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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - BATTLE (The molecular basis of NleB-mediated bacterial virulence)

Teaser

Summary

\"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) thatÂ was identified as the mayor cause of Â â€œsummer diarrhoeaâ€ 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.Â Newborn infants can acquire EPEC during the first days ofÂ lifeÂ mainly by two routes: maternal organisms ingested at the time of birth; or bacteria from other infants with Â diarrhoeaÂ 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.\"

Work performed

1. Obtain the three dimensional structure of Nleb1 and NleB1 complexed with UDP-GlcNAc by employing macromolecular crystallography methods.

- NleB1 was crystallised. Crystals were further optimised and good diffraction datasets were collected in the ESRF synchrotron radiation facility, Grenoble
- NleB1 protein structure was solved in complex with Mn atom coordinated by the DxD motif.

2. Obtain the three dimensional structure of NleB1 in complex with their Eukaryotic protein partner FADD-DD.

- NleB1 Structure provided the first clues about NelB1-FADD-DD interaction.
- The Critical role or FADD-DD Arginine 117 in the complex formation was shown.
- A Stable complex of Nelb1 and FADD-DD protein was obtained and purified.

3. Characterise the Arginine GlcNacylation mechanism at functional and atomic level.

- Site directed mutagenesis of NleB1 was performed to identify the key residues involved in Ump-GlcNAc transfer mechanism.
- One previously unappreciated NLeB1 residue was pointed out being critical in the GlcNAc transfer mechanism.

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.

- NleB1 Sugar binding properties were analysed and measured. Binding affinities and reaction kinetics were calculated.
- GlcNac analogues were tested with the aim to find better binders.
- NleB1 FAD-DD binding properties were characterised, including both full length protein and FADD-DD peptide assays.Binding Affinities were calculated.

Results obtained in points 1,2 and 3 are being prepared for being public diffused thought a multidisciplinar hight impact factor journal publications. Data obtained under the objective 4 allows the rational design of bi-substrate inhibitors by combining the sugar and the peptide components so, once available, will be delivered in specific chemical journals.

Final results

The molecular characterisation of a small bacterial cytoplasmic N-Glycosyl transferase designed to work inside eukaryotic cells has enormous potential as it will be required to design potential antimicrobials and data can be used, as well, to guide the engineering of a recombinant NleB able to recognise different protein partners. The latter with enormous potential to be used in cell signalling or even in other research fields by marking or permanently blocking the desired protein. However the further research directions are still far away from these ambitious objectives and basic steps must be first covered.

The Project has delivered the Structure of NleB1 and has finished with the biophysical characterisation of the enzyme. In addition the first attempts to cover the molecular mechanisms behind arginine GlcNAcylation and protein partner recognition have been delivered as well. However, the release of Three dimensional structures of FADD-DD and TRADD-DD complexes with NleB1 is still a pending task. To complete the picture of this interaction will allow a better understanding of the molecular process of protein partner recognition and will aid in the rational design of specific molecular probes against NleB1.

In a low time scale, the project has started the search for the optimal NleB1 inhibitor. As far as UDP-GlcNac is a promiscuous molecule expected to react with several critical hosts proteins, a more specific and directed approach by using bi-substrate inhibitors approach has been taken. Although the project wasn\'t able to deliver the first bi functional candidate molecules it explores the binding modes and opens the path to generate such kind of potential inhibitors. The clinical value of such a molecules is out of discussion but its has been notice during the project development that a new path to provide molecular probes to be used in molecular biology could be achievable as well.