Functional organization of the inverted repeats of IS30.

The mobile element IS30 has 26-bp imperfect terminal inverted repeats (IRs) that are indispensable for transposition. We have analyzed the effects of IR mutations on both major transposition steps, the circle formation and integration of the abutted ends, characteristic for IS30. Several mutants show strikingly different phenotypes if the mutations are present at one or both ends and differentially influence the transposition steps. The two IRs are equivalent in the recombination reactions and contain several functional regions. We have determined that positions 20 to 26 are responsible for binding of the N-terminal domain of the transposase and the formation of a correct 2-bp spacer between the abutted ends. However, integration is efficient without this region, suggesting that a second binding site for the transposase may exist, possibly within the region from 4 to 11 bp. Several mutations at this part of the IRs, which are highly conserved in the IS30 family, considerably affected both major transposition steps. In addition, positions 16 and 17 seem to be responsible for distinguishing the IRs of related insertion sequences by providing specificity for the transposase to recognize its cognate ends. Finally, we show both in vivo and in vitro that position 3 has a determining role in the donor function of the ends, especially in DNA cleavage adjacent to the IRs. Taken together, the present work provides evidence for a more complex organization of the IS30 IRs than was previously suggested

The construction and characterization of an effective transpositional system based on IS30

AbstractWe constructed an in vivo system to detect transpositional rearrangements induced by the insertion sequence IS30. The transposase protein expressed from the transposase producer plasmids catalyzed rearrangements on different target sequences presented in trans. High yields, up to 83%, of transpositional frequencies were observed. The frequency of rearrangements correlated with the amount of transposase protein produced and the attractivity of the target sequences. Alteration in the frequency of transposition was observed in the recA− E. coli strains JM109 and TG2. Remarkable structural and functional analogy was found with site-specific recombination systems

Vaccine potential of a nonflagellated virulence-plasmid cured, (fliD-, pSEV∆) mutant of Salmonella Enteritidis for chickens

The aim of these studies was to assess residual virulence and early protective capacity of a negatively markered live attenuated vaccine candidate Salmonella Enteritidis mutant against a highly virulent S. Enteritidis strain using a day old chicken model. Nonflagellated FliD negative mutants of Salmonella Enteritidis 11 (SE11) with and without the virulence-plasmid proved to be sufficiently attenuated (limited invasiveness in vitro/vivo) without reduced ability to colonise chicken gut. The early protective activity of a nonflagellated virulence plasmid cured (fliD-, pSEV∆) mutant against organ invasion, caecal colonisation and faecal shedding by the highly virulent challenge strain S. Enteritidis 147 NalR proved to be effective and safe. The innate and adaptive immunity was demonstrable during the first 4 weeks of life, and the serological response was clearly distinguishable from the response induced by the wild parental strain. In conclusion, we provided data for the first time about virulence plasmid cured nonflagellated mutant of S. Enteritidis to serve as a basis for development of a negatively markered potential live oral vaccine against virulent S. Enteritidis in chicken

Mutations in the carboxy-terminal part of IS30 transposase affect the formation and dissolution of (IS30)2 dimer

AbstractThe transposase of IS30 catalyses different transpositional rearrangements via the dimer (IS30)2 intermediate structure. Mutation analysis provides evidence that the C-terminal part of IS30 transposase is required for the formation and dissolution of (IS30)2 dimer. C-terminal mutants are also defective in transpositional fusion; however, this deficiency can be `suppressed' by addition of the final product of site-specific dimerisation, the core (IS30)2 intermediate structure. The transposase part studied shows significant homologies in three highly conserved regions to proteins of IS30-related mobile elements

The SAT protein of porcine parvovirus accelerates viral spreading through irreversible ER stress induction

International audienceThe SAT protein of porcine parvovirus (PPV) accumulates in the endoplasmic reticulum (ER) and SAT deletion induces "slow spreading" phenotype. The in vitro comparison of the wild type Kresse strain and its SAT(-) knockout mutant revealed that prolonged cell integrity and late viral release are responsible for the slower spreading of the SAT(-) virus. During PPV infection, regardless of the presence or absence of SATp, the expression of downstream ER stress response proteins (Xbp1 and CHOP) was induced. However, in the absence of SATp, significant differences were detected in the quantity and the localization of CHOP, suggesting a role of SATp in the induction of irreversible ER stress in infected cells. The involvement of irreversible ER stress induction in PT cell necrosis and the viral egress was confirmed by treatment of infected cells by ER stress inducing chemicals (MG132, DTT and Thapsigargin) that accelerated the egress and spreading both the wild type and the SAT(-) viruses. UV stress induction had no beneficial effect to PPV infectionunderscoring the specificity of ER stress pathways in the process. However, induction of CHOP and its nuclear translocation cannot alone be responsible for the biological effect of SAT, since nuclear CHOP could not complement the lack of SAT in a co-expression experiment.IMPORTANCE SATp is encoded in an alternative ORF of the PPV genome. Earlier we showed that SATp of the attenuated PPV-NADL-2 strain accumulates in the ER and accelerates virus release and spreading. Our present work revealed that "slow spreading" is a general feature of the SAT(-) PPV viruses and is the consequence of prolonged cell integrity. PPV infection induced ER stress in the infected cells regardless of SATp presence, as demonstrated by the morphological changes of the ER, and expression of the stress response proteins XBP1 and CHOP. However, the presence of SATp made the ER stress more severe and accelerated the cell death during infection as shown by the higher expression rate and the alteration of the localization of CHOP. The beneficial effect of irreversible ER stress on PPV spread was confirmed by the treatment of the infected cells with ER stress inducing chemicals

