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  • Opa50 has been prev shown to bind epith cells via surface associated proteoglycan receptos (HSPG), and mediates less binding to PMNs than do the other 10 Opa proteins expressed in the MS11 strain. Swanson et al found that gonococci recovered from males who were inoculated with Opa- strain were predominantly opaque, and >60% of the bacteria expressed the Opa52 protein homolog (so, they focused on this variant)Previous studies have also showed that gonococcal binding to PMNs is strongly enhanced by release of subcellular 2dary granule components to EC milieu.Fig. 1A: Gonococcal overlay of electroblotted PMN proteins reveals Opa52-mediated binding to specific proteins. Tested the hypothesis that these int’ns are mediated by a protein receptor expressed on the surface of neutrophils. Took whole cell lysates from isolated human PMNsboilw/BmeSDS-PAGEelectroblotted onto a transfer membraneprobed with a biotin labelled recomb gonococci expressing Opa50 , Opa52 and a strain deleted for the Opa50 expressing locus OpaC (-)-adherent bacteria were detected by incubation in streptavidin-conjugated HRP-bands at 90 and 180 kDa show how much bacteria bound 1B: subsequent probe of overlayed blot using CD66-specific monoclonal antibody; indicative of unbound CD66 antigensFig2: Opa is an integral membrane protein; affinity purified such that native opa conf’n is maintained-PMNs were surface labelled with membrane impermeant biotinylation reasgent NHS-LC-biotin (PMA also added to induce degranulation)detergent (TX100) soluble proteins were extracted and incubated with intact, recombinant gonococci expressing defined Opaswashpellet bacterial cells and see which PMN proteins were brought downPAGEWestern (SA-HRP)-biotinylated proteins detected by SA-HRP revealed recovery of 35, 90 and 180kDa proteins from Opa52-also probed for Opa to make sure that diffs in recovered protein was not due to diffs in recovered bacteriaenhanced recovery must be due to exoression of distinct opa, used CD66-family specific (non reactive to CGM6) Ab to detect 180kDa BGP (CD66a), Used NCA-specific Ab to detect CD66c, used CGM6-specific Ab to detect CD66b (not recovered)
  • Opa50 binds epithelial cells via cell surface proteoglycan receptors, irrespective of expressed CEACAMs cf. Opa52 can only attach to HeLa cells expressing CD66a, c, d, e and NOT CD66b (red box, CGM6, which was absent in Fig.2) as it looks like - ctl-also plated bacteria after saponin lysispics are reflective of colony forming units recovered-not sure if diffs in association in the diff cell lines are due to diffs in binding affinity or receptor expression level (but checked that by W. blot of hela line expressing CGM6)
  • Fig. 4: Adherence asay was done to check whether Opa50 expression could allow binding to CD66 family members, in addition to its previously described proteoglycan interactions (heparin competes for Opa50 binding with the heparin-sulfate proteoglycans). Parallel assay was done in the presence of poly-clonal anti-CEA sera to determine what level of Opa binding could be attributed specifically to the CD66receptors. Since the extracellular domain of CGM1 consists of only the N-term conserved domain common to all CEA family members, only Hela-CGM1 was used. heparin almost completely abrogated Opa50-mediated binding to HeLa-CGM1 but had no effect onthe adherence of Opa52-expressing gonococci. In contrast, the polyclonal antisera prevented binding by Opa52- but notthat of Opa50-expressing strains to HeLa-CGM1These assays indicate that Opa50 and Opa52 do possess distinct specificities for either HSPGs or CEA family antigens.
  • Distribution of cd66 antigen and bound gonococci on the cell surface was analysed by immuno cytochmeical staining and confocal laser scanning microscopy. Opa52 ecpressing gonococci appear to recruit CD66 family members to generate a focus of high receptor concentration at the site of bacterial adherence.No such phenomena occurs when opa50 expressing bacteria are employed (confirming that this is not a general cellular response to bacterial attachment). same results obtained using Hela-NCA, BGP, CEA cell lines; no recruitment was shown in hela-CGM6 with either opa50 or opa52
  • Wanted to analyse the potential for the CD66 family members to help uptake bound gonococci. Opa- and Opa50 expressing bacteria did not display significant levels of invasion. (Opa50-mediated uptake was really helped with serum 9but opa52 was not)HeLa-NCA did not take up as much bacteria as BGP, CGM1 or CEA proves that theres not nec. A strict correlation between binding and invation.
