Identifying Antimicrobial Components in the Allogenic Amnion-Chorion Membrane Using Proteomics

Objectives: The human derived composite amnion-chorion membrane (ACM) has been used to improve wound healing because it can promote cell proliferation. Its antimicrobial properties have not been well studied although some antimicrobial components have been identified in human placental tissues. Our goal was to assess the antimicrobial properties of ACM and identify potential antimicrobial components.
Methods: Antimicrobial properties of ACMs were assessed against porcine pericardium collagen membranes (PPCMs) as control via the agar diffusion assay. Two species, Streptococcus gordonii and Prevotella intermedia were plated on THB agar or blood agar respectively. Membranes were soaked in saline and then placed onto the agar plate. The plates were cultured anaerobically at 37⁰C for 24 to 48 hours. The clear zone free of bacterial growth around the members, if any, indicates the presence of antimicrobial components.
Three samples of ACM (1 mm x 1 mm) were processed and analyzed using a high performance liquid chromatography system (HPLC) coupled with an Orbitrap Fusion Tribrid mass spectrometer. Proteins were identified by Mascot searched against with Swissprot database and quantified with total ion count by Scaffold. Identified proteins were accepted with a false discovery rate (FDR) < 1.0%. Proteins sharing significant peptide evidence were grouped into clusters and annotated with gene ontology (GO) terms.

Results: There were clear zones of bacterial absence around ACM on the agar but there was no clear zone around the PPCM. Over nine hundred proteins were identified using proteomics. Keratin, plectin, fibronectin, vimentin, collagen and other structural proteins were dominant. Antimicrobial proteins including histone H2A/H2B, cathelicidin, lactoferrin and lysozyme were identified in ACM.

Conclusions: ACM has antimicrobial effects on oral bacteria and histones, cathelicidin, lactoferrin and lysozyme might execute the antimicrobial action. Further experiments will be performed to evaluate the killing mechanisms of these identified components.