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. 2017 Jul 12:5:51.
doi: 10.3389/fchem.2017.00051. eCollection 2017.

Buwchitin: A Ruminal Peptide with Antimicrobial Potential against Enterococcus faecalis

Linda B Oyama  1 , Jean-Adrien Crochet  1 , Joan E Edwards  1 , Susan E Girdwood  1 , Alan R Cookson  1 , Narcis Fernandez-Fuentes  1 , Kai Hilpert  2 , Peter N Golyshin  3 , Olga V Golyshina  3 , Florence Privé  1 , Matthias Hess  4 , Hilario C Mantovani  5 , Christopher J Creevey  1 , Sharon A Huws  6
Affiliations

Buwchitin: A Ruminal Peptide with Antimicrobial Potential against Enterococcus faecalis

Linda B Oyama et al. Front Chem. .

Abstract

Antimicrobial peptides (AMPs) are gaining popularity as alternatives for treatment of bacterial infections and recent advances in omics technologies provide new platforms for AMP discovery. We sought to determine the antibacterial activity of a novel antimicrobial peptide, buwchitin, against Enterococcus faecalis. Buwchitin was identified from a rumen bacterial metagenome library, cloned, expressed and purified. The antimicrobial activity of the recombinant peptide was assessed using a broth microdilution susceptibility assay to determine the peptide's killing kinetics against selected bacterial strains. The killing mechanism of buwchitin was investigated further by monitoring its ability to cause membrane depolarization (diSC3(5) method) and morphological changes in E. faecalis cells. Transmission electron micrographs of buwchitin treated E. faecalis cells showed intact outer membranes with blebbing, but no major damaging effects and cell morphology changes. Buwchitin had negligible cytotoxicity against defibrinated sheep erythrocytes. Although no significant membrane leakage and depolarization was observed, buwchitin at minimum inhibitory concentration (MIC) was bacteriostatic against E. faecalis cells and inhibited growth in vitro by 70% when compared to untreated cells. These findings suggest that buwchitin, a rumen derived peptide, has potential for antimicrobial activity against E. faecalis.

Keywords: Enterococcus faecalis; antibiotic resistance; antimicrobial activity; antimicrobial peptides; metagenomics; microbiome; rumen bacteria.

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Figures

Figure 1
Figure 1
SDS-PAGE analysis of purification steps of buwchitin protein expressed in E. coli TOP10 cells on a 20% denaturing polyacrylamide gel (4 h after induction with 1 mM IPTG). Lane 1, protein molecular weight marker; Lane 2, cell lysate; Lane 3, supernatant; Lane 4, Wash step; Lane 5, eluted buwchitin protein. The arrow indicates band of purified protein of interest. Expected size is 8.35 (±3–4 kDa from His-tag).
Figure 2
Figure 2
Growth rate of E. faecalis in presence of antibacterial agents. Growth rate was determined by monitoring cell density at OD600 nm in three independent measurements at 1 × MIC concentration. Error bars represent the standard deviation.
Figure 3
Figure 3
Representative transmission electron micrographs of E. faecalis. (A) Untreated E. faecalis cells at 1 h. (B) Buwchitin treated cells (200 μg/ml) at 1 h. (C) Untreated E. faecalis at 24 h. (D) Buwchitin treated cells (200 μg/ml) at 24 h. Scale bars on micrographs.
Figure 4
Figure 4
Structural model of buwchitin (gray) in cartoon and surface representation. Side chains of selected amino-acid colored according to atom type (N: blue; C: white; O: red). The N- terminus (Nt) and C- terminus (Ct) is also shown. Figure prepared using PyMol (Schrödinger, 2010).
See this image and copyright information in PMC

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