Modification by Sialic Acid of Neisseria gonorrhoeae Lipooligosaccharide Epitope Expression in Human Urethral Exudates: An Immunoelectron Microscopic Analysis

Abstract

Expression of lipooligosaccharide (LOS) antigenic determinants during human gonococcal infection was studied in secretions from seven men with gonococcal urethritis. Five monoclonal antibodies with distinct gonococcal LOS specificities and an H.8 lipoprotein monoclonal antibody were used in combination with immunogold electron microscopic analysis. The LOS epitope defined by antibody 6B7 was present on all seven strains in secretions and after in vitro growth. Gonococci from six of seven patients, when grown in vitro, expressed the 6B4 LOS epitope. The 6B4 epitope is a Gal beta 1-4-GlcNAc residue, which is immunochemically similar to the precursor of the human erythrocyte i antigen. This epitope was found unmodified on gonococcal LOS in urethral secretions from two patients. The unmodified epitope could not be demonstrated on organisms in five secretions. Neuraminidase digestion exposed the 6B4 epitope on organisms in these secretions and increased the 6B4 epitope density in the two secretions, which contained the unmodified epitope. These studies indicate that in vivo modification by sialylation of gonococcal LOS Gal beta 1-4-GlcNAc residue occurs during human infection.

A novel factor H-FC chimeric immunotherapeutic molecule against Neisseria gonorrhoeae

“…Newer modalities of treatment whose mechanism(s) of action differ from those of conventional agents provide hope that drug-resistance may be deterred when traditional mechanisms of selection are circumvented (3). Sialylation of gonococcal LOS is an important component of gonococcal pathogenesis, which occurs in humans (20,21) and also during experimental infection of mice (54). Gonococcal mutants that are incapable of LOS sialylation following deletion of the LOS sialyltransferase (lst) gene are less virulent in the mouse model of vaginal colonization (54).…”

“…We next tested binding of FHD1119G/human IgG1 Fc to 15 clinical isolates of N. gonorrhoeae (listed in Supplemental Table S1), including three contemporary ceftriaxone-resistant isolates: CTXr(Sp) (4), H041 (7), and NJ-60 and two isolates with elevated MICs to ceftriaxone (NJ-44 and NJ-48). All strains tested for binding of D1119G/Fc were grown in media supplemented with 2 μg/ml CMP-Neu5Ac to sialylate LOS, as occurs in vivo (20,21). Although binding of FHD1119G/HuFc varied across strains, it was seen to all sialylated strains that were tested in a flow cytometry assay (≈ 6 to 346-fold fluorescence increase over control values; Fig.…”

“…FH18-20/murine IgG2a Fc proteins that contained these individual mutations were expressed in CHO cells and purified from tissue culture supernatants; the molecular masses and relative purity of the proteins were determined by SDS-PAGE stained with Coomassie Blue (data not shown). Bacteria were grown in media containing CMP-Neu5Ac, which results in sialylation of LNT LOS, similar to sialylation that occurs in vivo (20). We compared binding of FH18-20/Fc mutant proteins to sialylated gonococci with binding of the wild-type (WT) FH18-20/Fc (Fig.…”

“…In vivo, gonococci scavenge 5′-cytidinemonophospho-N-acetylneuraminic acid (CMPNeu5Ac) from the host to sialylate their lipooligosaccharide (LOS) (20,21). The two LOS structures that can be sialylated are the nearly ubiquitously expressed lacto-N-neotetraose (LNT, Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-4HepI) structure and the less frequently encountered P K -like structure (Neu5Acα2-6Galα1-4Galβ1-4Glcβ1-4HepI) (22).…”

“…Many pathogens, including Ng, bind the complement inhibitor factor H (FH) to evade complement-dependent killing. Sialylation of gonococcal lipooligosaccharide, as occurs in vivo, augments binding of human FH through its domains [18][19][20]. We explored the utility of fusing FH18-20 with IgG Fc (FH18-20/Fc) to create a novel anti-infective immunotherapeutic.…”