  • Opa52 mediated binding and invasion ofo CD66 expressing HeLa cell lines by recombinant E.coli. Pattern of binding is reminiscient of that seen with Opa50 and Opa52 expressing gonococci. Opa50 expressing e coli bind equally well in hela-neo and helabgp, since binding to hspgopa52 expressing ecoli only adhere well to Hela BGP repeated with all hela lines and got similar results
  • Gentamycin assays using recombinant e coli strains
  • PMNs infected by Opa50-expressing gonococci show a distribution of CD66 antigen all over the cell surface, which is indinguishable for that seen in uninfected cells (not shown). Opa52 expressing bacteria cause a strong recruitment of CD66 proteins to the sites of interaction. Together, these obs support results obtained with the transfected HeLa cell lines which suggest that Opa52 mediates binding of CD66 antigens on PMNs, while Opa50 does not (re: fig 5)PMNs were degranulated due to large intracellular stores of CD66 in 2ndary and tertiaty granules holes (i.e. to reduce background CD66 staining)

Transcript

  • 1. CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseriagonorrhoeaeand human polymorphonuclear phagocytes
    Scott D.Gray-Owen, ChristophDehio,
    AnjaHaude, Fritz Grunert
    Thomas F.Meyer
  • 2. Background
    Nesseria gonorrhoeae are gram-negative bacterial pathogens highly adapted to survive in a single host population—humans
    • Colonize mucosal surfaces, route of transmission being the genito-urinary tract following sexual transmission of gonococci
    Opacity-Associated (Opa) Proteins
    • Integral outer-membrane proteins
    • 3. Secondary structure = 8 membrane spanning domains arranged as antiparallel β strands (membrane embedded β-barrel with 4 extracellular loops)
    • 4. Up to ~11 variant opa proteins can be harboured by a single gonoccocal strain
    • 5. Expression undergoes phase variation , where each gene can be independently switched to an ‘on’ or ‘off’ state
    • 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.
  • 7. Carcinoembryonic Antigen-related Cellular Adhesion Molecule
    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.
  • 8. Question
    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?
    Method
    -Use recombinant gonococci and E. coli to express defined Opa proteins
    -Transfect HeLa cell lines expressing individual members of the CEA family to analyse Opa-mediated interactions with each member in isolation.
  • 9. CD66a
    CEACAM1
    CD66c
    CEACAM6
    Replicate blots, using different antibodies, to identify which protein was actually recovered
    Fig. 2. Opa52 mediates the specific binding of distinct members of the CEA family of receptor proteins.
    Fig. 1. Gonococcal binding to electrophoretically separated PMN
    components by bacterial overlay assay.
  • 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.
    20μm
  • 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.
  • 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).
  • 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.
  • 14. Fig. 7. Opa-mediated interactions of recombinant E.coli with stably
    transfected HeLa cells expressing BGP (CD66a).
  • 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.
  • 16. Opa52 Recruits CD66 Antigens on Human PMNs
    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
    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.
  • 17. Summary of Conclusions
    -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.
    -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.
    -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
    -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.
  • 18. Opsonin-independent phagocytosis (Invasion and intracellular accomodation?)
  • 19. Additional References:
    Nat Rev Immunol. 2006 Jun;6(6):433-46.
    CEACAM1: contact-dependent control of immunity.
    Gray-Owen SD, Blumberg RS.
    Curr Opin Microbiol. 2003 Feb;6(1):43-9.
    'Small' talk: Opa proteins as mediators of Neisseria-host-cell communication.
    Hauck CR, Meyer TF.