Bases moleculares de la señalización mediada por dUTPasas triméricas de bacteriófagos

Stl es un represor maestro codificado por islas de patogenicidad de Staphylococcus aureus (SaPIs) que participa en la diseminación de estos elementos genéticos móviles en el cromosoma bacteriano. Después de la infección o inducción de un fago colaborador residente, las SaPIs son de-reprimidas por interacciones específicas de las proteínas del fago con el represor Stl, permitiendo a la isla replicarse e introducir su material genético en las cápsides del fago, aprovechando su maquinaria para conseguir una transferencia horizontal de genes cuando las partículas víricas con el material genético de la isla infectan otras células. Las SaPIs han desarrollado un mecanismo fascinante para asegurar su transferencia promiscua al dirigirse a proteínas estructuralmente no relacionadas que realizan funciones idénticas y conservadas para el fago, las dUTPasas diméricas y triméricas. Estas proteínas actuarían de manera análoga a las proteínas G protooncogénicas, donde su función moonligthing y señalizadora estaría regulada por dUTP, el cual actuaría de segundo mensajero permitiendo o impidiendo la unión del represor y, por tanto, la replicación y transferencia de los genes de la isla. En esta tesis desciframos el mecanismo molecular de esta elegante estrategia mediante ensayos mutacionales que, primeramente, delimitan las áreas implicadas en la interacción y, finalmente, determinando y validando la estructura del represor Stl de SaPIbov1 solo y en complejo con dUTPasas de diferentes fagos de S. aureus.Sorprendentemente, Stl de SaPIbov1 se trata de una proteína modular capaz de interaccionar con dUTPasas diméricas y triméricas mediante dominios y mecanismos diferentes, En el caso del complejo de Stl de SaPIbov1 con dUTPasas triméricas, el dominio intermedio del represor interviene de manera clave a través del centro activo de la enzima emulando al sustrato de ésta, el dUTP, e interaccionando con motivos conservados de la misma. Por otra parte, la formación del complejo del represor con dUTPasas diméricas se genera a través del motivo Cterminal, el cual no sólo interacciona con el centro activo de la enzima sino que, además, emula la superficie de dimerización de la dUTPasa, formando un heterodímero con ésta. La adquisición de multiespecificidad a través del reclutamiento de dominios propone a Stl de SaPIbov1 como un nuevo tipo de represor, el cual conserva su mecanismo de interacción con ADN pero permite una evolución de otras partes de la proteína con el fin de una difusión amplia de las SaPIs por la naturalezaStl is a master repressor encoded by Staphylococcus aureus pathogenicity islands (SaPIs) that participates in the dissemination of these mobile genetic elements on the bacterial chromosome. After infection or induction of a resident helper phage, the SaPIs are de-repressed by specific interactions of the phage proteins with the Stl repressor, allowing the island to replicate and introduce its genetic material into the phage capsids, taking advantage of its machinery to achieve a horizontal gene transfer when virus particles with genetic material from the island infect other cells. SaPIs have developed a fascinating mechanism to ensure their promiscuous transfer by targeting structurally unrelated proteins that perform identical and conserved functions for the phage, the dimeric and trimeric dUTPases. These proteins would act in a similar way to the proto-oncogenic G proteins, where their moonligthing and signaling function would be regulated by dUTP, which would act as a second messenger, allowing or preventing the binding of the repressor and, therefore, the replication and transfer of the genes of the island. In this thesis we decipher the molecular mechanism of this elegant strategy through mutational assays that, firstly, delimit the areas involved in the interaction and, finally, determining and validating the structure of the Stl repressor of SaPIbov1 alone and in complex with dUTPases from different S. aureus phages. Surprisingly, SaPIbov1 Stl is a modular protein capable of interacting with dimeric and trimeric dUTPases through different domains and mechanisms. In case of the SaPIbov1 Stl complex with trimeric dUTPases, the intermediate domain of the repressor plays a key role through the active center of the enzyme emulating its substrate, dUTP, and interacting with conserved motifs of the same. On the other hand, the formation of the repressor complex with dimeric dUTPases is generated through the Cterminal motif, which not only interacts with the active center of the enzyme but also emulates the dimerization surface of the dUTPase, forming a heterodimer. The adquisition of multispecificity through the recruitment of domains proposes Stl from SaPIbov1 as a new type of repressor, which preserves its mechanism of interaction with DNA but allows an evolution of other parts of the protein with the aim of a wide diffusion of the SaPI proteins

