Thrane, S. | Search bioRxiv

bioRxiv2022

Synergistic Activity of Repurposed Peptide Drug Glatiramer Acetate with Tobramycin Against Cystic Fibrosis Pseudomonas aeruginosa

Pseudomonas aeruginosa is the most common pathogen infecting the lungs of people with cystic fibrosis (CF), causing both acute and chronic infections. Intrinsic and acquired antibiotic resistance, coupled with the physical barriers resulting from desiccated CF sputum, allow P. aeruginosa to colonise and persist in spite of antibiotic treatment. As well as the specific difficulties in eradicating P. aeruginosa from CF lungs, P. aeruginosa is also subject to the wider, global issue of antimicrobial resistance. Glatiramer acetate (GA) is a peptide drug, used in the treatment of multiple sclerosis (MS), which has been shown to have moderate anti-pseudomonal activity. Other antimicrobial peptides (AMPs) have been shown to be antibiotic resistance breakers; potentiating the activities of antibiotics when given in combination restoring and/or enhancing antibiotic efficacy. Growth, viability, minimum inhibitory concentration (MIC)-determination and synergy analysis showed that GA improved the efficacy of TOB against reference strains of P. aeruginosa, reducing TOB MICs and synergising with the aminoglycoside. This was also the case for clinical strains from people with CF. GA significantly reduced the concentration of TOB required to inhibit 50% (MIC50) of viable cells (from 1.69 [95%CI 0.26-8.97] to 0.62 [95%CI 0.15-3.94] mg/L, p=0.002) and inhibit 90% (MIC90) (from 7.00 [95%CI 1.18-26.50] to 2.20 [95%CI 0.99-15.03] mg/L, p=0.001) compared with TOB-only. Investigating mechanisms of GA activity showed that GA resulted in significant disruption of outer membranes, depolarisation of cytoplasmic membranes and permeabilisation of P. aeruginosa and was the only agent tested (including cationic AMPs) to significantly affect all three.

microbiology↗

bioRxiv2019

Structure-guided design of a synthetic mimic of an EPCR-binding PfEMP1 protein

Structure-guided vaccine design provides a route to elicit a focused immune response against the most functionally important regions of a pathogen surface. This can be achieved by identifying epitopes for neutralizing antibodies through structural methods and recapitulating these epitopes by grafting their core structural features onto smaller scaffolds. In this study, we have conducted a modified version of this protocol. We focused on the PfEMP1 protein family found on the surfaces of erythrocytes infected with Plasmodium falciparum. A subset of PfEMP1 proteins bind to endothelial protein C receptor (EPCR), and their expression correlates with development of the symptoms of severe malaria. Structural studies revealed the PfEMP1 to present a helix-kinked-helix motif which forms the core of the EPCR binding site. Using Rosetta-based design we successfully grafted this motif onto a three-helical bundle scaffold. We show that this synthetic binder interacts with EPCR with nanomolar affinity and adopts the expected structure. We also assessed its ability to bind to antibodies found in immunized animals and in humans from malaria endemic regions. Finally, we tested its capacity to effectively elicit antibodies that prevent EPCR binding and analysed the degree of cross-reactivity of these antibodies across a diverse repertoire of EPCR-binding PfEMP1. This provides a case study of immunogen design, assessing the effect of designing a focused immunogen that contains the core features of a ligand binding site, rather than those of a neutralizing antibody epitope.

microbiology↗