Person: García Martínez, Begoña
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García Martínez
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Begoña
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Ciencias de la Salud
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0000-0002-0476-4900
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5048
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Publication Open Access The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus(Oxford University Press, 2018) Caballero Sánchez, Carlos; Menéndez Gil, Pilar; Catalán Moreno, Arancha; Vergara Irigaray, Marta; García Martínez, Begoña; Segura, Víctor; Irurzun Domínguez, Naiara; Villanueva San Martín, Maite; Ruiz de los Mozos Aliaga, Igor; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate PublikoaRNA-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.Publication Open Access A DIVA vaccine strain lacking RpoS and the secondary messenger c-di-GMP for protection against salmonellosis in pigs(BioMed Central, 2020) Gil Puig, Carmen; Latasa Osta, Cristina; García Ona, Enrique; Lázaro, Isidro; Labairu, Javier; Echeverz Sarasúa, Maite; Burgui Erice, Saioa; García Martínez, Begoña; Lasa Uzcudun, Íñigo; Solano Goñi, Cristina; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa; Gobierno de Navarra / Nafarroako Gobernua, IIM 13329.RI1Salmonellosis is the second most common food-borne zoonosis in the European Union, with pigs being a major reservoir of this pathogen. Salmonella control in pig production requires multiple measures amongst which vaccination may be used to reduce subclinical carriage and shedding of prevalent serovars, such as Salmonella enterica serovar Typhimurium. Live attenuated vaccine strains offer advantages in terms of enhancing cell mediated immunity and allowing inoculation by the oral route. However, main failures of these vaccines are the limited cross-protection achieved against heterologous serovars and interference with serological monitoring for infection. We have recently shown that an attenuated S. Enteritidis strain (ΔXIII) is protective against S. Typhimurium in a murine infection model. ΔXIII strain harbours 13 chromosomal deletions that make it unable to produce the sigma factor RpoS and synthesize cyclic-di-GMP (c-di-GMP). In this study, our objectives were to test the protective effects of ΔXIII strain in swine and to investigate if the use of ΔXIII permits the discrimination of vaccinated from infected pigs. Results show that oral vaccination of pre-weaned piglets with ΔXIII cross-protected against a challenge with S. Typhimurium by reducing faecal shedding and ileocaecal lymph nodes colonization, both at the time of weaning and slaughter. Vaccinated pigs showed neither faecal shedding nor tissue persistence of the vaccine strain at weaning, ensuring the absence of ΔXIII strain by the time of slaughter. Moreover, lack of the SEN4316 protein in ΔXIII strain allowed the development of a serological test that enabled the differentiation of infected from vaccinated animals (DIVA).Publication Open Access Relevant role of fibronectin-binding proteins in Staphylococcus aureus biofilm-associated foreign-body infections(American Society for Microbiology, 2009) Vergara Irigaray, Marta; Valle Turrillas, Jaione; Merino Barberá, Nekane; Latasa Osta, Cristina; García Martínez, Begoña; Ruiz de los Mozos Aliaga, Igor; Solano Goñi, Cristina; Toledo Arana, Alejandro; Penadés, José R.; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako GobernuaStaphylococcus 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.Publication Open Access Coordinated cyclic-di-GMP repression of salmonella motility through YcgR and cellulose(American Society for Microbiology, 2013) Zorraquino Salvo, Violeta; García Martínez, Begoña; Latasa Osta, Cristina; Echeverz Sarasúa, Maite; Toledo Arana, Alejandro; Valle Turrillas, Jaione; Lasa Uzcudun, Íñigo; Solano Goñi, Cristina; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: 1312/2010Cyclic di-GMP (c-di-GMP) is a secondary messenger that controls a variety of cellular processes, including the switch between a biofilm and a planktonic bacterial lifestyle. This nucleotide binds to cellular effectors in order to exert its regulatory functions. In Salmonella, two proteins, BcsA and YcgR, both of them containing a c-di-GMP binding PilZ domain, are the only known c-di-GMP receptors. BcsA, upon c-di-GMP binding, synthesizes cellulose, the main exopolysaccharide of the biofilm matrix. YcgR is dedicated to c-di-GMP-dependent inhibition of motility through its interaction with flagellar motor proteins. However, previous evidences indicate that in the absence of YcgR, there is still an additional