Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these molecular syringes for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells

Identification of epitopes recognised by mucosal CD4+ T-cell populations from cattle experimentally colonised with Escherichia coli O157:H7

Additional file 5. Sequence alignment of Intimin epitopes against Intimin sequences from non-O157 EHEC serotypes. Alignment of Intimin CD4+ T-cell epitope sequences with representative Intimin sequences from EHEC serotypes O145, O127, O26, O103, O121, O45 and O111. Percentage values indicate % similarity to the EHEC O157:H7 reference sequence

Transcriptomics of Haemophilus (Glässerella) parasuis serovar 5 subjected to culture conditions partially mimetic to natural infection for the search of new vaccine antigens

11 p.Haemophilus (Glässerella) parasuis is the etiological agent of Glässer’s disease in pigs. Control of this disorder has been traditionally based on bacterins. The search for alternative vaccines has focused mainly on the study of outer membrane proteins. This study investigates the transcriptome of H. (G.) parasuis serovar 5 subjected to in vitro conditions mimicking to those existing during an infection (high temperature and iron-restriction), with the aim of detecting the overexpression of genes coding proteins exposed on bacterial surface, which could represent good targets as vaccine candidates. The transcriptomic approach identified 13 upregulated genes coding surface proteins: TbpA, TbpB, HxuA, HxuB, HxuC, FhuA, FimD, TolC, an autotransporter, a protein with immunoglobulin folding domains, another large protein with a tetratricopeptide repeat and two small proteins that did not contain any known domains. Of these, the first six genes coded proteins being related to iron extraction. Six of the proteins have already been tested as vaccine antigens in murine and/or porcine infection models and showed protection against H. (G.) parasuis. However, the remaining seven have not yet been tested and, consequently, they could become useful as putative antigens in the prevention of Glässer’s disease. Anyway, the expression of this seven novel vaccine candidates should be shown in other serovars different from serovar 5.S

Attaching and effacing (A/E) lesion formation by enteropathogenic E. coli on human intestinal mucosa is dependent on non-LEE effectors

Enteropathogenic E. coli (EPEC) is a human pathogen that causes acute and chronic pediatric diarrhea. The hallmark of EPEC infection is the formation of attaching and effacing (A/E) lesions in the intestinal epithelium. Formation of A/E lesions is mediated by genes located on the pathogenicity island locus of enterocyte effacement (LEE), which encode the adhesin intimin, a type III secretion system (T3SS) and six effectors, including the essential translocated intimin receptor (Tir). Seventeen additional effectors are encoded by genes located outside the LEE, in insertion elements and prophages. Here, using a stepwise approach, we generated an EPEC mutant lacking the entire effector genes (EPEC0) and intermediate mutants. We show that EPEC0 contains a functional T3SS. An EPEC mutant expressing intimin but lacking all the LEE effectors but Tir (EPEC1) was able to trigger robust actin polymerization in HeLa cells and mucin-producing intestinal LS174T cells. However, EPEC1 was unable to form A/E lesions on human intestinal in vitro organ cultures (IVOC). Screening the intermediate mutants for genes involved in A/E lesion formation on IVOC revealed that strains lacking non-LEE effector/s have a marginal ability to form A/E lesions. Furthermore, we found that Efa1/LifA proteins are important for A/E lesion formation efficiency in EPEC strains lacking multiple effectors. Taken together, these results demonstrate the intricate relationships between T3SS effectors and the essential role non-LEE effectors play in A/E lesion formation on mucosal surfaces

Caracterización de nuevos cultivares tipo Picota en función de su perfil de compuestos bioactivos

Resumen del póster presentado al XII Simposio Nacional y X Ibérico de Maduración y Postcosecha (POST18), celebrado en Badajoz del 4 al 7 de junio de 2018.La Picota del Valle del Jerte es una cereza con Denominación protegida que se encuentra en peligro de sustitución por la introducción de variedades foráneas. La obtención de nuevos cruces que mantenga las características propias de las Picotas, sumándoles los beneficios que aportan otras variedades comerciales, es el objetivo del Programa de Mejora Genética de cerezo del Valle del Jerte, en el que se han utilizado como parentales las variedades autóctonas tipo Picota Ambrunés, Pico Negro y Pico Colorado junto con otras no Picota como Staccato, Utah Giant, Sweet Heart y Hudson. Entre otros parámetros típicos y conocidos, como el desprendimiento del pedúnculo al ser recolectadas, es necesario reunir aquellos caracteres que confieren a las Picotas un carácter propio y distintivo. Así, el objetivo de este trabajo ha sido fijar el perfil de polifenoles de los parentales tipo Picota y determinar cuáles de los cruces obtenidos se asemejan más a este patrón. Se analizaron 18 nuevas variedades y los parentales. Los polifenoles de 20 frutos extraídos con metanol/agua se analizaron por HPLC-MS. El análisis de componentes principales (PCA) elaborado a partir de 14 polifenoles permitió separar en un cuadrante los parentales tipo Picota de los otros, principalmente debido a los altos niveles de ácido clorogénico que contenían los primeros. Al realizar el PCA incluyendo las 18 nuevas variedades, se observó que tres de ellas, provenientes de cruce de Ambrunés con Staccato, se encuadraban en la misma área que las Picotas. En las variedades donde uno de los parentales era Utah Giant o Sweet Heart prevalecieron las características de éstos en cuanto a la composición en compuestos bioactivos. Se concluye que los parentales tipo Picota mostraron un patrón de polifenoles distinto al de otras cerezas. La cereza tipo Ambrunés es el que dio origen a nuevas variedades con un perfil de polifenoles que se asemejaba más al "patrón Picota".Peer Reviewe

