Investigaciones financiadas por la Unión Europea (OpenAire) - Europar Batasunak finantzatutako ikerketak (OpenAire)

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Open Access
Deletion of the GI-2 integrase and the wbkA flanking transposase improves the stability of Brucella melitensis Rev 1 vaccine
(BioMed Central, 2013) Mancilla, Marcos; Grilló Dolset, María Jesús; Miguel López, María Jesús de; López Goñi, Ignacio; San Román Aberasturi, Beatriz; Zabalza Baranguá, Ana; Moriyón Uría, Ignacio; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Brucella melitensis Rev 1 is the best vaccine available for the prophylaxis of small ruminant brucellosis and, indirectly, for reducing human brucellosis. However, Rev 1 shows anomalously high rates of spontaneous dissociation from smooth (S) to rough (R) bacteria, the latter being inefficacious as vaccines. This S-R instability results from the loss of the O-polysaccharide. To overcome this problem, we investigated whether some recently described mechanisms promoting mutations in O-polysaccharide genes were involved in Rev 1 S-R dissociation. We found that a proportion of Rev 1 R mutants result from genome rearrangements affecting the wbo O-polysaccharide loci of genomic island GI-2 and the wbkA O-polysaccharide glycosyltransferase gene of the wbk region. Accordingly, we mutated the GI-2 int gene and the wbk IS transposase involved in those arrangements, and found that these Rev 1 mutants maintained the S phenotype and showed lower dissociation levels. Combining these two mutations resulted in a strain (Rev 2) displaying a 95% decrease in dissociation with respect to parental Rev 1 under conditions promoting dissociation. Rev 2 did not differ from Rev 1 in the characteristics used in Rev 1 typing (growth rate, colonial size, reactivity with O-polysaccharide antibodies, phage, dye and antibiotic susceptibility). Moreover, Rev 2 and Rev 1 showed similar attenuation and afforded similar protection in the mouse model of brucellosis vaccines. We conclude that mutations targeting genes and DNA sequences involved in spontaneous O-polysaccharide loss enhance the stability of a critical vaccine phenotype and complement the empirical stabilization precautions taken during S Brucella vaccine production.
Open Access
Ecophysiological roles of abaxial anthocyanins in a perennial understorey herb from temperate deciduous forests
(Oxford University Press, 2015) Fernández Marín, Beatriz; Esteban Terradillos, Raquel; Míguez, Fátima; Artetxe, Unai; Castañeda Presa, Verónica; Pintó Marijuan, Marta; Becerril, José María; García Plazaola, José Ignacio; Natura Ingurunearen Zientziak; Ciencias del Medio Natural; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Accumulation of abaxial anthocyanins is an intriguing leaf trait particularly common among deeply shaded understorey plants of tropical and temperate forests whose ecological significance is still not properly understood. To shed light on it, possible ecophysiological roles of abaxial anthocyanins were tested in the perennial understorey herb of temperate deciduous forests Saxifraga hirsuta, chosen as a model species due to the coexistence of green and anthocyanic leaves and the presence of an easily removable lower anthocyanic epidermis. Anthocyanins accumulated during autumn, which temporally matched the overstorey leaf fall. Patterns of development of abaxial anthocyanins and direct measurements of photochemical efficiency under monochromatic light were not consistent with a photoprotective hypothesis. Enhancement of light capture also seemed unlikely since the back-scattering of red light towards the lower mesophyll was negligible. Seed germination was similar under acyanic and anthocyanic leaves. A relevant consequence of abaxial anthocyanins was the dramatic reduction of light transmission through the leaf. The dark environment generated underneath the Saxifraga canopy was enhanced by the horizontal repositioning of leaves, which occurs in parallel with reddening. This might play a role in biotic interactions by inhibiting vital processes of competitors, which may be of especial importance in spring before the overstorey leaves sprout.
