Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation
ABSTRACT
Gonorrhea, caused by the bacterium Neisseria gonorrhoeae (Gc), is characterized by neutrophilic influx to infection sites. Gc has developed mechanisms to resist killing by neutrophils that include modifications to its surface lipooligosaccharide (LOS). One such LOS modification is sialylation: Gc sialylates its terminal LOS sugars with cytidine-5′-monophosphate-N-acetylneuraminic acid, which is scavenged from the host using LOS sialyltransferase (Lst) since Gc cannot make its sialic acid. Sialylation enables sensitive strains of Gc to resist complement-mediated killing in a serum-dependent manner. However, little is known about the contribution of sialylation to complement-independent, direct Gc-neutrophil interactions. In the absence of complement, we found sialylated Gc expressing opacity-associated (Opa) proteins decreased the oxidative burst and granule exocytosis from primary human neutrophils. In addition, sialylated Opa+ Gc survived better than vehicle treated or Δlst Gc when challenged with neutrophils. However, Gc sialylation did not significantly affect Opa-dependent association with or internalization of Gc by neutrophils. Previous studies have implicated sialic acid-binding immunoglobulin-type lectins (Siglecs) in modulating neutrophil interactions with sialylated Gc. Blocking neutrophil Siglecs with antibodies that bind to their extracellular domains eliminated the ability of sialylated Opa+ Gc to suppress the oxidative burst and resist neutrophil killing. These findings highlight a new role for sialylation in Gc evasion of human innate immunity, with implications for the development of vaccines and therapeutics for gonorrhea.
IMPORTANCE
Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subverts protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampens neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.
INTRODUCTION
Neisseria gonorrhoeae (Gc) causes the bacterial sexually transmitted infection gonorrhea. Gc is designated as an urgent threat level pathogen by the Centers for Disease Control and Prevention, with approximately 82.4 million cases each year globally (1). Factors contributing to the prevalence of gonorrhea include increasing resistance to antibiotics, resistance to soluble and cellular innate immune components, and the lack of protective immunity elicited by prior infection (2, 3). Finding new approaches to treat or prevent Gc infection is of utmost importance.
The most abundant surface component on Gc is lipooligosaccharide (LOS). The length and composition of LOS is regulated by lgt (LOS glycosyltransferase) genes. Phase variation of lgt genes results in varying oligosaccharide compositions within a Gc population, including during infection (4–7). Gc isolated from uncomplicated urethral infection predominantly produces LOS that can be sialylated. Sialylation is the incorporation of sialic acid onto the alpha and/or beta chain terminal galactose of LOS, catalyzed by Gc LOS sialyltransferase (Lst) (8–10). Gc cannot synthesize sialic acid and instead scavenges cytidine-5′-monophosphate-N-acetylneuraminic acid (CMP-NANA from the host) (11). Lst is constitutively expressed (12, 13), is required for optimal genital tract infection (14–16), and uses diverse forms of CMP-sialic acid (17).
LOS sialylation is a form of molecular mimicry that is thought to enable Gc to evade immune recognition (18, 19). Sialylation was originally characterized due to conferring “unstable” serum resistance on Gc recovered from urethral secretions but not after laboratory passage (11, 20, 21). Sialylation has since been shown to inhibit all three complement activation pathways by decreasing C1 engagement and C4b deposition and increasing recruitment of factor H on Gc (17, 22, 23). Sialylated glycans enable the immune system to discriminate self from non-self, achieved in part through sialic acid-binding immunoglobulin-type lectins (Siglecs) (24–27). Siglecs are produced by most immune cells, including neutrophils, which are the predominant immune cells recruited in Gc infections and constitute the purulence in gonorrheal secretions (28). Human neutrophils express Siglec-5, -9, and -14. Siglec-5 and -9 contain cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that recruit SH2 domain-containing phosphatases to inhibit cellular activation (29–34). By contrast, Siglec-14 associates with the adapter protein DAP12, which contains an immunoreceptor tyrosine-based activating motif (ITAM) that once phosphorylated, recruits Syk kinase to activate downstream signaling (35, 36). The genes that encode Siglec-5 and Siglec-14 are adjacent and considered paired receptors since the proteins share almost complete sequence identity in their binding domains and similar glycan binding preferences (35, 37). 10%–70% of individuals in certain racial/ethnic groups harbor a SIGLEC14/5 fusion gene where Siglec-14 is not expressed but Siglec-5 is (38). Thus, on balance, signaling through neutrophil Siglecs dampens inflammatory signaling. Sialylated Gc has been reported to interact with the extracellular domains of these Siglecs as Fc chimeras (39).
