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The molecular basis of group B meningococcal serum resistance
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211- MOLECULAR IDENTIFICATION OF THE CRI-RELATED BLOOD GROUP ANTIGENS IMPLICATED IN MALARIAL INFECTION. Moulds, JM’, Zimmaman pz. Birmingham Ds, ~oumbo 0” Kassambara L’, Sagam I”, Atkinson JPs, Krych Ms. Hoarcade 8, Miller L6. ‘University of Texas-Houston Medical School, *Case Western Reserve University, %e Ohio State Uaiversity, %Joivershy of Mali, 5Washington University Medical School, %IAID-National I&totes of Health. The severity of malaria iofectioo correlates with the ability of Phmodium falciparum-infected erylhmqtes to form mm. The Knops blood group antigens {McCoy (Mc(? & MC@?, SwainLaogJey @I’) sod Viiieo (Vii)} located on complement receptor one (CRl) tineace this reaction. We sought to understand the role of CR1 io malaria pathogenesis by first locaking critical CR1 regions using RT-PCR he@nnhqlex analysis of DNA from donors having selected Knops types. In addition, 101 African donors were tested with anti-Mcc’, Mccb and Sl” and their genomic DNA analyzed. This included several tvnlies informative for inheritance of McCoy or Sl’. Two candidate positions were ide.ntiti~ aa# 1590 (bp 4795) was associated with McC while aa# 1601 (bp 4828) associated with Sla. CR1 deletions and substitutions were used to cotino these associations. Hemagglutioation of erythmcytes by the Knops antisera is inhibited by recombinaot CR1 Agglutination byanti-McC” and -SI’ was inhibited by CR1 constructs that carried wild type SCRs 22-30 but not SCRs l-7.8-14 or 15-21. Conshucts bearing wild type Lys 1590 inhiiited anti-M& while mutantGlu
1590 inhibited~&MC@. Similarly, wild type Arg 1601 inhibited an&W while mutantGly 1601 inhibitedand-W These findings indicate that SCR25 of CRl. a region not required for C3b- or Obbindii, plays a role in roaelting. for these mucitions will allow us populations in a&r to detena& antigens have in the pathogenesis
Development of PCR genotyping to screea large endemic what role the blood group of malaria.
COMPLEMENT AND THE MALARIA PARASITE IN THE MOSQUlTO VEYXOR G. Margos’, G.A. Butcheti, A. Davies’, R.E. Sinden’, and P.J.Lachmann’ Centre for Veterinary Science, ‘Microbial Immunology Grou University of Cambridge, and ?Department of Biology, Imperial College of Science, Technology and Medicine, London, U.K. We have shown that mosquito midgut stages of Phmodium bergki are ptectedagainst complement (C) active in the mosquito mid-gut, but become increasingly sensitive to C lysis during further development. Parasite C evasion mechanisms target various steps in the C cascade and may involve acquisition of host molecules or expression of inhibitors by the parasite. To investigate the mechanisms involved in protection of malaria parasites, midgut stages were stained for C3 deposition and for uptake of CD59 present on red blood cells. Pbs21, a surface pmtein specifically expressed by mosquito midgut stages, was used as marker m&cule to identify these stages. Parasite stages in samples taken at various time points between 30 minutes and 22 hours showed C3 positive fluorescence following exposure to rat C, even those which showed p&al protection against C lysis. Using an anti-rat CD59 antibody mosquito midgut stages wete negative for CD59 whereas rat red blood cells in the same sample stained positive. Furthermore, only a marginal difference was found in resistanceisusceptibili~y of parasites to normal rat sefum whether they originated from either mouse or rat, indicating that there is no species specilic protection against C lysis. In addition, exposure of parasites to rat C in the presence of antirat CD59 antibody did not increase lysis. These data suggest that parasite or host molecules other than CD59 are involved in protection of malaria parasites against C mediated lysis. tigoing research aims to identify these molecules.
