Engineering of Antimicrobial Surfaces by Using Temporin Analogs to Tune the Biocidal/antiadhesive Effect

Abstract

Proliferation of resistant bacteria on biomaterials is a major problem leading to nosocomial infections. Due to their broad-spectrum activity and their ability to disrupt bacterial membranes through a rapid membranolytic mechanism, antimicrobial peptides (AMPs) are less susceptible to the development of bacterial resistance and therefore represent good candidates for surface coating strategies to prevent biofilm formation. In this study, we report on the covalent immobilization of temporin-SHa, a small hydrophobic and low cationic antimicrobial peptide exhibiting broad-spectrum activity, and (SHa) analogs on modified gold surfaces. Several analogs derived from SHa with either a carboxamidated ([K³]SHa, d-[K³]SHa) or a carboxylated C-terminus ([K³]SHa-COOH) were used to achieve peptide grafting on gold surfaces modified by a thiolated self-assembled monolayer (SAM). Surface functionalization was characterized by polarization modulation infrared reflection absorption spectroscopy (PM-RAIRS) and X-ray photoemission spectroscopy (XPS). The antibacterial properties of the temporin-functionalized surfaces were tested against the Gram-positive Listeria ivanovii. Direct visualization of the peptide effects on the bacterial membrane was investigated by scanning electron microscopy equipped with a field emission gun (SEM-FEG). All active temporin analogs were successfully grafted and display significant antibacterial activity (from 80 to 90% killing efficiency) in addition to a 2-fold decrease of bacterial adhesion when all d-SHa analogs were used.

Keywords: SHa analogs; antibacterial/antiadhesive activity; antimicrobial peptides; gold surface functionalization; temporin-SHa.

Figure 1

Different grafting strategies of SHa analogs. (a) Grafting of C-terminal α-carboxamidated SHa analogs on carboxylic acid-terminated MUA SAMs. (b) Grafting of free C-terminal carboxylate SHa analogs on amine-terminated MUAM SAMs. Reactive functions of the MUA/MUAM SAM and of [K³]SHa are indicated in bold.

Figure 2

PM-RAIRS spectra of gold surfaces functionalized with (a) MUAM, (b) MUAM-[K³]SHa-COOH, (c) MUA and (d) MUA-[K³]SHa.

Figure 3

High resolution XPS spectra of gold surfaces functionalized with (a) MUAM, (b) MUAM-[K³]SHa-COOH, (c) MUA and (d) MUA-[K³]SHa for (A) the C1s region, (B) the N1s region, (C) the O1s region and (D) the S2p region.

Figure 4

Structural morphology of the Gram-positive bacteria Listeria ivanovii in contact with grafted surfaces compared to bare gold. (a) Au, (b) MUA, (c) [K³]SHa, (d) d-[K³]SHa, (e) [A^2,6,9, K³]SHa, (f) MUAM, (g) [K³]SHa-COOH and (h) [A^2,6,9, K³]SHa-COOH. The scale bare represents 1 μm.

Figure 5

Killing efficiency towards L. ivanovii bacteria compared to gold bare substrate for MUA and MUAM, to MUA for [K³]SHa, d-[K³]SHa, [A^2,6,9, K³]SHa, and to MUAM for [K³]SHa-COOH and [A^2,6,9, K³]SHa-COOH, respectively. The raw data (dark bars) are compared to the data corrected by the adhesion factor (light bars). The killing of X compared to its control is calculated using %Killing_X = [(CFU_CONTROL − CFU_X)/CFU_CONTROL)] × 100. Error bars indicate standard deviation from three independent CFU counts.

Figure 6

L. ivanovii bacterial adhesion (%) compared to gold bare substrate (100%), left bars. Water contact angle, WCA (°) measured on Au and modified surfaces, right bars.