Gc expresses numerous gene products that defend against neutrophil antimicrobial activities (28). Gc also varies its ability to interact non-opsonically with neutrophils through phase-variable expression of opacity-associated (Opa) proteins (40). Most Opa proteins bind one or more carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). In particular, binding to the granulocyte-restricted CEACAM3 elicits phagocytosis, reactive oxygen species production, and granule release (41–47).
In this study, we unexpectedly found that sialylation promotes Gc survival from human neutrophils in a complement-independent manner. Our results implicate Siglecs in dampening the neutrophil activation that is elicited by CEACAM-binding Opa proteins, bringing new insight into the ways that Gc modulates soluble and cellular innate immunity to persist in its obligate human host.
RESULTS
Gc Lst incorporates sialic acid onto LOS, which is retained upon infection of neutrophils
To investigate the role of LOS sialylation on direct, nonopsonic Gc interactions with neutrophils, we first assessed Lst- and CMP-NANA-dependent sialylation of Opa-expressing (Opa+) Gc. The wild-type (WT) Lst parent for these studies was a derivative of strain FA1090 1-81-S2 that constitutively expresses the OpaD protein in an otherwise Opa-negative background, hereafter termed OpaD (48). The OpaD protein binds CEACAMs 1 and 3 on human neutrophils, leading to rapid phagocytic killing (48, 49). This strain produces the sialylatable lacto-N-neotetraose (LNnT) alpha chain and lactose beta chain (50). OpaD was transformed with insertionally inactivated lst to generate an isogenic mutant that cannot be sialylated (OpaDΔlst) (15). Bacteria were incubated with CMP-NANA or vehicle control, then washed to remove residual sialic acid and processed for downstream analysis.
Monoclonal antibody (mAb) 6B4 binds the LNnT epitope of the alpha chain of Neisseria LOS; however, sialylation of its terminal galactose prevents 6B4 binding (8, 51). By Western blot, 6B4 reacted with lysates of OpaD, and binding was lost when OpaD was incubated with CMP-NANA (Fig. 1A). 6B4 reacted with OpaDΔlst lysates with or without CMP-NANA (Fig. 1A). However, the small size (<5 kDa) of LOS makes resolving and detecting it by Western blot a challenge. Therefore, to analyze sialylation on individual bacteria, we used imaging flow cytometry with 6B4. The fluorescence index of OpaD incubated with CMP-NANA was significantly lower than OpaD without CMP-NANA and OpaDΔlst with or without CMP-NANA; these three were statistically indistinguishable (Fig. 1B and C).
Fig 1
Beyond LNnT, Gc LOS can be sialylated on its Pk-like alpha chain (18, 52) and the lactose-bearing beta chain (50, 53). To directly detect Lst-dependent sialylation at any site on Gc LOS, independent of 6B4 exclusion, we applied copper click chemistry using azido-labeled sialic acid, followed by alkyne-FITC cycloaddition (54–57). OpaD incorporated CMP-Azido (Az)-NANA in a Lst-dependent manner (Fig. 1D and E), which competed with unmodified CMP-NANA at approximately equimolar concentration (Fig. 1F). Using Gc preincubated with CMP-Az-NANA, we monitored the retention of sialylation on OpaD in the presence of neutrophils. Representative images collected at 30 min post-infection showed alkyne-FITC-labeled bacteria that were both intracellularly and extracellularly attached to neutrophils (Fig. 1G). Thus imaging flow cytometry, in combination with immunofluorescence and click chemistry, can be used to track and quantify Gc LOS sialylation alone and in cells.
