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dUTPase Signalling: from Phage to Eukaryotes

Periodic Reporting for period 3 - DUT-signal (dUTPase Signalling: from Phage to Eukaryotes)

Reporting period: 2018-12-01 to 2020-05-31

Preserving DNA integrity is of vital importance for all organisms. Genomes of all free-living organisms and many viruses encode the enzyme dUTPase (dUTP pyrophosphatase; DUT; EC which cleaves dUTP into dUMP and pyrophosphate. Since most DNA polymerases cannot distinguish between thymine and uracil, it has been traditionally assumed that DUTs are essential for DNA integrity and viability in many organisms by reducing the dUTP pool, preventing incorporation of dUTP into the chromosome. Recently, however, DUTs have been identified as being involved in the control of relevant cellular processes. How these regulatory functions are controlled remains unsolved. Based on our recent data, this project pursues the hypothesis that DUTs are signalling molecules, from phage to eukaryotes, acting analogously to eukaryotic G-proteins. The proposed work is important as it identifies a putative new universal family of signalling proteins, which remarkably involves dUTP as a second messenger.
Nucleotide signalling molecules contribute to cell signalling in all forms of life. Among others, cAMP and GTP play pivotal roles by controlling a vast number of cellular pathways in eukaryotes. Alteration of these fine-tuned mechanisms is associated with multiple disease processes, including cancer. Prokaryotic signalling nucleotides such as cyclic di‐AMP (c‐di‐AMP), cyclic di‐GMP (c‐di‐GMP) and guanosine tetra- or pentaphosphate ((p)ppGpp) contribute to bacterial virulence. Here we propose that the ubiquitous cellular metabolite dUTP is a novel-signalling molecule conserved through evolution. Our exciting evidence supports dUTP binding to DUTs inducing conformational changes in the DUT proteins that promote interaction with cellular binding partners, creating novel functional units. The biological significance of DUTs and dUTP has been underestimated to date and the concept of DUTs as signalling molecules, involving dUTP as a second messenger, represents a paradigm shift requiring investigation. Dissection of the molecular basis of this novel concept will aid understanding of many biological systems from phage to eukaryotes.
The hypothesis will be tested in different systems, from phages to more complex prokaryotic and eukaryotic models. These models have been chosen because of the existence of DUTs with different extra motifs in closely related species. As a general strategy we will generate variant DUT proteins carrying specific mutations affecting either their dUTPase activity, the correct disposition of the C-terminal domain V (trimeric DUTs), the extra motifs (dimeric and trimeric DUTs) or interactions with partners. In addition, in the different model organisms the cognate dut genes will be substituted by dut genes expressing the aforementioned mutant proteins, by dut genes from other species with different extra motifs or even by dut genes expressing non-structurally related DUTs. If, as occurred in all previously tested models, only the correct expression of the cognate DUTs is compatible with a normal lifecycle of the organism, this result will clearly break the dogma that DUTs are exclusively metabolic enzymes and will definitively implicate DUTs in signalling. Once this has been established, and to identify the DUT interaction partners, we will use two complementary strategies: i) systematic yeast two-hybrid screens and ii) affinity chromatography to isolate in vivo assembled DUT-protein complexes, which will be analysed by mass spectrometry. The specificity of the identified interactions will be analysed by different proteomic, genetic (two-hybrid) or biophysical techniques. Finally, the role of the identified DUT partners, and how these interactions affect/control the biology of the organisms will be analysed in vivo and in vitro, depending on the identified proteins/interactions.
Five different aims were proposed in this project, and the major achievements accomplished to date related to the different aims have been:

Aim 1. Establishing the molecular basis of DUT signalling by unravelling the three-dimensional structure of trimeric and dimeric staphylococcal DUTs in complex with their bacterial and phage partners.

- We have solved the structure of different trimeric and dimeric staphylococcal Duts.
- Using complementary approaches, we have identified different partners interacting with the trimeric and dimeric Duts.
- We have solved the structure of the dimeric and trimeric staphylococcal DUTs in complex with the SaPIbov1 repressor Stl, and have determined structurally how the Duts induce the SaPIbov1 cycle (Nature Communication, in revision).

Aim 2. Dissecting the DUT-dependent regulatory pathways in bacteriophages.

