PYRUVYLIERUNG VON SEKUNDÄREN ZELLWANDPOLYMEREN ALS ESSENZIELLE LIGANDENMODIFIKATION ZUR ERKENNUNG VON SLH-PROTEINEN: LEKTIONEN VON PAENIBACILLUS ALVEI

4,6-pyruvateketal-modified β-D-N-acetylmannosamine present in cell wall glycopolymers (SCWP) is the basis of a predictably wide-spread and ancestral mechanism of protein cell surface display in Gram-positive bacteria, designed for proteins equipped with terminal surface layer homology (SLH) domains. The most prominent SLH-proteins are S-layer proteins; their maintenance in biological contexts depends on stable and flexible attachment to constantly changing cell surfaces. We characterized the structural basis of cell wall anchoring by SLH-domains by analyzing co-crystal structures of the three consecutive SLH domains from the S-layer protein SpaA of the model organism Paenibacillus alvei with synthesized monosaccharide and disaccharide building blocks of its SCWP, consisting of [-3)-4,6-Pyr-β-D-ManpNAc-(1-4)-β-D-GlcpNAc-(1-] repeats. SLH domains appear to be specific for the non-reducing-end Pyr-β-D-ManNAc moiety. This specificity is consistent with that of the Bacillus anthracis S-layer protein Sap for its SCWP that is pyruvylated only on the terminal ManNAc. While pyruvylation of β-D-ManNAc is essential for SCWP functionality, information on the underlying reaction is scarce. Notably, bacterial genomes encoding SLH proteins also encode a CsaB pyruvyltransferase. P. alvei SCWP biosynthesis involves the wall teichoic acid biosynthesis enzymes TagO and TagA, and CsaB for which we obtained evidence of activity on a lipid-bound disaccharide precursor. Initial homology modelling of P. alvei CsaB revealed several conserved amino acid residues possibly involved in catalysis. While intervening biosynthetic steps of SCWP elongation and export remain challenging, the P. alvei and B. anthracis genome harbor candidates for LytR-CpsA-Psr enzymes which transfer wall teichoic acid from lipid-linked substrates to peptidoglycan. We aim at gaining insight into CsaB-catalyzed β-D-ManpNAc pyruvylation and its stage and role in SCWP assembly, using P. alvei as a well tractable model. This project investigates biochemical, structural and biosynthetic aspects of SCWP sugar pyruvylation which due to its importance in Gram-positive cell wall assembly might be exploited as a novel antibacterial target.