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CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseriagonorrhoeaeand human polymorphonuclear...
Background<br />Nesseria gonorrhoeae are gram-negative bacterial pathogens highly adapted to survive in a single host popu...
Secondary structure = 8 membrane spanning domains arranged as antiparallel β strands (membrane embedded β-barrel with 4 ex...
Up to ~11 variant opa proteins can be harboured by a single gonoccocal strain
Expression undergoes phase variation , where each gene can be independently  switched to an ‘on’ or ‘off’ state
Mediate Tight secondary interactions with host epithelial cells, but can also invade endothelial cells, lymphocytes, and g...
Carcinoembryonic Antigen-related Cellular Adhesion Molecule<br />The human CEACAM family harbours the CD66 epitope, and co...
Question<br />Primary urogenital colonization by N. gonorrhoeae can lead to the production of purulent exudate that primar...
CD66a<br />CEACAM1<br />CD66c<br />CEACAM6<br />Replicate blots, using different antibodies, to identify which protein was...
Fig. 3. Phase-contrast pictures of Opa-mediated binding of N. gonorrhoeae strains to stably transfected HeLa cell lines ex...
Fig. 4. Differential effects of heparin and CD66-reactive polyclonal antibodies on the Opa-mediated binding of N.gonorrhoe...
Fig. 5. Co-localization of Opa-expressing N.gonorrhoeae and CD66 on the surface of stably transfected HeLa cells expressin...
Fig. 6. Opa-mediated internalization of N. gonorrhoeae  by CD66-expressing HeLa cell lines. Bactericidal concentration of ...
Fig. 7. Opa-mediated interactions of recombinant E.coli with stably<br />transfected HeLa cells expressing BGP (CD66a).<br />
Fig. 8. Internalization of recombinant E.coli expressing gonococcal opacity protein by CD66-expressing HeLa cell lines. Th...
Opa52 Recruits CD66 Antigens on Human PMNs<br />Fig. 9. Co-localization of CD66 antigens and Opa-expressing N.gonorrhoeae ...
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Nova seminar

