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A regra de não frustração da OPA e a aquisição do controlo

Vaz, João Cunha
Fonte: Universidade de Coimbra Publicador: Universidade de Coimbra
Tipo: Tese de Doutorado
POR
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37.75%
A temática do corporate governance continua muito actual face aos escândalos financeiros ocorridos na última década e à crise financeira em curso, destacando-se entre os seus mecanismos de controlo as ofertas públicas de aquisição (OPA), com uma actividade crescente nos últimos anos, e cujo papel pode ser determinante no quadro de uma eventual recuperação económica mundial. Neste contexto, assume particular importância um específico problema relativo ao governo da sociedade admitida à negociação alvo de uma OPA: a regulamentação das relações entre accionistas e membros do órgão de administração da sociedade visada, mais concretamente, a regra de não frustração da oferta (também conhecida por non-frustration ou passivity rule). Como principais objectivos propomo-nos então responder a duas questões essenciais: (i) Qual o modelo de governo societário relativo aos poderes do órgão de administração na adopção de medidas defensivas na pendência de uma OPA, capaz de assegurar uma protecção adequada aos accionistas e um mais eficiente mercado do controlo societário? (ii) Qual o nível mínimo de harmonização europeia desejável no domínio do corporate governance e, por outro lado, qual o papel da Directiva 2004/25/CE na aplicação de um regime uniforme das OPA...

CD66 receptor specificity exhibited by neisserial Opa variants is controlled by protein determinants in CD66 N-domains

Bos, Martine P.; Kuroki, Motomu; Krop-Watorek, Anna; Hogan, Daniel; Belland, Robert J.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 04/08/1998 EN
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27.73%
Neisseria gonorrhoeae strain MS11 is able to express 11 different opacity (Opa) proteins on its outer surface. A number of these Opa proteins have been shown to function as adhesins through binding of CD66 receptors present on human cells. CD66 antigens, or carcinoembryonic antigen family members, constitute a family of glycoproteins belonging to the immunoglobulin superfamily. Opa variants recognize this class of receptors in a differential manner such that certain Opa variants recognize up to four different CD66 receptors (CD66a, -c, -d, and -e), whereas others recognize only two (CD66a and -e) or none. We explored the basis for this receptor tropism in the present study. Our data show that glycoforms of CD66e and deglycosylated CD66e are recognized by gonococci in an Opa-specific manner. Binding by Opa variants of recombinant N-terminal domains of CD66 receptors expressed in Escherichia coli reflected the adherence specificities of Opa variants to HeLa cells expressing native CD66 molecules. These data indicate that recognition of CD66 receptors by Opa variants is mediated by the protein backbone of the CD66 N-domains. Furthermore, by using chimeric constructs between different CD66 N-domains we identified distinct binding regions on the CD66e N-domain for specific groups of Opa variants...

Opa Expression Correlates with Elevated Transformation Rates in Neisseria gonorrhoeae

Hill, Stuart A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /01/2000 EN
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27.7%
Neisseria gonorrhoeae is naturally competent for DNA transformation. Under most conditions encountered in vivo, gonococci express one or more opacity (Opa) proteins on their surfaces. Recently, it was shown that DNA preferentially binds to the surfaces of Opa-expressing organisms compared to those of isogenic Opa-negative strains, presumably due to the numerous cationic residues in the predicted surface-exposed loops of the Opa protein. This study examined whether Opa-DNA interactions actually influence DNA transformation of the gonococcus. The data show that Opa-expressing gonococci are more efficient recipients of DNA for transformation and are more susceptible to exogenous DNase I treatment at early stages during the DNA transformation process than non-Opa expressors. Furthermore, inhibition of the transformation process was demonstrable for Opa+ populations when either nonspecific DNA or the polyanion heparin was used. Overall, the data suggest that Opa expression, with its presumptive positive surface charge contribution, promotes DNA transformation by causing a more prolonged sequestration of donor DNA at the cell surface, which translates into more efficient transformation over time.

