Molecular Basis of Pyruvylated Cell Wall Polymer Interactions

Various Gram-positive bacteria employ surface layer homology (SLH) domain-mediated sorting of cell surface proteins and engage peptidoglycan-associated, pyruvylated secondary cell wall polymer (SCWP) as a cell wall ligand. While pyruvate ketal linked to beta-ManNAc of the SCWP backbone is regarded as an ancestral and indispensable epitope for the binding interaction, its molecular basis is currently unknown. We hypothesize that distinct structural features of exposed pyruvate ketal and the amino sugar residues in the SCWP repeats are crucial for the interaction with the SLH domains of proteins. To unravel the molecular basis of the binding interactions, the stoichiometrically defined SCWP of Paenibacillus alvei together with the 2D crystalline surface layer protein SpaA - a cognate, SLH domain-containing binding partner - serves as a well tractable model. The current perspective of the P. alvei SCWP and SpaA protein (projects P21954, P22791) is the basis for the proposed research: a) The SCWP consists of, on average, ten (3-beta-D-ManNAc-1,4-beta-D-GlcNAc-1) repeats with each ManNAc residue modified with a 4,6-linked pyruvate ketal. b) Initial modelling of the SLH region of SpaA, using the corresponding part of the Bacillus anthracis S-layer protein as a template, revealed a pseudotrimer with the critical amino acid motifs lining the grooves so that they would be accessible for SCWP binding. c) First crystals of the SLH region of SpaA have been obtained, providing the basis for the envisaged co-crystallization with SCWP. d) A pyruvyltransferase CsaB ortholog is encoded in the chromosomal SCWP biosynthesis locus of P. alvei and available in recombinant form. By employing a bottom-up approach involving a portfolio of synthetic SCWP fragments and truncated, soluble SpaA, a biological interaction unit shall be determined, characterized, and analyzed in a co-crystallized state. In the proposed research, chemical, biophysical, genetic, and crystallographic approaches will be syner- gistically employed in a series of in vitro experiments designed to elucidate the molecular basis of the binding interactions between pyruvylated SCWP and SLH domains. The contribution of pyruvate will be assessed by comparison with carboxyl-reduced substrates. Different SCWP fragments will be provided as possible pyruvyla- tion targets to investigate at which stage of polymer formation pyruvylation of ManNAc occurs. Our research will provide insight into bacterial cell surface display of SLH domain-containing proteins at the atomic level, thereby increasing our knowledge of how bacteria stick their cell wall together. At the same time it carries applied potential should the governing interactions be recruited to tailor bacterial cell surfaces for specific purposes and to develop therapeutic countermeasures against emerging pathogens. It might well be that the investigated binding mechanism is more prevalent than currently anticipated, since SCWPs might escape from detection of pyruvate due to the acid treatment frequently employed for their release from bacterial cell walls.

P27374 - Molecular interaction basis of pyruvylated cell wall polymer Bacteria have developed sophisticated strategies to display proteins on their cell surface; these mediate a variety to biological functions such as protection form adverse environmental effects or pathogenicity. This project investigates a predictably widespread but little understood mechanism of protein cell surface display, which might be exploited for the design of novel antibacterial strategies. In this mechanism, a distinct class of proteins harboring a so-called SLH-trimer fold interacts with a sugar chain that, in turn, is bound to the bacterial cell wall (cell wall glycopolymer). The cell wall glycopolymer is composed of repeats of two amino sugars, of which the N-acetylmannosamine residue is modified with an acid (pyruvate). This project was based on the assumption that this modification is pivotal for protein anchoring. Using a combination of protein-biochemical, cellular, analytical and crystallographic approaches in an international cooperation involving research teams of the University of Natural Resources and Life Sciences, Vienna, Austria, and the University of Victoria, Canada, this project succeeded in defining and characterizing at a molecular level, the smallest biological interaction unit of SLH proteins and the glycopolymer cell wall ligand. For this purpose, various biosynthesis intermediates of the cell wall glycopolymer form the model bacterium Paenibacillus alvei have been chemically synthesized and investigated for their interaction capability with a recombinant SLH cell surface protein of the bacterium. We showed that terminal, modified N-acetylmannosamine serves as a binding epitope for the protein's SLH domain trimer, of which several amino acids which are involved in the binding interaction have been identified. Notably, the highly conserved amino acid glycine at position 29 (SLH-Gly29) allows for the flexible attachment of the protein which may allow anchoring readjustment to relieve protein strain caused by cell growth and division. Based on the finding that the modification of the N-acetylmannosamine residue is pivotal for the binding interaction with the protein, the enzyme carrying out this modification - the pyruvyltransferase CsaB - was identified and characterized. For this purpose, we reconstituted the biosynthesis of a cell wall glycopolymer intermediate by virtue of the bacterium's recombinant biosynthesis enzymes TagA and CsaB. We demonstrated that CsaB is active on the stage of the lipid-linked cell wall glycopolymer repeat. Further, the genomic locus encoding the whole biosynthesis machinery of the cell wall glycopolymer was identified, which allows further studies. Given that the investigated protein cell surface display mechanism is also present in the pathogen Bacillus anthracis, the generated data may have application potential for the design of novel antibacterial countermeasures.