Lst-dependent sialylation of Opa+ Gc dampens human neutrophil activation and protects Gc from killing by neutrophils
We explored the contribution of Gc Lst-dependent sialylation to neutrophil activation. First, the release of reactive oxygen species (ROS) was measured. As previously reported, non-sialylated OpaD elicited robust neutrophil ROS production (Fig. 2A and B) (42, 45, 48). Sialylation significantly reduced the magnitude of the OpaD-elicited oxidative burst; ROS production was inversely correlated with the concentration of CMP-NANA for sialylation (Fig. 2A and B). Sialylation similarly reduced the oxidative burst in response to other Opa+ bacteria (Fig. 2C; Fig. S1A). OpaDΔlst elicited neutrophil ROS production regardless of CMP-NANA addition and was indistinguishable from non-sialylated OpaD (Fig. 2D; Fig. S1B).
Fig 2
While ROS does not directly contribute to neutrophil anti-gonococcal activity (58–60), ROS production requires intracellular signaling events and granule release (61). Neutrophil granules contain antimicrobial components, some of which are bactericidal for Gc (62–64). Thus we analyzed the effect of sialylation on the ability of OpaD to stimulate exocytosis of primary and secondary neutrophil granules as measured by CD63 and CD66b surface presentation, respectively (42). Sialylation of Gc significantly reduced neutrophil exocytosis of primary (Fig. 2E) and secondary (Fig. 2F) granules at 30 min post-infection. Together, these data indicate sialylation reduces the ability of Gc to activate neutrophils.
We next tested the contribution of Lst-dependent sialylation to bacterial survival in the presence of adherent, IL-8-primed primary human neutrophils without the addition of human serum (complement). Sialylated OpaD had a statistically significant increase in survival over non-sialylated bacteria at 15 and 30 min post-infection (Fig. 3A). OpaDΔlst phenocopied non-sialylated OpaD at these times, showing that both sialic acid and Lst were required for the increased survival of OpaD exposed to neutrophils. By 60 and 120 min post-infection, survival of sialylated OpaD was not statistically different from non-sialylated Gc (Fig. 3A). Sialylation also significantly increased the survival from neutrophils of CEACAM-binding Opa60 Gc, through 2-h infection (Fig. 3B). However, survival of Opaless Gc from neutrophils was unaffected by sialylation, which fits with our previous reports that Opa-negative Gc persists in the presence of neutrophils (48, 59, 65) (Fig. 3C). These results highlight a role for Lst-dependent sialylation in survival of Opa+ Gc from neutrophils.
Fig 3
Sialylation of LOS does not directly affect OpaD-CEACAM3 interactions
Given the importance of CEACAM3 to the phagocytic killing of Opa+ Gc in neutrophils, we tested the hypothesis that Gc sialylation disrupted OpaD-CEACAM3 engagement. First, sialylated or non-sialylated OpaD was incubated with a Glutathione S-transferase (GST)-tagged N-terminus of human CEACAM3 (N-CEACAM3), and CEACAM3 binding was measured by flow cytometry using anti-GST fluorescence (66). Lst-dependent sialylation did not significantly affect OpaD binding of N-CEACAM3 (Fig. 4A). Sialylation also did not affect the interaction of OpaD with N-CEACAM1 (Fig. S2). Opaless Gc, which does not bind CEACAMs, served as a negative control. Second, the association of sialylated or non-sialylated OpaD with human CEACAM3-transfected CHO cells was measured by imaging flow cytometry. Lst-dependent sialylation did not significantly affect bacterial association with hCEACAM3-CHO cells (Fig. 4B). These results indicate that contrary to our hypothesis, sialylation does not affect Opa protein-CEACAM engagement.
Fig 4
In a previous report, sialylation decreased the adherence of Opa+ Gc to neutrophils in suspension, but this effect was lost over time (67). We thus hypothesized that the mechanism by which sialylated Gc resisted killing by neutrophils was due to decreased association with and/or internalization by these immune cells. To test this hypothesis, we used imaging flow cytometry (68), in which extracellular Gc was discriminated from intracellular bacteria by the accessibility of a mAb against FA1090 PorB1b. The binding of this antibody to Gc was unaffected by sialylation (Fig. S3). Sialylation did not significantly affect OpaD association with (Fig. 4C) or internalization by (Fig. 4D) neutrophils through 30 min post-infection, when effects were measured by ROS production and bacterial survival. We conclude that sialylation does not directly affect Opa+ Gc interaction with neutrophil CEACAMs.
