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Final Report Summary - PGRECONST (Assembly and coordination of peptidoglycan synthesis complexes during bacterial growth and cell division)

This report summarizes the research activity performed by Dr. Manuel Pazos during the Marie Curie Intra-European Fellowship project PGRECONST at the Centre for Bacterial Cell Biology, Newcastle University under the supervision of Prof. Waldemar Vollmer.

Beta-lactam antibiotics, like penicillin, target peptidoglycan synthases (PBPs) and inhibit their transpeptidase sites inducing the lysis of the bacterial cell. Although structural modifications of the initially discovered penicillin have led to the development of antibiotics with higher affinity to the PBPs, there is an urgent need to find new drugs to fight bacterial antibiotic resistance. In Europe, 25,000 deaths have been reported as a result of antimicrobial resistance, with two thirds of these deaths being due to Gram-negative bacteria, and the societal costs of antimicrobial resistance is estimated €1.5 billion per year in Europe (according to the Office of Health Economics, April 2011). Studies addressing fundamental questions in bacterial cell growth have been published in the major discovery journals, indicating the relevance of the EU funded IEF-project PGRECONST.
Most bacteria surround their cytoplasmic membrane with an essential structural component, the peptidoglycan (PG) sacculus, to maintain their cell shape and to counteract the osmotic pressure of the cytoplasm. The sacculus is a remarkable molecule made of glycan chains crosslinked by short peptides, forming a thin, net-like and elastic single layer in the periplasm of Escherichia coli. Several enzymatic activities are required to enlarge the surface area of the PG sacculus during bacterial growth and cell division, i.e. to synthesize and attach new PG and hydrolyse bonds in the sacculus to open meshes allowing the insertion of the new PG. Although it is known that the activities within both PG synthesis complexes, called elongasome and divisome, are regulated by different proteins from the cytoplasm and periplasm, they are poorly characterized and when and where interactions between cell envelope proteins take place in the cell are still largely unknown. The knowledge about the essential proteins, biochemical activities and interactions during cell wall growth allow us to address the synthetic reconstruction of essential functions in the test tube, to support the results and conclusions obtained from the cellular studies.

During the IEF-PGRECONST project new interactions have been found involving proteins from both PG synthetic complexes. The interactions have been confirmed in vivo by Bimolecular Fluorescent Complementation (BiFC), co-immunoprecipitation and/or Fӧster Resonance Energy Transfer (FRET) assays. To characterize these interactions, purified membrane proteins were assayed using different approaches including Microscale Thermophoresis and crosslink-pulldown experiments, analytical ultracentrifugation and glycosyltransferase and transpeptidase activity assays. Preliminary data have been also obtained using liposomes and lipid nanoparticle reconstitution assays, and crystallization experiments are currently in progress. Genetic studies have been also performed to characterize the essentiality of proteins involved in the regulation of the major bifunctional synthases activity, as PBP1B.
Interesting results have been achieved from the main aims of the project during the fellowship. A new interaction between a PG synthesis protein and a Tol-Pal complex protein has been found, and the effect over the synthetic activity of the complex has been described. New interactions between proteins from elongasome and divisome complexes have been identified and their role in activity regulation has been quantified, supporting the functional redundancy of the major bifunctional synthases, PBP1A and PBP1B. These data also agrees with the current model in which the elongation and septation machineries interact at an intermediate stage before cell division takes place. It has also been found that PBP1B activity is not impaired in vivo in the absence of different effector proteins, and that the impairment of the outer membrane integrity may increase the cell septation. Overall, the results showed how robust the peptidoglycan synthetic process is through the functional redundancy of the major synthases.
Heretofore, the regulation of the PG synthesis complexes activity and their coordination with the outer membrane constriction during cell division have been explored only on few studies, and how the cell does the transition between PG synthesis during elongation to septation is still largely unknown. The main goal of the project was to identify and characterise new protein interactions between both complexes that help to clarify the PG synthesis process during the bacterial cell cycle.
With the research work done during this project we have evidenced that the main synthase involved in cell elongation, PBP1A, interact with two cell division proteins that enhance its synthetic activity. These finding will be submitted for publication to an international scientific journal in the near future. The new protein interactions within the septosome and the effects caused by the absence of some PBP1B-effector proteins on cell septation will allow us to further understand how the cell division and outer-membrane constriction are coordinated.
In the long term, identification of the proteins involved in cell growth and division and their effect over the required enzymatic activities will help us to screen for new antibiotic compounds and identify novel strategies to design them.

Scientist in charge:
Prof. Waldemar Vollmer
Institute for Cell and Molecular Biosciences
The Centre for Bacterial Cell Biology, Baddiley-Clark Building, Newcastle University, Richardson Road, Newcastle upon Tyne, NE2 4AX
Tel: +44 (0) 191 208 3216

Marie Curie Fellow:
Dr. Manuel Pazos Don Pedro
Institute for Cell and Molecular Biosciences
The Centre for Bacterial Cell Biology, Baddiley-Clark Building, Newcastle University, Richardson Road, Newcastle upon Tyne, NE2 4AX
Tel: +44 (0) 191 208 3211

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