Neisserial Lipooligosaccharide Is a Target for Complement Component C4b

We identified Neisseria meningitidis lipooligosaccharide (LOS) as an acceptor for complement component C4b (C4b). Phosphoethanolamine (PEA) residues on the second heptose (HepII) residue in the LOS core structure formed amide linkages with C4b. PEA at the 6-position of HepII (6-PEA) was more efficient than 3-PEA in binding C4b. Strains bearing 6-PEA bound more C4b than strains with 3-PEA and were more susceptible to complement-mediated killing in serum bactericidal assays. Deleting 3-PEA from a strain that expressed both 3- and 6-PEA simultaneously on HepII did not decrease C4b binding. Glycose chain extension of the first heptose residue (HepI) influenced the nature of the C4b-LOS linkage. Predominantly ester C4b-LOS bonds were seen when lacto-N-neotetraose formed the terminus of the glycose chain extension of HepI with 3-PEA on HepII in the LOS core. Related LOS species with more truncated chain extensions from HepI bound C4b via amide linkages to 3-PEA on HepII. However, 6-PEA in the LOS core bound C4b even when the glycose chain from HepI bore lacto-N-neotetraose at the terminus. The C4A isoform exclusively formed amide linkages, whereas C4B bound meningococci preferentially via ester linkages. These data may serve to explain the preponderance of 3-PEA-bearing meningococci among clinical isolates, because 6-PEA enhances C4b binding that may facilitate clearance of 6-PEA-bearing strains resulting from enhanced serum killing by the classical pathway of complement. We identified Neisseria meningitidis lipooligosaccharide (LOS) as an acceptor for complement component C4b (C4b). Phosphoethanolamine (PEA) residues on the second heptose (HepII) residue in the LOS core structure formed amide linkages with C4b. PEA at the 6-position of HepII (6-PEA) was more efficient than 3-PEA in binding C4b. Strains bearing 6-PEA bound more C4b than strains with 3-PEA and were more susceptible to complement-mediated killing in serum bactericidal assays. Deleting 3-PEA from a strain that expressed both 3- and 6-PEA simultaneously on HepII did not decrease C4b binding. Glycose chain extension of the first heptose residue (HepI) influenced the nature of the C4b-LOS linkage. Predominantly ester C4b-LOS bonds were seen when lacto-N-neotetraose formed the terminus of the glycose chain extension of HepI with 3-PEA on HepII in the LOS core. Related LOS species with more truncated chain extensions from HepI bound C4b via amide linkages to 3-PEA on HepII. However, 6-PEA in the LOS core bound C4b even when the glycose chain from HepI bore lacto-N-neotetraose at the terminus. The C4A isoform exclusively formed amide linkages, whereas C4B bound meningococci preferentially via ester linkages. These data may serve to explain the preponderance of 3-PEA-bearing meningococci among clinical isolates, because 6-PEA enhances C4b binding that may facilitate clearance of 6-PEA-bearing strains resulting from enhanced serum killing by the classical pathway of complement. Complement forms an important arm of the innate immune system that combats neisserial infections. Deficiencies of terminal complement components (C5-C9) predispose individuals to recurrent neisserial infections (1Ross S.C. Densen P. Medicine (Baltimore). 1984; 63: 243-273Crossref PubMed Scopus (554) Google Scholar, 2Fijen C.A. Kuijper E.J. te Bulte M.T. Daha M.R. Dankert J. Clin. Infect. Dis. 1999; 28: 98-105Crossref PubMed Scopus (122) Google Scholar). The classical pathway of complement is essential to mediate in vitro killing by serum (3Ingwer I. Petersen B.H. Brooks G. J. Lab. Clin. Med. 1978; 92: 211-220PubMed Google Scholar). Activation of the classical pathway is initiated by binding of the C1 complex to antibodies bound to an antigen on the bacterial surface. Activated C1s in the complex first cleaves C4 to C4b, which binds covalently to the bacterial surface, and then cleaves C2 that binds to C4b, leading to the formation of the C4b2a complex, which is the C3-convertase of the classical pathway. Binding of a C3 molecule to or close to the classical pathway C3-convertase imparts C5-convertase activity to the enzyme complex (reviewed in Ref. 4Walport M.J. N. Engl. J. Med. 2001; 344: 1058-1066Crossref PubMed Scopus (2427) Google Scholar). Activation of C4 results from the separation of 77 amino acids (C4a) from the N-terminal end of the C4 α-chain by C1s, resulting in the formation of the metastable C4b molecule. This results in the activation of the internal thioester bond of C4b (5Law S.K. Lichtenberg N.A. Levine R.P. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7194-7198Crossref PubMed Scopus (135) Google Scholar), so that the carbonyl group linked to sulfur (the native thioester) becomes more electrophilic (i.e. an electron acceptor) and reacts readily with nucleophilic groups (i.e. electron-donating groups) such as -OH to form an ester linkage or with -NH2 to form an amide linkage (6Dodds A.W. Ren X.D. Willis A.C. Law S.K. Nature. 1996; 379: 177-179Crossref PubMed Scopus (166) Google Scholar). Alternatively, the carbonyl group can react with water and become hydrolyzed. There are two isoforms of C4 present in normal human serum, called C4A and C4B (7Awdeh Z.L. Alper C.A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3576-3580Crossref PubMed Scopus (419) Google Scholar). A histidine residue in the α-chain at position 1106 imparts to C4B the ability to form ester linkages. The presence of an aspartic acid residue at position 1106 results in C4A functionality and preferential amide bond formation with target -NH2 groups (8Carroll M.C. Fathallah D.M. Bergamaschini L. Alicot E.M. Isenman D.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6868-6872Crossref PubMed Scopus (80) Google Scholar). C4A plays an important role in the clearance of immune complexes from serum, and deficiency of this molecule predisposes individuals to autoimmune disorders (9Hartmann D. Fremeaux-Bacchi V. Weiss L. Meyer A. Blouin J. Hauptmann G. Kazatchkine M. Uring-Lambert B. J. Clin. Immunol. 1997; 17: 176-184Crossref PubMed Scopus (24) Google Scholar, 10Bakkaloglu A. Pascual M. Schifferli J.A. Ozen S. Besbas N. Saatci U. Turk. J. Pediatr. 1995; 37: 147-151PubMed Google Scholar, 11Atkinson J.P. Clin. Exp. Rheumatol. 1989; 7: S95-S101PubMed Google Scholar). C4B deficiency was reported to be associated with an increased incidence of bacteremia and meningitis caused by encapsulated organisms (12Bishof N.A. Welch T.R. Beischel L.S. J. Infect. Dis. 1990; 162: 248-250Crossref PubMed Scopus (84) Google Scholar, 13Rowe P.C. McLean R.H. Wood R.A. Leggiadro R.J. Winkelstein J.A. J. Infect. Dis. 1989; 160: 448-451Crossref PubMed Scopus (64) Google Scholar), but other reports have not supported this finding (14Cates K.L. Densen P. Lockman J.C. Levine R.P. J. Infect. Dis. 1992; 165: 942-944Crossref PubMed Scopus (19) Google Scholar, 15Goddard E.A. Creemers P. Beatty D.W. Pediatr. Infect. Dis. J. 1994; 13: 661-662Crossref PubMed Scopus (2) Google Scholar). Whereas bacterial targets for C3b have been characterized (16Joiner K.A. Grossman N. Schmetz M. Leive L. J. Immunol. 1986; 136: 710-715PubMed Google Scholar, 17Joiner K.A. Goldman R. Schmetz M. Berger M. Hammer C.H. Frank M.M. Leive L. J. Immunol. 1984; 132: 369-375PubMed Google Scholar, 18Hetherington S.V. Patrick C.C. Hansen E.J. Infect. Immun. 1993; 61: 5157-5163Crossref PubMed Google Scholar, 19Alberti S. Alvarez D. Merino S. Casado M.T. Vivanco F. Tomas J.M. Benedi V.J. Infect. Immun. 1996; 64: 4726-4732Crossref PubMed Google Scholar, 20Bellinger-Kawahara C. Horwitz M.A. J. Exp. Med. 1990; 172: 1201-1210Crossref PubMed Scopus (108) Google Scholar, 21Edwards J.L. Apicella M.A. Cell Microbiol. 