Role of the Gonococcal Neisserial Heparin Binding Antigen in Microcolony Formation, and Serum Resistance and Adherence to Epithelial Cells

Abstract

The sexually transmitted infection gonorrhoea is on the rise worldwide and an increased understanding of the mechanisms of colonization and pathogenesis of Neisseria gonorrhoeae is required to aid development of new treatment and prevention strategies. In the current study, we investigate the neisserial heparin-binding antigen (NHBA) of N. gonorrhoeae and confirm its role in binding to several glycans, including heparin, and identify interactions of NHBA with both gonococcal and host cells. Furthermore, we report that a gonococcal nhba mutant displays decreased cell aggregation and microcolony formation, as well as reduced survival in human serum and reduced adherence to human cervical and urethral epithelial cells, relative to the wild-type strain. These data indicate that the gonococcal NHBA contributes to several aspects of the colonization and survival of N. gonorrhoeae and may be a target for new antimicrobial or vaccines.

Keywords: Neisseria gonorrhoeae; NHBA; adherence; aggregation; glycan; gonococcus; heparin; microcolony; neisserial heparin-binding Antigen; serum resistance; vaccine antigen.

Figure 1.

Neisserial heparin-binding antigen (NHBA) sequence features and expression. A, Schematic of Neisseria gonorrhoeae (Ng) strain 1291 NHBA and Neisseria meningitidis (Nm) strain MC58 NHBA proteins is shown, with the lipobox motif (gray box) and glycine stretch (black box) shown in the N-terminal, and the arginine-rich region (white box) shown in the central region of NHBA. The amino acid length of each protein is shown on the right. B, The N. gonorrhoeae and N. meningitidis NHBA proteins were aligned using ClustalW in MacVector software (version 17.0) (see Supplementary Methods). Identical amino acids are shown as dark gray vertical lines, mismatches light gray lines, and gaps as white. The amino acid sequences of the arginine-rich region in the N. gonorrhoeae and N. meningitidis NHBA proteins (boxed) and its flanking sequences are shown, with identical amino acids shaded gray and mismatches shown in white. The cleavage site of the meningococcal NalP upstream of the Arg-rich region, and human lactoferrin (hLf), kallikrein (hKl) and C3 convertase are shown. C, Western blot of whole-cell lysates of the N. gonorrhoeae 1291 wild-type (WT), NHBA knockout (ΔNHBA), and complemented (ΔNHBA_C) strains grown at 37˚C, using polyclonal anti-NHBA antibodies. Up-regulation of NHBA expression in the WT grown at 32˚C versus 37˚C is also shown. The periplasmic protein NGAG_01228 is shown as a loading control. D, Flow cytometry of whole cells of N. gonorrhoeae WT, ΔNHBA and ΔNHBA_C strains grown at 32˚C and 37˚C with the expression of NHBA on the cell surface being confirmed using polyclonal anti-NHBA antibody. The negative control is WT with secondary antibody only. Values represent geometric mean fluorescence.

Figure 2.

Gonococcal neisserial heparin-binding antigen (NHBA) is involved in cell aggregation. A, Growth and settling curves of Neisseria gonorrhoeae 1291 wild-type (WT), NHBA knockout (ΔNHBA), and complemented (ΔNHBA_C) strains in gonococcal broth, with absorbance measured at an optical density at 600 nm (OD₆₀₀). B, N. gonorrhoeae colony-forming units (CFUs) per milliliter in a culture at an OD₆₀₀ of 1, before and after trypsinization (0.25% typsin for 5 minutes). The CFU counts of WT and ΔNHBA_C strains increased 2.6-fold and 2.4-fold, respectively, after trypsin treatment (both P < . 001), whereas the ΔNHBA CFU count was not affected (P = .31). *P < .05; †P < .001 (comparison to WT). C, Flow cytometric analysis of recombinant N. gonorrhoeae NHBA (NHBA)_Ng binding to whole-cell N. gonorrhoeae (black represents N. gonorrhoeae only; white, N. gonorrhoeae plus labeled NHBA_Ng). D, Whole-cell enzyme-linked immunosorbent assay titration curve showing binding of NHBA_Ng to N. gonorrhoeae (black line). The antibody-only control curve (dotted line) indicates the absence of nonspecific interactions between whole-cell N. gonorrhoeae and the His-tag antibody.

