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Binding of C4b-binding protein to porin: a molecular mechanism of serum resistance of Neisseria gonorrhoeae.

Ram S, Cullinane M, Blom AM, Gulati S, McQuillen DP, Monks BG, O'Connell C, Boden R, Elkins C, Pangburn MK, Dahlbäck B, Rice PA - J. Exp. Med. (2001)

Bottom Line: Only recombinant C4bp mutant molecules containing the NH2-terminal alpha-chain short consensus repeat (SCR1) bound to both Por1A and Por1B gonococci, suggesting that SCR1 contained Por binding sites.C4bp alpha-chain monomers did not bind gonococci, indicating that the polymeric form of C4bp was required for binding.Using fAb fragments against C4bp SCR1, C4bp binding to Por1A and Por1B strains was inhibited in a complement-dependent serum bactericidal assay.

View Article: PubMed Central - PubMed

Affiliation: Evans Biomedical Research Center, Boston Medical Center, Boston, Massachusetts 02118, USA. sram@bu.edu

ABSTRACT
We screened 29 strains of Neisseria gonorrhoeae and found 16/21 strains that resisted killing by normal human serum and 0/8 serum sensitive strains that bound the complement regulator, C4b-binding protein (C4bp). Microbial surface-bound C4bp demonstrated cofactor activity. We constructed gonococcal strains with hybrid porin (Por) molecules derived from each of the major serogroups (Por1A and Por1B) of N. gonorrhoeae, and showed that the loop 1 of Por1A is required for C4bp binding. Por1B loops 5 and 7 of serum-resistant gonococci together formed a negatively charged C4bp-binding domain. C4bp-Por1B interactions were ionic in nature (inhibited by high salt or by heparin), whereas the C4bp-Por1A bond was hydrophobic. Only recombinant C4bp mutant molecules containing the NH2-terminal alpha-chain short consensus repeat (SCR1) bound to both Por1A and Por1B gonococci, suggesting that SCR1 contained Por binding sites. C4bp alpha-chain monomers did not bind gonococci, indicating that the polymeric form of C4bp was required for binding. Using fAb fragments against C4bp SCR1, C4bp binding to Por1A and Por1B strains was inhibited in a complement-dependent serum bactericidal assay. This resulted in complete killing of these otherwise fully serum resistant strains in only 10% normal serum, underscoring the importance of C4bp in mediating gonococcal serum resistance.

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MS11 Por1B loops 5 and 7 together participate in forming a C4bp-binding region. Transformation of F62 with pUNCH61 (reference 40) resulted in strains with hybrid Por molecules F62loop1MS11loop2–8 and F62loop1–4MS11loop5–8. F62 sequence is indicated by black bars; MS11 sequence is represented by hatched bars. Both hybrids bound C4bp, suggesting that the C4bp binding region in MS11 lay in a region encompassed by loops 5–8. The BbsI-BsgI fragment of F62 was cloned into pUNCH61 to obtain pBUMC1 and this plasmid was used to transform F62. This yielded a gonococcal strain with hybrid Por molecule MS11loop1 F62loop2–7MS11loop1–8, which did not bind C4bp, thus eliminating MS11 loop 8 as the C4bp binding loop. Using overlap extension PCR or site-directed mutagenesis on pBUMC1, we mutated loops 5, 6, or 7 either individually or in combination and then transformed F62. We noted that only hybrid Por molecules that contained both MS11 loop 5 and loop 7 bound C4bp. To further demonstrate the absolute requirement of MS11 loops 5 and 7, we mutated MS11 loops 5, 6, and 7 individually in pUNCH61 to resemble the corresponding F62 loop, and then transformed F62. Mutations of loop 5 (MS11loop1–4F62loop5MS11loop6–8) and loop 7 (MS11loop1–6F62loop7MS11loop8) resulted in complete abrogation of C4bp binding, whereas mutating loop 6 (MS11loop1–5F62loop6MS11loop7–8) did not influence C4bp binding. Collectively, these data suggest that MS11 loops 5 and 7 together participate in forming a C4bp-binding domain.
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Figure 4: MS11 Por1B loops 5 and 7 together participate in forming a C4bp-binding region. Transformation of F62 with pUNCH61 (reference 40) resulted in strains with hybrid Por molecules F62loop1MS11loop2–8 and F62loop1–4MS11loop5–8. F62 sequence is indicated by black bars; MS11 sequence is represented by hatched bars. Both hybrids bound C4bp, suggesting that the C4bp binding region in MS11 lay in a region encompassed by loops 5–8. The BbsI-BsgI fragment of F62 was cloned into pUNCH61 to obtain pBUMC1 and this plasmid was used to transform F62. This yielded a gonococcal strain with hybrid Por molecule MS11loop1 F62loop2–7MS11loop1–8, which did not bind C4bp, thus eliminating MS11 loop 8 as the C4bp binding loop. Using overlap extension PCR or site-directed mutagenesis on pBUMC1, we mutated loops 5, 6, or 7 either individually or in combination and then transformed F62. We noted that only hybrid Por molecules that contained both MS11 loop 5 and loop 7 bound C4bp. To further demonstrate the absolute requirement of MS11 loops 5 and 7, we mutated MS11 loops 5, 6, and 7 individually in pUNCH61 to resemble the corresponding F62 loop, and then transformed F62. Mutations of loop 5 (MS11loop1–4F62loop5MS11loop6–8) and loop 7 (MS11loop1–6F62loop7MS11loop8) resulted in complete abrogation of C4bp binding, whereas mutating loop 6 (MS11loop1–5F62loop6MS11loop7–8) did not influence C4bp binding. Collectively, these data suggest that MS11 loops 5 and 7 together participate in forming a C4bp-binding domain.

