Suchergebnisse

Molecular beacons (MBs) are oligonucleotide probes with a hairpin-like structure that are typically labelled at the 5′ and 3′ ends with a fluorophore and a quencher dye, respectively. The conformation of the MB acts as a switch for fluorescence emission. When the fluorophore is in close proximity to the quencher, fluorescence emission cannot be detected, meaning that the switch is in an OFF state. However, if the MB structure is modified, separating the fluorophore from the quencher, the switch turns ON allowing fluorescence emission. This property has been extensively used for a wide variety of applications including real-time PCR reactions, study of protein-DNA interactions, and identification of conformational changes in RNA structures. Here, we describe a protocol based on the MB technology to measure the RNA unfolding capacities of the CspA RNA chaperone from Staphylococcus aureus. This method, with slight variations, may also be applied for testing the activity of other RNA chaperones, RNA helicases, or ribonucleases. ; This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant Agreement No. 646869) and the Spanish Ministry of Economy and Competitiveness (BFU2014-56698-P). C.J.C. was supported by a predoctoral contract from the Public University of Navarre (UPNA), Spain.

Bacteria use two-component systems (TCSs) to sense and respond to their environments. Free-living bacteria usually contain dozens of TCSs, each of them responsible for sensing and responding to a different range of signals. Differences in the content of two-component systems are related with the capacity of the bacteria to colonize different niches or improve the efficiency to grow under the conditions of the existing niche. This review highlights differences in the TCS content between Staphylococcus aureus and Staphylococcus saprophyticus as a case study to exemplify how the ability to sense and respond to the environment is relevant for bacterial capacity to colonize and survive in/on different body surfaces. ; B.R is recipient of a PhD grant from Universidad Pública de Navarra. Work in the Laboratory of Microbial Pathogenesis is funded by the Spanish Ministry of Science, Innovation and Universities grant BIO2017-83035-R Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union. A.F.H. is supported by the European Research Council ERC under the European Union's Horizon 2020 research and innovation program Grant Agreement ERC-ADG-2014 Proposal no 670932 Dut-signal from EU awarded to José R. Penadés and was the recipient of a Tenovus Project Grant (S16-12). ; B.R is recipient of a PhD grant from Universidad Publica de Navarra. Work in the Laboratory of Microbial Pathogenesis is funded by the Spanish Ministry of Science, Innovation and Universities grant BIO2017-83035-R Agencia Espanola de Investigacio'n/Fondo Europeo de Desarrollo Regional, European Union. A.F.H. is supported by the European Research Council ERC under the European Union's Horizon 2020 research and innovation program Grant Agreement ERC-ADG-2014 Proposal no 670932 Dut-signal from EU awarded to Jose R. Penades) and was the recipient of a Tenovus Project Grant (S16-12).

Molecular beacons (MBs) are oligonucleotide probes with a hairpin-like structure that are typically labelled at the 5′ and 3′ ends with a fluorophore and a quencher dye, respectively. The conformation of the MB acts as a switch for fluorescence emission. When the fluorophore is in close proximity to the quencher, fluorescence emission cannot be detected, meaning that the switch is in an OFF state. However, if the MB structure is modified, separating the fluorophore from the quencher, the switch turns ON allowing fluorescence emission. This property has been extensively used for a wide variety of applications including real-time PCR reactions, study of protein-DNA interactions, and identification of conformational changes in RNA structures. Here, we describe a protocol based on the MB technology to measure the RNA unfolding capacities of the CspA RNA chaperone from Staphylococcus aureus. This method, with slight variations, may also be applied for testing the activity of other RNA chaperones, RNA helicases, or ribonucleases. ; This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant Agreement No. 646869) and the Spanish Ministry of Economy and Competitiveness (BFU2014-56698-P). C.J.C. was supported by a predoctoral contract from the Public University of Navarre (UPNA), Spain.

Staphylococcus aureus is a leading cause of prosthetic joint infections (PJI) characterized by bacterial biofilm formation and recalcitrance to immune-mediated clearance and antibiotics. The molecular events behind PJI infection are yet to be unraveled. In this sense, identification of polymorphisms in bacterial genomes may help to establish associations between sequence variants and the ability of S. aureus to cause PJI. Here, we report an experimental nucleotide-level survey specifically aimed at the intergenic regions (IGRs) of the icaADBCR locus, which is responsible for the synthesis of the biofilm exopolysaccharide PIA/PNAG, in a collection of strains sampled from PJI and wounds. IGRs of the icaADBCR locus were highly conserved and no PJI-specific SNPs were found. Moreover, polymorphisms in these IGRs did not significantly affect transcription of the icaADBC operon under in vitro laboratory conditions. In contrast, an SNP within the icaR coding region, resulting in a V176E change in the transcriptional repressor IcaR, led to a significant increase in icaADBC operon transcription and PIA/PNAG production and a reduction in S. aureus virulence in a Galleria mellonella infection model. In conclusion, SNPs in icaADBCR IGRs of S. aureus isolates from PJI are not associated with icaADBC expression, PIA/PNAG production and adaptation to PJI. ; This work was financially supported by the Spanish Ministry of Science and Innovation grant PID2020-113494RB-I00 to I.L. (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union), the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 754412 [MoRE2020—Region Västra Götaland], and CARe—Centre for Antibiotic Resistance Research at University of Gothenburg. L.M.-L. was supported by the European Union's H2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No 801586 (IberusTalent).

