Characterization of the alpha-haemolysin determinant from the human enteropathogenic Escherichia coli O26 plasmid pEO5

The 157-kb conjugative plasmid pEO5 encoding alpha-haemolysin in strains of human enteropathogenic Escherichia coli (EPEC) O26 was investigated for its relationship with EHEC-haemolysin-encoding plasmids of enterohaemorrhagic E. coli (EHEC) O26 and O157 strains. Plasmid pEO5 was found to be compatible with EHEC-virulence plasmids and did not hybridize in Southern blots with plasmid pO157 from the EHEC O157:H7 strain EDL933, indicating that both plasmids were unrelated. A 9227-bp stretch of pEO5 DNA encompassing the entire alpha-hlyCABD operon was sequenced and compared for similarity to plasmid and chromosomally inherited alpha-hly determinants. The alpha-hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of the murine E. coli alpha-hly plasmid pHly152, in particular, the structural alpha-hlyCABD genes (99.2% identity) and the regulatory hlyR regions (98.8% identity). pEO5 and alpha-hly plasmids of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural alpha-hlyCABD genes. The major difference found between the hly regions of pHly152 and pEO5 is caused by the insertion of an IS2 element upstream of the hlyC gene in pHly152. The presence of transposon-like structures at both ends of the alpha-hly sequence indicates that this pEO5 virulence factor was probably acquired by horizontal gene transfer.

Characterization of Vibrio Parahaemolyticus isolated from oysters and mussels in São Paulo, Brazil

Vibrio parahaemolyticus is a marine bacterium, responsible for gastroenteritis in humans. Most of the clinical isolates produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes respectively. In this study, twenty-three V. parahaemolyticus, previously isolated from oysters and mussels were analyzed by PCR using specific primers for the 16S rRNA and virulence genes (tdh, trh and tlh) and for resistance to different classes of antibiotics and PFGE. Nineteen isolates were confirmed by PCR as V. parahaemolyticus. The tlh gene was present in 100% of isolates, the tdh gene was identified in two (10.5%) isolates, whereas the gene trh was not detected. Each isolate was resistant to at least one of the nine antimicrobials tested. Additionally, all isolates possessed the blaTEM-116 gene. The presence of this gene in V. parahaemolyticus indicates the possibility of spreading this gene in the environment. Atypical strains of V. parahaemolyticus were also detected in this study.

Production of the refolded oligopeptide-binding protein (OppA) encoded by the citrus pathogen Xanthomonas axonopodis pv. citri

The oligopeptide-binding protein, OppA, binds and ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides expressed by several bacterial species. In the present study, we report the cloning, purification, refolding and conformational analysis of a recombinant OppA protein derived from Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The oppA gene was expressed in Escherichia coli BL21 (DE3) strain under optimized inducing conditions and the recombinant protein remained largely insoluble. Solubilization was achieved following refolding of the denatured protein. Circular dichroism analysis indicated that the recombinant OppA protein preserved conformational features of orthologs expressed by other bacterial species. The refolded recombinant OppA represents a useful tool for structural and functional analyses of the X. citri protein.

Enterotoxigenic and genetic profiles of Bacillus cereus strains of food origin in Brazil

In Brazil. the incidence of Bacillus cereus outbreaks is unknown, and there is little information about B. cereus occurrence in food. In addition, data on toxin production and genetic characterization of the B. cereus isolates cannot be found. This pathogen causes two distinct types of toxin-mediated foodborne illnesses known as diarrheal and emetic syndromes. Diarrheal syndrome has been linked to three different enterotoxins: two protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE); and an enterotoxic protein, cytotoxin K (cytK). Emetic syndrome is related to cereulide, a toxin encoded by the ces gene. In this study, NHE and HBL production capacities of 155 strains of B. cereus isolated from Brazilian food products were evaluated with an immunoassay. Strains were also tested for the presence of the genes of the HBL and NHE complexes, cytK, cytK-1, cytK-2, and ces, using PCR. HBL was detected in 105 (67.7%) strains and NHE in 154 (99.4%) strains. All the strains harbored at least one gene of the NHE complex, while 96.1% of them were positive for at least one of those of the HBL complex. Genes cytK1 and ces were not detected. All strains showed toxigenic capacity and could represent a risk for consumers if good practices are not followed. This is the first report on toxigenic and genetic profiles of B. cereus strains isolated in Brazil.

