Phosphotransferases in bacterial cell wall biosynthesis

The cell wall is an important, and sometimes overlooked, key facet of one of two major branches of bacteria, called the Gram-positives. Without the cell wall, the Gram-positive bacterial cell would become seriously compromised and the soluble contents of the cell would burst through the rather fragile single lipid membrane that encompasses the cell. The cell would die. The cell wall of these organisms comprises two major components, a mesh-like structure of cross-linked strands of peptidoglycan, which becomes attached to long, polymeric carbohydrates, such as the teichoic acids and the capsule. Both peptidoglycan and teichoic acid play key roles - peptidoglycan provides the necessary physical protection from the osmotic pressure of the cell, and its synthesis is co-ordinated with the growth of the cell so that the peptidoglycan is not limiting. Teichoic acids are required for the interaction of bacteria with their surroundings, for the regulation of other enzyme activities on the cell surface and contribute to the maintenance of the overall shape of the cell. Capsules are often required to protect pathogens against components of the immune system. Teichoic acids, capsular polysaccharides and peptidoglycan are synthesised in multiple, independent steps by a series of specific biological catalysts, called enzymes. Teichoic acids and capsular polysaccharides are synthesised inside the cell, whereas peptidoglycan is synthesised outside the cell. The lipid-linked teichoic acid precursor is transported across the membrane and transferred onto peptidoglycan, although the precise stage in the synthesis of peptidoglycan that this transfer step occurs is unknown. Only this attachment of the major anionic polymers (wall teichoic acid and capsule) to peptidoglycan builds the final, functional cell wall essential for bacterial lifestyle.

We have discovered the enzyme family, called LCP, which performs this transfer step, and in this proposal we seek to understand the molecular basis for its biochemical properties. Specifically, we will study the biochemical reaction catalysed by these enzymes and determine high resolution 3-dimensional structures of the enzymes in complex with the reaction components. We will also develop the synthesis, catalysed by the known enzymes in the pathway, of teichoic acid building blocks that will be better suited to the study of the biochemistry of these proteins than the commercially available components that we have used thus far.

Gram-positive bacteria have a single, cytoplasmic membrane and a thick cell wall to provide a protective, rigid layer. The cell wall, a complex glycopolymer, helps to protect the integrity of the cell from the osmotic pressure of the cytoplasm. The cell wall glycopolymer comprises peptidoglycan (PG) and covalently attached acidic/anionic polymers, such as wall teichoic acids (WTA) and capsular polysaccharides (CPS). The synthesis of PG and anionic/acidic polymers are linked and are co-ordinated by cytoskeletal elements. In contrast to PG, the WTAs/CPSs are polymerized inside the cell, at the inner face of the cytoplasmic membrane, before being transported across the membrane for attachment to PG. The precursors of both PG and the anionic/acidic polymers are linked to the same undecaprenol phosphate carrier lipid for transport across the membrane. In the last step of bacterial cell wall assembly a phosphodiester bond is formed between WTA/CPS and an N-acetyl muramic acid group of PG, connecting the major polymers to build the mature, functional cell wall. The subject of this application is the loading of PG by WTA, catalysed by a newly identified and widespread class of phosphotransferases, the LCP family of enzymes. Our preliminary data, mostly genetic and structural biology, and some preliminary biochemistry, which are all summarised within the underpinning of this proposal, indicate that the LCP family of proteins do not have regulatory roles in cell wall biosynthesis, as has been hypothesised; instead they encode the necessary enzymatic function to perform this biochemical reaction. In this proposal we seek to understand at a molecular level the transferase step, by the structure solution of enzymatically relevant complexes by crystallography, the enzymatic synthesis of more biologically relevant WTA mimics, the dissection of the reaction mechanism and the in vitro reconstitution of the decoration of PG by WTA in a lipid vesicle mimic of the cell.

Bacteria are the most abundant and diverse organisms known to man and our relationships with them are mostly beneficial. A complete understanding of bacterial physiology is therefore important if we wish to understand them, not only in their exploitation for mankind's benefit (in, for example, the "white" biotechnology sector and bio-fuels), but also as we strive to determine new means to disable disease-causing species and strains. This research, on a fundamental step in the biosynthesis of the cell wall of bacteria, will mostly impact upon other academics in the rather large fields of structural biology, biochemistry and microbiology. We anticipate interest from others who study the equivalent process in plants and fungi, organisms that also contain a significant cell wall structure. The methodologies that we will pursue are likely to be of interest to others who wish to exploit bacteria in the semi-synthetic manufacturing of biologicals.

Whilst the immediate outcome of this project is not to develop novel antibiotics, the enzymatic step that we wish to understand has potential in this regard. At an appropriate time in the duration of the project, we will contact the commercial development team of Newcastle University to assess further the potential for commercial exploitation. Moreover, Newcastle University has a spin-out biotech company, Demuris Ltd; we will discuss with representatives from Demuris, in the first instance, their likely interest in the development of inhibitors to this enzyme step.

New knowledge that is generated by the research proposal will be disseminated by its publication in high impact, international scientific journals. Both applicants have extensive experience in this regard. Should the discoveries be major, then we will co-ordinate press releases through the media office of the University, and summaries of the information placed on websites hosted by the University and RJL. Both applicants will also present the results at national and international conferences and in invited seminars at other institutions.