E-Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2021

Effect of Trypsin on the Antimicrobial Peptide Mel4 and Mel4-coated Contact Lenses (#317)

Parthasarathi Kalaiselvan 1 , Debarun Dutta 1 2 , Naresh Kumar 3 , Mark Willcox 1
  1. School of Optometry and Vision Science, UNSW , Sydney, NSW, Australia
  2. Optometry and Vision Science Research Group, Aston University, Birmingham, United Kingdom
  3. School of Chemistry, UNSW , Sydney, New South Wales, Australia

Purpose:  The purpose of this study was to examine the ability of trypsin to digest Mel4 when it is in solution or covalently attached to a contact lens surface.

Methods: Mel4 peptide in solution was incubated with trypsin overnight (1:100, trypsin:Mel4 peptide) in Muller Hinton Broth containing 0.2% w/v bovine serum albumin and 0.01% v/v acetic acid. After overnight incubation, 1.5ml of supernatant was transferred to a new glass vial and the minimal inhibitory concentration (MIC) was examined against P.aeruginosa and S.aureus. Three MACL were incubated with immobilised trypsin overnight in 0.1M ammonium bicarbonate buffer. Following overnight incubation, the lenses were washed three times with ammonium bicarbonate buffer and phosphate-buffered saline (PBS). All the lenses were incubated at 37°C overnight with either P.aeruginosa in PBS or S. aureus in 1/10 Tryptic Soy Broth, after which the numbers of viable bacteria on the lenses were determined by agar plate counts.

Results: Untreated Mel4 had MIC of 62.5 μg ml against P.aeruginosa and 125 μg ml against S.aureus. However, after trypsin digestion, the MIC of Mel4 increased by at least >64-fold against P.aeruginosa and >32-fold with S.aureus; at the highest concentration of Mel4 used (4000μg/ml) digested Mel4 did not inhibit the growth of either bacterium. Similarly, trypsin-treated MACL showed no reduction in the adhesion of P.aeruginosa (3.9 log10 cfu/lens) compared to control lenses (3.5 log10 cfu/lens) whereas untreated MACL showed 2.9 log reduction compared to control lenses. Likewise, trypsin-treated MACL showed no reduction in the adhesion of S.aureus (3.8 log10 cfu/lens) compared to control lenses (3.3 log10 cfu/lens) whereas untreated MACL showed 3.1 log reduction compared to control lenses. There was a significant difference in the reduction in bacterial adhesion between trypsin treated and untreated MACL (p <0.001).

Conclusion: This study has demonstrated that Mel4 peptide is susceptible to trypsin digestion when in solution and when covalently bound to lenses.