The Paracoccidioides brasiliensis gp70 antigen is encoded by a putative member of the flavoproteins monooxygenase family

Glycoprotein gp70 is an important intracellular antigen from Paracoccidioides brasillensis that elicits both humoral and cellular immune responses. Herein, the PbGP70 gene cloning from isolate Pb18 using internal peptide sequence information is reported. The deduced protein sequence bears two N-glycosylation sites, antigenic sites and two mouse T-cell epitopes. Anti-recombinant gp70 (rPbgp70) polyclonal antibodies reacted with a 70-kDa component in total cell extract of A brasiliensis, while MAbC5F11 and paracoccidioiclomycosis patients` sera recognized rPbgp70. Confocal microscopy with anti-rPbgp70 and MAbC5F11 showed intense staining and cytoplasmatic co-localization. The protein sequence belongs to the flavoprotein monooxygenase family which groups important anti-oxidative bioactive compounds. We found increased PbGP70 transcript accumulation under oxidative stress induced by H(2)O(2), during fungal growth and in macrophage phagocyted/bound yeasts. Therefore, gp70 might play a dual role in P. brasiliensis by both eliciting immune cellular and humoral responses in the host and protecting the fungus from oxidative stress generated by phagocytic cells. (c) 2009 Elsevier Inc. All rights reserved.

Mice expressing the E7 oncogene of HPV16 in epithelium show central tolerance, and evidence of peripheral anergising tolerance, to E7-encoded cytotoxic T-lymphocyte epitopes

In order to derive mice which expressed both the E7 open reading frame transgene of human papillomavirus type 16 in skin and MHC class 1 restriction elements for several E7-encoded cytotoxic T-lymphocyte (CTL) epitopes, K14.HPV16E7 mice which express E7 in basal keratinocytes were crossed to the F1 generation with A2.1 K-b transgenic mice which express the MHC binding cleft domains of human HLA A*0201, and murine H-2(b). F1 mice (denoted K14E7xA2.1) expressed E7 in the thymus at least as early as 2-5 days before birth. Immunisation of FVBxA2.1 control mice (transgenic for HLA A*0201 and H-2(b) but not for E7), with two HLA A*0201-restricted epitopes of E7 and one H-2(b)-restricted CTL epitope of E7, gave strong primary CTL responses recognising epitope-pulsed or constitutively E7-expressing syngeneic target cells. In contrast, in immunised K14E7xA2.1 mice, the CTL responses to the H-2(b) epitope and one of the HLA A*0201 CTL epitopes were strongly down-regulated, and to the other HLA A*0201 epitope, completely abolished, as demonstrated by percentage specific killing by bulk splenocyte cultures in cyrotoxicity assays, and by CTL precursor frequency analysis, In thymus-transplanted bone marrow radiation chimeras in which the immune system of K14E7xA2.1 mice was replaced by a FVBxA2.1 immune system, specific immunisation did not result in reemergence of strong E7-directed CTL responses. In agreement with these in vitro findings, specific immunisation failed to significantly alter the course of E7-associated tumour development in K14E7xA2.1 mice. These data are consistent with a model of central deletional CTL tolerance to E7-encoded epitopes recognised in the context of two distinct MHC class 1 restriction elements, and with the possibility of peripheral T-cell anergy maintained by expression of E7 in the skin. (C) 1998 Academic Press.