- We have demonstrated that these enzymes are required for phage replication by interacting with phage proteins (manuscript in preparation).
- As a consequence of the work we are performing with the phages and the Duts, we have reported the first new mode of natural transduction discovered in over 60 years, since Joshua Lederberg’s lab discovered classical generalized and specialized transduction in the 1950’s. In “lateral” transduction, DNA packaging initiates in situ from prophage genomes, and proceed for up to several hundred kb of the Staphylococcus aureus chromosome. Bacterial DNA is packaged at the efficiencies of viral genome packaging, and is transferred at astonishingly high frequencies that are 3-4 orders of magnitude greater than any known mechanism of transduction. To our knowledge, the observed efficiency and scale of host chromosomal DNA transfer is unprecedented in bacteriology.

Aim 3. Analysing the signalling capacity of trimeric and dimeric DUTs using prokaryotic models.

- We have established five different models: S. aureus phages, Escherichia coli, Enterococcus faecalis, Enterococcus faecium and Mycobacterium smegmatis.
- We have identified specific partners for each Duts in each model.
- We have obtained a set of mutants (both in the dut genes and in the genes encoding the interacting partners). To evaluate the impact of the dUTP in these processes we have also obtained inactive Dut mutants with the ability to bind to dUTP (or not).
- We have discovered the fascinating strategy used by the Staphylococcal pathogenicity islands to spread in nature. Analysing two different SaPIs, SaPIbov1 and SaPI2, encoding two different Stl repressors, we show that each SaPI Stl repressor has evolved to target a different conserved phage pathway. This occurs because the Stl repressors can interact with multiple, structurally unrelated phage proteins, from different phages, but in all cases these proteins perform the same conserved function for the phage. Even more surprising and exciting is the finding that these multiple interactions do not occur because the Stl repressors interact with a conserved domain present in all the proteins performing the same function. Remarkably, the SaPI repressors have evolved divergent domains to target these structurally unrelated proteins, allowing them to target the process rather than the protein. These findings reflect the highly evolved, fascinating and unprecedented biological strategy of the SaPIs, which ultimately enables promiscuous SaPI intra- and inter-generic transfer in nature.
- The work with E. coli has allowed us to identify SaPI-like elements in this species.

Aim 4. Determining the signalling capacity of monomeric DUTs.

- We have initiated the studies to obtain the 3D structure of the EBV DUT in complex with the ectodomain of TLR2

Aim 5. Expanding DUT signalling to eukaryotic models.

- We have obtained very exciting and promising data related to this aim. We have demonsrated that eukaryotic parasites’ DUT proteins carrying extra domains have cognate cellular partners with which they interact by a dUTP-dependent ON/OFF mechanism, thus controlling cellular processes. This has been demonstrated using two medically important parasites: Trypanosoma brucei and Leishmania mexicana. The results obtained here have the potential to break the dogma that eukaryotic encoded DUTs are exclusively metabolic enzymes and if successful will define the involvement of eukaryotic DUT in cell signalling.
There are two exciting results derived from this grant that are clearly beyond the state of the art:

1. The first scientific breakthrough has revealed a new way that bacteria evolves, thought to be at least 1,000 times more efficient than currently known mechanisms. The insights will help scientists to better understand how superbugs can rapidly evolve and become increasingly antibiotic resistant. This new process has been named Lateral Transduction, and now joins the two known methods of transduction: general and specialised transduction, both discovered by the American scientist Joshua Lederberg, who won the Nobel Prize in Physiology or Medicine for his work with bacteria. Working with the bacteria Staphylococcus aureus, we have been able to demonstrate that this new naturally occurring method of transduction was at least one thousand times more efficient than generalised transduction, the best currently known method. Due to the efficiency of lateral transduction, we hypothesise it is likely to be the most impactful type of transduction to occur in bacteria during its evolutionary process.

2. The second important result is the confirmation, using eukaryotic models, that the DUT enzymes are signalling molecules. Using Trypanosoma brucei and Leishmania mexicana as model organisms, the results already obtained in this grant propose that DUTs are also important signalling molecules in eukaryotes, acting as cellular regulators. This investigation is important, as it identifies a new and universal family of signalling proteins in eukaryotes, revealing their mechanism of action. Further confirmation of this hypothesis will have broad and substantial implications for biology. Further, since this enzyme is encoded by most human and animal pathogens, an understanding of the molecular basis involving DUTs in signalling, including the identification and characterisation of the cellular pathways controlled by these enzymes, may lead to the design of new approaches to control relevant infectious diseases.