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Research project (§ 26 & § 27)
Duration : 2019-01-01 - 2020-12-31

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.
Research project (§ 26 & § 27)
Duration : 2018-10-01 - 2022-09-30

Higher filamentous fungi decompose organic matter, interact with other organisms and produce metabolites, many of which are used by humankind in industry and medicine. They rely on absorptive nutrition and require a large surface area. Therefore, their body is shaped as a network of thin and long filaments, or hyphae. The tubular lifestyle, however, brings such biological problems as overexposure to the environment, the loss of water/nutrients and vulnerability to pathogens and abiotic stresses. Thus, the physicochemical properties of the hyphal surface are essential for the adaptation of a fungus to its ecological niche. In this project, we will focus on hydrophobins (HFB) that are small cysteine-rich proteins secreted exclusively by filamentous microbes. The amphiphilic nature of these proteins and their superior surface activity allow them to self-assemble in monolayers at liquid/air and liquid/solid interfaces to change the interface surface energy (hydrophobicity) and chemistry. This project hypothesizes that HFBs are crucially important for fungal fitness because they can modulate the physiochemical properties of the hyphal surface and thus play a role in the establishment of a fungus in its ecological niche. We propose using the genus Trichoderma because these fungi are ecologically versatile and have a high diversity of HFBs. The study will consist of (i) an in-depth molecular evolutionary analysis of HFBs in Trichoderma and related fungi. It will reveal possible mechanisms that resulted in the enrichment of HFB-encoding genes in Trichoderma; (ii) the homology modeling and heterologous production of the selected extant and resurrected Trichoderma HFBs. We will investigate the properties of HFBS and engineer of “tailor-made” HFBs; (iii) the physiochemical characterization of the produced HFBs by the standard proteomic techniques and with the use of a comprehensive toolkit of physiochemical and surface chemical methods; (iv) the analysis of HFB-gen regulation in different conditions of growth in the presence of plants, fungi, bacteria and on a diversity of natural polymers, at various developmental stages and under stress conditions. (v) The functional molecular biological analysis of the selected HFB-encoding genes in at least two Trichoderma spp. by gene deletion, gene overexpression, and expression of fluorescently tagged HFBs for microscopic detection. The applicability of the CRISPR/Cas9 system for hfb silencing will be tested. Thus, the novelty of our approach is the establishment of a systems biological view on HFBs in a given fungus using a combination of tools from fungal genomics, molecular biology, and physical chemistry. We hope to resolve the involvement of HFBs in the ecophysiological adaptations of fungi, i.e. in fungal fitness. This project will also lead to the detection of HFBs with unknown properties that may then be used for a diversity of applications in industry and medicine.
Research project (§ 26 & § 27)
Duration : 2018-01-01 - 2020-12-31

The HotCHPot project is aimed at synthesizing and studying the self-assembly behavior of Heterogeneously Charged Particles, also known as Inverse Patchy Colloids (IPCs). These are charged nano- or microparticles with polar patches with the opposite charge that repel each other and attract the rest of the particles, while the non-patch areas on the particles repel each other as well. The relative patch-non-patch charge ratio, the overall particle charge, the screening length and the spatial extent of the patches will be varied to tune the highly directional and selective interactions between these patchy particles. The assembly of these entities in two- and three-dimensional systems will be studied in situ through the combined use of Dynamic Light Scattering (DLS), gated-STED super-resolution microscopy, Ultra Small Angle X-ray Scattering (USAXS), Grazing-Incidence Small-Angle X-ray scattering (GISAXS) and cryo-electron microscopy. The experimental efforts and results will be guided by and compared to predictions made by numerical simulations. Associating experimental work and simulations will allow us to address the highly important question of the design of patchy particles, such that the collective behavior of the resulting material is consistent with the desired properties.

Supervised Theses and Dissertations