Mentions: 29 strains of N. gonorrhoeae were screened for binding to C4bp; these are listed in Table . Strain FA6616, containing the MS11 Por molecule reintroduced into an MS11 background using plasmid pUNCH61 40, was used in this study, and for convenience will be referred to as MS11 hereafter. Bacteria grown on chocolate agar supplemented with Isovitalex equivalent in 5% CO2 for 10 to 11 h 41 were suspended in HBSS containing 0.15 mM CaCl2 and 1 mM MgCl2 (HBSS2+) in C4bp binding assays. Alternatively, bacteria were harvested from chocolate agar plates after overnight growth and grown in gonococcal liquid media 41. Results of C4bp binding were equivalent when strains were grown in either media. Plasmids pUNCH61 and pUNCH62 contained the por1B and por1A genes of strains MS11 and FA19, respectively, plus ∼1 kB of gonococcal DNA 3′ to the por gene, respectively, containing a chloramphenicol-resistance marker (CmR [40, 42]). Strains MS11 and FA19 are resistant to killing (SR) by 33% nonimmune NHS. pUNCH61 and pUNCH62 were each used to transform the SS strain F62 to replace F62 Por with either MS11 Por (F62PorMS11) or FA19 Por (F62PorFA19), respectively. In addition to replacing Por completely, transformation with pUNCH61 resulted in hybrids F62loop1MS11loop2–8, and F62loop1–4MS11loop5–8 (see Fig. 4), and transformation of F62 with pUNCH62 yielded hybrids F62loop1FA19loop2–8 and F62loop1–4FA19loop5–8 (see Fig. 3). Because C4bp binding studies with these MS11/F62 hybrids suggested that the C4bp binding region in MS11 Por was contained in the COOH-terminal half of the Por molecule (see Results), we then constructed hybrid MS11loop1F62loop2–7MS11loop8 by replacing the BbsI-BsgI fragment of MS11 Por encompassing loops 2 through 7 in pUNCH61 with the corresponding BbsI-BsgI fragment of F62 Por. Using this plasmid, designated pBUMC1, we constructed hybrids that were mutated at loops 5, 6, or 7 individually or in combination, using either overlap extension PCR for loops 5 and 6, or site-directed mutagenesis for loop 7 (see below: Por mutagenesis). The amino acid sequences of the putative exposed regions of Por loops 5, 6, and 7 of F62 and MS11 are indicated in Table ; variations in sequence are indicated in bold type. The net charge of the surface exposed loop regions was calculated by assigning +1 for arginine (r) and lysine (K), and +0.5 for histidine (H), and –1 for aspartic acid (D) and glutamic acid (E), as described previously 43.