Carcinoembryonic Antigen Family Receptor Specificity of Neisseria meningitidis Opa Variants Influences Adherence to and Invasion of Proinflammatory Cytokine-Activated Endothelial Cells

Muenzner, Petra; Dehio, Christoph; Fujiwara, Taku; Achtman, Mark; Meyer, Thomas F.; Gray-Owen, Scott D.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/2000 EN
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27.7%
The carcinoembryonic antigen (CEA) family member CEACAM1 (previously called biliary glycoprotein or CD66a) was previously shown to function as a receptor that can mediate the binding of Opa protein-expressing Neisseria meningitidis to both neutrophils and epithelial cells. Since neutrophils and polarized epithelia have both been shown to coexpress multiple CEACAM receptors, we have now extended this work to characterize the binding specificity of meningococcal Opa proteins with other CEA family members. To do so, we used recombinant Escherichia coli expressing nine different Opa variants from three meningococcal strains and stably transfected cell lines expressing single members of the CEACAM family. These infection studies demonstrated that seven of the nine Opa variants bound to at least one CEACAM receptor and that binding to each of these receptors is sufficient to trigger the Opa-dependent bacterial uptake by these cell lines. The other two Opa variants do not appear to bind to either CEACAM receptors or heparan sulfate proteoglycan receptors, which are bound by some gonococcal Opa variants, thus implying a novel class of Opa proteins. We have also extended previous studies by demonstrating induction of CEACAM1 expression after stimulation of human umbilical vein endothelial cells with the proinflammatory cytokine tumor necrosis factor alpha...

Neisseria gonorrhoeae Coordinately Uses Pili and Opa To Activate HEC-1-B Cell Microvilli, Which Causes Engulfment of the Gonococci†

Griffiss, J. McLeod; Lammel, Claudia J.; Wang, Jun; Dekker, Nusi P.; Brooks, G. F.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/1999 EN
Relevância na Pesquisa
27.67%
This study was undertaken to examine concomitant roles of pili and colony opacity-associated proteins (Opa) in promoting Neisseria gonorrhoeae adherence to and invasion of human endometrial HEC-1-B cells. Adherence of N. gonorrhoeae to cultured HEC-1-B cells was saturable, even though organisms adhered to <50% of the cells. During 4 to 6 h of incubation, adherent mono- and diplococci formed microcolonies on the surfaces of the cells. Microvilli of the HEC-1-B cells adhered by their distal ends to individual cocci within the microcolonies. When the microcolonies grew from isogenic pilus-negative (P−) Opa−, P− Opa+, or P+ Opa− gonococci, microvilli did not elongate, and the colonies were not engulfed. In contrast, the microvilli markedly elongated during exposure to P+ Opa+ gonococci. The microvilli adhered to the organisms along their full lengths and appeared to actively participate in the engulfment of the microcolonies. Internalized microcolonies, with P+ Opa+ gonococci, contained dividing cocci and appeared to be surrounded by cell membrane but were not clearly within vacuoles. In contrast, degenerate individual organisms were within vacuoles. Low doses of chloramphenicol, which inhibits protein synthesis by both prokaryotes and eukaryotes...

Carcinoembryonic Antigen Family Receptor Recognition by Gonococcal Opa Proteins Requires Distinct Combinations of Hypervariable Opa Protein Domains

Bos, Martine P.; Kao, David; Hogan, Daniel M.; Grant, Christopher C. R.; Belland, Robert J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2002 EN
Relevância na Pesquisa
27.67%
Neisserial Opa proteins function as a family of adhesins that bind heparan sulfate proteoglycan (HSPG) or carcinoembryonic antigen family (CEACAM) receptors on human host cells. In order to define the CEACAM binding domain on Opa proteins, we tested the binding properties of a series of gonococcal (strain MS11) recombinants producing mutant and chimeric Opa proteins with alterations in one or more of the four surface-exposed loops. Mutagenesis demonstrated that the semivariable domain, present in the first loop, was completely dispensable for CEACAM binding. In contrast, the two hypervariable (HV) regions present in the second and third loops were essential for binding; deletion of either domain resulted in loss of receptor recognition. Deletion of the fourth loop resulted in a severe decrease in Opa expression at the cell surface and could therefore not be tested for CEACAM binding. Chimeric Opa variants, containing combinations of HV regions derived from different CEACAM binding Opa proteins, lost most of their receptor binding activity. Some chimeric variants gained HSPG binding activity. Together, our results indicate that full recognition of CEACAM receptors by Opa proteins requires a highly coordinate interplay between both HV regions. Furthermore...

