Enhanced factor H binding to sialylated Gonococci is restricted to the sialylated lacto-N-neotetraose lipooligosaccharide species: implications for serum resistance and evidence for a bifunctional lipooligosaccharide sialyltransferase in Gonococci

Abstract

We isolated serologically identical (by serovar determination and porin variable region [VR] typing) strains of Neisseria gonorrhoeae from an infected male and two of his monogamous female sex partners. One strain (termed 398078) expressed the L1 (Galalpha1 --> 4 [corrected] Galbeta1 --> 4Glcbeta1 --> 4HepI) lipooligosaccharide (LOS) structure exclusively; the other (termed 398079) expressed the lacto-N-neotetraose (LNT; Galbeta1 --> 4GlcNAcbeta1 --> 3Galbeta1 --> 4Glcbeta1 --> 4HepI) LOS structure. The strain from the male index case expressed both glycoforms and exhibited both immunotypes. Nuclear magnetic resonance analysis revealed that sialic acid linked to the terminal Gal of L1 LOS via an alpha2 --> 6 linkage and, as expected, to the terminal Gal of LNT LOS via an alpha2--> 3 linkage. Insertional inactivation of the sialyltransferase gene (known to sialylate LNT LOS) abrogated both L1 LOS sialylation and LNT LOS sialylation, suggesting a bifunctional nature of this enzyme in gonococci. Akin to our previous observations, sialylation of the LNT LOS of strain 398079 enhanced the binding of the complement regulatory molecule, factor H. Rather surprisingly, factor H did not bind to sialylated strain 398078. LOS sialylation conferred the LNT LOS-bearing strain complete (100%) resistance to killing by even 50% nonimmune normal human serum (NHS), whereas sialylation of L1 LOS conferred resistance only to 10% NHS. The ability of gonococcal sialylated LNT to bind factor H confers high-level serum resistance, which is not seen with sialylated L1 LOS. Thus, serum resistance mediated by sialylation of gonococcal L1 and LNT LOS occurs by different mechanisms, and specificity of factor H binding to sialylated gonococci is restricted to the LNT LOS species.

FIG. 1.

Effects of sialylation on LOS migration of wild-type strains and sialyltransferase (lst) mutants. Proteinase K-digested bacterial lysates were resolved on a 12% Bis-Tris gel using MES running buffer, and LOS was visualized by silver staining (A) and Western blotting (B). Strain 398 expressed both the 3F11 (4.5 kDa) and L1 (a lower-molecular-mass LOS species) epitopes. Strain 398079 expressed predominantly the 3F11 LOS species, while 398078 expressed only the L1 LOS. In all cases, migration of the 3F11 and L1 LOS species was retarded upon growth in CMP-NANA-containing media. Inactivation of lst resulted in loss of the ability of all strains to sialylate either LOS species.

FIG. 2.

Lst sequences from 6 meningococcal and 14 gonococcal strains were aligned using ClustalW. A representative alignment of the bifunctional Lsts from meningococcal strain 126E and gonococcal strain 398078 is shown. Species-specific variation occurred at 21 separate amino acid positions (boxed) distributed across the protein. Strain variation within a species (shaded) was noted at 11 meningococcal (above) and 7 gonococcal (below) amino acid positions. The isoleucine (Ile) residue at position 168, shown to be critical for bifunctionality of 126E Lst, is marked with an asterisk.

FIG. 3.

Factor H binds only to gonococcal strains (398 and 398079) bearing sialylated lacto-N-neotetraose (3F11) LOS, but not to strain 398078 bearing sialylated L1 LOS. (Upper panel) Factor H binding to strains 398 (LNT and L1 LOS), 398078 (L1 LOS), and 398079 (LNT LOS) grown either without CMP-NANA (histogram represented by thin solid line) or in media containing 1 μg/ml (bold line) or 100 μg/ml (gray shaded histogram) CMP-NANA was quantified by flow cytometry. (Lower panel) Factor H binding to lst mutants of 398, 398078, and 398079 grown with (100 μg/ml; gray shaded histogram) or without CMP-NANA. The x axis represents fluorescence, and the y axis denotes the number of events. Isotype controls (antibodies alone, no factor H) are shown in each plot by thin broken lines. One representative experiment of duplicate experiments is shown.

FIG. 4.

(A) Serum resistance conferred by sialylation of LNT LOS occurs at lower CMP-NANA concentrations compared to L1 LOS sialylation. Bacteria were grown in the presence of increasing concentrations of CMP-NANA, and serum bactericidal assays were performed in the presence of 10% NHS. Strain 398079 showed 88% survival when grown in the presence of 1 μg/ml CMP-NANA, while 398078 showed only 17% survival under similar growth conditions. A gradual increase in serum resistance was seen with 398078 as CMP-NANA concentrations were increased. The mean (±standard deviation) of one representative experiment performed in duplicate is shown. (B) Sialylation of lacto-N-neotetraose LOS confers a higher level of serum resistance than sialylation of L1 LOS. Strains 398, 398078, and 398079 were grown in media containing 100 μg/ml CMP-NANA in an attempt to maximize LOS sialylation. Bacteria were then subjected to serum bactericidal testing in the presence of increasing serum concentrations. The mean (±standard deviation) of one representative experiment performed in duplicate is shown.