212- ‘INTERACTION BETWEEN
213- C~MPLEMJSNT-GROWB MENIN~~C~CCAL INTERACTIONS: A STUDY USING ISOGENIC MUTANI STRAINS Ram S’, Vogel U*, Gulati S’, Heinze G*, Van der Ley P*, Frost M* and Rice P. A’. *Boston Medical Center, Boston, MA,*Universitit WUrzburg, Wibzburg, Germany and IRIVM, The Netherlands. Lipooligosaccharide (LOS) sialylation and 2&polysialic acid capsule (Cap) play a key role in mediating group B meningococcal serum resistance. The interactions of complemer (C) with isogenic mutant strains that lacked either Cap (siti-), LOS sialic acid (I&), or both @iaD-/lst-) were studied to elucidate serum resistance mechanisms. Though siaD- and siaD/lst- bound similar amounts of factor H (fH), C3 and C4 at 30 min, the former bound 50% less C5b-9, suggesting that LOS sialic acid probably regulates C at the level of CS-convertax formation. Cape strains [wild-type (wt) and Ist-] bound 2-fold less IgG, C3 and C4 compared to Cap- mutants. Using western blots, PorA was found to be the main acceptor molecule for C3b on Cap-t strains. Only -50% of C3b on all strains was processed to iC3b. Cap+ strains bound 33% less fH than Cap- strains. Meningococci bound comparatively small amounts of M (only about IN to 112 that seen with LOS-sialylated gonococci). The presence of LOS sialic acid did not enhance fH binding to Cap+ or Cap- strains. Deleting PorB from Cap+ seains decreased M binding 5-fold, while deleting PorA decreased fH binding 2 to 3 fold, suggesting that PorB was the main M acceptor on Cap+ strains. Deleting either Por molecule from Cap- strains did not influence fH binding, suggesting that either Par could bind M in the absence of Cap. None of the strains studied bound C4bp. Thus, unlike gonocccci, C regulatory molecules (M and C4bp) do not appear to play a major role in mediating serum resistance
214- THE MOLECULAR BASIS OF GROUP B MENINGOCOCCAL SERUM RESISTANCE Ram S*, Mackinnon FG’, Gulati S*, Vogel U’, Gorringe A’, ant Rice PA*. *Boston Medical Center. Boston, MA, ‘Oxford University, Oxford, U.K, IUniversitit Wiirzborg, Wibzburg, Germany and ‘CAMR, Salisbury, U.K. We studied the interaction of complement with 14 epidemiologically related group B meningococcal strains that differed only with respect to capsule (Cap) expression or lipooligosaccharide (LOS) immunotype. LOS sialylation of Cap strains resulted in resistance to 10% but not 50% normal serum. LOS sialylation did not enhance the amount of factor H binding nor decrease total C3 or C4 binding at 30 min, but did consistently reduce the rate of binding of C3 and C4 to bacteria. This resulted in 50% less C5b-9 binding, probably due to decreased CS-convertase formation. Less than 40% C3b was processed to iC3b regardless of LOS sialylation. Cap+ strains with sialylated LOS were highly resistant to even 50% serum, while those with nonsialylated (and truncated) LOS were significantly less resistant. Cap+ strains bound 1.5-fold less C3 and 2-fold less C4 compared to Cap- strains. Cap+ strains bound 30% less factor H than Cap- strains, suggesting that Cap (2,8linked polysialic acid; colominic acid) interfered with factor H binding to other surface receptor(s). Cap+ strains bound 2-fold less IgG, but 3 to 5-fold more I&l than Cap- strains. IgM bound exclusively to Cap on Cap+ strains, and binding was completely inhibited by pure colominic acid. Decreased C3 and C4 binding to Cap+ strains was not due to inaccessibility of C3 or C4 to surface target(s), because Cap specific mAb 2-2-B fixed complement very efficiently. Thus, LOS sialylation and Cap regulate complement at different stages in the complement cascade and mediate group B meningococcal serum resistance.