The bacteriophage–phage-inducible chromosomal island arms race designs an interkingdom inhibitor of dUTPases

Stl, the master repressor of the Staphylococcus aureus pathogenicity islands (SaPIs), targets phage-encoded proteins to derepress and synchronize the SaPI and the helper phage life cycles. To activate their cycle, some SaPI Stls target both phage dimeric and phage trimeric dUTPases (Duts) as antirepressors, which are structurally unrelated proteins that perform identical functions for the phage. This intimate link between the SaPI’s repressor and the phage inducer has imposed an evolutionary optimization of Stl that allows the interaction with Duts from unrelated organisms. In this work, we structurally characterize this sophisticated mechanism of specialization by solving the structure of the prototypical SaPIbov1 Stl in complex with a prokaryotic and a eukaryotic trimeric Dut. The heterocomplexes with Mycobacterium tuberculosis and Homo sapiens Duts show the molecular strategy of Stl to target trimeric Duts from different kingdoms. Our structural results confirm the participation of the five catalytic motifs of trimeric Duts in Stl binding, including the C-terminal flexible motif V that increases the affinity by embracing Stl. In silico and in vitro analyses with a monomeric Dut support the capacity of Stl to recognize this third family of Duts, confirming this protein as a universal Dut inhibitor in the different kingdoms of life

Bases moleculares de la señalización mediada por dUTPasas triméricas de bacteriófagos

Tesis doctoral, 328 p. Figuras y tablas.[EN] Stl is a master repressor encoded by Staphylococcus aureus pathogenicity islands (SaPIs) that participates in the dissemination of these mobile genetic elements on the bacterial chromosome. After infection or induction of a resident helper phage, the SaPIs are de-repressed by specific interactions of the phage proteins with the Stl repressor, allowing the island to replicate and introduce its genetic material into the phage capsids, taking advantage of its machinery to achieve a horizontal gene transfer when virus particles with genetic material from the island infect other cells. SaPIs have developed a fascinating mechanism to ensure their promiscuous transfer by targeting structurally unrelated proteins that perform identical and conserved functions for the phage, the dimeric and trimeric dUTPases. These proteins would act in a similar way to the proto-oncogenic G proteins, where their moonligthing and signaling function would be regulated by dUTP, which would act as a second messenger, allowing or preventing the binding of the repressor and, therefore, the replication and transfer of the genes of the island. In this thesis we decipher the molecular mechanism of this elegant strategy through mutational assays that, firstly, delimit the areas involved in the interaction and, finally, determining and validating the structure of the Stl repressor of SaPIbov1 alone and in complex with dUTPases from different S. aureus phages. Surprisingly, SaPIbov1 Stl is a modular protein capable of interacting with dimeric and trimeric dUTPases through different domains and mechanisms. In case of the SaPIbov1 Stl complex with trimeric dUTPases, the intermediate domain of the repressor plays a key role through the active center of the enzyme emulating its substrate, dUTP, and interacting with conserved motifs of the same. On the other hand, the formation of the repressor complex with dimeric dUTPases is generated through the Cterminal motif, which not only interacts with the active center of the enzyme but also emulates the dimerization surface of the dUTPase, forming a heterodimer. The adquisition of multispecificity through the recruitment of domains proposes Stl from SaPIbov1 as a new type of repressor, which preserves its mechanism of interaction with DNA but allows an evolution of other parts of the protein with the aim of a wide diffusion of the SaPI proteins.[ES] Stl es un represor maestro codificado por islas de patogenicidad de Staphylococcus aureus (SaPIs) que participa en la diseminación de estos elementos genéticos móviles en el cromosoma bacteriano. Después de la infección o inducción de un fago colaborador residente, las SaPIs son de-reprimidas por interacciones específicas de las proteínas del fago con el represor Stl, permitiendo a la isla replicarse e introducir su material genético en las cápsides del fago, aprovechando su maquinaria para conseguir una transferencia horizontal de genes cuando las partículas víricas con el material genético de la isla infectan otras células. Las SaPIs han desarrollado un mecanismo fascinante para asegurar su transferencia promiscua al dirigirse a proteínas estructuralmente no relacionadas que realizan funciones idénticas y conservadas para el fago, las dUTPasas diméricas y triméricas. Estas proteínas actuarían de manera análoga a las proteínas G protooncogénicas, donde su función moonligthing y señalizadora estaría regulada por dUTP, el cual actuaría de segundo mensajero permitiendo o impidiendo la unión del represor y, por tanto, la replicación y transferencia de los genes de la isla. En esta tesis desciframos el mecanismo molecular de esta elegante estrategia mediante ensayos mutacionales que, primeramente, delimitan las áreas implicadas en la interacción y, finalmente, determinando y validando la estructura del represor Stl de SaPIbov1 solo y en complejo con dUTPasas de diferentes fagos de S. aureus.Sorprendentemente, Stl de SaPIbov1 se trata de una proteína modular capaz de interaccionar con dUTPasas diméricas y triméricas mediante dominios y mecanismos diferentes, En el caso del complejo de Stl de SaPIbov1 con dUTPasas triméricas, el dominio intermedio del represor interviene de manera clave a través del centro activo de la enzima emulando al sustrato de ésta, el dUTP, e interaccionando con motivos conservados de la misma. Por otra parte, la formación del complejo del represor con dUTPasas diméricas se genera a través del motivo Cterminal, el cual no sólo interacciona con el centro activo de la enzima sino que, además, emula la superficie de dimerización de la dUTPasa, formando un heterodímero con ésta. La adquisición de multiespecificidad a través del reclutamiento de dominios propone a Stl de SaPIbov1 como un nuevo tipo de represor, el cual conserva su mecanismo de interacción con ADN pero permite una evolución de otras partes de la proteína con el fin de una difusión amplia de las SaPIs por la naturalezaPeer reviewe

