Affinity Comparison of p3 and p8 Peptide Displaying Bacteriophages Using Surface Plasmon Resonance

Ever increasing demands in sensitivity and specificity of biosensors have recently established a trend toward the use of multivalent bioreceptors. This trend has also been introduced in the field of bacteriophage affinity peptides, where the entire phage is used as a receptor rather than the individual peptides. Although this approach is gaining in popularity due to the numerous advantages, binding kinetics of complete phage particles have never been studied in detail, notwithstanding being essential for the efficient design of such applications. In this paper we used an in house developed fiber-optic surface plasmon resonance (FO-SPR) biosensor to study the affinity and binding kinetics of phages, displaying peptide libraries. By using either peptide expression on the p3 or on the p8 coat proteins, a corresponding density of 5 up to more than 2000 peptides on a single virus particle was obtained. Binding parameters of 26 different filamentous phages, displaying peptides selective for enhanced Green Fluorescent Protein (eGFP), were characterized. This study revealed a broad affinity range of phages for the target eGFP, indicating their potential to be used for applications with different requirements in binding kinetics. Moreover, detailed analysis of <i>k</i>_off and <i>k</i>_on values of several selected p3 and p8 phages, using the FO-SPR biosensor, clearly showed the correlation between the binding parameters and the density at which eGFP-peptides are being expressed. Consequently, although p3 and p8-based phages both revealed exceptionally high affinities for eGFP, two p8 phages were found to have the highest affinity with dissociation constants (<i>K</i>_d) in the femtomolar range

Alternating Current Electrophoretic Deposition for the Immobilization of Antimicrobial Agents on Titanium Implant Surfaces

One prominent cause of implant failure is infection; therefore, research is focusing on developing surface coatings that render the surface resistant to colonization by micro-organisms. Permanently attached coatings of antimicrobial molecules are of particular interest because of the reduced cytoxicity and lower risk of developing resistance compared to controlled release coatings. In this study, we focus on the chemical grafting of bioactive molecules on titanium. To concentrate the molecules at the metallic implant surface, we propose electrophoretic deposition (EPD) applying alternating current (AC) signals with an asymmetrical wave shape. We show that for the model molecule bovine serum albumin (BSA), as well as for the clinically relevant antifungal lipopeptide caspofungin (CASP), the deposition yield is drastically improved by superimposing a DC offset in the direction of the high-amplitude peak of the AC signal. Additionally, in order to produce immobilized CASP coatings, this experimental AC/DC-EPD method is combined with an established surface activation protocol. Principle component analysis (PCA) of time-of-flight secondary ion mass spectrometry (ToF-SIMS) data confirm the immobilization of CASP with higher yield as compared to a diffusion-controlled process, and higher purity than the clinical CASP starting suspensions. Scratch testing data indicate good coating adhesion. Importantly, the coatings remain active against the fungal pathogen <i>C. albicans</i> as shown by in vitro biofilm experiments. In summary, this paper delivers a proof-of-concept for the application of AC-EPD as a fast grafting tool for antimicrobial molecules without compromising their activities

Scanning electron microscopy images of 4 hours-old biofilms, grown in the presence or absence (untreated) of 11.8 μM rHsAFP1.

<p>Images at multiple magnifications (500x, 1000x and 2000x) are presented.</p

Three-dimensional structure of rHsAFP1.

<p>(<b>A</b>) A family of 20 lowest energy structures superimposed over all backbone heavy atoms; (<b>B</b>) A ribbon representation with disulfide bonds shown in yellow. The termini are labeled as N and C. Diagrams were generated using MOLMOL.</p

Structure-function relationship study of HsAFP1-derived fragments against <i>C</i>. <i>albicans</i> biofilms*.