T sejt rekonstitúciós vizsgálatok ZAP-70 deficiens egerekben

A ZAP-70 kináz (70kDa-os zéta lánc aszociált kináz) központi szerepet tölt be a T sejtek antigén receptoron keresztüli aktivációjának jeltovábbításában. A molekula fontosságát jól jelzi, hogy hiányában számos jelátviteli folyamat gátolt, illetve egérben és emberben is súlyos T sejtes immundeficiencia alakul ki. Ez utóbbi oka, hogy a ZAP-70 kináz a T sejt differenciáció során is kritikus szerepet tölt be, hiányában a T sejtek fejlődése blokkolódik a thymusban a kettős pozitív (CD4+CD8+) stádiumban, aminek következtében a perifériás nyirokszervekben nem találhatunk érett T sejteket. Munkánk során ZAP-70 deficiens egerekben tanulmányoztuk a T sejt hiány helyreállítás lehetőségeit. Ehhez adoptív transzfer vizsgálatokat hajtottunk végre, melyek során a ZAP-70-/- egereket vad típusú (azaz ZAP-70-et expresszáló) tetvéreik csontvelő illetve thymus sejtjeivel rekonstituáltuk intrahepatikus ill. intraperitonealis utakon. Eredményeink szerint mindkét transzfer technika hatékonynak bizonyult a T sejtek helyreállítátban. A sejtek transzferét követően 2 hetenként vérvétellel igazoltuk a T sejtek megjelenését a vérben. Azon állatok túlélése, melyekben T sejtek jelentek meg szignifikánsan meghaladta, az immundeficiens egerekét. A kísérletek végén (a transzfert követő végzett áramlási citometriás vizsgálatok illetve immunhisztokémia is azt igazolta, hogy T sejtek jelentek meg a transzferált egerek lépében, nyirokcsomóiban illetve a bélhez aszociált nyirokszövetekben. Továbbá a thymus sejt összetételének vizsgálata során megállapítottuk, hogy szignifikánsan emelkedett az érett CD4- vagy CD8 egyszeresen pozitív sejtek aránya, amely a T sejt érés normalizálódását jelezte. Sikerült kétféle transzfer módszerrel stabil kimérizmust létrehoznunk ZAP-70 hiányos egerekben. Bizonyítottuk, hogy mind a thymus mind a csontvelői eredetű sejtek képesek voltak a T sejt fejlődés helyreállítására

T CELL RECONSTITUTION STUDIES IN ZAP-70 DEFICIENT MICE

The ZAP-70 kinase (70kDa Zeta-Chain Associated Protein) plays a central role in signal transduction through the antigen receptor during T cell activation. The importance of the molecule is clearly demostrated when it is absent: several signaling pathways are inhibited, and severe T-cell immunodeficiency appears both in humans and mice. The reason of the latter is that ZAP-70 is indispensable in T cell differentiation: in its absence the maturation of T cells in the thymus is blocked in the double positive (CD4+CD8+) stage, and, as a consequence no mature T-cells can be found in the peripheral lymphoid organs. In our work we studied the possibilities of T cell reconstitution in ZAP-70 deficient mice. We performed adoptive transfer experiments, where ZAP-70-/- mice were reconstituted with bone marrow or thymus cells from their wild type (ZAP-70 expressing) siblings intrahepatically or intraperitoneally. According to our results both transfer techniques were effective in restoring T cells. After the cell transfers, blood was taken every 2 weeks to detect the presence of T cells in the blood. The survival of those animals which had T cells reconstituted exceeded significantly those which were immunodeficient. Both flow cytometric measurements and immunohistochemistrical staining performed after the experiments (following the transfers) proved that T cells appeared in the spleen, lymph nodes and gut associated lymphoid tissues of the animals. Furthermore, during the investigation of cell constitution of the thymus, we have found that the ratio of CD4+ or CD8+ single positive cells increased significantly, which indicated the normalisation of T-cell maturation. Thus, we managed to establish stable chimerism in ZAP-70 deficient mice with two methods. We proved that both thymus and bone marrow originated cells were able to restore the development of T-cells. This work was supported by the OTKA-K101493 research grant to Ferenc Boldizsár. Ferenc Boldizsár recieves Bolyai János Research Scholarship from the Hungarian Academy of Sciences