element that mediates motility impairment under high c-di-GMP levels. Here we have uncovered that cellulose per se is the factor that further promotes inhibition of bacterial motility once high c-di-GMP contents drive the activation of a sessile lifestyle. Inactivation of different genes of the bcsABZC operon, mutation of the conserved residues in the RxxxR motif of the BcsA PilZ domain, or degradation of the cellulose produced by BcsA rescued the motility defect of ΔycgR strains in which high c-di-GMP levels were reached through the overexpression of diguanylate cyclases. High c-di-GMP levels provoked cellulose accumulation around cells that impeded flagellar rotation, probably by means of steric hindrance, without affecting flagellum gene expression, exportation, or assembly. Our results highlight the relevance of cellulose in Salmonella lifestyle switching as an architectural element that is both essential for biofilm development and required, in collaboration with YcgR, for complete motility inhibition.Publication Open Access Evaluation of a Salmonella strain lacking the secondary messenger c-di-GMP and RpoS as a live oral vaccine(Public Library of Science, 2016) Latasa Osta, Cristina; Echeverz Sarasúa, Maite; García Ona, Enrique; Burgui Erice, Saioa; Casares, Noelia; Hervás Stubbs, Sandra; Lasarte, Juan José; Lasa Uzcudun, Íñigo; Solano Goñi, Cristina; García Martínez, Begoña; Gil Puig, Carmen; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIM 13329.RI1Salmonellosis 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 vaccinePublication Open Access Lack of the PGA exopolysaccharide in Salmonella as an adaptive trait for survival in the host(Public Library of Science, 2017) Echeverz Sarasúa, Maite; García Martínez, Begoña; Sabalza Baztán, Amaia; Valle Turrillas, Jaione; Gabaldón Estevan, Juan Antonio; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaMany bacteria build biofilm matrices using a conserved exopolysaccharide named PGA or PNAG (poly-β-1,6-N-acetyl-D-glucosamine). Interestingly, while E. coli and other members of the family Enterobacteriaceae encode the pgaABCD operon responsible for PGA synthesis, Salmonella lacks it. The evolutionary force driving this difference remains to be determined. Here, we report that Salmonella lost the pgaABCD operon after the divergence of Salmonella and Citrobacter clades, and previous to the diversification of the currently sequenced Salmonella strains. Reconstitution of the PGA machinery endows Salmonella with the capacity to produce PGA in a cyclic dimeric GMP (c-di-GMP) dependent manner. Outside the host, the PGA polysaccharide does not seem to provide any significant benefit to Salmonella: resistance against chlorine treatment, ultraviolet light irradiation, heavy metal stress and phage infection remained the same as in a strain producing cellulose, the main biofilm exopolysaccharide naturally produced by Salmonella. In contrast, PGA production proved to be deleterious to Salmonella survival inside the host, since it increased susceptibility to bile salts and oxidative stress, and hindered the capacity of S. Enteritidis to survive inside macrophages and to colonize extraintestinal organs, including the gallbladder. Altogether, our observations indicate that PGA is an antivirulence factor whose loss may have been a necessary event during Salmonella speciation to permit survival inside the host.Publication Open Access Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella(National Academy of Sciences, 2009) Solano Goñi, Cristina; García Martínez, Begoña; Latasa Osta, Cristina; Toledo Arana, Alejandro; Zorraquino Salvo, Violeta; Valle Turrillas, Jaione; Casals, Joan; Pedroso, Enrique; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaBacteria have developed an exclusive signal transduction system involving multiple diguanylate cyclase and phosphodiesterase domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) that modulate the levels of the same diffusible molecule, 3 -5 -cyclic diguanylic acid (c-di-GMP), to transmit signals and obtain specific cellular responses. Current knowledge about c-di- GMP signaling has been inferred mainly from the analysis of recombinant bacteria that either lack or overproduce individual members of the pathway, without addressing potential compensatory effects or interferences between them. Here, we dissected c-di-GMP signaling by constructing a Salmonella strain lacking all GGDEF-domain proteins and then producing derivatives, each restoring 1 protein. Our analysis showed that most GGDEF proteins are constitutively expressed and that their expression levels are not interdependent. Complete deletion of genes encoding GGDEFdomain proteins abrogated