Lacto- and ganglio-series glycolipids are adhesion receptors for Neisseria gonorrhoeae.

The role of glycolipids as adhesion receptors for Neisseria gonorrhoeae is examined. Serum-resistant isolates, piliated and nonpiliated isogenic variants, as well as gonococci deficient in lipooligosaccharide and protein II, bind specifically to terminal and internal GlcNAc beta 1-3Gal beta 1-4Glc and GalNAc beta 1-4Gal beta 1-4Gcl sequences in lacto- and ganglio-series glycolipids, respectively, as measured by overlaying glycolipid chromatograms with 125I-labeled organisms. The binding activity was not affected by changing the growth conditions of the organism, as the gonococci bound to both classes of glycolipids when grown anaerobically, microaerophilically on agar or in broth, or under iron-limited conditions. The gonococci do not bind to lacto-sylceramide (Gal beta 1-4Glc beta 1-1Cer) derived from lacto-N-triaosylceramide or from asialo-GM2 by treatment with N-acetyl-beta-hexosaminidase, or to other neutral glycolipids tested. Although N. gonorrhoeae bound weakly to some gangliosides on thin-layer chromatograms, including sialylparagloboside and GM1, in solid phase assays the gonococci bound with high avidity to the sequence GalNAc beta 1-4Gal beta 1-4Glc, with moderate avidity to the sequence GlcNAc beta 1-3Gal beta 1-4Glc, and not at all to gangliosides. Interestingly, the 4.8-kDa component of gonococcal lipooligosaccharide, which contains lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc), strongly inhibits gonococcal-specific agglutination of human erythrocytes and inhibits the binding of labeled organisms to human paragloboside and lacto-N-triaosylceramide on thin-layer chromatograms. Possibly, this binding specificity explains why gonococci autoagglutinate in vitro.

Gonorrhea is the most commonly reported infectious disease in the United States with as many as 2 million reported cases occurring each year (1). The organism responsible for this disease is Neisseria gonorrhoeae, an obligate human pathogen that infects diverse sites within its host including the mucous membranes of the genitourinary tract, rectum, oropharynx, and conjunctiva, although under certain conditions bacteremia and disseminated gonococcal infection also occur (2). Gonococcal infection begins with bacterial adhesion * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. which is host-specific (3-5), suggesting that specific eucaryotic cell receptors are involved. Many studies indicate the importance of pili as adhesins mediating this process (6-13), although several different gonococcal outer membrane proteins (proteins I and II) have also been proposed to be important in adhesion (14)(15)(16)(17)(18)(19). Recently, Shaw and Falkow (20) demonstrated that piliated (P') gonococci adhered in greater numbers to human endometrial cultured cells than nonpiliated (P-) gonococci; however, once bound, both were internalized with equal efficiency.
There is substantial evidence that the eucaryotic cell receptors for N. gonorrhoeae contain carbohydrate (8,12,21,22), and recently it has been shown that both piliated (P') and nonpiliated (P-) gonococci bind to glycolipids (22). We report here that both lacto-and ganglio-series glycolipids containing the minimum carbohydrate sequences GlcNAc/?l-3Gal/31-4Glc and GalNAcpl-4Gal/31-4Glc, respectively, can support adhesion of N. gonorrhoeae and that binding to both structures is not dependent on pili, protein II (PII),' or the presence of lipooligosaccharide (LOS). Interestingly, lacto-series structures are also found in some LOS derived from the outer membrane of N. gonorrhoeae (13), which may explain the well known phenomenon of autoagglutination seen with this organism. (asialo-GM*) were prepared as described (27). a-2, 3-Sialyllactoneotetraosylceramide (sialylparagloboside) was isolated from type 0 human erythrocytes (28). Lacto-N-neotetraosylceramide (paragloboside) was prepared by desialylation of erythrocyte sialylparagloboside with 1 M formic acid for 60 min at 100 'C. Lacto-N-triaosylceramide was prepared by digestion of paragloboside with ,+galactosidase as described for the preparation of asialo-GMs from asialo-GM, (27). The concentrations of glycolipids listed in Table I     ' Abbreviations used: P', piliated; P-, nonpiliated; Pll-, protein II deficient; LOS-, lipooligosaccharide deficient; MAb, monoclonal antibody. sodium phosphate, pH 7.4, containing 0.15 M sodium phosphate (PBS). Bacteria were centrifuged at 4 "C and 10,000 X g, and pellets were washed twice in PBS. The cells were radioiodinated as described (31) using 0.4 mCi of Na"'l (ICN Biomedicals). The iodination was terminated after 3 min by removing the cells from the reaction tube, followed by centrifugation and two washes in PBS. The labeled gonococci were resuspended to 2.5 x lo6 or 4 X lo6 cpm/ml in Hanks' balanced salt solution, pH 7.4, containing 1% bovine serum albumin (HBSS-BSA).