Gonococcal opacity: lectin-like interactions between Opa proteins and lipooligosaccharide.

Blake, M S; Blake, C M; Apicella, M A; Mandrell, R E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1995 EN
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Previous evidence from our laboratory suggested that the tight intercellular adhesions between the outer membranes of gonococci displaying the opacity colony phenotype occurred because Opa proteins expressed on one gonococcus adhered to the lipooligosaccharide (LOS) of the opposing bacterium (M.S. Blake, p. 51-66, in G. G. Jackson and H. Thomas, ed., The Pathogenesis of Bacterial Infections, 1985, and M. S. Blake and E. C. Gotschlich, p. 377-400, in M. Inouye, ed., Bacterial Outer Membranes as Model Systems, 1986). A noncompetitive inhibition assay used previously to determine the carbohydrate structures recognized by the major hepatic asialoglycoprotein receptor was modified to determine the gonococcal LOS structures that bind Opa proteins (R. T. Lee, Targeted Diagn. Ther. Ser. 4:65-84, 1991). The LOS carbohydrates used in these assays were LOS structures purified from pyocin LOS mutants of Neisseria gonorrhoeae 1291 described by K. C. Dudas and M. A. Apicella (Infect. Immun. 56:499-504, 1988) and further characterized by C. M. John et al. (J. Biol. Chem. 266:19303-19311, 1991). Purified gonococcal Opa proteins were incubated with each of the parent and mutant LOS, and the amount of binding of Opa proteins was measured by a direct enzyme-linked immunosorbent assay using the Opa-specific monoclonal antibody 4B12. The affinities of the Opa proteins for each of the LOS were determined indirectly by measuring the concentrations of Opa proteins that noncompetitively inhibited 50% of the binding of LOS-specific monoclonal antibodies. This concentration is inversely proportional to the affinity of the inhibitor (R. T. Lee...

Differential recognition of members of the carcinoembryonic antigen family by Opa variants of Neisseria gonorrhoeae.

Bos, M P; Grunert, F; Belland, R J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1997 EN
Relevância na Pesquisa
27.8%
Opacity (Opa) protein variation in Neisseria gonorrhoeae is implicated in the pathogenesis of gonorrhea, possibly by mediating adherence and entry of the bacteria into human tissues. One particular Opa protein mediates adherence to epithelial cells through cell surface proteoglycans. Recently, two other eukaryotic cell receptors for Opa proteins have been reported. These receptors are members of a subgroup of the carcinoembryonic (CEA) gene family that express CD66 antigens. CEA family members vary in their distribution in human tissues. In order to understand whether interactions between Opa and CEA-like molecules play any role in pathogenesis, we must investigate which CEA family members are able to serve as Opa receptors and which Opa proteins recognize CEA-like molecules. We therefore studied HeLa cells that were stably transfected with five different members of the CEA family, i.e., CEA, CEA gene family member 1a (CGM1a), CGM6, nonspecific cross-reacting antigen (NCA), and biliary glycoprotein a (BGPa). We infected these transfectants with all possible 11 Opa variants of gonococcal strain MS11 and determined the numbers of bacteria that were bound and internalized. To account for proteoglycan-mediated adherence, infection assays were also performed in the presence of heparin. Our results show that of the 11 Opa variants of MS11...

Selection of Opa+ Neisseria gonorrhoeae by limited availability of normal human serum.

Bos, M P; Hogan, D; Belland, R J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1997 EN
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27.81%
Experimental infections of human male volunteers with Neisseria gonorrhoeae have provided valuable insights into the early stages of gonorrheal disease. Bacterial variants expressing outer membrane opacity (Opa) proteins appear to be selected from the inoculum during a period in which total recoverable numbers of bacteria decrease rapidly. This apparent survival advantage occurs simultaneously with the onset of an inflammatory response, characterized by local production of interleukin 6 (IL-6) and IL-8 and the appearance of leukocytes in urine. Since the inflammatory response may also result in the presence of serum factors on the mucosal surface, we investigated the possibility that killing in normal human serum (NHS) leads to the selection of Opa+ variants. We therefore studied killing of separate populations and mixtures of Opa- and Opa+ N. gonorrhoeae MS11mk in NHS. Expression of an Opa protein conferred a survival advantage upon the organism; i.e., the Opa+ variants were more serum resistant than their isogenic Opa- counterparts, resulting in a selection for Opa+ phenotypes when a mixture of Opa+ and Opa- gonococci (GC) was exposed to submaximal doses of NHS. This selection was observed in three different lipooligosaccharide (LOS) backgrounds...