Another look at the mechanism involving trimeric dUTPases in Staphylococcus aureus pathogenicity island induction involves novel players in the party

13 páginas, 5 figuras, 3 tablas, material suplementario en NAR onlineWe have recently proposed that the trimeric staphylococcal phage encoded dUTPases (Duts) are signaling molecules that act analogously to eukaryotic G-proteins, using dUTP as a second messenger. To perform this regulatory role, the Duts require their characteristic extra motif VI, present in all the staphylococcal phage coded trimeric Duts, as well as the strongly conserved Dut motif V. Recently, however, an alternative model involving Duts in the transfer of the staphylococcal islands (SaPIs) has been suggested, questioning the implication of motifs V and VI. Here, using state-of the-art techniques, we have revisited the proposed models. Our results confirm that the mechanism by which the Duts derepress the SaPI cycle depends on dUTP and involves both motifs V and VI, as we have previously proposed. Surprisingly, the conserved Dut motif IV is also implicated in SaPI derepression. However, and in agreement with the proposed alternative model, the dUTP inhibits rather than inducing the process, as we had initially proposed. In summary, our results clarify, validate and establish the mechanism by which the Duts perform regulatory functions.Ministerio de Economia y Competitividad (Spain) [BIO2013-42619-P to A.M.]; Medical Research Council (UK) [MR/M003876/1]; ERC-ADG-2014 Dut-signal (from EU) [Proposal no 670932 to J.R.P]; CSIC JAE-Doc Postdoctoral contract (Programa «Junta para la Ampliación de Estudios»), European Social Fund (to E.M.); FPU13/02880 (to J.R.C.), FPI BES-2014-068617 Predoctoral Fellowships (to C.A.). Diamond Light Source block allocation group (BAG) Proposal [MX10121]; Spanish Synchrotron Radiation Facility ALBA Proposal [2014060897]; European Community's Seventh Framework Programme [FP7/2007-2013]; BioStruct-X [283570]. Funding for open access charge: Ministerio de Economia y Competitividad (Spain) [BIO2013-42619-P to A.M.]; Medical Research Council (UK) [MR/M003876/1]; ERC-ADG-2014 Dut-signal (from EU) [Proposal no 670932 to J.R.P].Peer reviewe