Sialylated Gc binds immunoregulatory Siglecs to dampen neutrophil activation and anti-gonococcal activity
The inhibitory effect of Gc sialylation on neutrophil activation and not phagocytosis, along with work from other groups (37, 39, 69, 70), led us to investigate the contribution of sialic acid immunoglobulin-type lectins (Siglecs) on neutrophils. Using imaging flow cytometry, we confirmed the presence of Siglec-9 and Siglec-5/14 on the surface of primary human neutrophils (Fig. 5A) compared to unstained (Fig. S4A). Staining ranged from small puncta to rings around the cell periphery, though there was minimal overlap in Siglec-9 vs Siglec-5/14 localization (Fig. 5B).
Fig 5
We hypothesized that sialic acid-Siglec engagement interfered with Opa-CEACAM binding to dampen the neutrophil response to Opa+ Gc. To test this, neutrophils were incubated with blocking antibodies against the extracellular domains of Siglec-9 and -5/14 (71), then exposed to sialylated or non-sialylated OpaD, and ROS production was measured. The addition of anti-Siglec antibodies significantly enhanced the neutrophil oxidative burst elicited by sialylated OpaD (solid green vs solid purple, Fig. 5C; Fig. S4B), restoring it to levels that were no different from unsialylated OpaD (open purple bar, Fig. 5C). The effect of Siglec-blocking antibodies on ROS production was specific to sialylated Gc since non-sialylated OpaD elicited similar levels of ROS from neutrophils whether or not the antibodies were present (green outline vs purple outline, Fig. 5C). These data implicate Siglecs in the ability of sialylated Opa+ Gc to reduce neutrophil activation.
The dampened ROS release in response to sialylated Opa+ Gc led us to examine whether blocking Siglecs restored the ability of neutrophils to control Opa+ bacteria. Neutrophils were incubated with blocking antibodies against Siglec-9 and -5/14 or left untreated, then exposed to sialylated or non-sialylated OpaD. The addition of anti-Siglec antibodies significantly reduced the survival of sialylated OpaD after infection of neutrophils at 15 and 30 min, to a level equivalent to non-sialylated OpaD (Fig. 5D). Non-sialylated OpaD were recovered similarly from neutrophils whether or not anti-Siglec antibodies were present, indicating that the effect of Siglecs on Gc-neutrophil interaction is specific for sialylated Gc.
From these results, we conclude that Gc uses sialylation of its LOS in a Siglec-mediated, complement-independent manner to impede neutrophil activation and anti-gonococcal responses, thereby promoting bacterial survival during infection.
DISCUSSION
Gc sialylation of LOS by Lst is crucial for complement resistance and pathogenesis in vivo (10). This study reveals an unexpected, complement-independent role of sialylation: its ability to restrain neutrophil activation in response to Opa+ Gc, improving Gc survival upon neutrophil challenge. Sialylation reduced granule mobilization and release of both oxidative and nonoxidative species in response to Opa+ bacteria. Unexpectedly, sialylation of the LOS did not have a significant effect on Opa-CEACAM binding and engagement. Instead, by blocking the immunoregulatory Siglecs on neutrophils, sialylated Opa+ Gc could no longer suppress neutrophil activation or killing capacity. These data suggest the exploitation of neutrophil self-associated molecular pattern recognition by Gc through LOS sialylation by Lst. Our findings add to the increasing understanding of how pathogens co-opt host factors to impair immune cell activation (72).
This study focused on Gc interactions with neutrophils, the main cell type found in human infectious secretions. We used human neutrophils that were adherent and treated with IL-8, mimicking post-migration behavior in these terminally differentiated, short-lived primary cells (59). Work by our group and others has investigated how Gc expression of phase-variable Opa proteins affects their survival from neutrophils (40–42, 45, 48, 63, 73). Most Opa proteins bind selected human CEACAMs. In particular, neutrophils and other granulocytes express CEACAM3, which contains an ITAM that can recruit the Src family and Syk tyrosine kinases to drive signaling pathways leading to phagocytosis, granule release, and ROS production (40). Given the negative charge conferred by sialic acids, we hypothesized that LOS sialylation would impair the phagocytic activity of neutrophils toward Gc. However, we found no significant difference in the ability of sialylated Opa+ Gc to bind the soluble N-domain of CEACAM, CEACAM-expressing cells, or primary human neutrophils. While our study only focused on Opa+ Gc that bind CEACAMs 1 and 3, future work can explore how sialylation affects other modes of interaction between Gc and neutrophils, including antibody- and complement-mediated association.
