Structural and mutational characterisation of Shigella Pathogenicity Factors
Annually 163 mio. people are affected by bacillary dysentery, an acute inflammatory bowel disease, caused by Shigella bacteria. Shigella have the ability to invade the colonic epithelium in humans, thereby causing an acute mucosal inflammation. The invasive phenotype is encoded on a virulence plasmi...
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|Summary:||Annually 163 mio. people are affected by bacillary dysentery, an acute inflammatory bowel disease, caused by Shigella bacteria. Shigella have the ability to invade the colonic epithelium in humans, thereby causing an acute mucosal inflammation. The invasive phenotype is encoded on a virulence plasmid. The expression of the so-called invasion genes is controlled by a virulence factor VirF. The translation of the virF mRNA depends on the activity of a tRNA modifying enzyme – the tRNA-guanine transglycosylase (TGT). TGT-mutants of Shigella are almost unable to penetrate host cells.
In the first part of this work, the pathogenicity factor genes ipaA, ipgB2, ospD1, spa15, and ipgE, which are required at different points of the Shigella invasion mechanism, were cloned into several plasmid vectors and expressed in E. coli. In case of IpaA, OspD1, and IpgB2 only insoluble protein was obtained while in case of IpgE a purification protocol could be successfully established. Purified IpgE was identified via mass spectrometry and crystallisation conditions were detected.
In the second part of this work, mutagenesis studies on a drug design target – the TGT – were performed. Bacterial tRNA-guanine transglycosylase (TGT) catalyses the exchange of guanine in the wobble position of particular tRNAs by the modified base preQ1. In vitro, however, the enzyme is also able to insert the immediate biosynthetic precursor, preQ0, into those tRNAs. This substrate promiscuity is based on a peptide switch in the active site, gated by the general acid/base Glu235. The switch alters the properties of the binding pocket to allow either the accommodation of guanine or preQ1. The peptide conformer recognising guanine, however, is also able to bind preQ0. To investigate selectivity regulation, kinetic data for Zymomonas mobilis TGT (ZmTGT) were recorded. They show that selectivity in favour of the actual substrate preQ1 over preQ0 is not achieved by a difference in affinity but via a higher turn-over rate. Moreover, a TGT(E235Q) variant was constructed. The mutation was intended to stabilise the peptide switch in the conformation favouring guanine and preQ0 binding. Kinetic characterisation of the mutated enzyme revealed that the Glu235Gln exchange has, with respect to all substrate bases, no influence on kcat. In contrast, KM(preQ1) is drastically increased while KM(preQ0) seemes to be decreased. Hence, regarding kcat/KM as an indicator for catalytic efficiency, selectivity of TGT in favour of preQ1 is abolished or even inverted in favour of preQ0 for TGT(E235Q). Crystal structures of the mutated enzyme confirm that the mutation strongly favours the binding pocket conformation required for the accommodation of guanine and preQ0. The way this is achieved, however, significantly differs from what was predicted based on crystal structures of wild type TGT.
tRNA-guanine transglycosylases (TGT) - present in all three domains of life - catalyse a base exchange reaction in tRNAs. In bacteria, the mechanistic pathway of the guanine exchange towards the modified base preQ1 is well characterised due to crystal structure analyses and elaborate kinetic studies of ZmTGT. As TGT plays a key role in pathogenicity of Shigella, the causative agent of bacterial dysentery, it has been established as a target for structure-based drug design. However, eukaryotes also possess a TGT with high homology to the bacterial one and most residues inside the binding pocket are conserved. In an analogous reaction the base queuine is incorporated into tRNA. Hence, it is of utmost importance to study selectivity-determining features. Since no crystal structure of an eukaryotic TGT is yet available, in this study based on ZmTGT a genetically engineered human TGT binding pocket was designed as a model system. Three amino acids specific for the human TGT binding pocket were introduced by site-directed mutagenesis of the ZmTGT gene, (C158V, (A232S), V233G). After purification, kinetic measurements and crystallisation were performed. Modified assays showed that - surprisingly – the Zm TGT variants have dramatically slowed down so that kinetic parameters could not be determined for incorporation of queuine into tRNA. Nevertheless, crystal structure analysis and molecular dynamics simulations indicate a possible binding of queuine to the active site of the variants. Additionally, it was observed that the wild type enzyme is able to excorporate labeled guanine from tRNA in the presence of queuine. MD simulations indicate that the enzyme is able to open up a subpocket that potentially could accommodate queuine.|