Engineering Bordetella pertussis BrkA Autotransporter for Chitin-Binding Domain Surface Expression: Exploring its Potential for Whole-Cell Immobilization

Abstract

The BrkA autotransporter is an important virulence factor in Bordetella pertussis that confers serum resistance and mediates cell adherence. As it is self-secreting and only one protein needs to be manipulated, this makes it a relatively simple secretion pathway that can be exploited to deliver proteins to the surface for applications such as whole-cell immobilization in fermentation bioreactors. In this context, the chitin-binding domain derived from Pseudomonas aeruginosa PAO1 chitinase C emerges as a promising candidate, given its strong affinity for chitin, an abundant polysaccharide, and its pH-sensitive characteristics that simplifies the process of retrieving cells after a bioreaction. As such, we aim to exploit the BrkA autotransporter secretion system to export the chitin-binding domain to the cell surface. By leveraging the binding properties of the chitin-binding domain to chitin, we also aim to explore its potential in whole-cell immobilization. In our study, we successfully engineered a recombinant plasmid (TAAK-A54) by substituting part of the BrkA passenger domain with the chitin-binding domain. A western blot indicated expression of the intracellular chitin-binding domain with a molecular weight of 52 kDa, and the extracellular processed form at 38 kDa. A trypsin accessibility assay confirmed the expression and export of the chitin-binding domain to the cell surface. Lastly, we observed through microscopy that Escherichia coli cells expressing the chitin-binding domain can interact with chitin. Samples had decreased turbidity following incubation with chitin resin, with a maximum 3-fold change relative to the negative control, suggesting that cells were able to be immobilized by chitin. This study enhances the understanding of the repertoire of heterologous proteins that the BrkA autotransporter system can secrete. We demonstrated the feasibility of leveraging the BrkA secretion system for the external presentation of the chitin-binding domain. Our results indicate that the chitin-binding domain is worthy of further study for applications in whole-cell immobilization.