Siglecs help the innate immune system to distinguish between non-self and self-associated molecular patterns (26). Many immune cell Siglecs contain cytoplasmic ITIM domains that transduce inhibitory signals to dampen inflammation (24). Other sialylated bacteria, including N. meningitidis and Group B Streptococcus, have been shown to manipulate cell activation via Siglec engagement (27, 69), and Gc can bind human Siglec-Fc chimera proteins (39). Consistent with published findings, we detected Siglec-9 and Siglec-5/-14 on the human neutrophil surface (29, 32, 37). Moreover, blocking Siglecs reversed the inhibitory activity of sialylation on neutrophil responses to Gc, uncovering a new role for Siglecs in thwarting neutrophil anti-gonococcal responses. Interestingly, human Siglec-Fc chimeras have also been reported to bind PorB on the surface of unsialylated Gc, which was enhanced by the production of a shorter, nonsialylatable LOS (39). Whether non-sialylated Gc interacts with Siglecs on neutrophils and the consequences of such interaction remain to be explored. Unlike Siglec-5 and Siglec-9, Siglec-14 does not have a cytoplasmic domain; instead, it associates with the ITAM-bearing DAP12 (35, 37). Siglec-5 and Siglec-14 have almost identical ligand binding domains due to ongoing gene conversion and are considered paired receptors, possibly to balance immune responses to pathogens (24, 35, 38). In a study of genetic variations in Siglecs among a human cohort with a high burden of gonorrhea, uninfected individuals were more likely to produce Siglec-14, though not reaching statistical significance (39). Future work can examine whether neutrophils from individuals with or without Siglec-14 respond differently to sialylated Gc.
The localization of Lst has recently been updated from an outer membrane protein to the inner face of the cytoplasmic membrane, where the OS is assembled (74, 75). Unlike the closely related microbe N. meningitidis, Gc cannot endogenously synthesize CMP-NANA and must scavenge it from the extracellular environment for sialylation. Lst has both α2,3 (alpha chain LNnT) and α2,6 sialyltransferase activity (alpha chain Pk-like OS and beta chain lactose) and has been shown to use different CMP-sialic acids as substrates (8, 17, 52, 53). This promiscuity suggests that sialic acid availability rather than enzymatic activity drives the dynamics of LOS sialylation. We exploited this feature of Lst to add azido-labeled CMP-NANA in the form of N-acetylneuraminic acid (Neu5Ac) to directly track sialylation on the Gc surface. Other Lst substrates include CMP-Neu5Gc, which is missing in humans due to the evolutionary loss of the responsible hydrolase (76), and CMP-legionaminic acid and CMP-ketodeoxynonulosonic acid, which do not confer complement resistance and could serve as anti-gonococcal therapeutics (17).
One key question prompted by this work is how neutrophils affect the dynamics of Gc sialylation. Neutrophils have been proposed as a source of CMP-NANA since lysates from polymorphonuclear phagocytes, including neutrophils, confer “unstable” serum resistance on Gc (77). How Gc obtains CMP-NANA, which is cytoplasmic, is currently unknown since intracellular Gc resides in phagolysosomes (65). Phagosomes may contain sialic acid transporters; in addition, neutrophils may release CMP-NANA by lysis or elaboration of neutrophil extracellular traps. It is also tantalizing to speculate that neutrophil sialyltransferases, found in the secretory pathway, could intersect with Gc- and sialic acid-containing compartments to sialylate the bacteria (78). Conversely, neutrophils have surface-exposed sialidases, which desialylate their glycans during diapedesis (79, 80) and other cells’ glycans for adhesion (81). While we did not detect any desialylation of Gc over the first 30 min of neutrophil exposure, the sialylation state of Gc over time with neutrophils remains unknown. Modification of neutrophil-like cells to synthesize azido-labeled CMP-NANA (82) would allow sialylation of Gc to be tracked over time and in different compartments in an Lst-dependent manner.