Open Access
Identification of lptA, lpxE, and lpxO, three genes involved in the remodeling of Brucella cell envelope
(Frontiers Media, 2018) Conde Álvarez, Raquel; Palacios Chaves, Leyre; Gil Ramírez, Yolanda; Salvador Bescós, Miriam; Bárcena-Varela, Marina; Aragón Aranda, Beatriz; Martínez Gómez, Estrella; Zúñiga Ripa, Amaia; Miguel López, María Jesús de; Bartholomew, Toby Leigh; Hanniffy, Sean; Grilló Dolset, María Jesús; Vences Guzmán, Miguel Ángel; Bengoechea Alonso, José Antonio; Arce Gorvel, Vilma; Gorvel, Jean-Pierre; Moriyón Uría, Ignacio; Iriarte, Maite; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE, and lpxO, three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA, which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi. Free-lipid analysis revealed that lpxO corresponded to olsC, the gene coding for the ornithine lipid (OL) acyl hydroxylase active in O. anthropi and B. microti, while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE, or olsC were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence of the intact homolog genes in O. anthropi and B. microti but not in other brucellae suggests that LptA, LpxE, or OL β-hydroxylase do not significantly alter the PAMP properties of Brucella LPS and free-lipids and are therefore not positively selected during the adaptation to intracellular life.
Open Access
The lipopolysaccharide core of Brucella abortus acts as a shield against innate immunity recognition
(Public Library of Science, 2012) Conde Álvarez, Raquel; Bargen, Kristine von; Grilló Dolset, María Jesús; Jerala, Roman; Brandenburg, Klaus; Llobet, Enrique; Bengoechea Alonso, José Antonio; Moreno, Edgardo; Moriyón Uría, Ignacio; Gorvel, Jean-Pierre; Arce Gorvel, Vilma; Iriarte, Maite; Mancek Keber, Mateja; Barquero-Calvo, Elías; Palacios Chaves, Leyre; Chacón Díaz, Carlos; Chaves Olarte, Esteban; Martirosyan, Anna; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.
Open Access
Noncontiguous operon is a genetic organization for coordinating bacterial gene expression
(National Academy of Sciences, 2019) Sáenz Lahoya, S.; Bitarte Manzanal, Nerea; García, Beñat; Burgui Erice, Saioa; Vergara Irigaray, Marta; Valle Turrillas, Jaione; Solano Goñi, Cristina; Toledo Arana, Alejandro; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
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.
Open Access
Novel blaROB-1-bearing plasmid conferring resistance to β-lactams in Haemophilus parasuis isolates from healthy weaning pigs
(American Society for Microbiology, 2015) Moleres Apilluelo, Javier; Santos López, Alfonso; Lázaro, Isidro; Labairu, Javier; Prat, Cristina; Ardanuy, Carmen; González Zorn, Bruno; Aragon, Virginia; Garmendia García, Juncal; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Haemophilus parasuis, the causative agent of Glässer’s disease, is one of the early colonizers of the nasal mucosa of piglets. It is prevalent in swine herds, and lesions associated with disease are fibrinous polyserositis and bronchopneumonia. Antibiotics are commonly used in disease control, and resistance to several antibiotics has been described in H. parasuis. Prediction of H. parasuis virulence is currently limited by our scarce understanding of its pathogenicity. Some genes have been associated with H. parasuis virulence, such as lsgB and group 1 vtaA, while biofilm growth has been associated with nonvirulent strains. In this study, 86 H. parasuis nasal isolates from farms that had not had a case of disease for more than 10 years were obtained by sampling piglets at weaning. Isolates were studied by enterobacterial repetitive intergenic consensus PCR and determination of the presence of lsgB and group 1 vtaA, biofilm formation, inflammatory cell response, and resistance to antibiotics. As part of the diversity encountered, a novel 2,661-bp plasmid, named pJMA-1, bearing the blaROB-1 β-lactamase was detected in eight colonizing strains. pJMA-1 was shown to share a backbone with other small plasmids described in the Pasteurellaceae, to be 100% stable, and to have a lower biological cost than the previously described plasmid pB1000. pJMA-1 was also found in nine H. parasuis nasal strains from a separate collection, but it was not detected in isolates from the lesions of animals with Glässer’s disease or in nontypeable Haemophilus influenzae isolates. Altogether, we show that commensal H. parasuis isolates represent a reservoir of β-lactam resistance genes which can be transferred to pathogens or other bacteria.