Anaerobic growth of gonococci does not alter their Opa-mediated interactions with human neutrophils.

Frangipane, J V; Rest, R F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1992 EN
Relevância na Pesquisa
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Gonococci grown anaerobically (anaerobic gonococci) in the presence of nitrite induce the expression of at least three novel outer membrane proteins (PANs 1 to 3). Although PAN 1 is expressed by gonococci during gonorrhea, the function of the PAN proteins remains unknown. In the absence of serum, gonococci possessing opacity-associated (Opa, formerly PII) outer membrane proteins adhere to, stimulate, and are phagocytically killed by human neutrophils. Gonococci lacking Opa proteins demonstrate none of these activities. We investigated whether the PAN proteins, or any other characteristics of anaerobic gonococci, altered the ability of nonpiliated, Opa+ or Opa- gonococci to adhere to, stimulate, or be phagocytically killed by neutrophils. The expression of Opa4 by strain F62, as determined by its relative mobility on sodium dodecyl sulfate-polyacrylamide gels, appeared to be unaltered by anaerobic growth, as seen previously (V. L. Clark, L. A. Campbell, D. A. Palermo, T. M. Evans, and K. W. Klimpel, Infect Immun. 55:1359-1364, 1987). Anaerobic and aerobic Opa+ gonococci adhered to and stimulated neutrophils to the same extent. Similarly, anaerobic and aerobic Opa- gonococci adhered to and stimulated neutrophils equally poorly. Finally...

Variable opacity (Opa) outer membrane proteins account for the cell tropisms displayed by Neisseria gonorrhoeae for human leukocytes and epithelial cells.

Kupsch, E M; Knepper, B; Kuroki, T; Heuer, I; Meyer, T F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1993 EN
Relevância na Pesquisa
27.77%
Opacity proteins (Opa) of Neisseria gonorrhoeae, a family of variant outer membrane proteins implicated in pathogenesis, are subject to phase variation. In strain MS11, 11 different opa gene alleles have been identified, the expression of which can be turned on and off independently. Using a reverse genetic approach, we demonstrate that a single Opa protein variant of strain MS11, Opa50, enables gonococci to invade epithelial cells. The remaining variant Opa proteins show no, or very little, specificity for epithelial cells but instead confer interaction with human polymorphonuclear neutrophils (PMNs). Thus, depending on the opa allele expressed, gonococci are capable of invading epithelial cells or of interacting with human leukocytes. The respective properties of Opa proteins are maintained independent of the gonococcal strain; thus, the specificity for epithelial cells or leukocytes is intrinsic to Opa proteins. Significant homology exists in the surface exposed variable regions of two invasion supporting Opa proteins from independent strains. Efficient epithelial cell invasion is favoured by high level Opa production, however, a 10-fold reduction still allows significant invasion by gonococci. In contrast, recombinant Escherichia coli expressing Opa proteins adhered or invaded poorly under similar experimental conditions...

In situ expression and localization of Neisseria gonorrhoeae opacity proteins in infected epithelial cells: apparent role of Opa proteins in cellular invasion

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/06/1991 EN
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27.75%
During natural infection, gonococcal opacity proteins (Opa) undergo rapid phase variation, but how this phenomenon contributes to the virulence of the bacteria is not well understood. In the present immunomorphological study we examined the actual Opa status of individual gonococci during various stages of gonococcal infection of Chang epithelial cells, by probing ultrathin sections of infected specimens with Opa-specific monoclonal antibodies. Our results demonstrate a heterogeneous Opa expression during the initial interaction of the bacteria, but an almost 100% expression of one of the probed Opas during their secondary attachment and entry into the host cells, suggesting a role for distinct Opas in cellular penetration. The association between Opa expression, tight attachment, and bacterial invasion into the host cells could be confirmed with isogenic variants that expressed different Opa proteins. Once inside the epithelial cells, both morphologically intact, Opa positive and morphologically disintegrated, Opa negative bacteria were observed. The loss of Opa immunoreactivity in intracellular gonococci could not be related to the presence of a particular Opa protein, but could be mimicked by incubating the organisms with extracts of sonicated uninfected epithelial cells...

