Glycointeractome of Neisseria gonorrhoeae: Identification of Host Glycans Targeted by the Gonococcus To Facilitate Adherence to Cervical and Urethral Epithelial Cells

Abstract

Neisseria gonorrhoeae is a significant threat to global health for which a vaccine and novel treatment options are urgently needed. Glycans expressed by human cells are commonly targeted by pathogens to facilitate interactions with the host, and thus characterization of these interactions can aid identification of bacterial receptors that can be exploited as vaccine and/or drug targets. Using glycan array analysis, we identified 247 specific interactions between N. gonorrhoeae and glycans representative of those found on human cells. Interactions included those with mannosylated, fucosylated, and sialylated glycans, glycosaminoglycans (GAGs), and glycans terminating with galactose (Gal), N-acetylgalactosamine (GalNAc), and N-acetylglucosamine (GlcNAc). By investigating the kinetics of interactions with selected glycans, we demonstrate that whole-cell N. gonorrhoeae has a high affinity for mannosylated glycans (dissociation constant [K_D ], 0.14 to 0.59 μM), which are expressed on the surface of cervical and urethral epithelial cells. Using chromatography coupled with mass spectrometric (MS) analysis, we identified potential mannose-binding proteins in N. gonorrhoeae Pretreatment of cells with mannose-specific lectin (concanavalin A) or free mannose competitor (α-methyl-d-mannopyranoside) substantially reduced gonococcal adherence to epithelial cells. This suggests that N. gonorrhoeae targets mannosyl glycans to facilitate adherence to host cells and that mannosides or similar compounds have the potential to be used as a novel treatment option for N. gonorrhoeaeIMPORTANCE Multidrug-resistant strains of Neisseria gonorrhoeae are emerging worldwide, and novel treatment and prevention strategies are needed. Glycans are ubiquitously expressed by all human cells and can be specifically targeted by pathogens to facilitate association with host cells. Here we identify and characterize the N. gonorrhoeae host-glycan binding profile (glycointeractome), which revealed numerous interactions, including high-affinity binding to mannosyl glycans. We identify gonococcal potential mannose-binding proteins and show that N. gonorrhoeae uses mannosyl glycans expressed on the surface of cervical and urethral epithelia to facilitate adherence. Furthermore, a mannose-binding lectin or a mannoside compound was able to reduce this adherence. By characterizing the glycointeractome of N. gonorrhoeae, we were able to elucidate a novel mechanism used by this important pathogen to interact with human cells, and this interaction could be exploited to develop novel therapeutics to treat antibiotic-resistant gonorrhea.

Keywords: Neisseria gonorrhoeae; adherence; carbohydrate; epithelial cells; glycobiology; gonorrhea; mannose.

FIG 1

Glycan binding by Neisseria gonorrhoeae. The heat map shows binding (black bars) by whole-cell N. gonorrhoeae strain 1291 to glycans on the array (average of results from three independent experiments). Glycans are clustered into classes based on their respective terminal sugars. The number and percentage of glycans bound within each class are indicated. The full data set of glycan binding is shown in Table S1 in the supplemental material.

FIG 2

Adherence of N. gonorrhoeae to cervical epithelial cells (tCX) and urethral epithelial cells (tUEC) in the presence and absence of lectin. Gonococcal adherence is reduced in cells pretreated with the mannose-specific lectin ConA. Results are presented as the percentage of adherent bacteria from triplicate lectin-treated samples relative to that of the no-treatment control [results for no-treatment controls set at 100% are (1.7 ± 0.15) × 10³ and (1.4 ± 0.06) × 10³ adherent CFU for tCX and tUEC, respectively]. Error bars represent ±1 standard deviation. *, P < 0.05, calculated using a two-tailed Student's (P = 0.00032 and P = 0.0002 for tCX and tUEC, respectively). The nonbinding lectin PNA did not reduce gonococcal adherence (P = 0.26 and P = 0.24 for tCX and tUEC, respectively). Experiments were performed on at least three occasions, and representative results are shown.

FIG 3

Adherence of N. gonorrhoeae to cervical epithelial cells (tCX) and urethral epithelial cells (tUEC) in the presence and absence of free glycan. Adherence of N. gonorrhoeae to tCX and tUEC is reduced in the presence of a mannose competitor, α-methyl d-mannoside. Results are presented as the percentage of adherent bacteria from triplicate glycan-treated samples relative to that of the no-treatment control [results for no-treatment controls set at 100% are (4.3 ± 0.3) × 10³ and (1.4 ± 0.06) × 10³ adherent CFU for tCX and tUEC, respectively]. Error bars represent ±1 standard deviation. *, P < 0.05, calculated using a two-tailed Student t test. Tn antigen is a negative-control nonbinding glycan that does not affect bacterial adherence (P > 0.19). Experiments were performed on at least three occasions, and representative results are shown.