Binding of C4b-binding protein to porin: a molecular mechanism of serum resistance of Neisseria gonorrhoeae.

Ram S, Cullinane M, Blom AM, Gulati S, McQuillen DP, Monks BG, O'Connell C, Boden R, Elkins C, Pangburn MK, Dahlbäck B, Rice PA - J. Exp. Med. (2001)

MS11 Por1B loops 5 and 7 together participate in forming a C4bp-binding region. Transformation of F62 with pUNCH61 (reference 40) resulted in strains with hybrid Por molecules F62loop1MS11loop2–8 and F62loop1–4MS11loop5–8. F62 sequence is indicated by black bars; MS11 sequence is represented by hatched bars. Both hybrids bound C4bp, suggesting that the C4bp binding region in MS11 lay in a region encompassed by loops 5–8. The BbsI-BsgI fragment of F62 was cloned into pUNCH61 to obtain pBUMC1 and this plasmid was used to transform F62. This yielded a gonococcal strain with hybrid Por molecule MS11loop1 F62loop2–7MS11loop1–8, which did not bind C4bp, thus eliminating MS11 loop 8 as the C4bp binding loop. Using overlap extension PCR or site-directed mutagenesis on pBUMC1, we mutated loops 5, 6, or 7 either individually or in combination and then transformed F62. We noted that only hybrid Por molecules that contained both MS11 loop 5 and loop 7 bound C4bp. To further demonstrate the absolute requirement of MS11 loops 5 and 7, we mutated MS11 loops 5, 6, and 7 individually in pUNCH61 to resemble the corresponding F62 loop, and then transformed F62. Mutations of loop 5 (MS11loop1–4F62loop5MS11loop6–8) and loop 7 (MS11loop1–6F62loop7MS11loop8) resulted in complete abrogation of C4bp binding, whereas mutating loop 6 (MS11loop1–5F62loop6MS11loop7–8) did not influence C4bp binding. Collectively, these data suggest that MS11 loops 5 and 7 together participate in forming a C4bp-binding domain.
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Figure 4: MS11 Por1B loops 5 and 7 together participate in forming a C4bp-binding region. Transformation of F62 with pUNCH61 (reference 40) resulted in strains with hybrid Por molecules F62loop1MS11loop2–8 and F62loop1–4MS11loop5–8. F62 sequence is indicated by black bars; MS11 sequence is represented by hatched bars. Both hybrids bound C4bp, suggesting that the C4bp binding region in MS11 lay in a region encompassed by loops 5–8. The BbsI-BsgI fragment of F62 was cloned into pUNCH61 to obtain pBUMC1 and this plasmid was used to transform F62. This yielded a gonococcal strain with hybrid Por molecule MS11loop1 F62loop2–7MS11loop1–8, which did not bind C4bp, thus eliminating MS11 loop 8 as the C4bp binding loop. Using overlap extension PCR or site-directed mutagenesis on pBUMC1, we mutated loops 5, 6, or 7 either individually or in combination and then transformed F62. We noted that only hybrid Por molecules that contained both MS11 loop 5 and loop 7 bound C4bp. To further demonstrate the absolute requirement of MS11 loops 5 and 7, we mutated MS11 loops 5, 6, and 7 individually in pUNCH61 to resemble the corresponding F62 loop, and then transformed F62. Mutations of loop 5 (MS11loop1–4F62loop5MS11loop6–8) and loop 7 (MS11loop1–6F62loop7MS11loop8) resulted in complete abrogation of C4bp binding, whereas mutating loop 6 (MS11loop1–5F62loop6MS11loop7–8) did not influence C4bp binding. Collectively, these data suggest that MS11 loops 5 and 7 together participate in forming a C4bp-binding domain.
Mentions: 29 strains of N. gonorrhoeae were screened for binding to C4bp; these are listed in Table . Strain FA6616, containing the MS11 Por molecule reintroduced into an MS11 background using plasmid pUNCH61 40, was used in this study, and for convenience will be referred to as MS11 hereafter. Bacteria grown on chocolate agar supplemented with Isovitalex equivalent in 5% CO2 for 10 to 11 h 41 were suspended in HBSS containing 0.15 mM CaCl2 and 1 mM MgCl2 (HBSS2+) in C4bp binding assays. Alternatively, bacteria were harvested from chocolate agar plates after overnight growth and grown in gonococcal liquid media 41. Results of C4bp binding were equivalent when strains were grown in either media. Plasmids pUNCH61 and pUNCH62 contained the por1B and por1A genes of strains MS11 and FA19, respectively, plus ∼1 kB of gonococcal DNA 3′ to the por gene, respectively, containing a chloramphenicol-resistance marker (CmR [40, 42]). Strains MS11 and FA19 are resistant to killing (SR) by 33% nonimmune NHS. pUNCH61 and pUNCH62 were each used to transform the SS strain F62 to replace F62 Por with either MS11 Por (F62PorMS11) or FA19 Por (F62PorFA19), respectively. In addition to replacing Por completely, transformation with pUNCH61 resulted in hybrids F62loop1MS11loop2–8, and F62loop1–4MS11loop5–8 (see Fig. 4), and transformation of F62 with pUNCH62 yielded hybrids F62loop1FA19loop2–8 and F62loop1–4FA19loop5–8 (see Fig. 3). Because C4bp binding studies with these MS11/F62 hybrids suggested that the C4bp binding region in MS11 Por was contained in the COOH-terminal half of the Por molecule (see Results), we then constructed hybrid MS11loop1F62loop2–7MS11loop8 by replacing the BbsI-BsgI fragment of MS11 Por encompassing loops 2 through 7 in pUNCH61 with the corresponding BbsI-BsgI fragment of F62 Por. Using this plasmid, designated pBUMC1, we constructed hybrids that were mutated at loops 5, 6, or 7 individually or in combination, using either overlap extension PCR for loops 5 and 6, or site-directed mutagenesis for loop 7 (see below: Por mutagenesis). The amino acid sequences of the putative exposed regions of Por loops 5, 6, and 7 of F62 and MS11 are indicated in Table ; variations in sequence are indicated in bold type. The net charge of the surface exposed loop regions was calculated by assigning +1 for arginine (r) and lysine (K), and +0.5 for histidine (H), and –1 for aspartic acid (D) and glutamic acid (E), as described previously 43.

