Functional Assays of Antimicrobial Peptides (AMP's) Piscidin 1 and Piscidin 3 from Hybrid Striped Bass

Lorraine Homem with Dr. Myriam Cotten and Dr. Mary Ellard-Ivey

Introduction

As a first line of defense against microbial invasion fish use their innate immune system. Westerman et al., (2002) discovered endogenous a-helical anitmicrobial peptides (AMPs) from the skin and gills of farmed hybrid stripped bass; the two isoforms were called Moronecidin 1 and Moronecidin 2.
Simultaneously, Silphaduang et al., (2001) discovered three isoforms of the same peptide, also from the skin and gills of hybrid striped bass. These same peptides were named Piscidins 1, 2 and 3. Our research group refers to the peptides as Piscidins. The structure of Piscidin 2 was confirmed by Westerman et al., (2002) using circular dichroism spectroscopy and is an amphipathic a-helical peptide. The highly conserved amino terminus is rich in histidine and phenylalanine. Its activity is believed to be a result of membrane disruption.
The peptide purified by Westerman et al., (2002) was a mixture of non-amidated and amidated peptide. The significance of the C-terminal amidation is unknown. The main objective this summer was to carry out functional assays of Piscidin 1 and Piscidin 3 in bacteria and to compare activity of amidated and non-amidated Piscidin 1 in both prokaryotic and eukaryotic cells. These objectives were mainly achieved using microtiter assays.

Microtiter Assay Protocol

Bacterial cultures of gram positive bacteria S. aureus (SA) and B. cereus (BC) and gram negative bacteria E. coli (EC) and P. vulgaris (PV) were prepared from plates by taking a single colony and inoculating 50ml of THB in labeled 250ml Erlenmeyer flasks. Cultures were grown overnight in a shaker at 37 C, approx. rpm 220.
0.5ml of each bacteria from the overnight cultures was placed in respective 250ml autoclaved Erlenmeyer flasks containing 50ml THB. This set up was placed in a shaker for 130mins at 37C, rpm 220.Working hood bench was wiped down with 70% EtOH and UV light kept on for 1/2 hr to sterilize area of the working hood. Bacteria culture was diluted further 1 in 1000.
Concentrations of 2,10, 20 and 30mM, were made up from a 200mM solution of the peptide dissolved in water. Peptide and bacteria were respectively pipetted into sterile labeled Microtiter Plates. Absorbance taken before and after incubation in a shaker at 37C, rpm 100 to measure levels of bacterial growth inhibition.

Conclusions

Overall the data shows that P1 is active at lower concentration than P3. A truncation of the P3 peptide does not appear to affect activity against Staphylococcus aureus, Bacillus cereus and Escherichia coli. However, it alters the activity of the peptide against Proteus vulgaris. The inhibitory activity of both P1 and P3 differs between gram positive and gram negative. This is most likely determined by the membrane structure of the bacteria. C-terminal amidation of P1 is important in its activity against eukaryotic cells but not against prokaryotic cells.