"Bacterial pathogens have evolved distinct ways of colonizing host cells and promote infection. Many human intestinal bacterial pathogens such as Salmonella, Shigella and enteropathogenic/ enterohemorrhagic Escherichia coli utilize type III secretion systems (T3SS) to deliver virulence effector proteins into the host to promote colonization and interfere with antimicrobial host response. The T3SS Escherichia coli Host strain O127:H6 is a human Enteropathogenic pathogen (EPEC) that was identified as the mayor cause of “summer diarrhoea” until the 1950s in developed countries and was associated with high mortality rates. Nowadays, in developed countries, EPEC strains are no longer consider important agents of diarrhoea. However, are still responsible for occasional outbreaks in paediatric wards and daycare centres. Newborn infants can acquire EPEC during the first days of life mainly by two routes: maternal organisms ingested at the time of birth; or bacteria from other infants with diarrhoea disease, commonly transmitted on the hands of nursery personnel or parents. Furthermore, the incipient raise of multi drug resistance bacteria linked with the present of T3SS constitutes a potencial threat against health. Among the T3SS effectors, the NleB protein has been shown to be critical for the enteric pathogens virulence and its directly link with the diarrhoea disease. NleB1 is a glycosyltransferase that has been shown to interact with host cell death-domain- containing proteins. O-GlcNacylation post translational modification is well known in eukaryotic cells for modulate several cellular processes such as transcriptional regulation, cycle control, stress response or signal transduction, among others, by controlling protein localization, activity and molecular interactions. However, serine or threonine O-GlcNAcylation can, potentially, be removed by the action of Eukaryotic enzymes. NleB1 overcomes this issue just by promoting arginine N-GlcNAcylation. Unlike the widely known asparagine N-glycosylation the modification of the arginine guanidine group with the addition of sugar is a recent discovery with a clear link with singling. Furthermore, this strategy completely blocks any host cell response against infection. Understanding the molecular mechanisms behind this ""novel"" mechanism of glycosylation will allow the design of chemical probes with potential to be used as antimicrobials or has the potential to be developed into new biological tools. Furthermore this new glycosylation mechanism opens a complete new research field within the pathogen-host interactions and the cell-signalling mechanisms.
BATTLE project is divided into four main objectives or aims.
1. Obtain the three dimensional structure of Nleb1 and NleB1 complexed with UDP-GlcNAc by employing macromolecular crystallography methods.
2. Obtain the three dimensional structure of NleB1 in complex with their Eukaryotic protein partner FADD-DD.
3. Characterise the Arginine GlcNacylation mechanism at functional and atomic level.
4. Start the process of designing efficient molecular probes and potential inhibitors against NleB activity with special emphasis in the search of bi-substrate inhibitory molecules."