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Nova seminar

  1. 1. CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseriagonorrhoeaeand human polymorphonuclear phagocytes<br />Scott D.Gray-Owen, ChristophDehio, <br />AnjaHaude, Fritz Grunert<br />Thomas F.Meyer<br />
  2. 2. Background<br />Nesseria gonorrhoeae are gram-negative bacterial pathogens highly adapted to survive in a single host population—humans<br /><ul><li>Colonize mucosal surfaces, route of transmission being the genito-urinary tract following sexual transmission of gonococci</li></ul>Opacity-Associated (Opa) Proteins<br /><ul><li>Integral outer-membrane proteins
  3. 3. Secondary structure = 8 membrane spanning domains arranged as antiparallel β strands (membrane embedded β-barrel with 4 extracellular loops)
  4. 4. Up to ~11 variant opa proteins can be harboured by a single gonoccocal strain
  5. 5. Expression undergoes phase variation , where each gene can be independently switched to an ‘on’ or ‘off’ state
  6. 6. Mediate Tight secondary interactions with host epithelial cells, but can also invade endothelial cells, lymphocytes, and granulocytes. Due to phase variation, the tropism of the bacterium for different cell types depends on what Opa proteins are expressed.</li></li></ul><li>
  7. 7. Carcinoembryonic Antigen-related Cellular Adhesion Molecule<br />The human CEACAM family harbours the CD66 epitope, and contributes to the adhesive properties of cells e.g. by homophilic binding to CEACAMs on adjacent cells. They are characterized by a single N-terminal immunoglobulin (Ig) variable-like domain, and a varying number (0-6) of IgC2 constant like domain. CD66e (CEACAM5) is not expressed on PMNs. <br />
  8. 8. Question<br />Primary urogenital colonization by N. gonorrhoeae can lead to the production of purulent exudate that primarily consists of polymorphonuclear granulocytes, many of which are associated with gonococci. The non-opsonized phagocytosis of gonorrhoeae and subsequent respiratory burst can be inhibited by Opa-specific monoclonal antibodies, suggesting Opa function was necessary for these processes to occur. Which individual members of the CEA family of proteins are capable of interacting specifically with those Opa that mediate binding to human PMNs? <br />Method<br />-Use recombinant gonococci and E. coli to express defined Opa proteins<br />-Transfect HeLa cell lines expressing individual members of the CEA family to analyse Opa-mediated interactions with each member in isolation.<br />
  9. 9. CD66a<br />CEACAM1<br />CD66c<br />CEACAM6<br />Replicate blots, using different antibodies, to identify which protein was actually recovered<br />Fig. 2. Opa52 mediates the specific binding of distinct members of the CEA family of receptor proteins.<br />Fig. 1. Gonococcal binding to electrophoretically separated PMN<br />components by bacterial overlay assay.<br />
  10. 10. Fig. 3. Phase-contrast pictures of Opa-mediated binding of N. gonorrhoeae strains to stably transfected HeLa cell lines expressing various members of the CD66 family.<br />20μm<br />
  11. 11. Fig. 4. Differential effects of heparin and CD66-reactive polyclonal antibodies on the Opa-mediated binding of N.gonorrhoeaestrains to stably transfected HeLa cell lines expressing CGM1 (CD66d), indicating that indicate that Opa50 and Opa52 do possess distinct specificities for either HSPGs or CEA family antigens. <br />
  12. 12. Fig. 5. Co-localization of Opa-expressing N.gonorrhoeae and CD66 on the surface of stably transfected HeLa cells expressing CGM1 (CD66d). Filamentous actin was labelled by fluorescein isothiocyanate (FITC) conjugatedphalloidin. Rabbit serum was directed against gonococci (green).<br />
  13. 13. Fig. 6. Opa-mediated internalization of N. gonorrhoeae by CD66-expressing HeLa cell lines. Bactericidal concentration of gentamycin was added 3h after infection to selectively kill extacellular gonococci.<br />
  14. 14. Fig. 7. Opa-mediated interactions of recombinant E.coli with stably<br />transfected HeLa cells expressing BGP (CD66a).<br />
  15. 15. Fig. 8. Internalization of recombinant E.coli expressing gonococcal opacity protein by CD66-expressing HeLa cell lines. The uptake of recombinant Opa52 expressing E. coli by HeLa-BGP parallels that seen with N. gonorrhoeae, indicating that opa52 is sufficient to mediate this internalization process. <br />
  16. 16. Opa52 Recruits CD66 Antigens on Human PMNs<br />Fig. 9. Co-localization of CD66 antigens and Opa-expressing N.gonorrhoeae on the surface of human polymorphonuclear phagocytes. PMNs adherent to glass coverslips were degranulated with PMA and subsequently infected with N.gonorrhoeae strains expressing either the epithelial cell<br />invasion-associated Opa protein which mediates interaction with cell surface-associated heparan sulfates (Opa50), or expressing an Opa protein mediating interaction with PMNs (Opa52) for 1 h at 37°C. <br />
  17. 17. Summary of Conclusions<br />-A subset of the CD66 antigen expressing CEA family, including CGM1, NCA and BGP are shown to be cellular receptors bound by the gonococcal Opa proteins which mediate interactions with human PMNs. In addition, the epithelial cell-associated CEA protein was also seen to mediate Opa52 binding and gonococcal internalization by stably transfected HeLa cells. <br />-Based on gonococcal binding patterns shown, Opa52 binding of the CD66 family members can be localized to a 108 amino acid N-terminal domain present on all these molecules. <br />-The binding mediated by Opa52 expression is not strictly correlated with the subsequent internalization of adherent gonococci by transfected HeLa cell lines expressing defined CD66 family members<br />-The gonococci’s ability to induce efficient phagocytosis in the absence of opsonins suggests that this process plays some role in helping wither bacterial survival or transmission of the pathogen to other human hosts. Thus, N. gonorrhoeae seems to have evolved the means to penetrate the interior of phagocytic cells while avoiding their bactericidal effects.<br />
  18. 18. Opsonin-independent phagocytosis (Invasion and intracellular accomodation?)<br />
  19. 19. Additional References: <br />Nat Rev Immunol. 2006 Jun;6(6):433-46.<br />CEACAM1: contact-dependent control of immunity.<br />Gray-Owen SD, Blumberg RS.<br />Curr Opin Microbiol. 2003 Feb;6(1):43-9.<br />'Small' talk: Opa proteins as mediators of Neisseria-host-cell communication.<br />Hauck CR, Meyer TF.<br />

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