C4bp binding to porin mediates stable serum resistance of Neisseria gonorrhoeae.

Screening of 29 strains of Neisseria gonorrhoeae revealed that 16/21 serum resistant strains and 0/8 serum sensitive strains bound C4bp, suggesting that C4bp binding to gonococci could contribute to serum resistance. C4bp bound to gonococci retained cofactor (C4b-degrading) function. Using allelic exchange to construct strains with hybrid Por1A/B molecules, we demonstrate that the N-terminal loop (loop 1) of Por1A is required for C4bp binding. Serum resistant Por1B gonococcal strains also bind C4bp via their Por molecule. Using allelic exchange and site-directed mutagenesis, we have shown that loops 5 and 7 together form a negatively charged C4bp binding domain. C4bp-Por1B interactions are ionic in nature (inhibited by high salt as well as by heparin), while the C4bp-Por1A bond is hydrophobic. mAbs directed against SCR1 of the alpha-chain of C4bp inhibit C4bp binding to both Por1A and Por1B. Furthermore, only recombinant C4bp mutant molecules that contain alpha-chain SCR1 bind both Por1A and Por1B gonococci, confirming that SCR1 contains Por binding sites. C4bp alpha-chain monomers do not bind strains with either Por molecule, suggesting that the polymeric form of C4bp is required for binding to gonococci. Inhibition of C4bp binding to serum resistant Por1A and Por1B strains in a serum bactericidal assay using fAb fragments against C4bp SCR1 results in complete killing at 30 min of otherwise fully serum resistant strains in only 10% normal serum, underscoring the role of C4bp in mediating gonococcal serum resistance.

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

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.

Strategies for mimicking Neisserial saccharide epitopes as vaccines.

Monoclonal antibody (mAb) 2C7 recognizes a conserved and widely expressed oligosaccharide (OS) epitope on Neisseria gonorrhoeae. This OS epitope evokes a significant bactericidal and opsonic immune response after natural infection and vaccination. The OS epitope structure represents an excellent target for a potential protective gonococcal vaccine. Because carbohydrate antigens are T-cell independent, inducing weak antibody responses, OS molecules are not useful immunogens. We developed and examined two different strategies to mimic the 2C7 OS epitope: (i) an anti-idiotope (mAb CA1); and (ii) a peptide (PEP-1). These surrogate immunogens elicited antibody responses in mice (CA1 and PEP-1) and rabbits (CA1) that were bactericidal in vitro against gonococci. Both CA1 and PEP-1 are true immunologic mimics of OS and may form a basis for the development of vaccine candidates for human immunization against N. gonorrhoeae.

Structural and immunochemical characterization of a Neisseria gonorrhoeae epitope defined by a monoclonal antibody 2C7; the antibody recognizes a conserved epitope on specific lipo-oligosaccharides in spite of the presence of human carbohydrate epitopes.

Lipo-oligosaccharides (LOS) produced by Neisseria gonorrhoeae are important antigenic and immunogenic components of the outer membrane complex. Previously, we showed that murine monoclonal antibody (mAb) 2C7 did not cross-react with human glycosphingolipids but identified the LOS epitope that is widely expressed in vivo and in vitro (Gulati, S., McQuillen, D. P., Mandrell, R. E., Jani, D. B., and Rice, P. A. (1996) J. Infect. Dis. 174, 1223-1237). In the present study, we analyzed the structure of gonococcal strain WG LOS containing the 2C7 epitope and investigated the structural requirements for expression of the epitope. We determined that the WG LOS components are Hep[1]-elongated forms of 15253 LOS that have a lactose on both Hep[1] and Hep[2] (Yamasaki, R., Kerwood, D. E., Schneider, H., Quinn, K. P., Griffiss, J. M., and Mandrell, R. E. (1994) J. Biol. Chem. 269, 30345-30351). In addition, we found that expression of the 2C7 epitope within the LOS is blocked when the Hep[2]-lactose is elongated. Based on the structural data of these LOS and the results obtained from immunochemical analyses, we conclude the following: 1) mAb 2C7 requires both the 15253 OS minimum structure and the N-linked fatty acids in the lipoidal moiety for expression of the epitope; 2) mAb 2C7 binds to the LOS that elongates the lactose on Hep[1] of the 15253 OS, but not the one on Hep[2]; and 3) the 2C7 epitope is expressed on gonococcal LOS despite the presence of human carbohydrate epitopes such as a lactosamine or its N-acetylgalactosaminylated (globo) form. Our study shows that the conserved epitope defined by mAb 2C7 could potentially be used as a safe site for the development of a vaccine candidate.

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn.

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.

Complement processing and immunoglobulin binding to Neisseria gonorrhoeae determined in vitro simulates in vivo effects.

