Fig 1.
Incubation with normal human serum decreases neutrophil anti-gonococcal activity.
(A) Adherent, IL-8-treated primary human neutrophils were exposed to Opaless Gc, OpaD+ Gc, or OpaD+ Gc that were preincubated in 25% normal human serum from healthy subjects (UVA). CFU were enumerated from neutrophil lysates at 0, 30, and 60 minutes (min) post infection, and bacterial survival is presented as the mean ± SEM of CFU at the indicated time point relative to the mean CFU for the same condition at 0 minutes for 8 independent experiments. Two-way ANOVA with Sidak’s post-hoc comparisons were used to compare each condition within each time point. In (B), OpaD+ Gc was mixed with the indicated percentage of normal human serum (UVA), and survival from neutrophils at 60 minutes was performed as in (A). One-way ANOVA followed by Tukey’s post-hoc comparisons was used to compare each condition to PBS alone (0% serum). *p<0.05, **p<0.01,***p<0.001. (C) OpaD+ Gc alone (PBS) or incubated in the indicated percentage of normal human serum (UVA) was exposed to neutrophils in suspension at an MOI of 100 in the presence of luminol. ROS production was measured over the course of 60 minutes (min) as the relative light units (RLU) generated by luminol-dependent chemiluminescence. (D) ROS production was measured as in (C) from OpaD+ Gc incubated in serum (UVA), complement component 3-depleted serum (“C3-depl serum”), or heat-inactivated serum (“HI serum” (56°C, 30 minutes)). (C-D) Results are one representative of 3 independent experiments.
Fig 2.
Characteristics of the serum component that suppresses Opa+ Gc induced neutrophil ROS.
(A-F) OpaD+ Gc were exposed to primary human neutrophils and ROS production was measured as in Fig 1C. Before addition to neutrophils, OpaD+ Gc was incubated with serum from the indicated species (final concentration, 25%) (A); the indicated molecular weight fraction of human serum (B); human serum that was intact (solid orange; UVA), treated with trypsin (dotted red), or treated sequentially with trypsin inhibitor then trypsin (purple) (C); the ≥ 100 kD human serum fraction from (B) (orange), or serum depleted of immunoglobulins G, M, and A (“Ig-depl serum”, dotted blue) (D); human serum fractions generated by anion exchange chromatography via elution with the indicated molarity of NaCl (E); or the indicated suppressive serum fraction from B and E that was pre-incubated with Gc (“Gc-depl”) or not (“no-depl”) (F). Incubation of OpaD+ Gc with PBS+ was used as a positive control for neutrophil ROS production in all conditions (grey).
Fig 3.
OpaD+ Gc was incubated with 25% normal human serum (orange; UVA), heat-inactivated (HI) serum (brown), heat-inactivated C4BP-depleted (depl) serum (blue), or purified C4BP (50 μg/ml; purple) for 20 minutes at 37°C. Gc was fixed and stained with DAPI for intact bacteria (blue) and for C4BP using anti-C4BP antibody followed by Alexa Fluor 488-coupled goat anti-rabbit IgG (green), and analyzed by imaging flow cytometry. (A) Representative images (image numbers 7449 and 19815) of a single serum-incubated (top) and PBS-incubated (bottom) bacterium. (B) is the percent C4BP-positive bacteria and (C) is the mean intensity of AF488 fluorescence of the total bacterial population. In B-C, results are the mean ± SEM of 3 independent experiments. Statistical analyses were performed using one-way ANOVA with Tukey’s post-hoc comparisons to PBS. *p<0.05, **p<0.01,****p<0.0001.
Fig 4.
C4BP is necessary and sufficient for serum-mediated suppression of neutrophil anti-gonococcal activity.
(A-C) Adherent, IL-8-treated neutrophils were exposed to OpaD+ Gc incubated in PBS+ (red) or the following (all at 25% final concentration) as in Fig 1A: (A) heat inactivated serum that was C4BP-replete (brown; Lund University) or C4BP-depleted (depl, blue); (B) C4BP-replete serum (orange; Lund Universtiy) or 50 μg/mL purified C4BP (purple); (C) C4BP at the indicated concentrations. In (A-B), two-way ANOVA with Sidak’s post-hoc test was used to compare each condition within each time point for 3 independent experiments; (C) used one-way ANOVA followed by Tukey’s post-hoc comparisons to compare each condition to PBS alone (0 μg/mL C4BP). (D-E) Neutrophil ROS production was measured as in Fig 1C in response to OpaD+ Gc that was incubated (D) in PBS, heat-inactivated C4BP-replete serum (brown; Lund University), heat inactivated C4BP-depleted serum (blue), or purified C4BP at 50 μg/mL (purple); or (E) in the indicated concentration of C4BP. (F) Neutrophils were exposed to OpaDporS-23 Gc or OpaDporKan Gc, with or without C4BP addition to the infection milieu (50 μg/mL), and bacterial survival after 60 minutes was measured as in (A). Results are the mean ± SEM of 3 independent experiments; two-way ANOVA with Sidak’s post-hoc comparisons were used to compare each condition. *p<0.05, **p<0.01,****p<0.0001. (G) OpaD+ Gc (grey solid lines), OpaDporS-23 Gc (black dotted lines), and OpaDporKan Gc (purple solid lines) alone (PBS; triangles), incubated with C4BP (open circles), or added to wells containing C4BP at a final concentration of 50 ug/mL (C4BP “in trans”; stars) were exposed to neutrophils, and ROS production was measured as in (E). Results in (D, E, and G) are one representative of 3 independent experiments.
