Periodic Reporting for period 5 - DUT-signal (dUTPase Signalling: from Phage to Eukaryotes)
Reporting period: 2021-12-01 to 2022-11-30
Genomes of all free-living organisms and many viruses encode the enzyme dUTPase (dUTP pyrophosphatase; DUT; EC 3.6.1.23) which cleaves dUTP into dUMP and pyrophosphate. 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. 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.
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.
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.
- 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.
- We have solved the structure of the SaPI1 Stl repressor in complex with its inducing protein Sri, and have determined structurally how Sri induces the SaPI1 cycle. These results have identified a new family of repressors, and have characterised their mechanism of action.
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.
- 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. We have named this mechanism “lateral” transduction. To our knowledge, the observed efficiency and scale of host chromosomal DNA transfer via lateral transduction is unprecedented in bacteriology.
- We have reported the existence of helper and satellite pathogenicty islands, and have demonstrated how they interact to promote their promiscuous transfer in nature.
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).
- 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 demonstrated the signalling capacity of the monomeric DUTs.
Aim 5. Expanding DUT signalling to eukaryotic models.
- 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.
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.
- 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.
- We have solved the structure of the SaPI1 Stl repressor in complex with its inducing protein Sri, and have determined structurally how Sri induces the SaPI1 cycle. These results have identified a new family of repressors, and have characterised their mechanism of action.
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.
- 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. We have named this mechanism “lateral” transduction. To our knowledge, the observed efficiency and scale of host chromosomal DNA transfer via lateral transduction is unprecedented in bacteriology.
- We have reported the existence of helper and satellite pathogenicty islands, and have demonstrated how they interact to promote their promiscuous transfer in nature.
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).
- 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 demonstrated the signalling capacity of the monomeric DUTs.
Aim 5. Expanding DUT signalling to eukaryotic models.
- 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 several 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.
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.