Homologue Scanning Mutagenesis Reveals Cd66 Receptor Residues Required for Neisserial Opa Protein Binding

Bos, Martine P.; Hogan, Daniel; Belland, Robert J.
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 02/08/1999 EN
Relevância na Pesquisa
27.73%
The immunoglobulin-like family of CD66 antigens, present on human neutrophils and epithelial cells, are used as receptors for adhesins expressed by the pathogenic Neisseriae. N. gonorrhoeae strain MS11 can express 11 isoforms of these adhesins, called opacity-related (Opa) proteins. Each MS11 Opa protein recognizes a distinct spectrum of CD66 receptors. CD66–Opa binding is mediated by the NH2-terminal domain of the receptor and occurs through protein–protein interactions. In this report, we have investigated the molecular basis for the binding between the CD66 and Opa protein families by mapping amino acids in CD66 receptors that determine Opa protein binding. We performed homologue scanning mutagenesis between CD66e, which binds multiple Opa variants, and CD66b, which binds none, and tested both loss-of-function by CD66e and gain-of-function by CD66b in solution assays and in assays involving full-length receptors expressed by epithelial cells. We found that three residues in the CD66e N-domain are required for maximal Opa protein receptor activity. Opa proteins that recognize the same spectrum of native CD66 molecules showed differential binding of receptors with submaximal activity, indicating that the binding characteristics of these Opa proteins are actually slightly different. These data provide a first step toward resolving the structural requirements for Opa–CD66 interaction.

Relative Contributions of Recombination and Mutation to the Diversification of the opa Gene Repertoire of Neisseria gonorrhoeae▿

Bilek, Nicole; Ison, Catherine A.; Spratt, Brian G.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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27.77%
To understand the rates and mechanisms of Neisseria gonorrhoeae opa gene variation, the 11 opa genes were amplified independently so that an opa allelic profile could be defined for any isolate from the sequences at each locus. The opa allelic profiles from 14 unrelated isolates were all different, with no opa alleles shared between isolates. Examination of very closely related isolates from sexual contacts and sexual networks showed that these typically shared most opa alleles, and the mechanisms by which recent changes occurred at individual opa loci could be determined. The great majority of changes were due to recombination among existing alleles that duplicated an opa allele present at another locus or resulted in a mosaic of existing opa alleles. Single nucleotide changes or insertion/deletion of a single codon also occurred, but few of these events were assigned to mutation, the majority being assigned to localized recombination. Introduction of novel opa genes from coinfecting strains was rare, and all but one were observed in the same sexual network. Changes at opa loci occurred at a greater rate than those at the porin locus, and the opa11 locus changed more rapidly than other opa loci, almost always differing even between recent sexual contacts. Examination of the neighboring pilE gene showed that changes at opa11 and pilE often occurred together...

Opa+ and Opa− Isolates of Neisseria meningitidis and Neisseria gonorrhoeae Induce Sustained Proliferative Responses in Human CD4+ T Cells▿ †

Youssef, Abdel-Rahman; van der Flier, Michiel; Estevão, Silvia; Hartwig, Nico G.; van der Ley, Peter; Virji, Mumtaz
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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T cells may interact with a number of bacterial surface antigens, an encounter which has the potential to downmodulate host immune responses. Neisseria meningitidis, a human colonizer and an agent of septicemia and meningitis, expresses Opa proteins which interact with the CEACAM1 receptor expressed on activated T cells. Since CEACAM1 can act as an inhibitory receptor and T cells in subepithelial tissues may encounter whole bacteria, which often express Opa proteins in vivo, this study assessed primarily if Opa proteins expressed on meningococci affect T-cell functions. In addition, Opa-containing outer membrane vesicles (OMV) have been used as vaccine antigens, and therefore Opa+ and Opa− OMV were also studied. While Opa+ bacteria adhered to CEACAM-expressing T cells, both the Opa+ and Opa− phenotypes induced no to a small transient depression, followed by a prolonged increase in proliferation as well as cytokine production. Such responses were also observed with heat-killed bacteria or OMV. In addition, while anti-CEACAM antibodies alone inhibited proliferation, on coincubation of T cells with bacteria and the antibodies, bacterial effects predominated and were Opa independent. Thus, while Opa proteins of N. meningitidis can bind to T-cell-expressed CEACAM1...

