Molecular basis of cell wall polymer pyruvylation

A distinct mechanism of protein cell surface display in Gram-positive bacteria relies on the presence of the amino sugar N-acetylmannosamine at exposed, terminal position of secondary cell wall polymers (SCWP) and its modification with pyruvate. Pyruvate-modified N-acetylmannosamine is tailored for specific binding interactions with designated structural motifs of proteins. Thereby it contributes to Gram-positive cell wall assembly and integrity. The mechanism underlying the pyruvate modification of the sugar epitope is widely uncharacterized, both within SCWP biosynthesis and from a biochemical and molecular point of view. This knowledge is essential for exploiting this reaction as a potential antibacterial target in the future. We propose that pyruvylation of N-acetylmannosamine catalyzed by the pyruvate transferase CsaB occurs within the bacterial cell at the stage of the lipid-linked SCWP precursor and might have implications on final ligation of SCWP to the cell wall matrix. We will use the well tractable model bacterium Paenibacillus alvei to unravel the molecular basis of N-acetylmannosamine modification within SCWP biosynthesis. The current perspective of the bacterium`s CsaB enzyme is the basis for the proposed research: a) CsaB is active on a lipid-linked disaccharide SCWP precursor, but not on lipid-free substrates. b) Mutation of an amino acid residues predictably involved in substrate binding yielded inactive CsaB. c) Crystallization screens with CsaB in complex with its substrate phosphoenolpyruvate yielded several hits of microcrystals. In the proposed research, chemical, biophysical, microbiological, genetic, and crystallographic approaches will be synergistically employed in a series of in vitro experiments designed to elucidate the molecular basis of N-acetylmannosamine modification with pyruvate and its status within SCWP biosynthesis. By using a bottom- up approach involving defined synthetic SCWP precursor fragments in concert with CsaB, the acceptor substrate for the enzymatic reaction will be defined allowing to infer the spatiotemporal organization of the sugar modification within SCWP biosynthesis. In combination with enzymes, which transfer SCWP onto the cell wall matrix, the final step of the SCWP biosynthesis will be unraveled, using the same synthetic precursor fragments. Initial insight into CsaB catalysis will be obtained using wild-type versus mutated enzyme in concert with the best acceptor substrate using an established pyruvylation assay. Data will be complemented by crystallography studies. This work marks an important step towards understanding how a microbiologically important sugar epitope is elaborated and at the same time informs about its role in SCWP tethering to the cell wall matrix of Gram-positive bacteria. This may aid drug discovery and development programs focused on this important cell- wall biosynthetic pathway. Main researchers within this project are Christina Schäffer (applicant; microbiology, genetics, biochemistry), Paul Kosma (national collaborator; organic chemistry) and Stephen V. Evans (international collaborator; crystallography).

Molecular basis of cell wall polymer pyruvylation - Unraveling bacterial surface architecture: A step toward new biotech and medical advances Researchers have made significant strides in understanding how bacteria build and maintain their protective outer layers, uncovering key mechanisms that could lead to new advances in biotechnology and antibacterial treatments. Using Paenibacillus alvei as a model organism, the study reveals how specialized proteins anchor to the bacterial cell wall and how certain enzymes contribute to the formation of these anchoring structures. The key to bacterial surface stability. Many bacteria form highly organized S-layers-self-assembling protein structures that play vital roles in structural integrity, nutrient transport, and interactions with hosts. In Gram-positive bacteria like P. alvei, these layers are anchored through specific interactions between S-layer homology (SLH) domains and specialized sugar-based molecules called secondary cell wall polymers (SCWPs). Using advanced crystallographic and calorimetric techniques, researchers discovered that the SLH domain trimer from the P. alvei S-layer protein SpaA (SpaASLH) binds predominantly to a unique, modified sugar-a pyruvylated N-acetylmannosamine moiety- at the SCWP's terminal end. This binding triggers a structural rearrangement in the S-layer protein, strengthening its attachment and allowing it to accommodate longer SCWP fragments. This process ensures that bacterial S-layers remain stable as the bacteria grow and divide, highlighting a sophisticated evolutionary adaptation for cell wall maintenance. Unveiling the role of key enzymes in the biosynthesis of SCWP protein anchors. The enzyme CsaB plays a crucial role in adding the pyruvylation modification to SCWPs, making them compatible for S-layer binding. Researchers developed a new color-based assay to measure CsaB activity, revealing its precise functional mechanisms and laying the foundation for potential strategies to disrupt bacterial growth. Another essential enzyme, MnaA, contributes to SCWP biosynthesis by generating UDP-ManNAc, a sugar precursor required for bacterial cell wall formation. By determining its three-dimensional structure and kinetic properties, scientists gained valuable insights into its function. Notably, unlike similar enzymes in other bacteria, P. alvei MnaA is resistant to the antibiotic tunicamycin-an important discovery for understanding and potentially overcoming antibiotic resistance. Potential implications for biotechnology and medicine. These findings deepen our understanding of bacterial surface architecture and open doors for various practical applications. By targeting S-layer anchoring mechanisms, new antimicrobial therapies to combat pathogenic bacteria with similar cell wall structures, such as Bacillus anthracis. could be developed. Additionally, the ability to manipulate S-layers could enhance the development of bacterial biosensors, drug delivery systems, and other biotechnological innovations. This research marks a significant step toward harnessing bacterial structures for human benefit, with promising implications for both healthcare and industry.