Fig 5.
C4BP is necessary and sufficient for serum-mediated decrease in neutrophil internalization of OpaD+ Gc.
Gc were labeled with Tag-IT Violet (TIV), treated as indicated in the following graphs, and incubated with adherent, IL-8-treated primary human neutrophils. At the indicated times, cells were fixed and stained with DyLight 650 (DL650)-labeled anti-Gc antibody without permeabilization to recognize extracellular bacteria. Neutrophils were analyzed via imaging flow cytometry. (A) Data from 12,000 neutrophils with untreated OpaD+ Gc at 60 min were collected. One of these cells, number 11715, is displayed as a representative, with images captured from channels for (left to right) phase contrast, intracellular bacteria (red), total associated bacteria (purple), and merge. (B-C) Neutrophils were exposed to Opaless Gc (gray), OpaD+ Gc (red), or OpaD+ Gc incubated in C4BP-replete serum (25%, orange; Lund University). (B) reports the percentage of single, focused, intact neutrophils with ≥1 cell-associated bacterium (TIV+); (C) reports the percentage of neutrophils with ≥1 phagocytosed bacterium (TIV+ DL650−). (D) Neutrophils were exposed to OpaD+ Gc in PBS (red), or heat-inactivated serum that was intact (brown; Lund University) or C4BP-depleted (blue, both at 25% final), and the percentage of phagocytosed bacteria was calculated as in (C). (E-F) Neutrophils were exposed to OpaD+ Gc in PBS (red), serum (25%, orange; Lund University), or purified C4BP (50 μg/ml, purple). In (E), the percentage of phagocytosed bacteria was calculated as in (C). In (F), the percentage of cell-associated Gc that are internalized was calculated. Results are the mean ± SEM from ≥3 independent experiments. Statistical analyses were performed by two-way ANOVA followed by Sidak’s multiple comparisons. (G) OpaDporS-23 Gc and OpaDporKan Gc were incubated with neutrophils alone (grey bars) or with 50 μg/mL C4BP added to the media immediately prior to infection (“in trans”) for 30 minutes. The percentage of neutrophils with ≥ 1 phagocytosed bacterium was calculated as in (C) from 3 independent experiments. Statistical analyses were performed by two-way ANOVA followed by Sidak’s multiple comparisons. *p<0.05, **p<0.01,***p<0.001, ****p<0.0001.
Fig 6.
Binding of C4BP prevents internalization of CEACAM-binding Gc by neutrophils.
(A) The indicated variants of Gc were incubated with C4BP (purple), or PBS (grey). The percentage of neutrophils with≥ 1 phagocytosed bacterium was calculated as in Fig 5C. Data represent the mean ± SEM of ≥3 independent experiments. Statistical analyses were performed by two-way ANOVA followed by Sidak’s multiple comparisons. (B) Opaless Gc was incubated with C4BP (50 ug/mL), opsonized in rabbit anti-Gc IgG (80 μg/mL, 20 minutes, 37°C), sequentially incubated with IgG then C4BP, or left untreated. Unopsonized Gc and IgG-opsonized Opaless Gc bound similar amounts of C4BP (S8E Fig). The percentage of neutrophils with ≥ 1 phagocytosed bacterium was calculated as in (A). Statistical analyses were performed by one-way ANOVA followed by Tukey’s multiple comparisons. Opaless Gc incubated with C4BP behaved similarly to Opaless Gc (not shown). *p<0.05, **p<0.01.
Fig 7.
C4BP in gonococcal pathogenesis.
Neutrophils are recruited to mucosal sites of Neisseria gonorrhoeae infection by extravasating from the blood stream and crossing the epithelia paracellularly (1). Inflammatory conditions of the infection as well as serum transudate bring N. gonorrhoeae into contact with C4b-binding protein (C4BP) (2). The ability of N. gonorrhoeae to bind to C4BP to inhibit complement-mediated lysis has been extensively documented (3). This work shows that C4BP that is bound to the surface of N. gonorrhoeae decreases phagocytic uptake by neutrophils and suppresses neutrophil reactive oxygen species (ROS) production, in a complement-independent manner (4). C4BP binding decreases the interaction of CEACAM-binding, opacity protein (Opa)-expressing N. gonorrhoeae with neutrophils, but not bacteria that are opsonized with IgG or that express Opa proteins that engage other ligands (5). As a consequence, C4BP enhances survival of N. gonorrhoeae from neutrophils, a novel function for this canonical complement inhibitor (6). Figure generated using Biorender (https://biorender.com/).