Open Access
Opening Pandora's box: cause and impact of errors on plant pigment studies
(Frontiers Media, 2015) Fernández Marín, Beatriz; Artetxe, Unai; Barrutia, Oihana; Esteban Terradillos, Raquel; Hernández, Antonio; García Plazaola, José Ignacio; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Today, there is an astonishing volume of scientific information available for researchers, which can be easily accessed through powerful search tools. Yet, the question now is whether all this vast amount of information is reliable. In this sense, a “bad science” controversy arose recently when many Open Access (OA) journals (more than a half) published a false, error-ridden paper, which had been submitted in order to test the publishing ethics of these journals (Bohannon, 2013). This fake article was published mainly by fraudulent journals, but it was also accepted by a number of OA journals of renowned publishers with peer-review systems. The failure to reject an article full of errors revealed that the system's gearbox is leaking somewhere. The carelessness of peer-reviews in a number of OA journals has opened a Pandora's Box, and what is more disconcerting, nobody can guarantee that it could not also affect regular journals (non OA). Traditionally, it has been assumed that scientific journals should detect and correct all these failings through the peer review before publication. Regrettably, as we show in this communication, the system is far from being perfect (Pulverer, 2010; Székely et al., 2014).
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 Publikoa
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.
Open Access
Spatial distribution and risk factors of brucellosis in Iberian wild ungulates
(BioMed Central, 2010) Muñoz Álvaro, Pilar María; Boadella, Mariana; Arnal, Maricruz; Miguel, María Jesús de; Revilla, Miguel; Martínez, David; Vicente, Joaquín; Acevedo, Pelayo; Oleaga, Álvaro; Ruiz Fons, Francisco; Marín, Clara M.; Prieto, José M.; Fuente, José de la; Barral, Marta; Barberán, Montserrat; Fernández de Luco, Daniel; Blasco Canet, José María; Gortázar, Christian; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Background: The role of wildlife as a brucellosis reservoir for humans and domestic livestock remains to be properly established. The aim of this work was to determine the aetiology, apparent prevalence, spatial distribution and risk factors for brucellosis transmission in several Iberian wild ungulates. Methods: A multi-species indirect immunosorbent assay (iELISA) using Brucella S-LPS antigen was developed. In several regions having brucellosis in livestock, individual serum samples were taken between 1999 and 2009 from 2, 579 wild bovids, 6, 448 wild cervids and4, 454 Eurasian wild boar (Sus scrofa), and tested to assess brucellosis apparent prevalence. Strains isolated from wild boar were characterized to identify the presence of markers shared with the strains isolated from domestic pigs. Results: Mean apparent prevalence below 0.5% was identified in chamois (Rupicapra pyrenaica), Iberian wild goat (Capra pyrenaica), and red deer (Cervus elaphus). Roe deer (Capreolus capreolus), fallow deer (Dama dama), mouflon (Ovis aries) and Barbary sheep (Ammotragus lervia) tested were seronegative. Only one red deer and one Iberian wild goat resulted positive in culture, isolating B. abortus biovar 1 and B. melitensis biovar 1, respectively. Apparent prevalence in wild boar ranged from 25% to 46% in the different regions studied, with the highest figures detected in South-Central Spain. The probability of wild boar being positive in the iELISA was also affected by age, age-by-sex interaction, sampling month, and the density of outdoor domestic pigs. A total of 104 bacterial isolates were obtained from wild boar, being all identified as B. suis biovar 2. DNA polymorphisms were similar to those found in domestic pigs. Conclusions: In conclusion, brucellosis in wild boar is widespread in the Iberian Peninsula, thus representing an important threat for domestic pigs. By contrast, wild ruminants were not identified as a significant brucellosis reservoir for livestock.

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