Sialylated Gc has reduced infectivity in the experimental challenge of the human male urethra (14) and the genital tract of female mice (15, 16). However, Gc isolated from male urethral gonorrheal exudates are sialylated (8, 11, 21). In addition, Gc isolated from cervicovaginal human secretions are less likely to be sialylated, due to sialidase activity conferred by members of the female genital microbiome (83). Given these dynamics, we anticipate that balancing the sialylation state allows Gc to resist cellular and humoral immunity—including by engaging Siglecs to thwart neutrophil attack—while maintaining infectivity of target mucosal surfaces. These findings can be exploited for novel therapies for gonorrhea since Lst expression is not a phase variable. For instance, CMP-NANA analogs that do not confer serum resistance aid complement killing of the multidrug-resistant Gc (17, 84). Similarly, sialic acid analogs that do not engage Siglecs could be developed to render Gc more sensitive to neutrophils. These approaches, in conjunction with antibiotics and vaccines, could enhance neutrophil and soluble host defenses to combat drug-resistant gonorrhea.
MATERIALS AND METHODS
Bacterial strains and growth conditions
Gc in this study is in the FA1090 background, constitutively expressing the pilin variant 1-81-S2 and with in-frame deletions of all opa genes (Opaless) (48). Opaless Gc with non-phase variable, constitutively expressed opaD has been described previously (OpaD) (48); Opa60 and OpaI were constructed similarly (49, 66).
Gc was grown overnight on gonococcal medium base (Difco) with Kellogg’s supplements (85) (GCB) at 37°C with 5% CO2. Growth of Gc in a rich liquid medium containing Kellogg’s supplements and NaHCO3 to enrich for piliated, live, mid-logarithmic phase bacteria has been previously described (86). For sialylation, 50 µg/mL of CMP-NANA (Nacalai) reconstituted in phosphate-buffered saline (PBS) was added for the final 2.5-h growth (87). Gc was washed with PBS + 5 mM MgSO4 prior to infection. CMP-Az-NANA (R&D Systems) was added in lieu of or in competition with unmodified CMP-NANA (54).
Isogenic OpaDΔlst was generated by spot-transforming OpaD with genomic DNA from MS11 GP300 lst::Kan (from A. Jerse, USUHS) (15). Transformants were selected on GCB with 50 µg/mL kanamycin and backcrossed three times. Loss of Lst was confirmed by PCR, and the inability to sialylate itself was verified by 6B4 exclusion (see below). OpaD expression was confirmed by Western blotting bacterial lysates with 4B12 mAb against Opa (88, 89).
Gc sialylation and detecting sialylated Gc
Western blot: SDS-PAGE and immunoblotting for 6B4 reactivity were conducted as previously described (17). In brief, lysates from Gc with or without CMP-NANA pre-incubation were resolved using a 4%–20% gradient SDS-polyacrylamide gel (Bio-Rad) and transferred in CAPS methanol buffer to polyvinylidene difluoride (PVDF). Membranes were blocked in PBS + 0.5% Tween-20 +5% dry skim milk and probed with 6B4 tissue culture supernatant (from S. Ram, UMass) followed by anti-mouse IgM-horseradish peroxidase (HRP) antibody (Jackson ImmunoResearch). Blots were developed with SuperSignal chemiluminescent substrate (ThermoFisher). For imaging flow cytometry, Gc (108 CFU/mL), incubated with or without 50 µg/mL CMP-NANA, was labeled with 5 µM Tag-IT Violet (TIV) (BioLegend) and then incubated with 6B4, followed by AlexaFluor647-coupled goat anti-mouse IgM (Jackson ImmunoResearch) before fixation and processing. Samples were examined using ImageStream X Mark II imaging flow cytometer and analyzed using INSPIRE and IDEAS v. 6.2 software packages (Amnis Luminex Corporation). Focused, singlet TIV+ Gc was gated as in reference (66). AF647+ gate was set in reference to TIV+ Gc without 6B4. Data are reported as fluorescence index (geometric mean fluorescence intensity × percent AF647+).
Click chemistry: Gc (108 CFU/mL) with or without CMP-Az-NANA were stained with TIV before fixation to analyze Gc alone, or used to infect primary human neutrophils at a multiplicity of infection (MOI) of 1 (see below). After fixation with 2% paraformaldehyde (PFA), neutrophils were incubated with H.5 mouse anti-PorB (from M. Hobbs, UNC) to detect extracellular Gc, then permeabilized with 0.2% saponin. Copper-catalyzed click chemistry was carried out for 1 h at room temperature with continuous shaking, using reaction reagents: 2.5 mM THPTA (tris-hydroxypropyltriazolylmethylamine) ligand (Lumiprobe), 0.06 mM FAM alkyne 5-isomer (5-Carboxyfluorescein) (Lumiprobe), 2.4 mM L-ascorbic acid (VWR), and 2 mM copper (II) sulfate pentahydrate all dissolved in UltraPure distilled water (Invitrogen) (90). Samples were thoroughly washed before analysis by imaging flow cytometry.