211- MOLECULAR IDENTIFICATION OF THE CRI-RELATED BLOOD GROUP ANTIGENS IMPLICATED IN MALARIAL INFECTION. Moulds, JM’, Zimmaman pz. Birmingham Ds, ~oumbo 0” Kassambara L’, Sagam I”, Atkinson JPs, Krych Ms. Hoarcade 8, Miller L6. ‘University of Texas-Houston Medical School, *Case Western Reserve University, %e Ohio State Uaiversity, %Joivershy of Mali, 5Washington University Medical School, %IAID-National I&totes of Health. The severity of malaria iofectioo correlates with the ability of Phmodium falciparum-infected erylhmqtes to form mm. The Knops blood group antigens {McCoy (Mc(? & MC@?, SwainLaogJey @I’) sod Viiieo (Vii)} located on complement receptor one (CRl) tineace this reaction. We sought to understand the role of CR1 io malaria pathogenesis by first locaking critical CR1 regions using RT-PCR he@nnhqlex analysis of DNA from donors having selected Knops types. In addition, 101 African donors were tested with anti-Mcc’, Mccb and Sl” and their genomic DNA analyzed. This included several tvnlies informative for inheritance of McCoy or Sl’. Two candidate positions were ide.ntiti~ aa# 1590 (bp 4795) was associated with McC while aa# 1601 (bp 4828) associated with Sla. CR1 deletions and substitutions were used to cotino these associations. Hemagglutioation of erythmcytes by the Knops antisera is inhibited by recombinaot CR1 Agglutination byanti-McC” and -SI’ was inhibited by CR1 constructs that carried wild type SCRs 22-30 but not SCRs l-7.8-14 or 15-21. Conshucts bearing wild type Lys 1590 inhiiited anti-M& while mutantGlu
1590 inhibited~&MC@. Similarly, wild type Arg 1601 inhibited an&W while mutantGly 1601 inhibitedand-W These findings indicate that SCR25 of CRl. a region not required for C3b- or Obbindii, plays a role in roaelting. for these mucitions will allow us populations in a&r to detena& antigens have in the pathogenesis
Development of PCR genotyping to screea large endemic what role the blood group of malaria.
COMPLEMENT AND THE MALARIA PARASITE IN THE MOSQUlTO VEYXOR G. Margos’, G.A. Butcheti, A. Davies’, R.E. Sinden’, and P.J.Lachmann’ Centre for Veterinary Science, ‘Microbial Immunology Grou University of Cambridge, and ?Department of Biology, Imperial College of Science, Technology and Medicine, London, U.K. We have shown that mosquito midgut stages of Phmodium bergki are ptectedagainst complement (C) active in the mosquito mid-gut, but become increasingly sensitive to C lysis during further development. Parasite C evasion mechanisms target various steps in the C cascade and may involve acquisition of host molecules or expression of inhibitors by the parasite. To investigate the mechanisms involved in protection of malaria parasites, midgut stages were stained for C3 deposition and for uptake of CD59 present on red blood cells. Pbs21, a surface pmtein specifically expressed by mosquito midgut stages, was used as marker m&cule to identify these stages. Parasite stages in samples taken at various time points between 30 minutes and 22 hours showed C3 positive fluorescence following exposure to rat C, even those which showed p&al protection against C lysis. Using an anti-rat CD59 antibody mosquito midgut stages wete negative for CD59 whereas rat red blood cells in the same sample stained positive. Furthermore, only a marginal difference was found in resistanceisusceptibili~y of parasites to normal rat sefum whether they originated from either mouse or rat, indicating that there is no species specilic protection against C lysis. In addition, exposure of parasites to rat C in the presence of antirat CD59 antibody did not increase lysis. These data suggest that parasite or host molecules other than CD59 are involved in protection of malaria parasites against C mediated lysis. tigoing research aims to identify these molecules.