Dissecting the link between the enzymatic activity and the SaPI inducing capacity of the phage 80α dUTPase

The trimeric staphylococcal phage-encoded dUTPases (Duts) are signalling molecules that induce the cycle of some Staphylococcal pathogenicity islands (SaPIs) by binding to the SaPI-encoded Stl repressor. To perform this regulatory role, these Duts require an extra motif VI, as well as the Dut conserved motifs IV and V. While the apo form of Dut is required for the interaction with the Stl repressor, usually only those Duts with normal enzymatic activity can induce the SaPI cycle. To understand the link between the enzymatic activities and inducing capacities of the Dut protein, we analysed the structural, biochemical and physiological characteristics of the Dut80α D95E mutant, which loses the SaPI cycle induction capacity despite retaining enzymatic activity. Asp95 is located at the threefold central channel of the trimeric Dut where it chelates a divalent ion. Here, using state-of-the-art techniques, we demonstrate that D95E mutation has an epistatic effect on the motifs involved in Stl binding. Thus, ion binding in the central channel correlates with the capacity of motif V to twist and order in the SaPI-inducing disposition, while the tip of motif VI is disturbed. These alterations in turn reduce the affinity for the Stl repressor and the capacity to induce the SaPI cycle

A polyamorous repressor: deciphering the evolutionary strategy used by the phage­inducible chromosomal islands to spread in nature

1 página con el abstract del póster presentado a 44th FEBS Congress Krakow, Poland. July 6-11, 2019Staphylococcus aureus pathogenicity islands (SaPIs) are a family of related 15­17Kb mobile genetic elements that carry and disseminate superantigen and other virulence genes. SaPIs reside passively in the bacterial chromosome, repressed by a master repressor called Stl, encoded by the own SaPI. The key feature of their mobility and spread is the induction by helper phages of their excision, replication, and efficient encapsidation into specific small­headed phage­like infectious particles. After infection or induction of a resident helper phage, SaPIs are de­repressed by the specific protein­protein interaction of phage proteins with Stl. SaPIs have developed a fascinating mechanism to ensure their promiscuous transfer by targeting with the Stl repressor structurally unrelated phage proteins performing the same conserved function. Combining structural biology approach and functional characterization in­vivo and in­vitro we decipher the molecular mechanism of this elegant strategy by which the SaPI hijacks the phage process to sense the starting of the lytic cycle. Our structural studies show that the Stl of the island SaPIbov1 combines a canonic HTH N­terminal domain to bind DNA, and sequentially acquires new domains which act as recognizing modules for the different phage proteins (antirepressors). Our in­vivo and in­vitro data deciphers the molecular mechanism that underlies the interaction between the Stl repressor and different phage coded antirepressors, showing how each Stl module mimics the substrate for each anti­repressor type. The interaction of Stl with different types of anti­repressor always disrupts the Stl dimer, implying the DNA dissociation and SaPI derepression. Our results establish the molecular mechanism of the interaction event that detonates the intra­ and inter­ generic transference of the clinically relevant SaPIs.Peer reviewe

Dissecting the link between the enzymatic activity and the SaPI inducing capacity of the phage 80α dUTPase

Abstract The trimeric staphylococcal phage-encoded dUTPases (Duts) are signalling molecules that induce the cycle of some Staphylococcal pathogenicity islands (SaPIs) by binding to the SaPI-encoded Stl repressor. To perform this regulatory role, these Duts require an extra motif VI, as well as the Dut conserved motifs IV and V. While the apo form of Dut is required for the interaction with the Stl repressor, usually only those Duts with normal enzymatic activity can induce the SaPI cycle. To understand the link between the enzymatic activities and inducing capacities of the Dut protein, we analysed the structural, biochemical and physiological characteristics of the Dut80α D95E mutant, which loses the SaPI cycle induction capacity despite retaining enzymatic activity. Asp95 is located at the threefold central channel of the trimeric Dut where it chelates a divalent ion. Here, using state-of-the-art techniques, we demonstrate that D95E mutation has an epistatic effect on the motifs involved in Stl binding. Thus, ion binding in the central channel correlates with the capacity of motif V to twist and order in the SaPI-inducing disposition, while the tip of motif VI is disturbed. These alterations in turn reduce the affinity for the Stl repressor and the capacity to induce the SaPI cycle