Local inflammation elicited by Neisseria gonorrhoeae correlates closely with sensitivity to killing by normal human serum. Serum-sensitive (SS) isolates are rendered resistant in vitro by lipooligosaccharide sialylation. Differences in C3b processing on N. gonorrhoeae in vitro were found to match findings at the cervical level in vivo. Nonsialylated SS gonococci bound 5-fold more C3b than did stably serum-resistant (SR) gonococci; most was processed to iC3b, yet significant C3b persisted. Sialylated SS gonococci bound 4-fold less total C3 antigen than did SR gonococci, which was promptly converted to iC3b. C3b bound later on stably SR gonococci but again was processed swiftly to iC3b. In vivo, the iC3b/C3 ratio of SS isolates more closely resembled nonsialylated SS isolates in vitro, implying heterogeneous sialylation or desialylation in vivo. In vitro, total IgM bound was unchanged by sialylation of SS isolates, but total C4 bound decreased by 75%, suggesting that sialylation may indirectly regulate the classical complement pathway.

The contrasting mechanisms of serum resistance of Neisseria gonorrhoeae and group B Neisseria meningitidis.

Neisseria gonorrhoeae and Neisseria meningitidis have evolved intricate mechanisms to evade complement-mediated killing. Sialylation of gonococcal lipooligosaccharide (LOS) results in conversion of previously serum sensitive strains to unstable serum resistance, which is mediated by factor H binding. Porin (Por) is also instrumental in mediating stable serum resistance in gonococci. The 5th loop of certain gonococcal PorlAs binds factor H, which efficiently inactivates C3b to iC3b. Factor H glycan residues may be essential for factor H binding to certain Por1A strains. Por1A strains can also regulate the classical pathway by binding to C4b-binding protein (C4bp) probably via the 1st loop of the Por molecule. Certain serum resistant Por1 B strains can also regulate complement by binding C4bp through a loop other than loop 1. Purified C4b can inhibit binding of C4bp to Por 1B, but not Por1A, suggesting different binding sites on C4bp for the two Por types. Unlike serum resistant gonococci, resistant meningococci have abundant C3b on their surface, which is only partially processed to iC3b. The main mechanism of complement evasion by group B meningococci is inhibition of membrane attack complex (MAC) insertion by their polysaccharide capsule. LOS structure may act in concert with capsule to prevent MAC insertion. Meningococcal strains with Class 3 Por preferentially bind factor H, suggesting Class 3 Por acts as a receptor for factor H.

Binding of complement factor H to loop 5 of porin protein 1A: a molecular mechanism of serum resistance of nonsialylated Neisseria gonorrhoeae.

Neisseria gonorrhoeae isolated from patients with disseminated infection are often of the porin (Por1A) serotype and resist killing by nonimmune normal human serum. The molecular basis of this resistance (termed stable serum resistance) in these strains has not been fully defined but is not related to sialylation of lipooligosaccharide. Here we demonstrate that Por1A bearing gonococcal strains bind more factor H, a critical downregulator of the alternative complement pathway, than their Por1B counterparts. This results in a sevenfold reduction in C3b, which is >75% converted to iC3b. Factor H binding to isogenic gonococcal strains that differed only in their porin serotype, confirmed that Por1A was the acceptor molecule for factor H. We identified a surface exposed region on the Por1A molecule that served as the binding site for factor H. We used gonococcal strains with hybrid Por1A/B molecules that differed in their surface exposed domains to localize the factor H binding site to loop 5 of Por1A. This was confirmed by inhibition of factor H binding using synthetic peptides corresponding to the putative exposed regions of the porin loops. The addition of Por1A loop 5 peptide in a serum bactericidal assay, which inhibited binding of factor H to the bacterial surface, permitted 50% killing of an otherwise completely serum resistant gonococcal strain. Collectively, these data provide a molecular basis to explain serum resistance of Por1A strains of N. gonorrhoeae.

A novel sialic acid binding site on factor H mediates serum resistance of sialylated Neisseria gonorrhoeae.

Factor H (fH), a key alternative complement pathway regulator, is a cofactor for factor I-mediated cleavage of C3b. fH consists of 20 short consensus repeat (SCR) domains. Sialic acid binding domains have previously been localized to fH SCRs 6-10 and 13. To examine fH binding on a sialylated microbial surface, we grew Neisseria gonorrhoeae in the presence of 5'-cytidinemonophospho-N-acetylneuraminic acid, which sialylates lipooligosaccharide and converts to serum resistance gonococci previously sensitive to nonimmune serum killing. fH domains necessary for binding sialylated gonococci were determined by incubating organisms with recombinant human fH (rH) and nine mutant rH molecules (deletions spanning the entire fH molecule). rH and all mutant rH molecules that contained SCRs 16-20 bound to the sialylated strain; no mutant molecule bound to serum-sensitive nonsialylated organisms. Sialic acid was demonstrated to be the fH target by flow cytometry that showed a fourfold increase in fH binding that was reversed by neuraminidase-mediated cleavage of sialic acid off gonococci. Functional specificity of fH was confirmed by decreased total C3 binding and almost complete conversion to iC3b on sialylated gonococci. Sialic acid can therefore bind fH uniquely through SCRs 16-20. This blocks complement pathway activation for N. gonorrhoeae at the level of C3.

