Abstract:
By utilizing x-ray crystallography and electron microscopy (EM), this thesis describes the structure determination of two novel proteinaceous macromolecules together with their characterization by microbiological infection assays. These two objects of study revolve around an abundant cell surface molecule, wall teichoic acid (WTA), of S. aureus and its variants. These two structures are for one TarM, a glycosyltransferase involved in the final steps of the in-vivo synthesis pathway of WTA, and secondly Gp45, a receptor binding protein located in the baseplate of siphovirus φ11, a molecule that recognizes WTA by an unknown mechanism facilitating phage adsorption. Both recently discovered proteins, TarM and Gp45, were recombinantly overproduced in E. coli, isolated and crystallized. Since no homologous structure was available to facilitate the usage of the measured structure factor amplitudes for molecular replacement calculations, an appropriate structure solution protocol was sought. TarM crystals were treated with iodide ions and Gp45 crystals were likewise soaked in Tantalbromide solution in order to introduce anomalous scatterers into the protein crystal lattice. Consecutively, applying a combination of single and multiple isomorphous replacement with anomalous scattering (SIRAS and MIRAS, respectively) could retrieve the respective structure factor phase information for the protein atoms. A final solvent flattening routine enabled the structure solution of TarM. Gp45 could be interpreted in a model building process only after a more complex electron density modification routine, combining histogram matching, solvent flattening and averaging protocols. These unbiased models were refined against high-resolution datasets of 2.2 Ångstroms each. The position of Gp45 in the bacteriophage baseplate was elucidated by using negative staining EM on φ11 particles and by superposing this x-ray structure model on the 3D reconstruction image derived thereform. Semi-quantitative infection assays involving WTA deficient in glycosylation using TarM/TarS knockouts or mutants in strain S. aureus RN4220 or by blocking the cell surface specifically with recombinant Gp45 were carried out. Additionally, phages blocked with antibodies against Gp45/Gp54 were used to investigate dependencies governing impact and interaction of TarM and Gp45 with WTA in- and outside the cell. S. aureus is responsible for many fatalities in clinical environments caused by infections. Treatments cannot only become tedious but are also a difficult economic factor. The fast resistance developing nature of this germ, of which many details are not well understood, is a major obstacle to overcome lethal infections and therefore requires scientific research. The cell envelope represents a major research field and is in some cases affiliated with the emergence of resistance development. This dissertation describes a structure-based research utilizing in-vitro and in-silico laboratory methods, covering a tiny portion of this wide topic by presenting the structures of TarM and Gp45, and is to be seen as a contribution to the understanding of the complicated processes in and around the cell surface of a potent Gram-positive pathogen. The general goal of these interdisciplinary endeavours is to find sensitive treatments by implementing the vast data coming from cell envelope studies into drug design targeted against Gram-positive bacteria, specifically targeting crucial switches of the metabolism or components of the architecture itself.
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