Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application

Background The insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A). Results The main findings can be summarized in the following statements: i) TrxIA-2ic expression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2ic/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2ic (55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2ic was properly identified by western blot and mass spectrometric analysis of proteolytic digestions (63.25 % total coverage), iv) excellent immunochemical behavior of properly folded full TrxIA-2ic was legitimized by inhibition or displacement of [35S]IA-2 binding from IA-2A present in Argentinian Type 1 Diabetic patients, v) great stability over time was found under proper storage conditions and vi) low cost and environmentally harmless ELISA methods for IA-2A assessment were developed, with colorimetric or chemiluminescent detection. Conclusions E. coli GI724 strain emerged as a handy source of recombinant IA-2ic, achieving high levels of expression as a thioredoxin fusion protein, adequately validated and applicable to the development of innovative and cost-effective immunoassays for IA-2A detection in most laboratories.Fil: Guerra, Luciano Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Faccinetti, Natalia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Trabucchi, Aldana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Rovitto, Bruno David. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Sabljic, Adriana Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Poskus, Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Iacono, Ruben Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Valdez, Silvina Noemi. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentin

The luxS mutation causes loosely-bound biofilms in Shewanella oneidensis

<p>Abstract</p> <p>Background</p> <p>The <it>luxS </it>gene in <it>Shewanella oneidensis </it>was shown to encode an autoinducer-2 (AI-2)-like molecule, the postulated universal bacterial signal, but the impaired biofilm growth of a <it>luxS </it>deficient mutant could not be restored by AI-2, indicating it might not have a signalling role in this organism.</p> <p>Findings</p> <p>Here, we provide further evidence regarding the metabolic role of a <it>luxS </it>mutation in <it>S. oneidensis</it>. We constructed a <it>luxS </it>mutant and compared its phenotype to a wild type control with respect to its ability to remove AI-2 from the medium, expression of secreted proteins and biofilm formation. We show that <it>S. oneidensis </it>has a cell-dependent mechanism by which AI-2 is depleted from the medium by uptake or degradation at the end of the exponential growth phase. As AI-2 depletion is equally active in the <it>luxS </it>mutant and thus does not require AI-2 as an inducer, it appears to be an unspecific mechanism suggesting that AI-2 for <it>S. oneidensis </it>is a metabolite which is imported under nutrient limitation. Secreted proteins were studied by iTraq labelling and liquid chromatography mass spectrometry (LC-MS) detection. Differences between wild type and mutant were small. Proteins related to flagellar and twitching motility were slightly up-regulated in the <it>luxS </it>mutant, in accordance with its loose biofilm structure. An enzyme related to cysteine metabolism was also up-regulated, probably compensating for the lack of the LuxS enzyme. The <it>luxS </it>mutant developed an undifferentiated, loosely-connected biofilm which covered the glass surface more homogenously than the wild type control, which formed compact aggregates with large voids in between.</p> <p>Conclusions</p> <p>The data confirm the role of the LuxS enzyme for biofilm growth in <it>S. oneidensis </it>and make it unlikely that AI-2 has a signalling role in this organism.</p

Taxonomic diversity and identification problems of oncaeid microcopepods in the Mediterranean Sea

The species diversity of the pelagic microcopepod family Oncaeidae collected with nets of 0.1-mm mesh size was studied at 6 stations along a west-to-east transect in the Mediterranean Sea down to a maximum depth of 1,000 m. A total of 27 species and two form variants have been identified, including three new records for the Mediterranean. In addition, about 20, as yet undescribed, new morphospecies were found (mainly from the genera Epicalymma and Triconia) which need to be examined further. The total number of identified oncaeid species was similar in the Western and Eastern Basins, but for some cooccurring sibling species, the estimated numerical dominance changed. The deep-sea fauna of Oncaeidae, studied at selected depth layers between 400 m and the near-bottom layer at >4,200 m depth in the eastern Mediterranean (Levantine Sea), showed rather constant species numbers down to ∼3,000 m depth. In the near-bottom layers, the diversity of oncaeids declined and species of Epicalymma strongly increased in numerical importance. The taxonomic status of all oncaeid species recorded earlier in the Mediterranean Sea is evaluated: 19 out of the 46 known valid oncaeid species are insufficiently described, and most of the taxonomically unresolved species (13 species) have originally been described from this area (type locality). The deficiencies in the species identification of oncaeids cast into doubt the allegedly cosmopolitan distribution of some species, in particular those of Mediterranean origin. The existing identification problems even of well-described oncaeid species are exemplified for the Oncaea mediacomplex, including O. media Giesbrecht, O. scottodicarloi Heron & Bradford-Grieve, and O. waldemari Bersano & Boxshall, which are often erroneously identified as a single species (O. media). The inadequacy in the species identification of Oncaeidae, in particular those from the Atlantic and Mediterranean, is mainly due to the lack of reliable identification keys for Oncaeidae in warm-temperate and/or tropical seas. Future efforts should be directed to the construction of identification keys that can be updated according to the latest taxonomic findings, which can be used by the non-expert as well as by the specialist. The adequate consideration of the numerous, as yet undescribed, microcopepod species in the world oceans, in particular the Oncaeidae, is a challenge for the study of the structure and function of plankton communities as well as for global biodiversity estimates

Identification of a Key Amino Acid of LuxS Involved in AI-2 Production in Campylobacter jejuni

Autoinducer-2 (AI-2) mediated quorum sensing has been associated with the expression of virulence factors in a number of pathogenic organisms and has been demonstrated to play a role in motility and cytolethal distending toxin (cdt) production in Campylobacter jejuni. We have initiated the work to determine the molecular basis of AI-2 synthesis and the biological functions of quorum sensing in C. jejuni. In this work, two naturally occurring variants of C. jejuni 81116 were identified, one producing high-levels of AI-2 while the other is defective in AI-2 synthesis. Sequence analysis revealed a G92D mutation in the luxS gene of the defective variant. Complementation of the AI-2− variant with a plasmid encoded copy of the wild-type luxS gene or reversion of the G92D mutation by site-directed mutagenesis fully restored AI-2 production by the variant. These results indicate that the G92D mutation alone is responsible for the loss of AI-2 activity in C. jejuni. Kinetic analyses showed that the G92D LuxS has a ∼100-fold reduced catalytic activity relative to the wild-type enzyme. Findings from this study identify a previously undescribed amino acid that is essential for AI-2 production by LuxS and provide a unique isogenic pair of naturally occurring variants for us to dissect the functions of AI-2 mediated quorum sensing in Campylobacter