Project number: QLK3-CT-2001-00277
EC contribution: 2.016,129 € (euro)
Duration: 36 months
Starting date: (01/01/2002)
Contract type: shared cost
Summary: For a novel strategy to combat bacterial multidrug resistance we have selected as targets the bacterial apoptotic systems (BASs) (Fig. 1). They have a stable toxin and an unstable anti-toxin as their key elements that may be chromosomally encoded or encoded by multidrug-resistance plasmids (Fig. 2). Apoptotic systems differ in bacteria and eukaryotes, thus BASs and their checkpoint elements are an attractive and a so-far unexplored family of targets for the development of antibiotics. The novelty of this approach lies in the development of assays to highlight the deregulation of the BASs, with a subsequent triggering of cell death. High-throughput screening (HTS) and detailed structure-function knowledge of the BASs will enable the design of synthetic modifications necessary to enhance the specificity of targeting.
Problem: The need to discover novel antibacterial compounds to combat new multi-drug resistant strains is urgent, because of the rapid rise in the number of pathogenic bacteria that have acquired resistance to all commonly used drugs. This problem requires immediate action along new paths. We therefore decided to exploit BASís as a new class of targets for the development of novel anti-microbial compounds and anti-infective interventions.
Aims: To engineer bacterial strains for in vivo screening and in vitro methods to identify novel compounds that may deregulate the BASs. To develop cellular assays to monitor the activity of the BASs and to render them suitable for HTS of chemical libraries and natural compounds collections. The checkpoint elements and the cellular targets of the toxins will be analysed by the use of genomics and proteomics. Biochemical and biophysical methodologies will be used to characterise the details of toxin-antitoxin protein complexes.
Expected results: The work will involve an integrated programme involving: i) the production of novel, robust, easy to run, and cheap in vivo assays to screen for the deregulation of the BASs; ii) the identification of bacterial growth inhibitors that cross the cell membrane(s); iii) the detailed structural mechanistic, analytical, and engineering studies on the relevant BASs system; iv) the development of biochemical and biophysical methodologies to providing data on the interactions of the system components, which will be used in the rational selection of compounds and tailored drug design, and v) the validation of the highlighted positive hits in vitro. The results will be combined to design engineered bacteria which will be used to identify lead compounds. Employment of biochemical and biophysical methodologies yielding data on the details of the interactions of the system components for possible use in rational selection of compounds and tailored drug design will be pursued.
Potential applications: The identification of the BASís check point elements and the biochemical and biophysical analysis of the toxin/ target interactions, and understanding the functional mechanisms of BASs will allow to develop means to activate the toxins, leading to bacterial cell death. This, in turn, should lead to the identification of potential novel antimicrobial compounds that may constitute the next generation of antimicrobials. The scientific results will be implemented for the construction of engineered bacterial strains that will be used as starting points for industry to identify new natural compounds that blocks cell proliferation. The validated compounds will be used to build the desired compound that target BASs. The industrial participant has the expertise and commitment to develop new antimicrobials.
Fig. 1: A model of E. coli RelBE-mediated cell death. The antitoxin (RelB) neutralises the toxin (RelE) by forming a TA complex. The TA complex binds to the operator in the promoter region and represses transcription. A cellular protease degrades the antitoxin thereby leaving an activated toxin. The question mark indicates that it is not yet known if a free toxin or a TA with a different stoichiometry binds to the target.
Fig. 2: Genetic organisation of the chromosomal (A) and plasmid-based (B) TA loci. The upstream gene specify for an antitoxin (in purple) and the downstream for a toxin (in blue) and, in all cases, form an operon. The bent arrow denotes direction of transcription. Under condition of continuous expression both products are synthesised