The biochemical characterisation of pivotal enzymes involved in mycobacterial peptidoglycan biosynthesis

At least 30 million individuals worldwide will have died from tuberculosis (TB) in the last decade of the 20th century. In the UK the steady decline in TB cases over the whole of the last century halted in the mid 1980s and there has been alarming signs of increased numbers of cases in certain communities. The situation is compounded by the AIDS epidemic and by the emergence of Mycobacterium tuberculosis strains that are resistant to virtually all the drugs that would normally be used to treat TB. It can be argued that, globally, M. tuberculosis is the single most important infectious agent affecting mankind. All bacteria have cells that, like plants, are enclosed in a cell wall. This protects the organism from its immediate environment and, fortuitously, presents an important target for drugs, like penicillin, that can be used to treat bacterial infections. However, M. tuberculosis has a distinctive cell wall that differs in composition from that of other bacteria. Although there are drugs that affect the unique M. tuberculosis cell wall, the current treatment for tuberculosis lasts 6 months and is potentially toxic to patients who often cease treatment early. Moreover, the efficacy of treatment is threatened by the emergence of drug-resistant strains of M. tuberculosis. There is a great need for new and better drugs to treat TB. The work proposed here therefore fulfils the clear need to extend our understanding of the bacterial physiology of the tubercle bacillus in the hope of priming novel therapeutic approaches to this ancient human adversary. Communication to the general public will be channelled through the University Press Office at Birmingham (http://www.newscentre.bham.ac.uk/office.htm) which has established contacts in the local and national media. The applicants will also highlight there research through personal University based Web pages, which has open access.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), has a unique cell envelope which accounts for its unusual low permeability and hence, contributes to resistance against common antibiotics. One of the main structural elements consists of a cross-linked network of peptidoglycan (PG), and access to comprehensive knowledge of the underlying genetic and biochemical profiles of key enzymes in PG biosynthesis is urgently needed. We plan to achieve this by following a multi-pronged approach that will involve the detailed biochemical and structural characterisation of M. tuberculosis Mur ligases and Mt-DapE, D-glutamate metabolism, and the generation of conditional mutants. More specifically the following objectives will be intensively investigated: (a) The regulation and pathway flux of M. tuberculosis PG biosynthesis and characterisation of Mt-Mur ligases; (b) How does phosphorylation control the M. tuberculosis PG biosynthetic pathway; (c) The importance of M. tuberculosis PG stem-peptide modifications and re-engineering of PG biosynthesis on entering stationary phase growth; and (d) Biochemical and structural characterisation of key enzymes involved in DAP (Mt-DapE) and D-glutamate biosynthesis. Answering these questions will provide fundamental knowledge on M. tuberculosis PG and cell wall biosynthesis. In addition, it is anticipated that the above functional and structural studies, will ultimately prove useful for screening for inhibitors, potentially generating valuable leads towards new anti-TB drugs.