Inhibition of NK cells by murine CMV-encoded class I MHC homologue m144

Murine cytomegalovirus (CMV)-encoded protein m144 is homologous to class I MHC heavy-chain and is thought to regulate NK-cell-mediated immune responses in vivo. To examine the effects of m144 on Nh cytotoxicity in vitro, various cell lines were transfected with wild-type m144 or a chimeric construct in which the cytoplasmic domain of m144 was replaced with green fluorescence protein. Burkitt lymphoma line Raji expressed a significant level of m144 as determined by anti-m144 mAb binding or the green fluorescence of the fusion protein. The level of m144 expression was relatively low compared with that of transfected murine class I MHC Dd. However, m144 on Raji cells partially inhibited antibody-dependent cell-mediated cytotoxicity of IL-2-activated NK cells. NK cells from the CMV-susceptible BALB/c as well as those from the resistant C57BL/6 mice were inhibited by m144. Antibodies against the known murine NK inhibitory receptors Ly-49A, C, G, and I did not affect the inhibitory effect of m144. These results suggest that the murine CMV class I MHC homologue m144 partially inhibits MZ cells by interacting with a novel inhibitory receptor. (C) 1999 Academic Press.

Structure of a putative ancestral protein encoded by a single sequence repeat from a multidomain proteinase inhibitor gene from Nicotiana alata

Background: The ornamental tobacco Nicotiana alata produces a series of proteinase inhibitors (Pls) that are derived from a 43 kDa precursor protein, NaProPl. NaProPl contains six highly homologous repeats that fold to generate six separate structural domains, each corresponding to one of the native Pls. An unusual feature of NaProPl is that the structural domains lie across adjacent repeats and that the sixth Pl domain is generated from fragments of the first and sixth repeats. Although the homology of the repeats suggests that they may have arisen from gene duplication, the observed folding does not appear to support this. This study of the solution structure of a single NaProPl repeat (aPl1) forms a basis for unravelling the mechanism by which this protein may have evolved, Results: The three-dimensional structure of aPl1 closely resembles the triple-stranded antiparallel beta sheet observed in each of the native Pls. The five-residue sequence Glu-Glu-Lys-Lys-Asn, which forms the linker between the six structural domains in NaProPl, exists as a disordered loop in aPl1. The presence of this loop in aPl1 results in a loss of the characteristically flat and disc-like topography of the native inhibitors. Conclusions: A single repeat from NaProPl is capable of folding into a compact globular domain that displays native-like Pl activity. Consequently, it is possible that a similar single-domain inhibitor represents the ancestral protein from which NaProPl evolved.

m144, a murine cytomegalovirus (MCMV)-encoded major histocompatibility complex class I homologue, confers tumor resistance to natural killer cell-mediated rejection

Until now, it has been unclear whether murine cytomegalovirus (MCMV)-encoded protein m144 directly regulates natural killer (NK) cell effector function and whether the effects of m144 are only strictly evident in the context of MCMV infection. We have generated clones of the transporter associated with antigen processing (TAP)-2-deficient RMA-S T lymphoma cell line and its parent cell line, RMA, that stably express significant and equivalent levels of m144. In vivo NK cell-mediated rejection of RMA-S-m144 lymphomas was reduced compared with rejection of parental or mock-transfected RMA-S clones, indicating the ability of m144 to regulate NK cell-mediated responses in vivo. Significantly, the accumulation of NK cells in the peritoneum was reduced in mice challenged with RMA-S-m144, as was the lytic activity of NK cells recovered from the peritoneum. Expression of m144 on RMA-S cells also conferred resistance to cytotoxicity mediated in vitro by interleukin 2-activated adherent spleen NK cells. In summary, the data demonstrate that m144 confers some protection from NK cell effector function mediated in the absence of target cell class I expression, but that in vivo the major effect of m144 is to regulate NK cell accumulation and activation at the site of immune challenge.

Function of CMV-encoded MHC class I homologues

Homologues of MHC class I proteins have been identified in the genomes of human, murine and rat cytomegaloviruses (CMVs). Given the pivotal role of the MHC class I protein in cellular immunity, it has been postulated that the viral homologues subvert the normal antiviral immune response of the host, thus promoting virus replication and dissemination in an otherwise hostile environment. This review focuses on recent studies of the CMV MHC class I homologues at the molecular, cellular and whole animal level and presents current hypotheses for their roles in the CMV life cycle.

