Initial Characterization of WxL Proteins Derived From Lactiplantibacillus pentosus KW1

Abstract

Lactic acid bacteria (LAB) are a gram-positive, non-sporing group of bacteria that have shown potential as vaccine delivery vectors by anchoring heterologous antigens on their cell surface. Various anchoring strategies have been developed to achieve surface exposure of these antigens. Proteins harbouring a Trp-X-Leu motif, named WxL proteins, have been suggested to bind non-covalently to peptidoglycan, making them promising candidates for surface display. However, most existing studies are limited to in silico analyses of the WxL proteins, highlighting the need for experimental validation. In this thesis, the main goal it to provide an initial characterization of selected WxL proteins from Lactiplantibacillus pentosus KW1. The characterization includes determining the conditions in which these proteins are produced, elucidating their potential functions, evaluating possible binding capabilities, and identifying potential interaction partners. To investigate under which conditions these proteins were produced, vectors harbouring the predicted promoters upstream of WxL clusters, were cloned upstream of a reporter gene (mCherry). To investigate binding properties, the sequences of WxL1, WxL2A and WxL3A, were selected and cloned into vectors for overexpression in Escherichia coli. WxL1 was successfully purified, and binding experiments were conducted under different conditions to test external binding affinity to bacterial surface and purified peptidoglycan. Potential binding was assessed using SDS-PAGE, flow cytometry and fluorescence microscopy. In silico analysis of one selected gene cluster (c1) was performed to explore potential cooperative interactions among the encoded proteins in the cluster. Based on the in silico findings, interaction studies were conducted to evaluate potential interactions between WxL1 and the downstream gene, named Tmem1, in the c1 gene cluster. The results indicate that native WxL production is tightly regulated, possibly influenced by promoters responsive to nutrient availability, suggesting a nutrient-dependent expression profile. WxL proteins show affinity for both intact L. pentosus KW1 cells and purified peptidoglycan, with binding occurring under specific conditions such as defined pH levels and ionic strength, and potentially the presence of calcium ions (CaCl₂). Additionally, WxL1 was found to reduce growth and induce aggregation in E. coli, implying a potential role in cell-cell interactions. WxL1 also indicated a high affinity interaction with Tmem1 encoded in the same cluster, indicating a cooperative function potentially facilitating surface localization and enhancing the binding activity of WxL1