The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and proteinbased biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections. © 2014, American Society for Microbiology. ; J. Valle was supported by Spanish Ministry of Science and Innovation "Ramón y Cajal" contract. This research was supported by grants ERANET Pathogenomic (GEN2006-27792-C2-1-E/PAT), BIO2011-30503- C02-02, and AGL2011-23954 from the Spanish Ministry of Economy and Competitivity and IIQ14066.RI1 from Innovation Department of the Government of Navarra. We thank Enrique Calvo from the Proteomic Service of CNIC for his expert help in protein identification and Victor Segura from the Genomics, Proteomics and Bioinformatics Unit at the Center for Applied Medical Research, University of Navarra, for microarray data analysis. ; Peer Reviewed

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Modification of the biomaterial surface topography is a promising strategy to prevent bacterial adhesion and biofilm formation. In this study, we use direct laser interference patterning (DLIP) to modify polystyrene surface topography at sub-micrometer scale. The results revealed that three-dimensional micrometer structures have a profound impact on bacterial adhesion. Thus, line- and pillar-like patterns enhanced S. aureus adhesion, whereas complex lamella microtopography reduced S. aureus adhesion in static and continuous flow culture conditions. Interestingly, lamella-like textured surfaces retained the capacity to inhibit S. aureus adhesion both when the surface is coated with human serum proteins and when the material is implanted subcutaneously in a foreign-body associated infection model. ; J. Valle was supported by Spanish Ministry of Science and Innovation "Ramón y Cajal" contract. This research was supported by grants AGL2011-23954 and BIO2011-30503-C02-02 from the Spanish Ministry of Economy and Competitivity and IIQ14066. RI1 from Innovation Department of the Government of Navarra. A. Lasagni, D. Langhenirich, and R. Helbig thank the Deutsche Forschungsgemeinschaft (DFG) for the financial support of the project "Mechanically stable anti-adhesive polymer surfaces" (LA-2513 4-1). ; Peer Reviewed

Incluye dos ficheros de datos ; The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and proteinbased biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections. ; J. Valle was supported by Spanish Ministry of Science and Innovation "Ramón y Cajal" contract. This research was supported by grants ERANET Pathogenomic (GEN2006-27792-C2-1-E/PAT), BIO2011-30503- C02-02, and AGL2011-23954 from the Spanish Ministry of Economy and Competitivity and IIQ14066.RI1 from Innovation Department of the Government of Navarra.

Esta es la versión no revisada del artículo: Valle, J., Burgui, S., Langheinrich, D., Gil, C., Solano, C., Toledo-Arana, A., et al. (2015). Evaluation of Surface Microtopography Engineered by Direct Laser Interference for Bacterial Anti-Biofouling. Macromolecular Bioscience, 15(8), 1060–1069. Se puede consultar la versión publicada en http://doi.org/10.1002/mabi.201500107 ; Biofilm formation by bacterial pathogens on the surface of medical and industrial settings is a 25 serious health problem. Modification of the biomaterial surface topography is a promising 26 strategy to prevent bacterial attachment and biofilm development. However, fabrication of 27 functional biomaterials at large scale with periodic network-topology is still problematic. In this 28 study, we use direct laser interference (DLIP), an easily scalable process, to modify polystyrene 29 surface (PS) topography at sub-micrometer scale. The resulting structure surfaces were 30 interrogated for their capacity to prevent adhesion and biofilm formation of the major human 31 pathogen Staphylococcus aureus. The results revealed that three-dimensional micrometer 32 periodic structures on PS have a profound impact on bacterial adhesion capacity. Thus, line- 33 and pillar-like topographical patterns enhanced S. aureus adhesion, whereas complex lamella 34 microtopography reduced S. aureus adhesion both in static and continuous flow culture 35 conditions. Interestingly, lamella-like textured surfaces retained the capacity to inhibit S. aureus 36 adhesion both when the surface is coated with human serum proteins in vitro and when the 37 material is implanted subcutaneously in a foreign-body associated infection model. Our results 38 establish that the DLIP technology can be used to functionalize polymeric surfaces for the 39 inhibition of bacterial adhesion to surfaces. ; J. Valle was supported by Spanish Ministry of Science and Innovation "Ramón y Cajal" 369 contract. This research was supported by grants AGL2011-23954 and BIO2011-30503-C02-02 370 from the Spanish Ministry of Economy and Competitivity and IIQ14066.RI1 from Innovation 371 Department of the Government of Navarra.

