Molecular analysis of the fur (ferric uptake regulator) gene of a pathogenic Edwardsiella tarda strain

The gene encoding the Edwardsiella tarda ferric uptake regulator (Fur(Et)) was cloned from a pathogenic E. tarda strain isolated from diseased fish. Fur(Et) shares 90% overall sequence identity with the Escherichia coli Fur (Fur(Ec)) and was able to complement the mutant phenotype of a fur(Ec)-defective E. coli strain. Mutational analysis indicated that C92S and C95S mutations inactivated Fur(Et) whereas E112K mutation resulted in a superactive Fur(Et) variant. Fur(Et) negatively regulated its own expression; interruption of this regulation impaired bacterial growth, altered the production of certain outer membrane proteins, and attenuated bacterial virulence.

EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD

Edwardsiella tarda is a Gram-negative enteric pathogen that causes disease in both humans and animals. Recently, a type III secretion system (T3SS) has been found to contribute to Ed. tarda pathogenesis. EseB, EseC and EseD were shown to be secreted by the T3SS and to be the major components of the extracellular proteins (ECPs). Based on sequence similarity, they have been proposed to function as the 'translocon' of the T3SS needle structure. In this study, it was shown that EseB, EseC and EseD formed a protein complex after secretion, which is consistent with their possible roles as translocon components. The secretion of EseB and EseD was dependent on EscC (previously named Orf2). EscC has the characteristics of a chaperone; it is a small protein (13 kDa), located next to the translocators in the T3SS gene cluster, and has a coiled-coil structure at the N-terminal region as predicted by COILS. An in-frame deletion of escC abolished the secretion of EseB and EseD, and complementation of Delta escC restored the export of EseB and EseD into the culture supernatant. Further studies showed that EscC is not a secreted protein and is located on the membrane and in the cytoplasm. Mutation of escC did not affect the transcription of eseB but reduced the amount of EseB as measured by using an EseB-LacZ fusion protein in Ed. tarda. Co-purification studies demonstrated that EscC formed complexes with EseB and EseD. The results suggest that EscC functions as a T3SS chaperone for the putative translocon components EseB and EseD in Ed. tarda.

Dissection and localization of the immunostimulating domain of Edwardsiella tarda FliC

Bacterial flagellin is known to induce potent immune response in vertebrate systems via the toll-like receptor (TLR) 5. As a result, flagellin has been studied extensively as a vaccine adjuvant. In a previous study, we examined the vaccine and adjuvant potentials of the flagellin (FliC) of the fish pathogen Edwardsiella tarda. We found that E. tarda FliC induced low protective immunity by itself but could function as a molecular adjuvant and potentiate the specific immune response induced by the E. tarda antigen Eta6. Since FliC is a large protein and organized into distinct structural domains, we wondered whether the immunostimulating effect observed with the full-length protein could be localized to a certain region. To investigate this question, we in the present study dissected the FliC protein into several segments according to its structural features: (i) N163, which consists of the conserved N-terminal 163 residues of FliC; (ii) M160, which consists of the variable middle 160 residues; (iii) C94, which consists of the conserved C-terminal 94 residues; (iv) NC257, which is an artificial fusion of N163 and C94. To examine the adjuvanticity of the FliC fragments, DNA vaccine plasmids expressing FliC fragments in fusion with Eta6 were constructed and used to immunize Japanese flounder. The results showed that N163 produced the best adjuvant effect, which, in respect to improvement in the relative percent survival of the vaccinated fish, was comparable to that of the full-length FliC. None of the other FliC fragments exhibited apparent immunopotentiating effect. Further analysis showed that N163 enhanced the production of serum specific antibodies and, like full-length FliC, significantly upregulated the expression of the genes that are possibly involved in innate and adaptive immunity. These results indicate that N163 is the immunodominant region of FliC and suggest that E. tarda FliC may induce immune responses in Japanese flounder via mechanisms alternative to that involving TLR5. (C) 2010 Elsevier Ltd. All rights reserved.

Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain

Edwardsiella tarda is the etiological agent of edwardsiellosis, a systematic disease that affects a wide range of marine and freshwater fish cultured worldwide. In order to identify E. tarda antigens with vaccine potential, we in this study conducted a systematic search for E. tarda proteins with secretion capacity. One of the proteins thus identified was Esa1, which contains 795 amino acid residues and shares extensive overall sequence identities with the D15-like surface antigens of several bacterial species. In silico analyses indicated that Esa1 localizes to outer membrane and possesses domain structures that are conserved among bacterial surface antigens. The vaccine potential of purified recombinant Esa1 was examined in a Japanese flounder (Paralichthys olivaceus) model, which showed that fish vaccinated with Esa1 exhibited a high level of survival and produced specific serum antibodies. Passive immunization of naive fish with antisera raised against Esa1 resulted in significant protection against E. tarda challenge. Taking advantage of the secretion capacity of Esa1 and the natural gut-colonization ability of a fish commensal strain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Western immunoblot and agglutination analyses showed that FP3/pJsa1 produces outer membrane-localized Esa1 and forms aggregates in the presence of anti-Esa1 antibodies. Vaccination analyses showed that FP3/pJsa1 as an intraperitoneal injection vaccine and an oral vaccine embedded in alginate microspheres produced relative percent survival rates of 79% and 52%, respectively, under severe challenging conditions that resulted in 92-96% mortality in control fish. Further analyses showed that following oral vaccination, FP3/pJsa1 was able to colonize in the gut but unable to disseminate into other tissues. Together these results indicate that Esa1 is a protective immunogen and an effective oral vaccine when delivered by FP3/pJsa1 as a surface-anchored antigen. (c) 2010 Elsevier Ltd. All rights reserved.

Isolation and analysis of the vaccine potential of an attenuated Edwardsiella tarda strain

Edwardsiella tarda is an important aquaculture pathogen that can infect a wide range of marine and freshwater fish worldwide. In this study, a modified E. tarda strain, TX5RM, was selected by multiple passages of the pathogenic E. tarda strain TX5 on growth medium containing the antibiotic rifampicin. Compared to the wild type strain, the rifampicin-resistant mutant TX5RM (i) shows drastically increased median lethal dose and reduced capacity to disseminate in and colonize fish tissues and blood; (ii) exhibits slower growth rates when cultured in rich medium or under conditions of iron depletion; and (iii) differs in the production profile of whole-cell proteins. The immunoprotective potential of TX5RM was examined in a Japanese flounder (Paralichthys olivaceus) model as a vaccine delivered via intraperitoneal injection, oral feeding, bath immersion, and oral feeding plus immersion. All the vaccination trials, except those of injection, were performed with a booster at 3-week after the first vaccination. The results showed that TX5RM administered via all four approaches produced significant protection, with the highest protection levels observed with TX5RM administered via oral feeding plus immersion, which were, in terms of relative percent of survival (RPS), 80.6% and 69.4% at 5- and 8-week post-vaccination, respectively. Comparable levels of specific serum antibody production were induced by TX5RM-vaccinated via different routes. Microbiological analyses showed that TX5RM was recovered from the gut, liver, and spleen of the fish at 1-10 days post-oral vaccination and from the spleen, liver, kidney, and blood of the fish at 1-14 days post-immersion vaccination. Taken together, these results indicate that TX5RM is an attenuated E. tarda strain with good vaccine potential and that a combination of oral and immersion vaccinations may be a good choice for the administration of live attenuated vaccines. (C) 2010 Elsevier Ltd. All rights reserved.

迟缓爱德华氏菌(Edwardsiella tarda) LuxS/AI-2群体感应系统研究

从患病牙鲆中分离出迟缓爱德华氏菌株TX1，经报告菌株检测发现TX1有AI-2活性。用梯度PCR和Genome walking的方法克隆了TX1 luxS基因，将luxS基因在大肠杆菌DH5α中表达，证明其具有功能活性。在TX1中，luxS的表达与AI-2的活性基本是一致的，二者均受生长时期和生长条件的调节，即在glucose存在的条件下luxS表达和AI-2活性升高，而在高温条件下luxS表达和AI-2活性降低。glucose对AI-2活性以及luxS表达的影响经过荧光定量PCR，启动子活性检测，AI-2活性检测以及凝胶滞缓等一系列的实验证实是由cAMP-CRP复合物介导的，该复合物可以通过与luxS启动子相互作用而抑制luxS的表达。RNA干扰表明，TX1中luxS表达被干扰以后，对细菌产生了多方面的影响，包括：(1) 降低AI-2水平；(2) 降低细菌的生长能力；(3) 降低Ⅲ型分泌系统相关基因的表达水平以及生物膜的形成能力；(4) 减弱细菌毒力。外源AI-2的添加可以回复Ⅲ型分泌系统相关基因的表达水平以及生物膜的形成，但是并不能修复生长状况，表明LuxS在TX1中具有双重功能，即参与细胞代谢以及群体感应信号传导。基于LuxS/AI-2群体感应系统对细菌毒力的重要性，设计并筛选了一个该系统的阻遏因子5411。Pull-down实验证明5411可以和LuxS特异性结合。研究表明5411在TX1中表达导致细菌毒力显著下降。将5411克隆至牙鲆共生菌FP3中，发现5411可以被分泌到胞外并能被TX1吸收。将表达5411的共生菌导入牙鲆，发现其能够有效阻遏TX1对牙鲆的侵染。 这些结果表明：(1) TX1中AI-2的活性受控于LuxS，而后者则受生长时期和生长条件的调控；(2) luxS的正常表达对于细菌的正常生长和侵染是必需的；(3) LuxS/AI-2群体感应系统调控Ⅲ型分泌系统相关毒力因子的表达；(4) 通过阻遏LuxS/AI-2群体感应系统来抑制病原菌侵染是一种具有潜力的新型病害防控方法。

