Browsing by Person "Hau, Jann-Louis"

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Conformational dynamics of proteins in bacterial pathogens and the innate immune response
(2024) Hau, Jann-Louis; Fritz, Günter
Global health is facing two major threads, the rapid rise of multi-drug resistant bacterial pathogens and the increase autoimmune diseases. Therefore, studying the components of host-pathogen interactions is crucially important for the development of novel drugs and diagnostic tools to fight these threats. Both, virulence factors of pathogenic bacteria and the components of the host’s innate immune response are build up by multimeric protein complexes. In order to understand how these systems respond to different conditions and environments and to uncover the underlying molecular mechanisms, the conformational dynamics of these proteins must be resolved. This study focuses on the two important examples of a host-defence protein, S100A8/A9, and a bacterial virulence factor, the Na+-translocating NADH:ubiquinone oxidoreductase (NQR). The heterodimeric protein complex S100A8/A9 is secreted in very high amounts by immune cells at sites of infection and tissue damage. S100A8/A9 is a key element of the innate immune system. It strongly activates inflammatory response and exhibits antimicrobial properties by binding of essential transition metal nutrients. Therefore it is defined as a danger-associated molecular pattern (DAMP). Its role as a DAMP during inflammation and absence in healthy tissue defines S100A8/A9 is used as an excellent biomarker for a variety of inflammatory diseases in human and veterinary medicine. Only the human S100A8/A9 is thoroughly characterised so far. Imaging of S100A8/A9 by a tracer molecule would allow for sensitive and accurate imaging of pathogenic or sterile inflammation. Here, a new protocol for fast and and efficient isolation of human and porcine S100A8/A9 is established, which is also applicable to S100A8/A9 of other species. The characterisation of porcine S100A8/A9 revealed similar properties with respect to structure and metal binding to human S100A8/A9, while the antimicrobial properties of the porcine protein are less pronounced than in the human orthologue. The structures of human S100A9 in complex with different tracer molecules were determined by X-ray crystallography, revealing that the molecular benzimidazole core of the tracer binds into a dynamically adapting pocket of S100A9. Applying a newly established tryptophan fluorescence-based assay, it was shown that the affinity of the tracer is not affected by different imaging tags attached to the benzimidazole core. Since the bezimidazole core exhibits non-favourable pharmacokinetic properties, novel lead compounds for the targeting of S100A8/A9 in inflammation have to be established. New lead structures, which could serve as novel molecular cores, were identified by fragment-based crystallographic screening. As exemplified by the benzimidazole imaging tracers, S100A8/A9 ligands have to fulfil the requirements of the dynamic binding pocket. The identified molecules represent an excellent starting point for the development of a novel class of imaging probes for the sensitive detection of inflammation. The NQR is part of the respiratory chain of the human pathogen Vibrio cholerae. It couples the oxidation of NADH and the reduction of ubiquinone to the translocation of Na+ from the cytoplasm to the periplasm. The resulting Na+ electrochemical gradient, the sodium motive force, is vital for V. cholerae and drives several processes critical for pathogenesis, like e.g. movement of the flagellum and efflux of antibiotics. NQR-activity is linked to the expression of pathogenic factors including the cholera toxin and is therefore classified as a virulence factor itself. Since the NQR has no homologs in humans and is widespread among Gram-negative pathogenic bacteria, it is a promising target for the development of novel classes of antibiotics. However, the mechanism of electron transfer and the coupled sodium translocation as well as the quinone binding site had been elusive. Based on first structural information on the NQR, which exhibited unusual large distances between redox cofactors, drastic conformational changes had been proposed (Steuber et al., 2014). In this study, the structure of the NQR with substrates or inhibitors bound was determined by single particle cryo electron microscopy. The structures reveal that the coupling of electron transfer to conformational changes in the NQR subunits NqrC and NqrF are governed by the redox state of the intramembrane [2Fe-2S] cluster between NqrD/E, defining the NQR as a conformationally coupled redox pump. Furthermore, the binding site of ubiquinone and the NQR inhibitor HQNO was located in NqrB. Sodium ions were identified bound in NqrB, which could represent exit sites of the sodium translocation path. These findings were confirmed for the NQR of Prevotella byrantii by homology modelling. Taken together, the molecular mechanism described for the V. cholerae NQR applies also to NQRs from other organisms and homologous complexes. The described NQR mechanism and substrate binding sites lay the foundation for the structure-based design of NQR-inhibitors which could serve as new antibacterial drugs. In summary, the findings presented in this study promote the development of novel diagnostic tools and new antibiotics to combat the emerging threats of autoimmune diseases and multidrug resistant bacterial pathogens.
The low mutational flexibility of the EPSP synthase in Bacillus subtilis is due to a higher demand for shikimate pathway intermediates
(2023) Schwedt, Inge; Schöne, Kerstin; Eckert, Maike; Pizzinato, Manon; Winkler, Laura; Knotkova, Barbora; Richts, Björn; Hau, Jann-Louis; Steuber, Julia; Mireles, Raul; Noda‐Garcia, Lianet; Fritz, Günter; Mittelstädt, Carolin; Hertel, Robert; Commichau, Fabian M.
Glyphosate (GS) inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that is required for aromatic amino acid, folate and quinone biosynthesis in Bacillus subtilis and Escherichia coli. The inhibition of the EPSP synthase by GS depletes the cell of these metabolites, resulting in cell death. Here, we show that like the laboratory B. subtilis strains also environmental and undomesticated isolates adapt to GS by reducing herbicide uptake. Although B. subtilis possesses a GS-insensitive EPSP synthase, the enzyme is strongly inhibited by GS in the native environment. Moreover, the B. subtilis EPSP synthase mutant was only viable in rich medium containing menaquinone, indicating that the bacteria require a catalytically efficient EPSP synthase under nutrient-poor conditions. The dependency of B. subtilis on the EPSP synthase probably limits its evolvability. In contrast, E. coli rapidly acquires GS resistance by target modification. However, the evolution of a GS-resistant EPSP synthase under non-selective growth conditions indicates that GS resistance causes fitness costs. Therefore, in both model organisms, the proper function of the EPSP synthase is critical for the cellular viability. This study also revealed that the uptake systems for folate precursors, phenylalanine and tyrosine need to be identified and characterized in B. subtilis.