212- ‘INTERACTION BETWEEN
213- C~MPLEMJSNT-GROWB MENIN~~C~CCAL INTERACTIONS: A STUDY USING ISOGENIC MUTANI STRAINS Ram S’, Vogel U*, Gulati S’, Heinze G*, Van der Ley P*, Frost M* and Rice P. A’. *Boston Medical Center, Boston, MA,*Universitit WUrzburg, Wibzburg, Germany and IRIVM, The Netherlands. Lipooligosaccharide (LOS) sialylation and 2&polysialic acid capsule (Cap) play a key role in mediating group B meningococcal serum resistance. The interactions of complemer (C) with isogenic mutant strains that lacked either Cap (siti-), LOS sialic acid (I&), or both @iaD-/lst-) were studied to elucidate serum resistance mechanisms. Though siaD- and siaD/lst- bound similar amounts of factor H (fH), C3 and C4 at 30 min, the former bound 50% less C5b-9, suggesting that LOS sialic acid probably regulates C at the level of CS-convertax formation. Cape strains [wild-type (wt) and Ist-] bound 2-fold less IgG, C3 and C4 compared to Cap- mutants. Using western blots, PorA was found to be the main acceptor molecule for C3b on Cap-t strains. Only -50% of C3b on all strains was processed to iC3b. Cap+ strains bound 33% less fH than Cap- strains. Meningococci bound comparatively small amounts of M (only about IN to 112 that seen with LOS-sialylated gonococci). The presence of LOS sialic acid did not enhance fH binding to Cap+ or Cap- strains. Deleting PorB from Cap+ seains decreased M binding 5-fold, while deleting PorA decreased fH binding 2 to 3 fold, suggesting that PorB was the main M acceptor on Cap+ strains. Deleting either Por molecule from Cap- strains did not influence fH binding, suggesting that either Par could bind M in the absence of Cap. None of the strains studied bound C4bp. Thus, unlike gonocccci, C regulatory molecules (M and C4bp) do not appear to play a major role in mediating serum resistance
214- THE MOLECULAR BASIS OF GROUP B MENINGOCOCCAL SERUM RESISTANCE Ram S*, Mackinnon FG’, Gulati S*, Vogel U’, Gorringe A’, ant Rice PA*. *Boston Medical Center. Boston, MA, ‘Oxford University, Oxford, U.K, IUniversitit Wiirzborg, Wibzburg, Germany and ‘CAMR, Salisbury, U.K. We studied the interaction of complement with 14 epidemiologically related group B meningococcal strains that differed only with respect to capsule (Cap) expression or lipooligosaccharide (LOS) immunotype. LOS sialylation of Cap strains resulted in resistance to 10% but not 50% normal serum. LOS sialylation did not enhance the amount of factor H binding nor decrease total C3 or C4 binding at 30 min, but did consistently reduce the rate of binding of C3 and C4 to bacteria. This resulted in 50% less C5b-9 binding, probably due to decreased CS-convertase formation. Less than 40% C3b was processed to iC3b regardless of LOS sialylation. Cap+ strains with sialylated LOS were highly resistant to even 50% serum, while those with nonsialylated (and truncated) LOS were significantly less resistant. Cap+ strains bound 1.5-fold less C3 and 2-fold less C4 compared to Cap- strains. Cap+ strains bound 30% less factor H than Cap- strains, suggesting that Cap (2,8linked polysialic acid; colominic acid) interfered with factor H binding to other surface receptor(s). Cap+ strains bound 2-fold less IgG, but 3 to 5-fold more I&l than Cap- strains. IgM bound exclusively to Cap on Cap+ strains, and binding was completely inhibited by pure colominic acid. Decreased C3 and C4 binding to Cap+ strains was not due to inaccessibility of C3 or C4 to surface target(s), because Cap specific mAb 2-2-B fixed complement very efficiently. Thus, LOS sialylation and Cap regulate complement at different stages in the complement cascade and mediate group B meningococcal serum resistance.