Figure 3.

Gonococcal neisserial heparin-binding antigen (NHBA) is involved in microcolony formation. Scanning electron microscopy shows Neisseria gonorrhoeae 1291 wild-type (WT), NHBA knockout (ΔNHBA), and complemented (ΔNHBA_C) strains grown for 5 hours on glass sides (top panel) or on human urethral epithelial cell monolayers on glass slides (bottom panel). Aggregates and microcolonies can be seen for the WT and ΔNHBA_C strains, whereas the ΔNHBA strain is seen as single colonies or diplococci. Images were acquired at a magnification of ×5000, and the scale bar at the bottom of each box represents 5 µm.

Figure 4.

Gonococcal neisserial heparin-binding antigen (NHBA) binds to several glycans with high affinity. Surface plasmon resonance analysis of recombinant N. gonorrhoeae recombinant N. gonorrhoeae NHBA (NHBA_Ng) (binding to glycosaminoglycans (GAGs) (A) and non-GAG glycans (B). The name, structure and sulfation patterns (S) of each glycan is shown, along with the dissociation constant (K_D) of NHBA_Ng binding to each glycan reported as mean (standard deviation). K_D values are also shown for recombinant N. meningitidis NHBA (NHBA)_Nm interactions [13], to enable comparison. NHBA_Nm has higher affinity for β-Glc6P (K_D, 0.056 [0.025] µmol/L). Results were analyzed using BIAcore T200 software 2.0.2. Abbreviations: NB, no concentration-dependent binding; NS, non sulfated.

Figure 5.

Gonococcal recombinant neisserial heparin-binding antigen (NHBANg) binds to epithelial cells. A, Confocal fluorescent images acquired under ×40 magnification of NHBA_Ng binding to cervical epithelial (tCX) cells. Left, Extended focus (top left) and xyz (bottom left) images showing cross-sections of cells. Right, Antibody control (no NHBANg) images of untreated cells (top right) and cells treated with Recombinant N. gonorrhoeae NHBA (NHBANg) and secondary antibody (bottom right). Arrows indicate protein localizing on the cell surface. B, Flow cytometric analysis of binding of recombinant NHBA_Ng to human tCX and urethral epithelial (tUEC) cells.

Figure 6.

Gonococcal neisserial heparin-binding antigen (NHBA) contributes to survival in human serum and adherence to human epithelial cells. A, Survival of Neisseria gonorrhoeae 1291 wild-type (WT), NHBA knockout (ΔNHBA), and complemented (ΔNHBA_C) strains after 60 minutes in 10% (vol/vol) normal human serum. ‘+ Heparin’ indicates that bacteria were preincubated with heparin prior to addition of normal human serum. Data represent the average percentage survival for triplicate samples as a percentage of the inoculum and are shown relative to the WT (results for the WT strain, set at 100%, are 5.5 × 10⁴ colony-forming units [CFUs]). There was no significant difference in survival of the WT in serum according to the absence or presence of heparin. B, Adherence of N. gonorrhoeae to human cervical epithelia (tCX) and human urethral epithelial (tUEC) cells with N. gonorrhoeae 1291 WT, ΔNHBA, and ΔNHBA_C strains. C, Adherence of N. gonorrhoeae 1291 WT with tCX cells that were either untreated (no treatment) or pretreated with recombinant N. gonorrhoeae NHBA (NHBA_Ng) (1–100 µg/mL) or peanut agglutinin (PNA) as a negative control (100 µg/mL). Data represent the average percent adherence for triplicate samples as a percentage of that for the inoculum and are shown relative to the WT; the results for the WT, set at 100%, are 1.1 × 10⁵ (tCX) and 1.7 × 10⁵ (tUEC) (B) and 6.5 × 10⁴ adherent CFUs (C). Error bars represent standard deviations. Experiments were performed ≥3 times, and representative results are shown.*P < .05; †P < .01; ‡P < .001 (relative to untreated WT strain; 2-tailed Student t test).