Shedding light on the pyruvylation of cell wall polymers – an ancestral reaction for protein cell surface display in bacteria
Abstract
Several bacteria from almost all phyla are covered with a two-dimensional crystalline array of self-assembling (glyco)proteins, termed cell surface (S)-layer. This S layer has nanometer-scale periodicity and, thus, provides the basis for envisaged applications in nanotechnology and biomedicine (1). However, without detailed knowledge of how the anchoring of the S-layer to the underlying peptidoglycan cell wall of bacteria is elaborated. Driven by the research on the pathogen Bacillus anthracis and the model organism Paenibacillus alvei CCM2051T, most knowledge is available on the situation in Gram-positive bacteria, where so-called surface layer homology (SLH) domains serve as cell wall targeting modules for S-layer proteins. SLH domains, in turn, interact with a species-specific, peptidoglycan-bound secondary cell wall polymer (SCWP) that serves as a cell wall ligand (2-4). While it is obvious that this protein cell surface strategy holds promises for therapeutic intervention, a mechanistic understaniding of the underlying principle is still missing. These SCWPs fall into the category of non-classical SCWPs, since structurally different form the well known teichoic acids (3). The structure of the P. alvei SCWP was fully elucidated in our laboratory to be a polymer composed of eleven [→3)-β-D-ManpNAc-(1→4)-β-D-GlcpNAc-(1→] disaccharide repeats where every D-β-ManNAc residue is modified with a 4,6-linked pyruvate ketal (2) contributing to the anionic character of this SCWP. Importantly, the pyruvylation of D-β-ManNAc, which is also present at terminal position of the B. anthracis SCWP (4), is regarded an indispensable and ancestral epitope in this protein cell surface display mechanism (5-7). The functional coupling of SLH domain containing proteins (SLH proteins) and SCWP pyruvylation is substantiated by the finding that several SLH protein syntheisizing bacteria with a pyruvate containing cell wall have an ortholog of the CsaB enzyme predicted to catalyze the transfer of pyruvate ketal to β-D-ManNAc (5). Considering the predictably wide-spread occurrence of this protein cell surface display mechanism in both pathogenic and non-pathogenic bacteria it is surprising how little is known about the biosynthesis of pyruvylated SCWPs and the involved enzymes. This study is designed to shed light on the pyruvyltransferase CsaB of P. alvei, predicted to catalyze a key step in the biosynthesis of the P. alvei SCWP which s the basis for the cell surface display of a specific class of proteins (SLH proteins). This includes the determination of the enzymes's substrate specificity, kinetics and the identification amino acids involved in substrate binding. Further, interaction of CsaB with other enzymes form the P. avlei SCWP biosynthesis gene locus will be investigated, thereby contributing to our general understanding of how SCWPs are biosynthesized.
Publikationen
Project staff
Fiona Hager-Mair
Dipl.-Ing. Fiona Hager-Mair Ph.D.
fiona.hager@boku.ac.at
Tel: +43 1 47654-80058
Project Leader
01.01.2019 - 31.12.2020