Engineered Escherichia coli Biofilms Produce Adhesive Nanomaterials Shaped by a Patterned 43 kDa Barnacle Cement Protein

Abstract

Barnacles integrate multiple protein components into distinct amyloid-like nanofibers arranged as a bulk material network for their permanent underwater attachment. The design principle for how chemistry is displayed using adhesive nanomaterials, and fragments of proteins that are responsible for their formation, remains a challenge to assess and is yet to be established. Here, we use engineered bacterial biofilms to display a library of amyloid materials outside of the cell using full-length and subdomain sequences from a major component of the barnacle adhesive. A staggered charged pattern is found throughout the full-length sequence of a 43 kDa cement protein (AACP43), establishing a conserved sequence design evolved by barnacles to make adhesive nanomaterials. AACP43 domain deletions vary in their propensity to aggregate and form fibers, as exported extracellular materials are characterized through staining, immunoblotting, scanning electron microscopy, and atomic force microscopy. Full-length AACP43 and its domains have a propensity to aggregate into nanofibers independent of all other barnacle glue components, shedding light on its function in the barnacle adhesive. Curliated Escherichia coli biofilms are a compatible system for heterologous expression and the study of foreign functional amyloid adhesive materials, used here to identify the c-terminal portion of AACP43 as critical in material formation. This approach allows us to establish a common sequence pattern between two otherwise dissimilar families of cement proteins, laying the foundation to elucidate adhesive chemistries by one of the most tenacious marine fouling organisms in the ocean.

Comparative analysis of stalked and acorn barnacle adhesive proteomes

“…The AaCrCP group has a low number of predicted sites for either type of glycosylation. The minor clusters of P. pollicipes bulk proteins that show enrichment for either proline or threonine and valine also have a high number of predicted O-linked (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27) and a low number of predicted N-linked (less than four) glycosylation sites (data not shown), similar to the GrCP groups.…”

“…As omic sequencing continues to improve, both in the types of species that are sequenced and the quality of the sequencing data, this work provides a roadmap to reveal the fundamental biomolecular contributors to one of nature's most robust marine adhesives. Quantification of the amino acid composition of the entire adhesive for each species [ 91 ] and the abundance of each type of protein, as well as in vitro experiments examining species specific differences in fibril formation [ 18 , 19 ] or other traits are additional steps that will significantly contribute to understanding the role of bulk proteins in adhesion and whether these proteins have become more specialized across barnacle lineages. Finally, continued improvements in sample preparation, for both gDNA/mRNA and proteins, and the application of standard collection and sample processing methods to the adhesive from different species will enhance the ability for direct comparison of different species, highlighting both the similarities and interspecies differences in the adhesive proteome.…”

Adhesive Materials Inspired by Barnacle Underwater Adhesion: Biological Principles and Biomimetic Designs

“…(A–C) Adapted from Liang et al (2015) . (D–F) Adapted with permission from Estrella et al (2021) . Copyright 2021, American Chemical Society.…”

Design of a genetically programmed barnacle-curli inspired living-cell bioadhesive

“…The unique molecular design of amino acids has been proven to endow cp19k with high interfacial activity and enable it to self-assemble into amyloid fibrils in seawater analogs, exhibiting higher adhesion than before self-assembly [ 12 ]. Recently, some researchers have used engineered E. coli curli as a host system to display barnacle adhesion component AACP43, which can be exported through the periplasmic space and aggregated into fibrous structures in the biofilm [ 13 ]. Therefore, the use of barnacle cement protein-functionalized curli system is expected to make a new type of living glue.…”