Las espigas de aparición tardía en trigo : condiciones que determinan su ocurrencia y consecuencias sobre el rendimiento

p.35-45

Aspectos morfológicos y biológicos de la oruga militar tardía (Spodoptera frugiperda (J.E. Smith) (Lep.: Noctuidae)

p.193-199

Immigració, incorporació tardana, diversitat, llengua i escola. Inmigración, incorporación tardía, diversidad, lengua y escuela'.

Expone la problemática que supone el hecho de la inmigración en la comunidad autónoma de las Islas Baleares. Problemática derivada de la falta de adecuación de las infraestructuras institucionales para la incorporación, la adaptación y el conocimiento de los ejes fundamentales de esta realidad particular. También existe una confusión sobre la manera a utilizar para integrar a las personas que llegan, las estrategias a seguir y la lengua de acogida, teniendo presente que la lengua tiene que ser una puerta de entrada hacia la diversidad y no una barrera. Ante la convivencia intercultural, se tiene que aprender a gestionar los conflictos, que de esta se deriven para que sea positiva y enriquecedora. También se analiza el programa de educación compensatoria, el cual pretende ayudar para la plena integración escolar de los niños y niñas con problemas de marginación social: que pertenecen a minorías culturales, padecen condiciones sociales desfavorables o inician tarde su escolarización.

Estratègies per a un pla d'acolliment dirigit a l'alumnat d'incorporació tardana. 'Estrategias para un plan de acogida dirigido al alumnado de incorporación tardía'.

Las escuelas reciben muchos alumnos que provienen de diferentes países y culturas, los cuales se incorporan de manera tardía al sistema educativo y tienen un desconocimiento de las lenguas oficiales de la Comunidad Autónoma lo que provoca más dificultades. Ante esta situación la escuela tiene que aplicar una estrategia compensadora a través de la educación intercultural, fomentando valores de tolerancia, respecto y solidaridad, respectado el maraco legal y las necesidades educativas. Ante esta situación en la reforma del sistema educativo se contempló un cambio para el tratamiento de las lenguas. Se tienen que enseñar el uso de la lengua que permita al alumno comunicarse en cualquier situación de la vida cuotidiana. La lengua propia del país de llegada es una parte fundamental del proceso formativo si se pretende la integración de la persona, y se tiene que enseñar partiendo del aprendizaje comunicativo y con todo el apoyo lingüístico necesario.

Alumnes d'incorporació tardana, aula d'acollida. 'Alumnos de incorporación tardía, aula de acogida'.

La primera Jornada de intercambio de experiencias en torno al Plan de acogida lingüística y cultural, conocido como PALIC, tuvo lugar el 31 de marzo del 2003

L'alumnat d'incorporació tardana : reflexions entorn l'ensenyament-aprenentatge de la llengua. 'El alumnado de incorporación tardía : reflexiones en torno a la enseñanza-aprendizaje de la lengua'.

Resumen de la autora en catalán

Projecte d'incorporació tardana de l'IES Josep Brugulat de Banyoles. 'Proyecto de incorporación tardía del IES Josep Brugulat de Banyoles'.

Resumen basado en el de los autores

Funcionament de l'aula d'incorporació tardana. 'Funcionamiento del aula de incorporación tardía'.

Resumen de la autora en catalán

Quan comen??a la tarda, llegim. 'Cuando empieza la tarde, leemos'

Este art??culo forma parte de la monograf??a 'Editar avui' (Editar hoy)