Development of novel antimicrobial agents, and revealing their mechanism of action

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1. Please give a brief summary of your work.

Since their discovery, antimicrobial peptides (AMPs) have been seen as novel promising antimicrobial therapeutics because of their wide-range of activities, long evolutionary history and low potential of developing resistance against them. AMPs offer ideal templates to design novel biocidal agents, however, many naturally occurring AMPs are not optimized and require modifications to improve their therapeutic potential. In our lab, we design AMPs based on wheat antimicrobial proteins called puroindolines (PINs) with improved activity and biocompatibility properties. Our designed peptides have many potential applications in agriculture. We also investigate the mechanism of action of these PIN-based peptides.

2. Describe your approach and broader findings.

AMPs are an integral part of the innate immune system of many organisms. Many AMPs show broad-spectrum activity against bacteria, fungi, viruses, protozoa, and some are also effective against cancer cells. At present, more than 2500 natural antimicrobial peptides have been registered into the antimicrobial peptide database (APD). However, there have been only few cases of successful use of AMPs in agriculture, food industry, aquaculture and animal husbandry. There are several difficulties that need to be overcome to produce stable, functional and cost effective AMPs. Short AMPs with broad spectrum antimicrobial activity, metabolic stability and low cytotoxicity on normal mammalian cells are highly desired. Naturally occurring peptides with all these attributes are very rare. Analogs or synthetic derivatives have successfully been developed on the basis of natural peptide patterns.


I started working on AMPs when I was a Master student at Swinburne University in 2013 and then, I was awarded a Swinburne University Postgraduate Research Award (SUPRA) to work on my PhD project. The title of my research project is “Development of novel antimicrobial agents” under the supervision of Prof. Enzo Palumbo, Prof. Mrinal Bhave and Assoc. Prof. Andrew Clayton. I rationally designed a number of Tryptophan-rich AMPs (TRPs) based on the unique Trp-rich domain (TRD) of the wheat grain PIN proteins. The peptides displayed potent antimicrobial activities and maintained these activities under physiological conditions.

AMPs apply simultaneous and multiple, independent or cooperative actions that are probably resulted in their generally quick and potent antimicrobial activities. The mechanism by which AMPs act is a complex issue and there is a significant gap in our understanding of how they exert their microbicidal activities. We applied advanced and unique biophysical techniques such as time-lapse fluorescence lifetime imaging microscopy (FLIM) and time-lapse fluorescence microscopy, in combined with biochemical and microbiological techniques to study the mechanism of PuroA peptide (a synthetic Peptide based on PIN protein, which displays potent antimicrobial activities) on living microorganism cells. Our results disclosed the interaction kinetics of PuroA peptide with its biological targets, the detailed sequence of events during attack of the pathogenic Candida albicans cells by PuroA, and the main target that causes cell death.

*The attached video shows seventy five minutes of time-lapse imaging of a C.albicans cell that is attacking with PuroA, the cell nucleus stained red before addition of FITC-PuroA (green fluorescent colour). Injection of FITC-PuroA occurs at t = 0 min.

3. What is the wider contribution, or impact, to your scientific field(s)?

Their potency, efficacy and specificity to microbe cells and lack of cytotoxicity activities toward mammalian cells indicates the designed peptides, which are coming from wheat, could be highly valuable in many agricultural areas.


Additionally, understanding the mechanism of action of AMPs helps in development of novel and efficient antimicrobial agents that specifically target pathogen cells, particularly drug-resistance pathogens.

4. Are there any potential ideas you would like to explore to take this research further?

5. Please share a link for researchers to access a relevant publication, data-set, or thesis.

Application of PIN-based peptides: Anti-biofilm and sporicidal activity:

Shagaghi, N, Alfred, RL, Clayton, AHA, Palombo, EA & Bhave, M 2016, 'Anti-biofilm and sporicidal activity of peptides based on wheat puroindoline and barley hordoindoline proteins', Journal of Peptide Science, vol. 22, no. 7, pp. 492-500.


Properties and applications of archetypal TRPs:

Shagaghi, N, Palombo, EA, Clayton, AHA & Bhave, M 2016, 'Archetypal tryptophan-rich antimicrobial peptides: properties and applications', World Journal of Microbiology and Biotechnology, vol. 32, no. 2, pp. 1-10.


Revealing the mechanism of action of PuroA peptide:

Shagaghi, N, Bhave, M, Palombo, EA & Clayton, AHA 2017, 'Revealing the sequence of interactions of PuroA peptide with Candida albicans cells by live-cell imaging', Scientific Reports, vol. 7, no. 43542; doi: 10.1038/srep43542.


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