Neutrophil isolation
Human subjects research was conducted in accordance with a protocol approved by the University of Virginia Institutional Review Board for Health Sciences Research (#13909). Informed, formal written consent was obtained from each participant. Neutrophils were isolated from venous blood collected from healthy human subjects via dextran sedimentation followed by a Ficoll gradient as described (86). Neutrophils were resuspended in Dulbecco’s PBS (DPBS; without calcium and magnesium; Thermo Scientific) with 0.1% dextrose and used within 1 h after isolation. Samples were >95% neutrophils and >99% viable.
Neutrophil ROS production
Neutrophils were resuspended in Morse’s Defined Medium (MDM) (91) containing 20 µM luminol. Gc was added at an MOI of 100 and the generation of reactive oxygen species was measured every 3 min for 1 h via chemiluminescence (86). Negative controls included Opaless-infected and uninfected neutrophils (48). Line graphs are representative of three or more biological replicates. Bar graphs present all biological replicates’ area under the curve (AUC) values.
Neutrophil granule exocytosis
Surface expression of neutrophil granule proteins was measured as in reference (42), using phycoerythrin (PE)-CD63 for primary granules and allophycocyanin (APC)-CD66b for secondary granules (or isotype controls PE-IgG1 and APC-IgM) (BioLegend). Data were acquired using a Cytek Northern Lights spectral flow cytometer and analyzed using FCS Express (De Novo Software). The median fluorescence of each sample was normalized to unstimulated neutrophils as negative control.
Gc survival in the presence of neutrophils
Infection of adherent, IL-8 treated human neutrophils in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (HI-FBS) was conducted as in reference (86), except Gc was pre-incubated with or without CMP-NANA. After centrifugation to synchronize infection and incubation at 37˚C, 5% CO2, neutrophils were lysed with 1% saponin, serially diluted, and plated for overnight growth on GCB. Results are reported as a percentage of CFUs enumerated from lysates at the indicated timepoint, divided by CFUs enumerated at 0 min.
Imaging flow cytometric analysis of Gc association with and internalization by neutrophils
Infection of primary human neutrophils with TIV+ Gc and evaluation by imaging flow cytometry was performed as described (42, 68, 92). Because sialylation reduced bacterial binding of the goat anti-Gc antibody (Biosource) to almost undetectable levels (data not shown), extracellular Gc was recognized instead with H.5 anti-PorB, followed by AF647-coupled goat anti-mouse antibody.
N-CEACAM1 and N-CEACAM3 binding of Opa-expressing Gc
GST-tagged N-terminal domains of human CEACAMs 1 and 3 were purified and utilized as in reference (66). In brief, non-sialylated or sialylated Gc were incubated with GST-N-CEACAM1 or -N-CEACAM3 for 30 min at 37°C with end-over-end rotation. Gc was washed and incubated with mouse anti-GST antibody p1A12 (BioLegend) followed by an AF488-conjugated goat anti-mouse antibody (Jackson ImmunoResearch). The percentage of AF488+ (N-CEACAM-bound) Gc was measured using imaging flow cytometry.
Gc association with CHO-hCEACAM-1 and -3 cells
hCEACAM1-, hCEACAM3-, and Control-CHOs were generated using heparan-sulfate proteoglycan-deficient Chinese hamster ovary (CHO) cell strain PgsD-677 (ATCC). Cells were grown in Ham’s F-12K medium (ATCC) with HI-FBS and 1× Antibiotic-Antimycotic (Gibco) at 37°C, 5% CO2. Plasmids were prepared from VectorBuilder Escherichia coli stocks using Plasmid Miniprep (Qiagen) and ampicillin selection: human CEACAM1 (VB900000-5687rxc), human CEACAM3 (VB900000-7471zkb), or an empty control (VB010000-9288rhy) vector under a CAG promoter. CHOs were transfected using Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. After 48 h, transfectants were selected using 10 µg/mL puromycin (Gibco). A non-transfected control was used to confirm selection. Transfectants were maintained under puromycin selection throughout culturing and passaged at 70%–90% confluency; the medium was replaced with an antibiotic-free medium 2 h prior to infection. Surface expression of CEACAM on pooled CEACAM1- and 3-CHO transfectants and absence on Control-CHOs were confirmed by immunofluorescence microscopy using an anti-Pan-CEACAM antibody (Abcam, clone D14HD11).
