Biology subjects

Elliott, A. G.

Publications and source records attributed to Elliott, A. G..

3 recordsLinked to original sources

bioRxiv2026

Harnessing Diacylglycerol-Terminated Cationic Oligomers for Next-Generation Antibacterial Therapeutics

Cationic polymers, which mimic the structure of antimicrobial peptides (AMPs), are increasingly recognized as promising antimicrobial materials. Here, we report the synthesis and evaluation of a new class of cationic lipid-terminated oligomers (CLOs), comprised of 2C18-hydrophobic lipid tails, and short oligomeric cationic chains synthesised via Cu(0)-mediated reversible-deactivation radical polymerization (RDRP). Two 2-vinyl-4,4-dimethyl-5-oxazolone (VDM) oligomers with degrees of polymerization (DP) of 20 or 50 were synthesized using the lipid functional initiator (R)-3-((2-bromo-2-methylpropanoyl) oxy)propane-1,2-diyl dioctadecanoate (2C18-Br). Post-polymerization modification of the pendant oxazolone moieties was carried out using reactive amines, including N-Boc-ethylenediamine (BEDA) and N,N-dimethylethylenediamine (DMEN). Subsequent deprotection of the BEDA groups and quaternization of DMEN groups enabled the synthesis of six functional CLOs exhibiting distinct cationic functionalities. Antimicrobial assays against a panel of WHO bacterial and fungal priority pathogens (methicillin-resistant Staphylococcus aureus [MRSA], Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Candida albicans, and Cryptococcus neoformans) revealed that these CLOs exhibited potent and selective structure-dependent antibacterial activity, particularly against MRSA, with minimum inhibitory concentrations (MICs) in the clinically relevant range, below 4 {micro}g mL-1, comparable to antibiotics vancomycin and colistin. Among these, BEDA-functionalized CLOs demonstrated the strongest antimicrobial profile, which was significantly increased by increasing DP, as evidenced by a reduction in MIC values from 64 {micro}g mL-1 (for DP20) to [≤] 4 {micro}g mL-1 (for DP50) against A. baumannii. Biocompatibility assays against red blood cells and HEK293 cells indicated negligible toxicity, with haemolytic (HC50) and cytotoxic (CC50) values exceeding 512 {micro}g mL-1 across all CLOs. All CLOs displayed minimal activity against C. albicans (MIC [≥] 512 {micro}g mL-1). In contrast, activity against C. neoformans was influenced by both cationic functionality and DP, with DMEN-based CLOs exhibited superior antifungal activity at higher DP relative to their BEDA-based counterparts. Most CLOs displayed high selectivity (SI) toward MRSA (SI >128), while 2C18-O(BEDA)50 exhibited the broadest spectrum, showing potent antimicrobial activity and high selectivity against E. coli (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), A. baumannii (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), and MRSA (MIC [≤] 4 {micro}g mL-1, SI [≥] 128), along with moderate activity against P. aeruginosa (MIC = 32 {micro}g mL-1, SI > 16). Taken together, these findings elucidate the combined influence of end-group lipidation, cationic functionality, and polymer length in modulating antimicrobial activity, thereby establishing 2C18-terminated CLOs as a rationally tunable and biocompatible platform for antimicrobial material development.

microbiology↗

bioRxiv2024

Characterisation of in vitro resistance selection against second-/last-line antibiotics in methicillin-resistant Staphylococcus aureus

SYNOPSISO_ST_ABSBackgroundC_ST_ABSThe increasing occurrence of MRSA clinical isolates harbouring reduced susceptibility to mainstay antibiotics has escalated the use of second and last line antibiotics. Hence, it is critical to evaluate the likelihood of MRSA developing clinical resistance to these antibiotics. ObjectivesOur study sought to identify the rate in which MRSA develop resistance to vancomycin, daptomycin and linezolid in vitro and further determine the mechanisms underpinning resistance. MethodsMRSA was exposed to increasing concentrations of vancomycin, daptomycin, and linezolid for 20 days, with eight replicates for each antibiotic conducted in parallel. The resulting day 20 (D20) isolates were subjected to antimicrobial susceptibility testing, whole genome sequencing, autolysis assays, and growth curves to determine bacterial fitness. ResultsExposure to vancomycin or linezolid for 20 days resulted in a subtle two-fold increase in the MIC, whereas daptomycin exposure yielded daptomycin-nonsusceptible isolates with up to 16-fold MIC increase. The MIC increase was accompanied by variable changes in relative fitness and reduced resistance to autolysis in some isolates. D20 isolates harboured mutations in genes commonly associated with resistance to the respective antibiotics (e.g. walK for vancomycin, mprF and rpoB for daptomycin, rplC for linezolid), along with several previously unreported variants. Introduction of key mutations to these identified genes in the parental strain via allelic exchange confirmed their role in the development of resistance. ConclusionsIn vitro selection against vancomycin, daptomycin, or linezolid resulted in the acquisition of mutations similar to those correlated with clinical resistance, including the associated phenotypic alterations.

microbiology↗

bioRxiv2018

The macrocyclizing protease butelase 1 remains auto-catalytic and reveals the structural basis for ligase activity

Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform seed storage protein maturation upon cleavage dependent auto-activation in the low pH environment of storage vacuoles. AEPs have attracted attention for their macrocyclization reactions and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP, we show butelase 1 retains cleavage functions in vitro and in vivo. The in vivo rescue was incomplete, consistent with some trade-off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEPs, suggesting the many differences in its peptide binding region are the source of its efficient macrocyclization. All considered, it seems either butelase 1 has not fully specialized or a requirement for auto-catalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs.

biophysics↗