Molecular Typing of Neisseria gonorrhoeae Isolates by Opa-Typing and Ribotyping in New Delhi, India

Khaki, Pejvak; Bhalla, Preena; Fayaz, Ahmad Mir; Moradi Bidhendi, Sohiela; Esmailzadeh, Majid; Sharma, Pawan
Fonte: Hindawi Publishing Corporation Publicador: Hindawi Publishing Corporation
Tipo: Artigo de Revista Científica
EN
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Control and preventive measures for gonococcal infections are based on precise epidemiological characteristics of N. gonorrhoeae isolates. In the present study the potential utility of opa-typing and ribotyping for molecular epidemiological study of consecutive gonococcal strains was determined. Sixty gonococcal isolates were subjected to ribotyping with two restriction enzymes, AvaII and HincII, and opa-typing with TaqI and HpaII for epidemiological characterization of gonococcal population. Ribotyping with AvaII yielded 6 ribotype patterns while twelve RFLP patterns were observed with HincII. Opa-typing of the 60 isolates revealed a total 54 opa-types, which 48 were unique and 6 formed clusters. Fifty-two opa-types were observed with TaqI-digested PCR product while opa-typing with HpaII demonstrated 54 opa-types. The opa-types from isolates that were epidemiologically unrelated were distinct, whereas those from the sexual contacts were identical. The results showed that opa-typing is highly useful for characterizing gonococcal strains from sexual contacts and has more discriminatory than ribotyping that could differentiate between gonococci of the same ribotype. The technique even with a single restriction enzyme has a high level of discrimination (99.9%) between epidemiologically unrelated isolates. In conclusion...

Construction and Characterization of a Derivative of Neisseria gonorrhoeae Strain MS11 Devoid of All opa Genes

LeVan, Adriana; Zimmerman, Lindsey I.; Mahle, Amanda C.; Swanson, Karen V.; DeShong, Philip; Park, Juhee; Edwards, Vonetta L.; Song, Wenxia; Stein, Daniel C.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2012 EN
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To better understand the role of Opa in gonococcal infections, we created and characterized a derivative of MS11 (MS11Δopa) that had the coding sequence for all 11 Opa proteins deleted. The MS11Δopa bacterium lost the ability to bind to purified lipooligosaccharide (LOS). While nonpiliated MS11Δopa and nonpiliated Opa-expressing MS11 cells grew at the same rate, nonpiliated MS11Δopa cells rarely formed clumps of more than four bacteria when grown in broth with vigorous shaking. Using flow cytometry analysis, we demonstrated that MS11Δopa produced a homogeneous population of bacteria that failed to bind monoclonal antibody (MAb) 4B12, a MAb specific for Opa. Opa-expressing MS11 cells consisted of two predominant populations, where ∼85% bound MAb 4B12 to a significant level and the other population bound little if any MAb. Approximately 90% of bacteria isolated from a phenotypically Opa-negative colony (a colony that does not refract light) failed to bind MAb 4B12; the remaining 10% bound MAb to various degrees. Piliated MS11Δopa cells formed dispersed microcolonies on ME180 cells which were visually distinct from those of piliated Opa-expressing MS11 cells. When Opa expression was reintroduced into MS11Δopa, the adherence ability of the strain recovered to wild-type levels. These data indicate that Opa contributes to both bacterium-bacterium and bacterium-host cell interactions.