Identification and characterization of a G protein-coupled receptor homolog encoded by murine cytomegalovirus

This report describes the identification of a murine cytomegalovirus (MCMV) G protein-coupled receptor (GCR) homolog. This open reading frame (M33) is most closely related to, and collinear with, human cytomegalovirus UL33, and homologs are also present in human herpesvirus 6 and 7 (U12 for both viruses). Conserved counterparts in the sequenced alpha- or gammaherpesviruses have not been identified to date, suggesting that these genes encode proteins which are important for the biological characteristics of betaherpesviruses. We have detected transcripts for both UL33 and M33 as early as 3 or 4 h postinfection, and these reappear at late times. In addition, we have identified N-terminal splicing for both the UL33 and M33 RNA transcripts. For both open reading frames, splicing results in the introduction of amino acids which are highly conserved among known GCRs. To characterise the function of the M33 in the natural host, two independent MCMV recombinant viruses were prepared, each of which possesses an M33 open reading frame which has been disrupted with the beta-galactosidase gene. While the recombinant M33 null viruses showed no phenotypic differences in replication from wild-type MCMV in primary mouse embryo fibroblasts in vitro, they showed severely restricted growth in the salivary glands of infected mice. These data suggest that M33 plays an important role in vivo, in particular in the dissemination to or replication in the salivary gland, and provide the first evidence for the function of a viral GCR homolog in vivo.

A polydnavirus-encoded protein of an endoparasitoid wasp is an immune suppressor

The molecular mechanism by which polydnaviruses of endoparasitoid wasps disrupt cell-mediated encapsulation reactions of host insects is largely unknown. Here we show that a polydnavirus-encoded protein, produced from baculovirus and plasmid expression vectors, prevents cell surface exposure of lectin-binding sites and microparticle formation during immune stimulation of haemocytes. The inactivation of immune-related cellular processes by this protein was analysed using a specific lectin and annexin V and shown to be virtually identical to polydnavirus-mediated effects on haemocytes. Cytochalasin D application has similar effects on haemocytes, suggesting that the immune suppression by the polydnavirus protein is caused by the destabilization of actin filaments. Since the exposure of cell surface glycoproteins and the formation of microparticles are part of an immune response to foreign objects or microorganisms and a prerequisite for cell-mediated encapsulation of microorganisms and parasites, the virus-encoded protein may become an important tool for the inactivation of cellular immune reactions in insects and an essential component in understanding immune suppression in parasitized host insects.

Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface-exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

RelB nuclear translocation mediated by C-terminal activator reigons of Epstein Barr virus-encoded latent membrane protein 1 and its effect on antigen-presenting function in B cells

Previous studies have shown that Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is uniquely able to up-regulate the expression of the peptide transporters (referred to as TAP-1 and TAP-2) and major histocompatibility complex (MHC) class I in Burkitt's lymphoma (BL) cell lines. This up-regulation is often accompanied by a restoration of antigen-presenting function as measured by the ability of these cells to present endogenously expressed viral antigen to cytotoxic T lymphocytes. Here we show that the expression of LMP1 resulted in up-regulation and nuclear translocation of RelB that were coincident with increased expression of MHC class I in BL cells. Deletion of the C-terminal activator regions (CTARs) of LMP1 significantly impaired the abilities of LMP1 to translocate RelB into the nucleus and to up-regulate the expression of antigen-processing genes. Further analysis with single-point mutations within the CTARs confirmed that the residues critical for NF-kappaB activation directly contribute to antigen-processing function regulation in BL cells. This LMP1-mediated effect was blocked following expression of either dominant negative IkappaBalpha S32/36A, an NF-kappaB inhibitor, or antisense RelB. These observations indicate that upregulation of antigen-presenting function in B cells mediated by LMP1 is signaled through the NF-kappaB subunit RelB. The data provide a mechanism by which LMP1 modulates immunogenicity of Epstein-Barr virus-infected normal and malignant cells.

Preparing encoded states in an oscillator

Recently a scheme has been proposed for constructing quantum error-correcting codes that embed a finite-dimensional code space in the infinite-dimensional Hilbert space of a system described by continuous quantum variables. One of the difficult steps in this scheme is the preparation of the encoded states. We show how these states can be generated by coupling a continuous quantum variable to a single qubit. An ion trap quantum computer provides a natural setting for a continuous system coupled to a qubit. We discuss how encoded states may be generated in an ion trap.