The structure of a polygamous repressor reveals how phage-inducible chromosomal islands spread in nature

Stl is a master repressor encoded by Staphylococcus aureus pathogenicity islands (SaPIs) that maintains integration of these elements in the bacterial chromosome. After infection or induction of a resident helper phage, SaPIs are de-repressed by specific interactions of phage proteins with Stl. SaPIs have evolved a fascinating mechanism to ensure their promiscuous transfer by targeting structurally unrelated proteins performing identically conserved functions for the phage. Here we decipher the molecular mechanism of this elegant strategy by determining the structure of SaPIbov1 Stl alone and in complex with two structurally unrelated dUTPases from different S. aureus phages. Remarkably, SaPIbov1 Stl has evolved different domains implicated in DNA and partner recognition specificity. This work presents the solved structure of a SaPI repressor protein and the discovery of a modular repressor that acquires multispecificity through domain recruiting. Our results establish the mechanism that allows widespread dissemination of SaPIs in nature

Another look at the mechanism involving trimeric dUTPases in Staphylococcus aureus pathogenicity island induction involves novel players in the party

We have recently proposed that the trimeric staphylococcal phage encoded dUTPases (Duts) are signaling molecules that act analogously to eukaryotic G-proteins, using dUTP as a second messenger. To perform this regulatory role, the Duts require their characteristic extra motif VI, present in all the staphylococcal phage coded trimeric Duts, as well as the strongly conserved Dut motif V. Recently, however, an alternative model involving Duts in the transfer of the staphylococcal islands (SaPIs) has been suggested, questioning the implication of motifs V and VI. Here, using state-of the-art techniques, we have revisited the proposed models. Our results confirm that the mechanism by which the Duts derepress the SaPI cycle depends on dUTP and involves both motifs V and VI, as we have previously proposed. Surprisingly, the conserved Dut motif IV is also implicated in SaPI derepression. However, and in agreement with the proposed alternative model, the dUTP inhibits rather than inducing the process, as we had initially proposed. In summary, our results clarify, validate and establish the mechanism by which the Duts perform regulatory functions

A fascinating example of convergent evolution involves Staphylococcus aureus phage encoded dimeric and trimeric dUTPases in signaling

Póster presentado al XL SEBBM Congress. Barcelona, 23-26 de octubre de 2017The dUTPase (Dut) enzymes prevent the misincorporation of uracil into the DNA and are encoded by almost all free-living organisms and some viruses. We have previously showed that phage-encoded trimeric Duts mediates the Staphylococcus aureus pathogenicity island (SaPIs) transfer by interacting to the SaPI-encoded repressor Stl, proposing that these Duts are regulatory proteins. Some S. aureus phages encode structurally unrelated dimeric Duts instead trimeric Duts. Surprisingly, a recent work, has involved one of these predicted dimeric Duts in the transfer of SaPIs by interacting with the same Stl repressor. With the aim of decipher the molecular basis of SaPI induction by dimeric Duts and to compare with the mechanism reported for trimeric Duts, we examined the SaPI mobilization capacity of dimeric Duts in vivo as well as its binding capacity to Stl repressor in vitro. We analyzed the effect of the dUTP in the Stl-Dut interaction, and finally, we solved the 3-D structure by X-ray crystallography of several dimeric Duts in different activation states. Our results show that dimeric and trimeric Duts from S. aureus phages present a striking parallelism in the mechanism of SaPI mobilization. These similarities would confirm the role of dUTP as new nucleotide with second messenger function. However, some differences suggest peculiarities in the molecular mechanism of Stl recognition and binding for each type of Duts. Our results support the idea that the signalling role of the Dut proteins is an important force driving evolution and speciation.J.Rafael Ciges-Tomas. PhD student, fellowship FPU13/02880 awarded by Ministry of Education, Culture and SportPeer reviewe