Immunogenicity of Neisseria gonorrhoeae lipooligosaccharide epitope 2C7, widely expressed in vivo with no immunochemical similarity to human glycosphingolipids.

Natural infection with Neisseria gonorrhoeae may elicit a substantial antibody response directed against gonococcal lipooligosaccharide. Monoclonal antibody (MAb) 2C7 recognized a gonococcal lipooligosaccharide epitope, identified the epitope directly in 94% of 68 consecutive culture-positive genital secretions, and recognized 95% of 101 randomly chosen fresh (second-passage) gonococcal isolates. The epitope was stably maintained after multiple in vitro passages and did not compete with any of the known cross-reactive human glycosphingolipid structures. MAb 2C7 mediated in vitro killing and phagocytosis by human polymorphonuclear leukocytes of 1 serum-sensitive (sialylated or not) and 1 stably serum-resistant gonococcal isolate that expressed the epitope. Gonococcal endometritis and disseminated infection elicited increases (6.5-fold IgM, 4.4-fold IgG; 18-fold IgM, 17-fold IgG, respectively) in anti-2C7 epitope antibody. Immunization with a gonococcal outer membrane vaccine elicited a mean 44.5-fold increase in IgG anti-2C7 epitope antibody in 20 of 28 subjects. The epitope identified by MAb 2C7 may represent an excellent target for a potentially protective gonococcal vaccine candidate.

Experimental immunization with a monoclonal anti-idiotope antibody that mimics the Neisseria gonorrhoeae lipooligosaccharide epitope 2C7.

An anti-idiotope monoclonal antibody (MAb), called CA1 (Ab2), was produced in mice against MAb 2C7, which recognizes a widely in vivo-expressed gonococcal lipooligosaccharide (LOS) epitope. Mice immunized with MAb CA1 initially had a 2.5-fold increase in IgG (12-fold after a booster) but no increase in IgM anti-LOS (Ab1') antibody. Control mice immunized with LOS had a 4.5-fold rise in IgG and 4-fold rise in IgM anti-LOS antibody. In rabbits, MAb CA1 elicited a 9-fold rise in IgG and a 3.3-fold rise in IgM anti-LOS (Ab1') antibody. Ab1' antibody bactericidal activity was 1-2 logs greater than that produced by immunization with LOS. Ab1' mediated complete human polymorphonuclear leukocyte phagocytosis of 2C7 epitope-positive (but not 2C7 epitope-negative) gonococci. MAb CA1 acts as a molecular surrogate (Ab2beta) for the nominal LOS antigen and may form the basis for vaccine candidates for human immunization against Neisseria gonorrhoeae.

Functional characterization of a sialyltransferase-deficient mutant of Neisseria gonorrhoeae.

Previous studies indicate that sialylation of lipopolysaccharide (LPS) by host CMP-N-acetylneuraminic acid (CMP-NANA) catalyzed by bacterial sialyltransferase rendered gonococci resistant to killing by phagocytes, to entry into epithelial cell lines, to killing by immune serum and complement, and to absorption of complement component C3. These results have been confirmed by comparing a sialyltransferase-deficient mutant (strain JB1) with its parent (strain F62) in appropriate tests. In contrast to F62, JB1 was very susceptible to killing by human polymorphonuclear phagocytes in opsonophagocytosis tests and incubation with CMP-NANA did not decrease the level of killing. The inherent resistance of F62 in these tests was probably due to LPS sialylation by CMP-NANA and lactate present in the phagocytes. A JB1 variant expressing the invasion-associated Opa protein was as able to enter Chang human conjunctiva epithelial cells as an Opa-positive variant of F62, suggesting that the sialyltransferase is not required for Opa-mediated entry. After incubation with CMP-NANA, the number of F62 variant gonococci entering cells but not that of JB1 variant gonococci was drastically reduced. Both JB1 and F62 were killed by incubation with rabbit antibody to gonococcal major outer membrane protein, protein I, and human complement, but only F62 was rendered resistant to the killing by incubation with CMP-NANA. Finally, both JB1 and F62 absorbed similar amounts of complement component C3 and the binding was decreased by incubation with CMP-NANA only for the wild type, F62.

Serum resistance of Neisseria gonorrhoeae. Does it thwart the inflammatory response and facilitate the transmission of infection?