2002; 4: 585-598Crossref PubMed Scopus (48) Google Scholar), no detailed studies on C4b binding to microbial surfaces have been carried out. The classical pathway is important to initiate complement activation on neisseriae (3Ingwer I. Petersen B.H. Brooks G. J. Lab. Clin. Med. 1978; 92: 211-220PubMed Google Scholar). In this study, we have identified neisserial LOS 1The abbreviations used are: LOSlipooligosaccharideMOPS3-morpholinopropanesulfonic acidPEAphosphoethanolamineAbantibodymAbmonoclonal antibodyNHSnormal human serumHBSSHanks' balanced salt solutionOSoligosaccharideMSmass spectrometryESelectrosprayCEcapillary electrophoresisTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. as an acceptor for C4b. We have characterized (in detail) the role of the position of phosphoethanolamine (PEA) on the distal heptose (HepII) chain, and the influence of glycose extensions of HepI chain in determining the nature of the C4b-LOS linkage. We speculate that use of PEA to initiate complement activation may enable the host to circumvent molecular mimicry of host structures by neisserial LOS hexose substitutions (22Mandrell R.E. Apicella M.A. Immunobiology. 1993; 187: 382-402Crossref PubMed Scopus (141) Google Scholar) and restrict activation of the cascade to the bacterial surface. These findings may serve to explain how LOS phase variation affects complement activation on the bacterial surface. To our knowledge, this represents the first reported study detailing C4b interactions with a microbe. lipooligosaccharide 3-morpholinopropanesulfonic acid phosphoethanolamine antibody monoclonal antibody normal human serum Hanks' balanced salt solution oligosaccharide mass spectrometry electrospray capillary electrophoresis N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Bacterial Strains and Plasmids—Strains of N. meningitidis and their relevant characteristics are listed in Table I. LOS structures and resultant phenotypic features of the mutants are listed in Table II. Every strain or its mutant derivative possessed a single dominant LOS species as by not data by simultaneously expressed LOS meningitidis strains used in this study and their phenotypic lacto-N-neotetraose PEA at 3- and of LOS 3-PEA on LOS at and PEA at 6-position of LOS PEA at 6-position of LOS 3-PEA on lacto-N-neotetraose in a Table of N. meningitidis mutants used in this LOS not phosphoethanolamine 3-PEA on HepII in a strain was by of the as M. C. Meyer Proc. Natl. Acad. Sci. U. S. A. 1989; PubMed Scopus Google Scholar). The of strains and were as A a that the was as U. M. R. M. 1997; Google Scholar). the system R. D. R. Meyer 1993; PubMed Scopus Google Scholar, M. U. PubMed Scopus Google Scholar), the was at position of the resulting in strains and were with The of and U. M. R. M. 1997; Google Scholar). We first a derivative of U. M. R. M. 1997; Google Scholar) that the of a called strain at the of was at position of the the system R. D. R. Meyer 1993; PubMed Scopus Google Scholar). The resulting was and was used to the of strain to the mutant called in this The for the enzyme for LOS called was as U. G. M. Infect. Immun. 1999; PubMed Google Scholar). LOS mutants with a single residue on the HepI chain to were by of the as A. Infect. Immun. PubMed Scopus (19) Google Scholar) was the of mutants were to D.W. M. Microbiol. 1995; PubMed Scopus Google Scholar) with the as the the of a and were from the The the and To the by we a the the from that was then a in the of N. meningitidis were by with R. C.A. J.M. Infect. Immun. 1984; PubMed Google Scholar, R.E. Infect. Immun. PubMed Google Scholar) in and by of LOS by G. PubMed Scopus Google Scholar). To in to the we from the Neisseria strain with and This was the of J.C. J. 1996; PubMed Google Scholar). A was this at the of the and This an We a of the that to P. 1984; PubMed Scopus Google Scholar) and with the this such that the not the of the PEA at the of HepII was by of the phosphoethanolamine The of and its mutant 3-PEA on HepII been J.C. R. D.W. J.C. Microbiol. 2002; PubMed Scopus Google Scholar). from was used to the and of to the The presence of a single PEA in the core of LOS from the mutants was by mass of LOS and Complement human serum was from with no of or infections or and at C4A or C4B and was from individuals in other complement as in M. M. A. S. V. S. Clin. Infect. Dis. 2001; PubMed Scopus Google Scholar). C4b was from for use as a in C4 was used in that is for the HepI LOS R. C.A. J.M. Infect. Immun. 