Cells were seeded in a six-well plate and grown for 48 h to confluency (~106 cells/well). TIV +Gc were added at an MOI of 1 to cells on ice, centrifuged to synchronize infection, washed, and incubated for 30 min. Cells were lifted with Versene solution (Gibco) at 37°C, 5% CO2 for 10 min. Cells were then fixed with 2% PFA, collected with a rubber cell scraper, and filtered through 50 µm nylon mesh. Cells were blocked in PBS + 10% normal goat serum and incubated with anti-Pan-CEACAM antibody followed by goat anti-mouse AF555-conjugated antibody (Invitrogen). Cells were analyzed using imaging flow cytometry and IDEAS software. Focused cells were gated by gradient root mean square for image sharpness >50. Single cells were gated by a high aspect ratio and area >200 and <1,000. Single stained controls were used to set gates for AF555+ and TIV+ populations.
Siglec staining and blockade
Adherent, IL-8 primed neutrophils were fixed and lifted with a cell scraper then cells were washed in PBS and blocked in 10% normal goat serum. Next, neutrophils were incubated with anti-Siglec-9-APC (Clone 191240) and/or anti-Siglec-5/14-AF488 (Clone 194128) (R&D Systems) for 30 min. Samples were analyzed using imaging flow cytometry as above. To block Siglecs, isolated neutrophils were incubated with the unconjugated forms of anti-Siglec-9 and/or anti-Siglec-5/-14 for 30 min at 37°C + 5% CO2 before infection.
Statistics
Results are depicted as the means ± standard errors for >3 independent experiments. Statistics were calculated using GraphPad Prism; a P value of ≤ 0.05 was considered significant. Analysis of variance (ANOVA) or mixed model analysis was used for multiple comparisons of parametric data.
ACKNOWLEDGMENTS
We thank Louise Ball and Samuel Clark for preliminary experiments that informed this project, and Asya Smirnov and Linda Columbus for guidance in methods development. We thank Mike Solga of the UVA Flow Cytometry Core Facility (RRid: SCR_017829) and Hazel Ozuna (University of Louisville) for advice, Ann Jerse and Marcia Hobbs for strains and antibodies, and Sanjay Ram for reagents and discussions. We are grateful to the human subjects who made this research possible.
This work was supported by NIH R01AI097312, NIH U19AI144180, and NIH U01AI162457 (A.K.C.). A.J.C. was supported in part by NIH F31AI157528 and NIH 3R01AI097312-07S1. A.J.C. and M.W.B. were supported in part by NIH T32AI007046. M.M.P. and N.J.F. were supported by NIH R35GM124893. G.A.J. and C.M.J. were supported by the Merit Review Award BX000727 and the Research Career Scientist Award from the Research Service of the U.S. Department of Veterans Affairs.
Conceptualization: A.J.C. and A.K.C. Formal analysis: A.J.C, K.S.T., and N.J.F. Funding acquisition: A.K.C. and A.J.C. Project Administrator: A. K.C. Investigation: A.J.C., K.S.T., M.W.B., and N.J.F. Methodology: A.J.C., M.W.B., N.J.F., and M.M.P. Supervision: G.A.J., C.M.J., M.M.P., and A.K.C. Visualization: A.J.C. Writing—original draft: A.J.C. and A.K.C. Writing—review & editing: A.J.C., K.S.T., M.W.B., N.J.F., C.M.J., G.A.J., and A.K.C.
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mBio
Volume 15 • Number 5 • 8 May 2024
eLocator: e00119-24
Editor: M. Stephen Trent, University of Georgia, Athens, Georgia, USA
PubMed: 38587424
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© 2024 Cardenas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
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Received: 14 January 2024
Accepted: 12 March 2024
Published online: 9 April 2024
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M. Stephen Trent
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University of Georgia, Athens, Georgia, USA
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Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation. mBio
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