Las valoraciones de los/las profesionales que intervienen en casos de violencia de género en la pareja

Le problème de l'impunité et de la dévalorisation des droits des femmes qui vivent des violences machistes dans le couple (intimate Partner Violence) continue à être présent dans les systèmes pénaux européens. À travers une recherche innovante effectuée en Espagne, en Italie, en Angleterre et en Roumanie, à partir d'entretiens avec des femmes et des personnes travaillant dans le système pénal, d'observations ethnographiques des tribunaux et d'analyses des dossiers judiciaires, nous montrons comment la violence machiste dans le couple est traitée dans le système pénal

Inhibitory effect of IGF-I on type 2 nitric oxide synthase expression in Ins-1 cells and protection against activation-dependent apoptosis: involvement of phosphatidylinositol 3-kinase

Challenge of Ins-1 cells, a rat beta-pancreatic cell line, with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) promoted the expression of type 2 nitric oxide synthase (NOS-2) in a cooperative way. Treatment of Ins-1 cells with IGF-I significantly inhibited the expression of NOS-2, especially at subsaturating concentrations of LPS and IFN-gamma. The inhibitory effect of IGF-I on NOS-2 expression was abrogated when cells were incubated with wortmannin or LY294002, two inhibitors of phosphatidylinositol 3-kinase. Transient expression of the p110 subunit of phosphatidylinositol 3-kinase impaired the LPS and IFN-gamma-dependent NOS-2 promoter activity in cells transfected with a 1-kb fragment corresponding to the 5'-flanking region of the NOS-2 gene. However, expression of a dominant negative form of p85 abolished the inhibitory action of IGF-I on the NOS-2 promoter activity. Analysis of the decreased NOS-2 promoter activity in cells incubated with IGF-I showed a lower nuclear factor KB binding as determined by electrophoretic mobility shift assays. The synthesis of NO, produced after LPS and IFN-gamma challenge, triggered an apoptotic response in these cells. IGF-I reduced apoptosis mainly through the decreased synthesis of NO. However, in activated cells treated with N-[3-(aminomethyl)benzyl]acetamidine, a specific NOS-2 inhibitor, IGF-I completely abolished the NO-independent apoptosis. This protection from apoptosis was dependent on phosphatidylinositol 3-kinase activity. These results suggest an important anti-inflammatory and anti-apoptotic role for IGF-I in beta-pancreatic cells, with both actions depending on the activation of phosphatidylinositol 3-kinase.This work was supported by grant PM98-0120 from Dirección General de Investigación Científica y Técnica, Spain.Peer Reviewe

Analysis of metallic traces from the biodegradation of endomedullary AZ31 alloy temporary implants in rat organs after long implantation times

AZ31 alloy has been tested as a biodegradable material in the form of endomedullary implants in female Wistar rat femurs. In order to evaluate the accumulation of potentially toxic elements from the biodegradation of the implant, magnesium (Mg), aluminium (Al), zinc (Zn), manganese (Mn) and fluorine (F) levels have been measured in different organs such as kidneys, liver, lungs, spleen and brain. Several factors that may influence accumulation have been taken into account: how long the implant has been in place, whether or not the bone is fractured, and the presence of an MgF2 protective coating on the implant. The main conclusions and the clinical relevance of the study have been that AZ31 endomedullary implants have a degradation rate of about 60% after 13 months, which is fully compatible with fracture consolidation. Neither bone fracture nor an MgF2 coating seems to influence the accumulation of trace elements in the studied organs. Aluminium is the only alloying element in this study that requires special attention. The increase in Al recovered from the sampled organs represents 3.95% of the amount contained in the AZ31 implant. Al accumulates in a statistically significant way in all the organs except the brain. All of this suggests that in long-term tests AZ31 may be a suitable material for osteosynthesis.The authors are grateful to the MINECO, Spain, for financial support by projects MAT 2008-06719-C03-01-03 and MAT 2011-29152-C02-01.Peer reviewe