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Abstract

Over the last century, antibiotics have played a pivotal role in global health. The ever-growing threat of multidrug-resistant organisms, coupled with a declining antibiotic pipeline, has led to an urgent need for novel antimicrobial agents. One bacterial pathogen of concern is Pseudomonas aeruginosa. The World Health Organisation has declared it a "critical priority" pathogen for developing new antibiotics against it. It is intrinsically resistant to many antibiotics and is an opportunistic pathogen.

Antimicrobial peptides (AMPs) have been touted as potential novel antimicrobial agents. They are present in all kingdoms of life and play a vital role in the innate immunity of mammals. Two streams of antimicrobial peptides were evaluated. First, in silico, artificially designed short peptides (nine amino acids in length), which were predicted to be bioactive against P. aeruginosa and be non-haemolytic, were selected. Second, naturally occurring peptides (4 – 17 amino acids in length) from the Antimicrobial Peptide Database 3 (APD3) database, classified as antibacterial, were chosen.

Each library was synthesised using the Spot technique and screened against a luminescent P. aeruginosa strain and human erythrocytes. Promising candidates were identified with potent antimicrobial activity against P. aeruginosa (MIC 4 – 8 μg/mL) and low haemolytic toxicity (HC50 200 – >512 μg/mL). To create new libraries, three peptides from the novel and one from the natural peptide stream were subjected to amino acid substitutions. The libraries were screened as above, with an additional screen in human serum and a multidrug-resistant Escherichia coli isolate, which revealed no promising candidate. Further engineering strategies were applied to a novel peptide which varying success.

Mode of action studies revealed that both novel and natural AMPs rapidly kill P. aeruginosa. Biological Small-Angle X-Ray Scattering (BioSAXS) revealed that peptides and conventional antibiotics mechanistic actions could be differentiated upon exposure to bacteria. Visualisation of bacteria treated with natural and novel peptides revealed disruption to the membrane and intracellular changes. Furthermore, resistant studies revealed that a novel peptide, peptide 7, does not easily induce bacterial resistance.

Peptides were evaluated in the presence of simulated human physiological conditions to predict systemic in vivo activity. In vivo evaluation revealed only mild toxicity at high concentrations but unfortunately could not rescue the wax moth larvae, Galleria mellonella, from P. aeruginosa infection, although it did delay the infection.

In conclusion, this study demonstrated that using the Spot synthesis technology can identify potent peptides against P. aeruginosa and minimal mammalian cell toxicity in vitro. Moreover, bioinformatic analysis and machine learning can help predict new potent peptides. In addition, this study showed a potential of a novel application of the BioSAXS to differentiate modes of action for P. aeruginosa and enable faster decision making to develop novel antimicrobials more efficiently.

Publication Type:	Thesis (Doctoral)
Subjects:	R Medicine > R Medicine (General) R Medicine > RB Pathology R Medicine > RM Therapeutics. Pharmacology
Departments:	School of Health & Medical Sciences > Infection and Immunity Research Institute School of Health & Medical Sciences > School of Health & Medical Sciences Doctoral Theses Doctoral Theses

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