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

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

Hijacking the hijackers: Escherichia coli pathogenicity islands redirect helper phage packaging for their own benefit

Phage-inducible chromosomal islands (PICIs) represent a novel and universal class of mobile genetic elements, which have broad impact on bacterial virulence. In spite of their relevance, how the Gram-negative PICIs hijack the phage machinery for their own specific packaging and how they block phage reproduction remains to be determined. Using genetic and structural analyses, we solve the mystery here by showing that the Gram-negative PICIs encode a protein that simultaneously performs these processes. This protein, which we have named Rpp (for redirecting phage packaging), interacts with the phage terminase small subunit, forming a heterocomplex. This complex is unable to recognize the phage DNA, blocking phage packaging, but specifically binds to the PICI genome, promoting PICI packaging. Our studies reveal the mechanism of action that allows PICI dissemination in nature, introducing a new paradigm in the understanding of the biology of pathogenicity islands and therefore of bacterial pathogen evolution

Pirating conserved phage mechanisms promotes promiscuous staphylococcal pathogenicity island transfer

Targeting conserved and essential processes is a successful strategy to combat enemies. Remarkably, the clinically important Staphylococcus aureus pathogenicity islands (SaPIs) use this tactic to spread in nature. SaPIs reside passively in the host chromosome, under the control of the SaPI-encoded master repressor, Stl. It has been assumed that SaPI de-repression is effected by specific phage proteins that bind to Stl, initiating the SaPI cycle. Different SaPIs encode different Stl repressors, so each targets a specific phage protein for its de-repression. Broadening this narrow vision, we report here that SaPIs ensure their promiscuous transfer by targeting conserved phage mechanisms. This is accomplished because the SaPI Stl repressors have acquired different domains to interact with unrelated proteins, encoded by different phages, but in all cases performing the same conserved function. This elegant strategy allows intra- and inter-generic SaPI transfer, highlighting these elements as one of nature’s most fascinating subcellular parasites

dUTPasas, una nueva familia de proteínas señalizadoras

Tesis doctoral, 385 p., figuras y tablasLas dUTPasas (Duts) se clasifican en tres familias: monoméricas, diméricas y triméricas. Recientemente se han descrito funciones alternativas asociadas a estas enzimas y en algunos casos está asociada a inserciones no requeridas para la actividad catalítica, como es el caso de las Duts triméricas de fagos de S. aureus. Estas Duts inducen islas de patogenicidad de S. aureus (SaPIs) mediante la interacción con el represor de SaPI, la proteína Stl. En esta tesis identificamos fagos de S. aureus que codifican para Dut dimérica en lugar de trimérica, y que también tienen capacidad para movilizar SaPIs. El estudio del mecanismo molecular que rige la interacción entre Dut dimérica y Stl revela paralelismos entre Duts triméricas y diméricas, siendo el dUTP un inhibidor de la formación de complejo, y Stl un inhibidor de la actividad enzimática en ambas Duts. A pesar de estas similitudes los resultados confirman que Stl es un proteína modular y emplea diferentes dominios para la interacción con Dut dimérica y trimérica, mimetizando en ambos casos las interacciones del dUTP en el centro activo. El empleo de diferentes dominios permite la especialización. En ambos casos la formación de complejo requiere la ruptura del dímero de Stl. Sin embargo, la Dut trimérica mantiene el trímero en el complejo mientras que la interacción con Dut dimérica requiere de la ruptura del dímero de Dut dando lugar a dos heterodímeros, en el que Stl emula la superficie de dimerización de Dut. Por otra parte, se identifican fagos que no codifican para Dut pero sí para una enzima de la familia MazG. La caracterización enzimática de estas proteínas revela que presentan actividad dUTPasa, pero una cinética enzimática de carácter cooperativo, a diferencia de las Duts, quienes presentan una cinética michaeliana clásica. Las estructuras de MazG de fagos revelan un ensamblaje tetramérico, en una organización de dímeros de dímeros ,con un total de cuatro centros activos por tetrámero, estando cada centro formado por residuos pertenecientes a tres monómeros. Estas enzimas presentan un extremo C-terminal desordenado en ausencia de ligando, y que se posiciona sobre el centro activo tras la unión del sustrato. Se ha identificado un segundo cambio estructural asociado a la unión de ligando que afecta a la disposición relativa de los dímeros, presentando una forma relajada en la forma apo y un tetrámero más compacto en presencia de nucleótido. Finalmente se estudian Duts triméricas codificadas por bacterias del orden Lactobacilles, quienes presentan dos inserciones específicas en su secuencia y que no son requeridas para actividad enzimática. Una de estas inserciones presenta la misma posición en el trímero que el motivo VI de Duts de fagos, a pesar de presentar una posición diferente en la secuencia. Una segunda inserción, de seis residuos implica un cambio en la organización del centro activo, el cual está formado por solo dos monómeros del trímero, a diferencia de la organización canónica de DutsPeer reviewe

Identification of a guanine-specific pocket in the protein N of SARS-CoV-2

9 páginas, 5 figuras, 1 tabla. The online version contains supplementary materialavailable at https://doi.org/10.1038/s42003-022-03647-8The SARS-CoV-2 nucleocapsid protein (N) is responsible for RNA binding. Here we report the crystal structure of the C-terminal domain (NCTD) in open and closed conformations and in complex with guanine triphosphate, GTP. The crystal structure and biochemical studies reveal a specific interaction between the guanine, a nucleotide enriched in the packaging signals regions of coronaviruses, and a highly conserved tryptophan residue (W330). In addition, EMSA assays with SARS-CoV-2 derived RNA hairpin loops from a putative viral packaging sequence showed the preference interaction of the N-CTD to RNA oligonucleotides containing G and the loss of the specificity in the mutant W330A. Here we propose that this interaction may facilitate the viral assembly process. In summary, we have identified a specific guanine-binding pocket in the N protein that may be used to design viral assembly inhibitors.This work was supported by the COVID research grant COV20/01265 awarded to M.V. from the Instituto de Salud Carlos III (ISCIII; Spain) and by the EuropeanCommission–NextGenerationEU (Regulation EU 2020/2094), through CSIC’s GlobalHealth Platform (PTI Salud Global). X-ray diffraction data collection was supported bythe Spanish Synchrotron Radiation Facility ALBA through the COVID Proposal2020074407 awarded to J.R.C.-T. and M.VPeer reviewe

Identification of a guanine-specific pocket in the protein N of SARS-CoV-2

The SARS-CoV-2 nucleocapsid protein (N) is responsible for RNA binding. Here we report the crystal structure of the C-terminal domain (NCTD) in open and closed conformations and in complex with guanine triphosphate, GTP. The crystal structure and biochemical studies reveals a specific interaction between the guanine, a nucleotide enriched in the packaging signals regions of coronaviruses, and a highly conserved tryptophan residue (W330). In addition, EMSA assays with SARS-CoV-2 derived RNA hairpin loops from a putative viral packaging sequence showed the preference interaction of the N-CTD to RNA oligonucleotides containing G and the loss of the specificity in the mutant W330A. Here we propose that this interaction may facilitate the viral assembly process. In summary we have identified a specific guanine-binding pocket in the N protein that may be used to design viral assembly inhibitors.This work was supported by the COVID research grant COV20/01265 awarded to M.V. from the Instituto de Salud Carlos III (ISCIII; Spain) and by the European Commission–NextGenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global). X-ray diffraction data collection was supported by the Spanish Synchrotron Radiation Facility ALBA through the COVID Proposal 2020074407 awarded to J.R.C.-T. and M.V.N

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

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