Bacterial genes are typically grouped into operons defined as clusters of adjacent genes encoding for proteins that fill related roles and are transcribed into a single polycistronic mRNA molecule. This simple organization provides an efficient mechanism to coordinate the expression of neighboring genes and is at the basis of gene regulation in bacteria. Here, we report the existence of a higher level of organization in operon structure that we named noncontiguous operon and consists in an operon containing a gene(s) that is transcribed in the opposite direction to the rest of the operon. This transcriptional architecture is exemplified by the genes menE-menC-MW1733-ytkD-MW1731 involved in menaquinone synthesis in the major human pathogen Staphylococcus aureus. We show that menE-menC-ytkD-MW1731 genes are transcribed as a single transcription unit, whereas the MW1733 gene, located between menC and ytkD, is transcribed in the opposite direction. This genomic organization generates overlapping transcripts whose expression is mutually regulated by transcriptional interference and RNase III processing at the overlapping region. In light of our results, the canonical view of operon structure should be revisited by including this operon arrangement in which cotranscription and overlapping transcription are combined to coordinate functionally related gene expression. ; This work was supported by the Spanish Ministry of Economy and Competitiveness Grants BIO2014-53530-R and BIO2017-83035-R (Agencia Española de Investigacion/Fondo Europeo de Desarrollo Regional, European Union). A.T.-A. is supported by the European Research Council under the European Union's Horizon 2020 research and innovation programme Grant Agreement 646869.

Salmonellosis is one of the most important bacterial zoonotic diseases transmitted through the consumption of contaminated food, with chicken and pig related products being key reservoirs of infection. Although numerous studies on animal vaccination have been performed in order to reduce Salmonella prevalence, there is still a need for an ideal vaccine. Here, with the aim of constructing a novel live attenuated Salmonella vaccine candidate, we firstly analyzed the impact of the absence of cyclic-di-GMP (c-di-GMP) in Salmonella virulence. Cdi- GMP is an intracellular second messenger that controls a wide range of bacterial processes, including biofilm formation and synthesis of virulence factors, and also modulates the host innate immune response. Our results showed that a Salmonella multiple mutant in the twelve genes encoding diguanylate cyclase proteins that, as a consequence, cannot synthesize c-di-GMP, presents a moderate attenuation in a systemic murine infection model. An additional mutation of the rpoS gene resulted in a synergic attenuating effect that led to a highly attenuated strain, referred to as ΔXIII, immunogenic enough to protect mice against a lethal oral challenge of a S. Typhimurium virulent strain. ΔXIII immunogenicity relied on activation of both antibody and cell mediated immune responses characterized by the production of opsonizing antibodies and the induction of significant levels of IFN-γ, TNF-α, IL-2, IL-17 and IL-10. ΔXIII was unable to form a biofilm and did not survive under desiccation conditions, indicating that it could be easily eliminated from the environment. Moreover, ΔXIII shows DIVA features that allow differentiation of infected and vaccinated animals. Altogether, these results show ΔXIII as a safe and effective live DIVA vaccine. ; SB is a predoctoral fellow from the Public University of Navarra. CG and BG are recipients of a postdoctoral contract under Grants IIM 13329.RI1 and BIO2011-30503-C02-02, respectively. This work was supported by grant IIM 13329.RI1 from the Departamento de Innovación, Empresa y Empleo, Government of Navarra and grants BIO2011-30503-C02-02 and BIO2014-53530-R from the Spanish Ministry of Economy and Competitiveness. ; Peer Reviewed

The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors. ; This work was supported by the BIO2005-08399 and ERA-Net pathogenomics GEN2006-27792-C2-1-E/PAT grants from the Spanish Ministerio de Educación y Ciencia and grant LSHM-CT-2006-019064 from the European Union.