N. gonorrhoeae differentially subvert the effectiveness of complement (C) and alter the inflammatory responses elicited in human infection. Disseminated (DGI) isolates typically resist killing by normal serum (are serum-resistant), inactivate more C3b (to iC3b preferentially bound via amide linkages), generate less C5a, and result in less inflammation at local sites. Pelvic inflammatory disease isolates are serum-sensitive, inactivate less C3b (while maintaining active C3b via stable amide linkages), generate more C5a, and result in more inflammation at local sites. Sialylation of SS gonococci, presumed to occur in vivo, converts them to serum-resistant, but it does not change the patterns of C3b inactivation and therefore may not affect local inflammation. IgG antibody directed against gonococcal reduction modifiable protein (Rmp) blocks C-mediated killing of N. gonorrhoeae. Anti-Rmp blocking antibodies may harbor specificity for OmpA sequences shared with other neisserial species or Enterobacteriaceae or may be directed against unique Rmp upstream cysteine loop specific sequences, or both. Preexisting antibodies directed against Rmp facilitate transmission of gonococcal infection to exposed women; exclusion of highly immunogenic Rmp antigens from vaccine candidates may be important.

Human vaccination with Escherichia coli J5 mutant induces cross-reactive bactericidal antibody against Neisseria gonorrhoeae lipooligosaccharide.

The lipopolysaccharides of enteric gram-negative bacteria and the lipooligosaccharides (LOS) of Neisseria gonorrhoeae may share antigenic determinants that are targets of bactericidal antibody. Natural (disseminated) infection with a serum-resistant gonococcal strain and immunization with Escherichia coli J5 stimulated bactericidal IgG anti-LOS antibodies that recognize different serum-resistant gonococcal LOS epitopes. In bactericidal assays, convalescent serum from disseminated infection killed only the homologous strain while post-J5 vaccination serum killed 6 of 9 additional strains. Both convalescent and post-J5 vaccination sera mediated marker (51Cr) release from liposomes sensitized with serum-resistant gonococcal LOS (homologous strain), linking acquired killing activity to cross-reacting anti-LOS antibody. Post-J5 IgG mediated 51Cr release more effectively than did convalescent IgG. Thus, bactericidal antibody elicited by J5 vaccination is more efficacious and broadly cross-reactive against serum-resistant gonococci than is antibody elicited by natural infection. Moreover, multiple LOS epitopes may serve as bactericidal targets on serum-resistant gonococci.

Invasive infections due to Candida krusei: report of ten cases of fungemia that include three cases of endophthalmitis.

Candida krusei has become an increasingly important invasive pathogen, particularly in immunocompromised patients. Previous experimental and clinical experience suggest that C. krusei has a low propensity for hematogenously infecting the eye. We report 10 cases of fungemia due to C. krusei at our institutions, including three cases of endophthalmitis due to C. krusei. Fungemia was associated with nodular skin lesions in all seven patients with neutropenia and occurred despite administration of antifungal prophylaxis or empirical therapy. None of the patients apparently died as a direct result of C. krusei fungemia. Treatment with amphotericin B resulted in resolution of endophthalmitis, although one patient required vitrectomy. Early institution of aggressive therapy with amphotericin B may alter the course and improve the prognosis of C. krusei infection, particularly in immunocompromised patients.

Pneumocystis carinii pneumonia complicating somatostatin therapy of Cushing's syndrome in a patient with metastatic pancreatic islet cell carcinoma and Zollinger-Ellison syndrome.

Described is the case of a 73-yr-old woman with metastatic pancreatic islet carcinoma that manifested initially as Zollinger-Ellison syndrome followed by onset of endogenous Cushing's syndrome, who developed Pneumocystis carinii pneumonia while on therapy with a long-acting somatostatin analog. Although P. carinii pneumonia has been observed in patients with Cushing's syndrome associated with other conditions, this is the first reported case in a patient with Zollinger-Ellison syndrome. Heightened awareness of the possibility of opportunistic infections in patients receiving somatostatin therapy for Cushing's syndrome of any cause, particularly Zollinger-Ellison syndrome, may be warranted.

Cellular response to human acetylcholine receptor in patients with myasthenia gravis.

A preparation of human skeletal muscle acetylcholine receptor (AchR) was used in vitro as an antigen to stimulate lymphocytes from patients with myasthenia gravis (MG). Clinical data obtained from the patients included duration and severity of disease; history of steroid treatment or prior thymectomy; and the presence of thymoma. Lymphocytes from patients with MG showed a significantly higher response to human AchR antigen than did lymphocytes from control subjects. Previous studies of cellular response to AchR have used receptor prepared from eel or ray electric organs. By stimulating lymphocytes from MG patients with a preparation of human AchR, we have come one step closer to documenting a possible contribution of a cellular immune response to the pathogenesis of MG.