<p>* BIC50 values were determined by CTB assay; mean ± SEM for n ≥ 3 independent experiments is presented; BIC50, minimum inhibitory concentration that is required to inhibit biofilm formation by 50% as compared to control treatment. Unpaired Student t-tests were performed to analyse significant differences between the effect of the linear fragments and rHsAFP1; the significance level is presented (*, ** and *** represent <i>P</i><0.05, <i>P</i><0.01 and <i>P</i><0.001, respectively; NS, no significant difference).</p><p>Structure-function relationship study of HsAFP1-derived fragments against <i>C</i>. <i>albicans</i> biofilms*.</p

Representation of the HsLin peptides imposed on the rHsAFP1 structure, according to the amino acid sequence.

<p>HsLin peptides are shown as a thick blue line in the same orientation as rHsAFP1; other residues of rHsAFP1, not present in the HsLin peptide, are shown as a thin blue line. Note that (i) the cysteine residues are replaced by α-aminobutyric acid to avoid formation of disulfide bonds and that (ii) the CSαβ scaffold is not present in the HsLin peptides, and therefore, the peptides do not adopt the same conformation as the mature rHsAFP1.</p

Secondary shift analysis of rHsAFP1, pH 4.0 at 298 K.

<p>Regions of α-helix and β-strand are indicated at the top of the figure.</p

Synergy between caspofungin and HsLin06 for biofilm inhibition.

<p>Metabolic activity was measured using CTB. Sigmoidal curves were generated using data of at least three independent experiments (n ≥ 3), using the model <i>Y = Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope)</i>) in GraphPad Prism. Dose response curves of caspofungin in the presence of synergistic concentrations of HsLin06 are presented. Black arrows represent synergy. Coloured lines represent different HsLin doses, as follows: brown: 43.75 μM; orange: 21.88 μM; dark yellow: 10.94 μM; green: 5.47 μM; blue: 1.5; purple: 0.75 μM μM and black: 0 μM.</p

Sequence alignment of HsAFP1 with other plant defensins.

<p>(<b>A</b>) Amino acid sequence alignment of NaD1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref006" target="_blank">6</a>], Psd1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref005" target="_blank">5</a>], MtDef4 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref061" target="_blank">61</a>], RsAFP1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref062" target="_blank">62</a>], RsAFP2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref062" target="_blank">62</a>] and HsAFP1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref032" target="_blank">32</a>], matching their cysteine residues (numbered I-VIII). Multiple alignment was performed using the COBALT alignment tool [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref063" target="_blank">63</a>]. Cysteine-pairing is shown at the top of the figure. Highly conserved residues are shown in grey; (-) denote gaps in the alignment. Blue boxes represent peptide fragments that exhibit antifungal activity similar to the parental peptide, and hence, are important for antifungal activity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref004" target="_blank">4</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref064" target="_blank">64</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref066" target="_blank">66</a>]. The orange box indicates the position of the γ-core. (<b>B</b>) Amino acid sequence alignment of HsAFP1 and the HsAFP1 linear peptide fragments (HsLin01-HsLin06). Multiple alignment was performed using the COBALT alignment tool [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132701#pone.0132701.ref063" target="_blank">63</a>]. Highly conserved residues are shown in grey; (-) denote gaps in the alignment. The orange box indicates the position of the γ-core.</p

Synergy between rHsAFP1 and caspofungin or amphotericin B, for (A) biofilm inhibition, as determined by CTB assay; (B) biofilm eradication, as determined by CTB assay; and (C) growth inhibition of planktonic cultures.

<p>Growth was analysed by measuring the OD₄₉₀. Sigmoidal curves were generated using data of at least three independent experiments (n ≥ 3), using the model <i>Y = Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*HillSlope)</i>) in GraphPad Prism. Dose response curves of caspofungin in the presence of synergistic concentrations of rHsAFP1 are presented. Black arrows represent synergy. Coloured lines represent different rHsAFP1 doses, as follows: brown: 16.8 μM; red: 8.4 μM; orange: 4.2 μM; dark yellow: 2.1 μM; green: 1.05 μM; turquois: 0.53 μM; blue: 0.26 μM and black: 0 μM.</p