Bottom Line: Only recombinant C4bp mutant molecules containing the NH2-terminal alpha-chain short consensus repeat (SCR1) bound to both Por1A and Por1B gonococci, suggesting that SCR1 contained Por binding sites.C4bp alpha-chain monomers did not bind gonococci, indicating that the polymeric form of C4bp was required for binding.Using fAb fragments against C4bp SCR1, C4bp binding to Por1A and Por1B strains was inhibited in a complement-dependent serum bactericidal assay.

View Article: PubMed Central - PubMed

Affiliation: Evans Biomedical Research Center, Boston Medical Center, Boston, Massachusetts 02118, USA. sram@bu.edu

ABSTRACT
We screened 29 strains of Neisseria gonorrhoeae and found 16/21 strains that resisted killing by normal human serum and 0/8 serum sensitive strains that bound the complement regulator, C4b-binding protein (C4bp). Microbial surface-bound C4bp demonstrated cofactor activity. We constructed gonococcal strains with hybrid porin (Por) molecules derived from each of the major serogroups (Por1A and Por1B) of N. gonorrhoeae, and showed that the loop 1 of Por1A is required for C4bp binding. Por1B loops 5 and 7 of serum-resistant gonococci together formed a negatively charged C4bp-binding domain. C4bp-Por1B interactions were ionic in nature (inhibited by high salt or by heparin), whereas the C4bp-Por1A bond was hydrophobic. Only recombinant C4bp mutant molecules containing the NH2-terminal alpha-chain short consensus repeat (SCR1) bound to both Por1A and Por1B gonococci, suggesting that SCR1 contained Por binding sites. C4bp alpha-chain monomers did not bind gonococci, indicating that the polymeric form of C4bp was required for binding. Using fAb fragments against C4bp SCR1, C4bp binding to Por1A and Por1B strains was inhibited in a complement-dependent serum bactericidal assay. This resulted in complete killing of these otherwise fully serum resistant strains in only 10% normal serum, underscoring the importance of C4bp in mediating gonococcal serum resistance.

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Related in: MedlinePlus