Interaction and interdependency of GlmM and DacA - two essential enzymes required for methicillin resistance in Staphylococcus aureus

Staphylococcus aureus is frequently found on the skin, nares or other mucosal surfaces of healthy individuals without causing any symptoms or harm. However, S. aureus can also cause a wide variety of infections ranging from small skin infections to life-threatening invasive diseases. Alarmingly, many S. aureus strains have acquired resistance to multiple antimicrobials and as a result S. aureus is still one of the leading causes of antibiotic-resistant healthcare-associated infections worldwide. The resulting difficulty in treating these infections is a major public health concern. Therefore, it is of interest to study biosynthesis pathways and enzymes that are required for bacterial growth to ultimately develop new antimicrobials. The purpose of this study is to characterize the interaction and interdependency of the two staphylococcal enzymes GlmM and DacA. Both enzymes are extremely important for the growth of S. aureus. At the first glance there is no apparent connection between them as GlmM is required for the production of an essential cell wall precursor and DacA is responsible to the production of a recently discovered signalling nucleotide c-di-AMP. However, recent work has indicated that there is a direct protein/protein contact between these two enzymes, suggesting that the pathways that they regulate are somehow interconnected. DacA and GlmM are therefore potentially central players in coordinating the synthesis of the cell wall polymer peptidoglycan with pathways that are regulated by c-di-AMP. The aims of this project are to provide an image of the DacA/GlmM complex in order to show exactly how these two proteins interact, find out how the activity of one enzyme influences the activity of the other and how this interaction affects the growth and methicillin resistance of the cell. In summary, the work will yield valuable information on the interdependency of two important bacterial enzymes, which interconnect two essential cellular processes in the Gram-positive bacterial pathogen S. aureus. Due to the direct link of S. aureus to human infection, the output of this work on essential cellular pathways can potentially offer future opportunities for the development of novel therapeutic approaches interfering with the growth of S. aureus and potentially a number of other Gram-positive pathogens.

The cell wall in Staphylococcus aureus is composed of a thick peptidoglycan layer, cell surface proteins, teichoic acids and carbohydrate polymers. Proper assembly of the different structures is not only essential for the pathogenesis of S. aureus but also for bacterial growth and several antimicrobial agents target the synthesis of key cell wall components. GlmM is a phosphoglucosamine mutase required for the production of UDP-N-acetylglucosamine, an essential cytoplasmic precursor for the synthesis of peptidoglycan. In S. aureus and many other Gram-positive bacteria, glmM forms a three-gene operon with dacA and ybbR, which encode the c-di-AMP synthase DacA and the membrane protein YbbR that is thought to regulate the activity of cylcase, respectively. c-di-AMP is a recently discovered signalling molecule that is essential for growth of S. aureus and many other Gram-positive bacteria in standard laboratory conditions. Recent work has highlighted a physical interaction between GlmM and DacA, suggesting that these enzymes are central players in coordinating peptidoglycan synthesis with the metabolic pathways that are regulated by c-di-AMP. These include in S. aureus potassium and osmolyte uptake as well as pH homeostasis. The aims of this project are to characterize the interdependency of the DacA and GlmM enzyme activities using in vitro assay systems, to determine the structure of the DacA/GlmM complex, to investigate the in vivo dynamics and the functional consequence of the DacA/GlmM interaction on the metabolism and methicillin resistance of S. aureus and to conclusively determine if GlmM is essential for the growth of S. aureus to resolve conflicting data. All together this work will allow us to propose a model for the molecular mechanism by which binding of GlmM can influence the activity of the c-di-AMP cyclase, provide insight into the biological significance of this protein interaction and uncover new avenues to interfere with the growth of S. aureus.

For details please see pathways to impact document. In brief, the outcomes of this research project will provide information on the structures and molecular basis by which two central Staphylococcus aureus enzymes, DacA and GlmM, interact to coordinate the essential cellular processes of c-di-AMP signalling and peptidoglycan synthesis. Both enzymes are conserved and present in a large number of other Gram-positive bacteria and hence information gained from this work will have an impact on a variety of different academic research fields. Many S. aureus strains have acquired resistance to multiple antimicrobials and S. aureus remains a leading cause of antibiotic-resistant healthcare-associated infections worldwide. The resulting difficulty in treating these infections is a major public health concern. Therefore, it is of interest to study biosynthesis pathways and enzymes such as described in this application that are required for bacterial growth to ultimately develop new antimicrobials. Therefore, this work will also be of interest to pharma as well as health care professionals.