Construction of Opa-Positive and Opa-Negative Strains of Neisseria meningitidis to Evaluate a Novel Meningococcal Vaccine

Sadarangani, Manish; Hoe, J. Claire; Callaghan, Martin J.; Jones, Claire; Chan, Hannah; Makepeace, Katherine; Daniels-Treffandier, Hélène; Deadman, Mary E.; Bayliss, Christopher; Feavers, Ian; van der Ley, Peter; Pollard, Andrew J.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 12/12/2012 EN
Relevância na Pesquisa
27.81%
Neisseria meningitidis is a major global pathogen causing invasive disease with a mortality of 5–10%. Most disease in developed countries is caused by serogroup B infection, against which there is no universal vaccine. Opacity-associated adhesin (Opa) proteins are major meningococcal outer membrane proteins, which have shown recent promise as a potential novel vaccine. Immunisation of mice with different Opa variants elicited high levels of meningococcal-specific bactericidal antibodies, demonstrating proof in principle for this approach. Opa proteins are critical in meningococcal pathogenesis, mediating bacterial adherence to host cells, and modulating human cellular immunity via interactions with T cells and neutrophils, although there are conflicting data regarding their effects on CD4+ T cells. We constructed Opa-positive and Opa-negative meningococcal strains to allow further evaluation of Opa as a vaccine component. All four opa genes from N. meningitidis strain H44/76 were sequentially disrupted to construct all possible combinations of N. meningitidis strains deficient in one, two, three, or all four opa genes. The transformations demonstrated that homologous recombination of exogenous DNA into the meningococcal chromosome can occur with as little as 80 bp...

Constitutively Opa-Expressing and Opa-Deficient Neisseria gonorrhoeae Strains Differentially Stimulate and Survive Exposure to Human Neutrophils

Ball, Louise M.; Criss, Alison K.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/2013 EN
Relevância na Pesquisa
27.81%
The Neisseria gonorrhoeae (the gonococcus [Gc]) opacity-associated (Opa) proteins mediate bacterial binding and internalization by human epithelial cells and neutrophils (polymorphonuclear leukocytes [PMNs]). Investigating the contribution of Opa proteins to gonococcal pathogenesis is complicated by high-frequency phase variation of the opa genes. We therefore engineered a derivative of Gc strain FA1090 in which all opa genes were deleted in frame, termed Opaless. Opaless Gc remained uniformly Opa negative (Opa−), whereas cultures of predominantly Opa− parental Gc and an intermediate lacking the “translucent” subset of opa genes (ΔopaBEGK) stochastically gave rise to Opa-positive (Opa+) bacterial colonies. Loss of Opa expression did not affect Gc growth. Opaless Gc survived exposure to primary human PMNs and suppressed the PMN oxidative burst akin to parental, Opa− bacteria. Notably, unopsonized Opaless Gc was internalized by adherent, chemokine-primed, primary human PMNs, by an actin-dependent process. When a non-phase-variable, in-frame allele of FA1090 opaD was reintroduced into Opaless Gc, the bacteria induced the PMN oxidative burst, and OpaD+ Gc survived less well after exposure to PMNs compared to Opa− bacteria. These derivatives provide a robust system for assessing the role of Opa proteins in Gc biology.

Interactions of Neisseria gonorrhoeae with human neutrophils: studies with purified PII (Opa) outer membrane proteins and synthetic Opa peptides.

Naids, F L; Belisle, B; Lee, N; Rest, R F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1991 EN
Relevância na Pesquisa
27.83%
We investigated the role of gonococcal outer membrane protein PII (also called Opa protein) in nonopsonic adherence to human neutrophils. Gonococcal outer membranes, purified Opa in detergent (Opa), purified Opa in liposomes (Opa+ lips), and peptides composing the second hypervariable (HV2) region of OpaB (strain FA1090) in liposomes (pepHV2 lips) were tested for their abilities to inhibit subsequent gonococcal adherence to human neutrophils. Outer membranes from gonococci possessing adherent Opa, liposomes containing adherent Opa, purified adherent Opa, and two of three liposome preparations (pepHV2 lips) containing peptides from the HV2 region of an adherent Opa inhibited subsequent adherence to neutrophils of homologous Opa+ gonococci. On the other hand, outer membranes from Opa- gonococci, outer membranes containing a nonadherent Opa (OpaA from strain FA1090), purified OpaA, and OpaA lips had little or no inhibitory effect. Outer membranes containing adherent Opas, purified adherent Opas, and liposomes containing such Opas all bound to neutrophils, whereas preparations containing OpaA or no Opa protein did not. The results indicate that (i) Opa proteins can bind to neutrophils in a partially purified or purified form and (ii) the HV2 region of Opa appears to at least partially mediate Opa's biological role.