1984; PubMed Google Scholar) was used as C4b bound to was C4 antibody and bound to were or from in Hanks' balanced salt solution and were with for in a of for at were in and two that were with C4b bound to targets via ester and amide or for at in a of which but not C4b S.K. Lichtenberg N.A. Levine R.P. J. Immunol. 1980; Google Scholar, Clin. Exp. Immunol. PubMed Scopus Google Scholar, J. J. Exp. Med. 165: PubMed Scopus Google Scholar). of the C4b is not because be from acceptor surfaces its were then with and on for at was as J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). C4b was at a of in and as 1984; 136: PubMed Scopus Google Scholar). LOS was of C4b, and bound to that were with for was carried as S. M. A. S. D. B. C. R. C. M. B. P. J. Exp. Med. 2001; PubMed Scopus Google Scholar). of was from bacterial by the as F. 7: Scopus Google Scholar). LOS was as J. J.C. 2002; PubMed Scopus Google Scholar) in from the were to the LOS was at for in was by and the solution was core oligosaccharide in were carried as J. J.C. 2002; PubMed Scopus Google Scholar). was on a to an mass via a were on were at in to with the the of internal at was bactericidal were as S. 1994; PubMed Scopus Google Scholar). of to the phase were in balanced salt solution and was to a as The of the was of the were at the of the and at was expressed as a of at to bacterial at A the for for C4b and the we that C4b targets on strains and LOS M. C. Microbiol. PubMed Scopus Google Scholar, J. Clin. Microbiol. Google Scholar) and their mutants LOS not that were with were in not the of C4b binding S. U. S. S. P. Neisseria Scholar). We to use the and of group strain These mutants bound the of C4b in more than the of data not The and mutants of strain were with and to bacterial were with to but not C4b from its to and with C4 that were with and then in as with their linkages to C4b that with the C4b target a molecular mass that (the mass of the of C4b, as seen in the as and mass of the C4 as seen in the The formed with than the complexes seen with or This that LOS was the acceptor because the mutants in their LOS with the mutant the and the mutant the in the of the C4b to organisms The C4b on the strains were by and of the C4b bound to this strain was via amide linkages, as by C4b The seen at and that formed amide linkages with C4b LOS the for C4b as as results that LOS was the acceptor for C4b. To we used for the LOS of the to LOS with on the presence of at the as and organisms with serum complement no at that did not the of the that with the A and was seen at This LOS bound to a form of C4b, called S. C. J. Immunol. 1980; Google Scholar). is by of the of C4b from the end by and and the ability to form M. C. S. 1986; PubMed Scopus Google Scholar). The of that is to the target the N-terminal and the of the of the C4b molecule. were to the LOS of the strain with the for the lacto-N-neotetraose M.A. C.A. D.E. Infect. Immun. PubMed Google Scholar) but were not because C4b or the was seen to the molecular mass at This on LOS to We in the organisms at by an in the of which was by The molecular mass of this was with an formed the of and the LOS molecule. This was in a that this with N. Google Scholar) not PEA on LOS the in with on LOS that can in an amide linkage with C4b is To the that PEA was the acceptor on LOS for C4b, we C4b binding to the and mutants of (the 3-PEA from its J.C. R. D.W. J.C. Microbiol. 