against model ...Gram‐positive (methicillin‐resistant Staphylococcus aureus) and/or Gram‐negative (Escherichia coli) bacteria of clinical interest. Through a combination of solution‐state, gas‐phase, solid‐state and in silico measurements, we determine 14 different physicochemical parameters for each of these 50 structurally related compounds. These parameter sets are then used to identify molecular structure‐physicochemical property‐antimicrobial activity relationships for our model Gram‐negative and Gram‐positive bacteria, while simultaneously providing insight towards the elucidation of SSA mode of antimicrobial action.
The antimicrobial efficacy of 50 supramolecular self‐associating salts (SSAs) and related compounds was established against MRSA and E. coli. In addition, 14 different physicochemical properties were determined experimentally for each compound within this library. From these data we identified relationships between compound structure, physiochemical property, and antimicrobial activity.
Since their discovery, antimicrobial compounds have been vital for the treatment and prevention of disease; making many previously fatal diseases treatable or at worst, manageable conditions. The ...inappropriate use of these compounds has led to the rapid development of resistance mechanisms within bacteria to the majority of compounds currently marketed. A recent UK governmental review predicted that by 2050 global deaths caused by antimicrobial resistant bacteria will outnumber those attributed to cancer 1. As new resistance mechanisms emerge and resistance within microbial populations increases, so does the need to further understand the molecular basis of resistance, develop new antimicrobial molecules and use better strategies to manage their use 2. In response to this, we discovered a novel class of antimicrobials and have created 50 structurally related members of this class 3-6. We sought to understand the structure-activity relationships which will result in the determination of the mode of action of these molecules. Consequently, each variant was screened against
Staphylococcus aureus
and
Escherichia coli
and the minimum inhibitory concentration was calculated for effective compounds. This will enable us to identify predictive tools that will aid the synthesis of the next generation of these novel therapeutic molecules. We will present our latest findings in the ongoing analysis of the antimicrobial activity for each variant of this new class of antimicrobial compound. In addition, we will discuss the insights provided by the detailed structure-function analysis. This project is in collaboration with Public Health England and NHS East Kent Trust.
As a result of the looming antimicrobial resistance crisis, there is an urgent need for novel antimicrobial treatments. This is particularly true for hard‐to‐treat Gram‐negative bacteria, as many ...antimicrobial agents are unable to cross the cell membrane to gain access to the cell interior, and thus elicit a therapeutic response. Herein, evidence is provided of the use of anionic supramolecular self‐associating amphiphiles (SSAs) as antimicrobial efficacy enhancers for commonly used antimicrobial agents, to which there is known resistance, against Gram‐negative bacteria. The co‐administration of the SSAs with antimicrobials is shown to sensitize traditionally hard to treat Pseudomonas aeruginosa to both rifampicin and novobiocin, from which structure activity relationships can be elucidated. Quantitative fluorescence microscopy is performed, indicating membrane permeabilization to be the likely mode of action of drug efficacy enhancement by the SSAs. These results offer an alternative strategy in antimicrobial adjuvant design, expanding focus beyond cationic peptides and into the realm of anionic small molecules. Finally, the self‐assembly of the SSAs in the presence of these antimicrobials is investigated through a combination of quantitative NMR, tensiometry, dynamic light scattering, and zeta potential studies, demonstrating the impact of these agents on SSA self‐association events.
Antimicrobial resistance is reducing the number of therapies available to effectively treat bacterial infection. Here, a novel class of anionic, supramolecular self‐associating amphiphiles is reported to act as efficacy enhancers for the antibiotics rifampicin and novobiocin against Gram‐negative bacteria—specifically P. aeruginosa. Additionally, fluorescence microscopy is used to provide preliminary evidence of mode of action.
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