Staphylococcus aureus can establish chronic infections on implanted medical devices due to its capacity to form biofilms. Analysis of the factors that assemble cells into a biofilm has revealed the occurrence of strains that produce either a polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG) exopolysaccharide- or a protein-dependent biofilm. Examination of the influence of matrix nature on the biofilm capacities of embedded bacteria has remained elusive, because a natural strain that readily converts between a polysaccharide- and a protein-based biofilm has not been studied. Here, we have investigated the clinical methicillin (meticillin)-resistant Staphylococcus aureus strain 132, which is able to alternate between a proteinaceous and an exopolysaccharidic biofilm matrix, depending on environmental conditions. Systematic disruption of each member of the LPXTG surface protein family identified fibronectin-binding proteins (FnBPs) as components of a proteinaceous biofilm formed in Trypticase soy broth-glucose, whereas a PIA/PNAG-dependent biofilm was produced under osmotic stress conditions. The induction of FnBP levels due to a spontaneous agr deficiency present in strain 132 and the activation of a LexA-dependent SOS response or FnBP overexpression from a multicopy plasmid enhanced biofilm development, suggesting a direct relationship between the FnBP levels and the strength of the multicellular phenotype. Scanning electron microscopy revealed that cells growing in the FnBP-mediated biofilm formed highly dense aggregates without any detectable extracellular matrix, whereas cells in a PIA/PNAG-dependent biofilm were embedded in an abundant extracellular material. Finally, studies of the contribution of each type of biofilm matrix to subcutaneous catheter colonization revealed that an FnBP mutant displayed a significantly lower capacity to develop biofilm on implanted catheters than the isogenic PIA/PNAG-deficient mutant. ; This work was supported by the BIO2005-08399 and ERA-NET Pathogenomics (GEN2006-27792-C2-1-E/PAT) grants from the Spanish Ministerio de Ciencia, the Euroinnova Navarra-INNOVATIC program from the Gobierno de Navarra, and the Staph Dynamics LSHM-CT-2006-019064 grant from the European Union.

RNA-binding proteins (RBPs) are essential to fine-tune gene expression. RBPs containing the cold-shock domain are RNA chaperones that have been extensively studied. However, the RNA targets and specific functions for many of them remain elusive. Here, combining comparative proteomics and RBP-immunoprecipitation-microarray profiling, we have determined the regulon of the RNA chaperone CspA of Staphylococcus aureus. Functional analysis revealed that proteins involved in carbohydrate and ribonucleotide metabolism, stress response and virulence gene expression were affected by cspA deletion. Stress-associated phenotypes such as increased bacterial aggregation and diminished resistance to oxidative-stress stood out. Integration of the proteome and targetome showed that CspA post-transcriptionally modulates both positively and negatively the expression of its targets, denoting additional functions to the previously proposed translation enhancement. One of these repressed targets was its own mRNA, indicating the presence of a negative post-transcriptional feedback loop. CspA bound the 5′UTR of its own mRNA disrupting a hairpin, which was previously described as an RNase III target. Thus, deletion of the cspA 5′UTR abrogated mRNA processing and auto-regulation. We propose that CspA interacts through a U-rich motif, which is located at the RNase III cleavage site, portraying CspA as a putative RNase III-antagonist. ; European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme [646869]; Spanish Ministry of Economy and Competitiveness [BFU2011-23222, BIO2014-53530-R, BFU2014-56698-P]; Spanish National Research Council [CSIC-PII-201540I013]; C.J.C. was supported by predoctoral contract from the Public University of Navarre (UPNA), Spain. Funding for open access charge: European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme [646869]. ; Peer reviewed

RNA-binding proteins (RBPs) are essential to finetune gene expression. RBPs containing the coldshock domain are RNA chaperones that have been extensively studied. However, the RNA targets and specific functions for many of them remain elusive. Here, combining comparative proteomics and RBPimmunoprecipitation- microarray profiling, we have determined the regulon of the RNA chaperone CspA of Staphylococcus aureus. Functional analysis revealed that proteins involved in carbohydrate and ribonucleotide metabolism, stress response and virulence gene expression were affected by cspA deletion. Stress-associated phenotypes such as increased bacterial aggregation and diminished resistance to oxidative-stress stood out. Integration of the proteome and targetome showed that CspA posttranscriptionally modulates both positively and negatively the expression of its targets, denoting additional functions to the previously proposed translation enhancement. One of these repressed targets was its own mRNA, indicating the presence of a negative post-transcriptional feedback loop. CspA bound the 5 UTR of its own mRNA disrupting a hairpin, which was previously described as an RNase III target. Thus, deletion of the cspA 5 UTR abrogated mRNA processing and auto-regulation. We propose that CspA interacts through a U-rich motif, which is located at the RNase III cleavage site, portraying CspA as a putative RNase III-antagonist. ; European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme [646869]; Spanish Ministry of Economy and Competitiveness [BFU2011-23222, BIO2014-53530-R, BFU2014-56698-P]; Spanish National Research Council [CSIC-PII-201540I013]; C.J.C. was supported by predoctoral contract from the Public University of Navarre (UPNA), Spain. Funding for open access charge: European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme [646869].