2002; PubMed Scopus Google Scholar). Strains were with and with or and was as serum was used to C4b that C4b-LOS on the were and because 3-PEA on HepII binds C4b than 6-PEA were seen in the but not when were with The C4b-LOS on In of the mutant in the of C4b that C4b bound to this strain via ester linkages. data that C4b forms amide linkages with PEA (in this on HepII of of LOS of C4b Binding via to PEA on the HepII of C4b that was to the LOS of the mutant of with the C4b to mutant the that this strain possessed an PEA residue or the position of the PEA from that of We the LOS of strain to the and position of the PEA LOS was by and were carried by and of the of the A by the of the core to be and a with the presence of two PEA oligosaccharide was and by the studies on the on the both PEA to the HepII residue of and These studies were and by that by with data J. J.C. 2002; PubMed Scopus Google Scholar), that the HepII residue was at both the 3- and simultaneously by PEA not and data and of LOS and core from N. meningitidis to in mass by of the mass by from to in mass by of the mass by from in a capillary mass of core oligosaccharide from N. meningitidis strain of is The at and the of the oligosaccharide are as The at is for two PEA a heptose residue J. J.C. 2002; PubMed Scopus Google Scholar). 6-PEA the for finding of PEA residues simultaneously at the 3- and of HepII to the of of PEA residues in C4 binding. We binding of C4b to and at the 3- and of with their mutant by and that of 3-PEA did not decrease C4b binding and binding to the was the not This that 6-PEA was the more efficient C4b acceptor or more readily to C4b. We the nature of the linkage C4b and LOS was by of C4b-LOS bonds no in the or the nature of C4b-LOS linkages the and mutants and their We C4b binding to strains that a 3-PEA and a strain a 6-PEA and or a 3-PEA and 6-PEA simultaneously strain to C4b binding to and binding were to that in C4b binding were not by in binding. We used because the presence of C4 binding to meningococci S. U. S. S. P. Neisseria Scholar). we an because HepI chain substitutions have an on binding of C4b to and binding of C4b to PEA in and mutants HepI and HepI Strains with a 6-PEA bound the of C4b There was no the of or and C4b binding the The of the strains the of C4 strains with a 6-PEA were in whereas the and in data that 6-PEA-bearing strains more C4b than strains with 3-PEA and are more to the bactericidal of position of PEA on HepII and to bactericidal of normal human of PEA on from two or more in at and mutants a single HepI and mutants a single HepI and mutants a single HepI and mutants a single HepI and mutants a single HepI and from two or more in and mutants a single HepI in a of the HepI LOS on C4b HepI chain of LOS is phase in because of the of residues in the M. B. P. 1999; PubMed Scopus Google Scholar). We the influence of in HepI chain on binding of C4b to Strains and and were by and were to the of the glycose of HepI to the and that C4b binding to the LOS of the mutant of via ester linkages, as by the of the of C4b bound to LOS A decrease in HepI was associated with a amide linkage formation LOS and C4b in strain 6-PEA-bearing strains and amide linkages were even in the presence of the lacto-N-neotetraose LOS species (i.e. in the C4 the of the C4b and isoforms of called C4A and in human C4A forms amide linkages with amino whereas C4B preferentially forms ester linkages with groups on targets (8Carroll M.C. Fathallah D.M. Bergamaschini L. Alicot E.M. Isenman D.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6868-6872Crossref PubMed Scopus (80) Google Scholar, D.E. J. Immunol. 1986; 136: Google Scholar). We used serum that C4A or C4B and the nature of the targets formed C4b and was with or serum, and linkages were by which both C4 as a There was no of ester linkages formed C4A and as by the of C4b of with serum C4A serum formed ester linkages, C4b-LOS were seen These data for amide linkages C4b and LOS and the acceptor of C4 isoforms on neisserial The of the classical pathway in was a (reviewed in Ref. of the Complement Google Scholar). The of the complement pathway been in However, to our knowledge, targets for C4b on microbial surfaces and the nature of the linkages formed C4b and have been serum killing of neisseriae of complement activation by the classical pathway (3Ingwer I. Petersen B.H. Brooks G. J. Lab. Clin. Med. 1978; 92: 211-220PubMed Google Scholar). In this study, we have identified LOS as a acceptor for C4b. The finding is the of PEA residues as targets for C4b. of PEA in complement activation on the bacterial may have The LOS of neisseriae host structures (22Mandrell R.E. Apicella M.A. Immunobiology. 