Biology subjects

Hind, C.

Publications and source records attributed to Hind, C..

4 recordsLinked to original sources

bioRxiv2024

Construction of a drug sensitised Candida auris strain

Candida auris is an emerging pathogenic fungus causing outbreaks of invasive disease in healthcare facilities worldwide with the majority of clinical isolates demonstrating intrinsic resistance to multiple drug classes. Therefore, there is a pressing need for new antifungals, but screening in wild-type C. auris strains may miss scaffolds which would be good subjects for med-chem projects as antifungals. Work presented here indicates that deletion of CDR1 and MDR1 in C. auris, respectively encoding an ATP-binding Cassette and a Major Facilitator Superfamily efflux pump, generates a strain that is hypersensitised to drugs and can act as a platform for antifungal drug screening. The cdr1{Delta}mdr1{Delta} mutant was 64- and 8- times more sensitive to fluconazole and voriconazole, respectively, compared with the wild-type strain. In a pilot study using a 2,000 compound library, use of the cdr1{Delta}mdr1{Delta} mutant identified over twice as many compounds compared to wild-type. When assessing the capacity to form biofilms, there was a modest difference in biomass between the wild-type and cdr1{Delta}mdr1{Delta} mutant, but there was little to no statistically significant difference observed when using a colorimetric dye that measured metabolic activity. Little to no statistically significant difference was observed between wild-type and the efflux pump mutant in virulence, as judged by the larval Galleria mellonella infectivity assay. Accordingly, the cdr1{Delta}mdr1{Delta} mutant will be useful when screening for drugs that limit formation of biofilms or limit fungal virulence, as these two properties are unaffected when drug efflux is compromised.

microbiology↗

bioRxiv2024

C7-substituted Quinolines as Potent Inhibitors of AdeG Efflux Pumps in Acinetobacter baumannii

Efflux, mediated by a series of multidrug efflux pumps, is a major contributor to antibiotic resistance in Gram-negative bacteria. Efflux pump inhibitors (EPIs), which can block efflux, have the potential to be used as adjuvant therapies to re-sensitize bacteria to existing antibiotics. In this study, 36 quinoline-based compounds were synthesized as potential EPIs targeting Resistance Nodulation Division (RND) family pumps in the multidrug-resistant pathogen Acinetobacter baumannii . In A. baumannii strains with overexpressed AdeFGH (chloramphenicol-adapted) and AdeABC (AYE, Ab5075-UW), these compounds enhanced Hoechst dye accumulation, indicating general efflux inhibition, and potentiated chloramphenicol which is an AdeG substrate. The research focused on two generations of quinoline compounds, with modifications at the C-7 position of first-generation compounds to improve hydrophobic interactions with the Phe loop in the AdeG efflux pump, to generate second-generation compounds. The modified quinolines showed strong pump inhibition and significant chloramphenicol potentiation, with MIC reductions of 4- to 64-fold. Notably, compounds 1.8 and 3.8 exhibited the highest inhibitory activity, while compounds 1.3 and 3.3 showed up to 64-fold potentiation, highlighting the importance of specific structural features at the C-7 position for efflux pump inhibition. The study also revealed selective inhibition of AdeFGH over AdeABC, with no potentiation observed for gentamicin, showing the specificity of these quinoline- based inhibitors. Importantly, the compounds showed no toxicity in a Galleria mellonella model at a 50 mg/kg dose level, highlighting their suitability as potential antibiotic adjuvants for combating bacterial resistance.

microbiology↗

bioRxiv2023

A cell-free strategy for profiling intracellular antibiotic sensitivity and resistance

Antimicrobial resistance (AMR) is a pandemic spread across multiple priority infectious disease threats. While the cell envelope plays a key role in AMR, this also makes it challenging to study how antibiotics function inside the cell. Herein, we present a Klebsiella pneumoniae cell-free gene expression (CFE) platform for the rapid profiling of intracellular antibiotic sensitivity and resistance. This cell-free approach provides the unique macromolecular and metabolite components from this microbe, which include multiple antibiotic targets from transcription, translation, and metabolic processes. First, we compare the K. pneumoniae CFE system to whole cell antimicrobial assays. We find that several antibiotic classes show higher sensitivity in the CFE system, suggesting limitations in antibiotic transport in the whole cell assay. Next, we evolved K. pneumoniae strains with resistance to specific antibiotics and use whole genome sequencing analysis for genotyping. As an exemplary case, we show that a single RNA polymerase beta subunit variant H526L (also frequently found in multidrug resistant Mycobacterium tuberculosis) confers a 58-fold increase in CFE resistance to rifampicin. Overall, we describe a safe (i.e., non-living, non-pathogenic) platform suitable for studying an infectious disease model in a Containment Level 1 laboratory. Our CFE strategy is generalisable to laboratory and clinical K. pneumoniae strains and provides a new experimental tool to profile intracellular AMR variants. In conclusion, our CFE tool provides a significant advance towards understanding AMR and complements wider infectious disease studies.

microbiology↗

bioRxiv2018

Parallel evolution of frog antimicrobial peptides produces identical conformations but subtly distinct membrane and antibacterial activities

Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Despite the high similarity in physical properties and preference for adopting secondary amphipathic, -helix conformations in membrane mimicking milieu, their spectrum of activity and potency often varies considerably. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of species specific effective antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, -helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes; the greater conformational flexibility and the higher amplitude channel conductance induced offers a rationale for the greater potency and broader spectrum of activity of temporin L over aurein 2.5. Specific contributions from individual residues are identified that define the mechanisms of action of each AMP. Our findings suggest AMPs in frogs are examples of parallel evolution whose utility is based on apparently similar but subtly distinct mechanisms of action.

biophysics↗