virulence, motility, long-term survival, and cellulose and fimbriae synthesis. Separate restoration revealed that 4 proteins from Salmonella and 1 from Yersinia pestis exclusively restored cellulose synthesis in a c-di-GMP–dependent manner, indicating that c-di-GMP produced by different GGDEF proteins can activate the same target. However, the restored strain containing the STM4551-encoding gene recovered all other phenotypes by means of gene expression modulation independently of c-di-GMP. Specifically, fimbriae synthesis and virulence were recovered through regulation of csgD and the plasmid-encoded spvAB mRNA levels, respectively. This study provides evidence that the regulation of the GGDEF-domain proteins network occurs at 2 levels: a level that strictly requires c-di-GMP to control enzymatic activities directly, restricted to cellulose synthesis in our experimental conditions, and another that involves gene regulation for which c-di-GMP synthesis can be dispensable.Publication Open Access Salmonella biofilm development depends on the phosphorylation status of RcsB(American Society for Microbiology, 2012) Latasa Osta, Cristina; García Martínez, Begoña; Echeverz Sarasúa, Maite; Toledo Arana, Alejandro; Valle Turrillas, Jaione; Campoy Sánchez, Susana; García del Portillo, Francisco; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIM13329.RI1The Rcs phosphorelay pathway is a complex signaling pathway involved in the regulation of many cell surface structures in enteric bacteria. In response to environmental stimuli, the sensor histidine kinase (RcsC) autophosphorylates and then transfers the phosphate through intermediary steps to the response regulator (RcsB), which, once phosphorylated, regulates gene expression. Here, we show that Salmonella biofilm development depends on the phosphorylation status of RcsB. Thus, unphosphorylated RcsB, hitherto assumed to be inactive, is essential to activate the expression of the biofilm matrix compounds. The prevention of RcsB phosphorylation either by the disruption of the phosphorelay at the RcsC or RcsD level or by the production of a nonphosphorylatable RcsB allele induces biofilm development. On the contrary, the phosphorylation of RcsB by the constitutive activation of the Rcs pathway inhibits biofilm development, an effect that can be counteracted by the introduction of a nonphosphorylatable RcsB allele. The inhibition of biofilm development by phosphorylated RcsB is due to the repression of CsgD expression, through a mechanism dependent on the accumulation of the small noncoding RNA RprA. Our results indicate that unphosphorylated RcsB plays an active role for integrating environmental signals and, more broadly, that RcsB phosphorylation acts as a key switch between planktonic and sessile life-styles in Salmonella enterica serovar Typhimurium.Publication Open Access Biofilm matrix exoproteins induce a protective immune response against Staphylococcus aureus biofilm infection(American Society for Microbiology, 2014) Gil Puig, Carmen; Solano Goñi, Cristina; Burgui Erice, Saioa; Latasa Osta, Cristina; García Martínez, Begoña; Toledo Arana, Alejandro; Lasa Uzcudun, Íñigo; Valle Turrillas, Jaione; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIQ14066.RI1The 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.Publication Open Access Sensory deprivation in Staphylococcus aureus(Springer Nature, 2018) Villanueva San Martín, Maite; García Martínez, Begoña; Valle Turrillas, Jaione; Rapún Araiz, Beatriz; Ruiz de los Mozos Aliaga, Igor; Solano Goñi, Cristina; Martí, Miguel; Penadés, José R.; Toledo Arana, Alejandro; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako InstitutuaBacteria use two-component systems (TCSs) to sense and respond to environmental changes. The core genome of the major human pathogen Staphylococcus aureus encodes 16 TCSs, one of which (WalRK) is essential. Here we show that S. aureus can be deprived of its complete sensorial TCS network and still survive under growth arrest conditions similarly to wild-type bacteria. Under replicating conditions, however, the WalRK system is necessary and sufficient to maintain bacterial growth, indicating that sensing through TCSs is mostly dispensable for living under constant environmental conditions. Characterization of S. aureus derivatives containing individual TCSs reveals that each TCS appears to be autonomous and self-sufficient to sense and respond to specific environmental cues, although some level of cross-regulation between non-cognate sensor-response regulator pairs occurs in vivo. This organization, if confirmed in other bacterial species, may provide a general evolutionarily mechanism for flexible bacterial adaptation to life in new niches.