The importance of adaptor proteins and cell signalling in the control of peptidoglycan synthesis

Abstract

Ph. D. Thesis.Bacterial cell division is a complex process that requires tight co-ordination and regulation of chromosome replication and segregation, and the synthesis and remodelling of the bacterial cell wall. The peptidoglycan sacculus is synthesised by peptidoglycan synthases and it is a vital component of the bacterial cell structure, to withstand the turgor of the cell to prevent its lysis FtsZ is one of the first proteins to localise at midcell during cell division where it forms a ring-like structure, the Z-ring, at the inner face of the cytoplasmic membrane. After Z-ring formation, the divisome is recruited to the division site to form the septum and the elongasome is positioned at the side wall for elongation. Both the elongasome and the divisome are dynamic macromolecular complexes composed of numerous proteins at unknown stoichiometries and it is thought there are proteins in both complexes that are yet to be discovered. Understanding bacterial cell division at the molecular and cellular level is an important area of research to enable the development of novel antibiotics as well as comprehending one of biology’s fundamental questions. GpsB is a conserved Gram-positive cytosolic protein that plays an important role in the elongation-division cycle by acting as a scaffold to control the relative spatial arrangements of peptidoglycan synthases (PBPs). In this thesis the molecular interactions of GpsB with cytoplasmic mini-domains of PBPs from two human pathogens (Listeria monocytogenes and Streptococcus pneumoniae) are described using structural and biochemical techniques. The importance of the critical interacting residues for the GpsB:PBP interaction for cell wall growth and viability of L. monocytogenes and S. pneumoniae were analysed in collaboration with the Halbedel, and Massidda and Winkler groups, respectively. A novel function of PBP binding was introduced into DivIVA, a cell division regulator and GpsB homolog, by protein engineering in an attempt to understand the functional divergence between GpsB and DivIVA. Potential building blocks for the development of GpsB:PBP inhibitors were identified in the form of small fragments by an X-ray crystallography-based fragment screening experiment at Diamond Light Source. Eukaryote-like serine/threonine protein kinases (eSTPKs) and partner phosphatases (eSTPs) are conserved in Gram-positive bacteria. They consist of an intracellular N-terminal kinase domain and an extracellular sensing region linked by a short transmembrane helix. The external regulatory region is comprised of three or four PASTA domains that bind to ii peptidoglycans and compounds containing beta-lactams. eSTPKs are involved in the regulation of many cellular processes including development of regulation, control of cell growth, stress response, virulence and sporulation. The eSTPK/eSTP pair from L. monocytogenes are PrkA and PrpC. This thesis concerns the importance of autophosphorylation for the function of the kinase domain of PrkA (PrkA-KD) and that phosphorylation of serine 173 is crucial for activation, agreeing with the mechanism of activation of Stk1 in Staphylococcus aureus. The previously uncharacterised phosphoprotein Lmo1503 (renamed ReoM) is a homologue of IreB from Enterococcus faecalis, a negative regulator of cephalosporin resistance. It is confirmed that ReoM is a substrate of the PrkA/PrpC pair and the crystal structure of the full length ReoM protein is presented. Isothermal titration calorimetry determined the interaction of ReoM and PrkA-KD is within the nanomolar range and there is a ten-fold reduction in affinity with a PrkAKDS173A mutation. The Halbedel group have linked ReoM phosphorylation to the activation of ClpCP-dependent degradation of the primary UDP-GlcNAc 1-carboxyvinyltransferase in L. monocytogenes, MurA. We therefore propose that cell wall integrity sensing by PrkA is coupled to the first committed step of peptidoglycan synthesis through the intermediate proteins ReoM and ClpCP