Research

Theoretical framework: Coproporphyrin III is an important virulence factor associated with the skin disease acne vulgaris, which is caused by an infection with the monoderm actinobacterium Cutibacterium acnes. Monoderm bacteria utilize the so-called “coproporphyrin-dependent” heme biosynthesis pathway. In this pathway several enzymes catalyse the formation of the final product heme b, two of those (coproporphyrin ferrochelatase, CpfC; coproheme decarboxylase, ChdC) are uniquely found to be fused in one open reading frame in pathogenic C. acnes strains. Objectives: Employing high-end biochemical and biophysical methods to study different constructs of pathogenic C. acnes CpfC-ChdC fusion protein (full length; N-terminal CpfC domain only; C-terminal ChdC domain only), but also of CpfC and ChdC (not fused) of non-pathogenic C. acnes strains will provide in-depth knowledge of protein folding, interaction and enzymatic capabilities. Further, state-of-the art structural studies will be used to describe the enzyme in the most complete way, in order to understand steric constraints of the overall structure and active site architecture, which may lead to the secretion of coproporphyrin III. Methods: Biochemical and biophysical characterization of the selected CpfC-ChdC constructs and variants will be performed using multiple high-end spectroscopic methods, steady-state and pre-steady state kinetic characterizations. Further we will employ state-of the art structural biology methods (X-ray crystallography, Cryo-EM, SAXS) to gain profound knowledge of the enzyme’s structure to the very detail. Innovation: Combining our long-standing expertise on CpfCs and ChdCs is the perfect starting point to investigate the molecular mechanisms and enzymatic shortcomings that lead to the production and secretion of the very important virulence factor coproporphyrin III. Knowledge of this underlying biochemical features is a precondition for further studies, including future drug development.

S-layers are 2D crystalline cell envelope structures of many prokaryotes and are potential targets for therapeutic inhibition. Many S-layers are glycosylated and in Gram-positive bacteria, can be attached through the interaction of an S-layer homology (SLH) domain trimer with peptidoglycan-linked secondary cell wall glycopolymer (SCWP). Current insight in SLH domain trimer-SCWP interactions stems from recombinant, non-glycosylated protein and short, synthetic SCWP fragments. As demonstrated for the model organism Paenibacillus alvei, a terminal pyruvylated N-acetylmannosamine residue of SCWP is essential for binding and two active binding grooves are provided by the SLH domain trimer in a mutually exclusive manner. Our hypothesis is that the native glycosylation of the P. alvei SLH domain trimer located in the two binding grooves influences the molecular logic of S-layer anchoring to the cell wall. We furthermore hypothesize that the terminal pyruvylated N-acetylmannosamine S-layer binding epitope for SCWP is an ideal starting point to design inhibitors of proper cell wall assembly. We will analyse the influence of the glycosylation of the SLH domain trimer on binding to SCWP will be studied by analysing its interaction with the SCWP ligand in a bottom-up approach of increasing complexity, in vitro and in vivo. We aim to identify small molecule inhibitors of the SLH domain trimer-SCWP interaction. The project uses chemical synthesis, protein- and glycan-engineering, biophysical protein-carbohydrate interaction analyses, X-ray crystallography, and cryo-electron tomography/ microscopy, and is supported by molecular modelling and simulation. This interdisciplinary project will yield a detailed mechanistic model of S-layer anchoring in Gram-positives. Understanding the governing glycoprotein-carbohydrate interactions at a molecular level will open avenues for their disruption–a field of increasing importance in the context of bacterial pathogens.

Dental caries is one of the most prevalent global chronic diseases. It is intimately linked to the establishment of a dysbiotic oral biofilm consortium on the tooth surface (dental plaque) among which Streptococcus mutans plays a key role, and many of the bacterium’s macromolecules involved in biofilm establishment have been investigated. Mutanofactin is a very recently identified secondary metabolite of S. mutans suggested to increase its cell surface hydrophobicity thereby promoting cell adhesion and biofilm formation. However, detailed insight into the molecular mechanism of mutanofactin action and its scope has not been achieved. This project will explore a new molecular mechanism underlying cariogenic biofilm development triggered by S. mutans-produced mutanofactin. It is pivotal to understand the physicochemical and microbiological implications of mutanofactin to obtain a comprehensive picture. This requires setting up biological systems of step-wise increasing complexity using defined components and methods for analytics and monitoring from the molecular to the biofilm level. The project design is based on a unique combination of synthetic, microbiological and physicochemical approaches, including de novo synthesis and novel techniques to characterize bacterial cell surface properties and biofilm matrix interactions. This project might lead to effective anti-cariogenic strategies urgently needed for human health and to reduce the global economic burden of tooth decay.