Assay of N. gonorrhoeae
Binding to Glycolipids-Gonococci bound to glycolipids separated by high performance thin-layer chromatography were detected as described (31). The binding of gonococci to purified glycolipids immobilized in microtiter plates (Falcon 3912-111, Bectin-Dickinson) was measured as previously described (31).

Immunostaining
Glycolipids with Anti-LOS Monoclonal Antibody-Authentic paragloboside was detected on thin-layer chromatograms by immunostaining followed by autoradiography for 24 h (32).
In control experiments gonococci \vere incubated kvith auxiliary lipids only to crlrrect for nonspecific binding ttvpically <l "P of the total radioactivity added).

N-neotetraosylceramide (paragloboside).
Possibly, this discrepency is related to the source and/or fatty acid contents (22) of the glycolipids used by these investigators. In some experiments, N. gonorrhoeae bound weakly to some of the gangliosides (Table I) including sialylparagloboside (Fig. lB,  lane %) and G\i, (Fig. lB, lane I); however, this observation was not always reproducible (Fig. %R, lane 1), and none of the gangliosides demonstrated concentration-dependent binding in microtiter plates (Fig. 3). Binding of N. gonorrhoeae to Purified Ganglio-series Glycolipids on Chromatograms-In agreement with the findings of Stromberg et al. (22), P' and P-, PI1 variants of N. gonorrhoeae, bind to gangliotetraosylceramide (asialo-Gb,,) and gangliotriaosylceramide (asialo-GMJ (Fig. 2). The organism did not bind to lactosylceramide derived from asialo-GM? by treatment with @galactosidase (Fig. 2B, lane 4) suggesting that the Bl-4-linked GalNAc, which is positioned internally in asialo-GM, and terminally in asialo-G,vy, is required for binding (Table I). As has been shown for many pulmonary pathogenic bacteria, including Pseudomonas aeruginosa, Haemophilus influenzae, and Streptococcus pneumoniae (27), all of the variants of N. gonorrhoeae tested (Table II) preferred GalNAc/%4Gal/31-4Glc sequences unsubstituted with sialyl residues for binding (Fig. 2R, lane 1). Interestingly, P. aeruginosa, H. influenzae, and S. pneumoniae also bind avidly to paragloboside." Binding patterns similar to those shown in Figs. 1B and 2B were also observed when the plates were incubated with all other gonococcal variants used in this study (Table II).
Quantitative Binding of N. gonorrhoeae to Immobilized Glycolipids and Inhibition of Binding by Glycolipids-Binding of N. gonorrhoeae to purified glycolipids adsorbed on microtiter plates was examined to further define the binding specificity and to compare the relative avidities of the lacto-and ganglioseries receptors. As shown in Fig. 3, the gonococci bind better to asialo-GM2 than to paragloboside and not at all to sialylparagloboside, G.Mlr or lactosylceramide. Binding to both asialo-GX,, (data not shown) and asialo-GM? is half-maximal at 0.2 Fg, about 7 times better than to paragloboside (or lacto-N-triaosylceramide, data not shown), suggesting a higher avidity for the ganglio-series glycolipids. Binding of P' and Pm variants of N. gonorrhoeae to both asialo-GM, and paragloboside was not affected by changing the growth conditions of the organism, as the gonococci bound equally well to both glycolipids when grown anaerobically, microaerophilically on agar or in broth, or under iron limited conditions (data not shown).
Biological Significance-N. gonorrhoeae agglutinate human erythrocytes (33) and adhere to and are phagocytosed by neutrophils (34). The receptor that mediates binding of these cells to the gonococci is probably paragloboside and lacto-hrtriaosylceramide which are present in substantial amounts in both cell types (23,27,35,36). Lacto-series also constitute the major glycolipid component of various tissues and organs and are glycolipid precursors of the major blood group antigens. Interestingly, Gal~l-4GlcNAc~1-3Gal~l-4Glc sequences are also present in many gonococcal LOS (13). As shown in Fig. 4  binding of N. gonorrhoeae to at least t,he lacto-series glycolipids is probably not dependent on the fatty acid in ceramide as has been reported for other bacteria (39).
Asialo-G11? has been reported to occur in cultured human endocervical cells (22), a relevant target tissue for infection, and asialo-gangliosides also occur in other human tissues, although in lower amounts (27,40,41). Both asialo-GUI and asialo-Glrlz, however, bind N. gonorrhoeae with highest avidity (Fig. 3). Recently, Paruchuri et al. (42) have identified the gene encoding for an adhesin which binds to asialo-GbI, and asialo-GXIz and have shown that the adhesin is a 36-kDa protein that is not associated with gonococcal pili. As mutants that do not express this adhesin retain their ability to agglutinate human erythrocytes (42), this adhesin probably is distinct from the paragloboside-binding specificity we describe here. Thus, more than one type of gonococcal adhesin may mediate binding to different human cell types and individually or coordinately contribute to the pathogenesis of disease.