Poster N6 - 57 amino acid peptide in Bap1 bacterial adhesin protein-mediated underwater adhesion

Introduction: Bacterial colonies develop biofilms to attach, thrive, and proliferate on various surfaces.1,2 These films aid in colonizing hosts and provide antibiotic resistance from human pathogens.3,4 The biofilm consists of multiple adhesive proteins known as ‘adhesins’, such as Bap1 and RbmC.5,6 Using Vibrio cholerae as a model biofilm-forming organism, which is the causal agent of pandemic cholera, a recent study showed that a 57-amino acid (aa) long peptide sequence in Bap1 mediates adhesion to lipids and abiotic surfaces.5,7 Here, we report the pull-off forces of the isolated 57-aa peptide on a model inorganic substrate - mica, using the Surface Forces Apparatus (SFA), a multiple beam interferometry-based force spectroscopy technique, to quantify its adhesion strength and potential use as a wet biomedical adhesive.
We compared the maximum pull-off forces of physisorbed 57 aa peptide films onto mica surfaces with that of Mussel foot protein-5 (Mefp-5) on mica.8,9 Mefp-5 has been studied extensively for decades, with known high dihydroxiphenylalanine (DOPA) content, in part, responsible for mediating robust adhesion to rocks in marine environments. On the other hand, the 57 aa peptide lacks DOPA, but possesses a high hydrophobic moiety content. This study will allow us to understand novel amino acid sequences that mediate robust wet adhesion and help inform the design of novel adhesives capable of performing in water and high ionic strength environments.

Materials and

Methods: A peptide containing a crucial 57 aa sequence identified in Bap1 was chemically synthesized and purchased from Atlantic Peptides. The peptide aa sequence is YLGLEWKTKTVPYLGVEWRTKTVSYWFFGWHTKQVAYLAPVWKEKTIPYAVPVTLSK-NH2. The peptide stock solution (150 µM) was stored in M9 buffer at 4C before use. Approx. 50 nm thick silver films were deposited on freshly cleaved mica using thermal evaporation. The back silvered mica surfaces were then glued onto cylindrical glass disks with a radius of curvature, R ~ 2 cm, and arranged in the SFA in a crossed cylinder geometry to construct an optical interferometer. The lower surface is suspended on a double cantilever spring (K ~1000 N/m). The interference fringes were used to measure the absolute separation distances (D) between the two surfaces and the normal force from the spring deflection. For the symmetric configuration, 0.5 µl of 150 µM peptide was deposited in a 50 µl droplet of M9 buffer (pH 7) between the two mica surfaces and allowed to physisorb for 30 mins at room temperature. For the asymmetric configuration, the peptide of the same concentration was deposited only on the bottom mica surface, and the other mica surface was left untreated, followed by rinsing to remove excess peptide. The surfaces were brought into contact during each measurement, held for 2 mins, and then separated at a constant speed (15-25 nm/s). The maximum pull-off force measured during separation was denoted as the adhesion force, -Fad/R. Adhesion energies were calculated using the JKR theory: Ead = -Fad/1.5πR.12

Results, Conclusions, and Discussions: First, thin films of the 57 aa peptide at 1.5 µM concentration in M9 buffer (ionic strength of 170 mM) were deposited in a symmetric configuration. The maximum pull-off forces, Fadh/R of -16 ± 2 mN/m (or adhesion energy, Eadh ~ 3.9 mJ/m2), and film thicknesses of 1.2 ± 0.4 nm at 60 mN/m compression forces were measured. For the same configuration and contact time, the thin films of Mefp-5 in pH 3 between mica surfaces show maximum pull-off forces of -7 mN/m (or adhesion energy, 1.5 mJ/m2), almost half of that measured with the bacterial peptide.8 The corresponding hardwall of Mefp-5 films were measured to be 3-4 nm.
For the asymmetric configuration of the 57 aa peptide at the same concentration, the peak pull-off forces were, Fadh/R of -20 ± 4 mN/m (or adhesion energy, Eadh ~ 4.2 mJ/m2), and film thickness of 0.8 ± 0.4 nm at 60 mN/m compression. In comparison, a similar configuration with Mefp-5 at pH 2.6 and ionic strength of 100 mM, adhesion energy, Eadh of 9 mJ/m2 and a hard wall of ~1 nm were measured, indicating a monolayer of the Mefp-5 was formed on one of the mica surfaces. Therefore, the adhesion energies measured with monolayer thickness of Mefp-5 protein is approximately twice than that measured with the bacterial peptide in our study. Since our measured pull-off forces for both configurations were not significantly different, even in asymmetric configuration, we interpret our results as being due to cohesive forces of the system. Hydropathy analysis of the 57 aa peptide shows that it consists of 47.375% hydrophobic residues, and therefore, hydrophobic interactions between the peptide molecules may contribute to the cohesive mechanism. Furthermore, the aromatic rings could be participating in π-cation, and π-π interactions that contribute to both inter and intra-molecular interactions within the peptide films. The jump-out distances for both configurations are 4-5 nm, which is greater than the hard wall distances measured but similar to the onset of interactions, indicating that the peptide film is compliant and elastic. These peptides can potentially be used to develop biofilm-inspired adhesives for biomedical applications.

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