1993; 187: 382-402Crossref PubMed Scopus (141) Google Scholar). the lacto-N-neotetraose LOS The use of bacterial PEA by the host to C4b and initiate activation of the complement cascade may be a to circumvent host This complement to be on the bacterial surface, to host which is to PEA because is a is not on host but in such as becomes on and been to the pathway of complement R.H. G. C. J. Clin. 1993; 92: PubMed Scopus Google Scholar). The presence of ester linkages C4b and the LOS of but not the more truncated LOS of the that C4b may to -OH of the terminal from the oligosaccharide extension at The presence of complexes a C4b molecule of C4b or were on as by at that with C4b The nature and of molecular mass complexes to be We a at The of this with an not that LOS binds to C3b is to The preponderance of amide linkages C4b and LOS of a detailed of PEA residues in the core We that strain bore PEA residues simultaneously at the 3- and of which is not of of the LOS that was the presence of the 6-PEA than the two on that was for the preponderance of amide linkages C4b and The dominant role of the 6-PEA in amide linkages in the presence of a lacto-N-neotetraose HepI chain is in the and and no in the of amide linkages were seen strains for the C4b binding ability of 6-PEA strains is on the that strains with a 6-PEA on HepII and bound more C4b than strains with a 3-PEA on HepII and an that was of the of or binding the data of the role of 6-PEA in binding C4b. The of enhanced C4b binding by the 6-PEA-bearing strains and was by the enhanced of strains to complement-mediated killing Binding of C4b to 3-PEA was influenced by HepI hexose In strains with a 3-PEA on HepII as of the C4b bound to LOS via ester linkages no when was to However, of the oligosaccharide from HepI a in C4b linkages from ester to the 6-PEA and to be even in the presence of lacto-N-neotetraose extension from as by of C4b when the were The preponderance of 3-PEA among clinical may a role for this residue in This is supported by the that PEA on bound more and C4 in in a for both than on not with C4b was more to the bactericidal of than in A of the interactions C4b and the lacto-N-neotetraose or substitutions on HepI and a 3- or a 6-PEA on is in These findings may serve to explain of strains from a 3-PEA on HepII (i.e. the and LOS and the and with are The that a at the of HepII is expressed in N. meningitidis and is seen with the LOS strain in this The a of and is to phase The (i.e. when is in with and a at the of HepII J.C. R. D.W. J.C. Microbiol. 2002; PubMed Scopus Google Scholar). The presence of a at the of HepII not decrease the of C4b binding strain to the ability of present 6-PEA to form amide linkages with C4b binding were seen with and 6-PEA on and 6-PEA on data not isoforms of called C4A and are present in forms amide linkages, whereas preferentially forms ester linkages with its targets (8Carroll M.C. Fathallah D.M. Bergamaschini L. Alicot E.M. Isenman D.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6868-6872Crossref PubMed Scopus (80) Google Scholar, D.E. J. Immunol. 1986; 136: Google Scholar). the of and binding to the with to serum bactericidal activity and binding to to be in our studies we used the isoforms to the results of the in amide ester linkages C4b and the bond formed and LOS was not by In formed ester linkages with the and be from its targets by In data on an important of classical pathway activation on of how complement is on on the of and may to that on have that of complement activation (i.e. the of complement may be an important in determining serum bactericidal activity M. Kuijper E.J. N. Dankert J. Infect. Immun. 2002; PubMed Scopus Google Scholar). is that the target for C4b is by of and this may explain in bactericidal by such is N. N. meningitidis as C3b may be resulting in a no killing complement We and Frank for for a detailed on the of strain and for of the

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