Neutrophil CEACAMs and the Inflammatory Response to Neisseria gonorrhoeae

Abstract

Background

Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are proteins that mediate intercellular adhesion, intracellular signaling, and microbial recognition. Of the CEACAMs made by human neutrophils, CEACAMs 1, 3, and 6 are receptors for opacity-associated (Opa) outer membrane proteins of the bacterial pathogen Neisseria gonorrhoeae, which drive their non-opsonic phagocytosis.

Summary

This review describes what is currently known about CEACAM subcellular localization, bacterial recognition, and downstream signaling events in human neutrophils in response to N. gonorrhoeae and the experimental systems used to elucidate them. It highlights the consequences of these activities on neutrophil activation and killing of N. gonorrhoeae, particularly those downstream of CEACAM3. The potential cross-talk between CEACAMs and with other neutrophil receptors, and how these signals may impact responses to N. gonorrhoeae, are also covered.

Key Messages

Most Opa proteins of N. gonorrhoeae bind CEACAM1, with a subset also binding CEACAM3. Neutrophil CEACAM binding stimulates signaling events that drive phagocytosis of Opa-expressing N. gonorrhoeae, accompanied by degranulation, oxidant production, and proinflammatory signaling and transcriptional responses. In particular, CEACAM3 ligation leads to rapid phagocytic killing of N. gonorrhoeae. N. gonorrhoeae can avoid CEACAM3-mediated clearance by phase variation to turn off Opa protein expression or producing non-CEACAM3-binding Opa proteins. It can also bind host-derived complement C4b-binding protein (C4BP) and sialic acid, to impede CEACAM3-Opa interaction and engage sialic acid-binding immunoglobulin-like lectins (Siglecs) that transduce inhibitory signals to dampen CEACAM3 signaling, respectively.

Introduction

The carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), also known as CD66 family members, are proteins that mediate intercellular adhesion, intracellular signaling, and microbial recognition (recently reviewed in Ref. [1]). Predominantly expressed on epithelial and immune cells, CEACAMs are composed of one or more immunoglobulin (Ig)-like extracellular domains and are anchored to the cell surface via transmembrane or glycosylphosphatidylinositol (GPI) anchors, through which they directly or indirectly initiate signaling events.

Humans make 12 CEACAMs. CEACAM1, the prototype of the family, engages in cis- and trans-homotypic interactions, and its major splice variant has an immunotyrosine-based inhibitory motif (ITIM) to dampen self-activation [2]. An array of pathogens bind CEACAM1 and other family members to facilitate their colonization and infection. In response, it is postulated that humans and other primates evolved immunotyrosine-based activation motif (ITAM)-containing CEACAMs 3 and 4 as phagocytic, pathogen-clearing receptors [3, 4].

Neutrophils are critical in the early host response to infection or injury, where they deploy antimicrobial products including peptides, proteases, nutritional immunity proteins, and enzymes that make reactive oxygen species (ROS) [5]. These products are localized in different subsets of cytoplasmic granules, which include primary (azurophilic), secondary (specific), and tertiary (gelatinase) granules, and secretory vesicles. Primary granules, containing the most toxic cargo, require the strongest intracellular signals for their release [6]. Granules can fuse with phagosomes to deliver their contents to engulfed microbes, or with the plasma membrane by exocytosis (termed degranulation); granule contents also appear on neutrophil extracellular traps [5]. Human neutrophils express CEACAMs 1, 3, 4, 6, and 8 [1]. CEACAMs 3 and 4 are constitutively expressed on the neutrophil membrane, whereas CEACAMs 1, 6, and 8 are localized to granules and can be presented on the membrane upon degranulation [7, 8]. CEACAMs 3, 4, and 8 are unique to granulocytes (i.e., neutrophils), whereas CEACAMs 1 and 6 have broader distribution across human tissues. Several pathogenic bacteria can bind CEACAMs 1, 3, and 6, whereas CEACAMs 4 and 8 have no reported bacterial binding partners at the time of writing this review [9–14].

Despite neutrophils’ antimicrobial arsenal, some pathogens survive in neutrophil-rich environments, including inside neutrophils (reviewed in Ref. [15]). One such pathogen is Neisseria gonorrhoeae (Gc), the cause of the sexually transmitted infection gonorrhea [16]. In the controlled human urethral infection model of gonorrhea (summarized in Refs. [17, 18]), neutrophils appear 2–4 days after bacterial inoculation, which coincides with the onset of symptoms. Exudates from individuals with gonorrhea are replete with neutrophils, a subset of which contain large numbers of intact bacteria [19–23]. The main route of non-opsonic phagocytosis of Gc is CEACAM-dependent and requires bacterial opacity-associated (Opa) proteins as their ligands [24–27].

In this review, we will discuss what is currently known about CEACAM-mediated interactions with Opa-producing Gc, including the signals elicited downstream of CEACAMs on neutrophils, the phase variability and receptor-binding profiles of Opa proteins, interactions between CEACAMs and with other receptors on neutrophils, and the consequences of Opa-CEACAM engagement on neutrophil functionality. We will particularly discuss CEACAM3 for its role in phagocytic killing and proinflammatory responses and how Gc attempts to thwart them to enhance its survival.

Neutrophil CEACAMs

CEACAM extracellular regions consist of between one and six Ig-like domains (Fig. 1) [1, 2]. The N-terminal Ig-like variable (IgV) domain, whose sequence varies among CEACAMs, is a binding site for bacterial ligands. Some CEACAMs including CEACAM1 use the IgV domain to mediate homo- and hetero-dimerization. Between the IgV and membrane bound regions, most CEACAMs have a variable number of Ig-like constant (IgC) domains. To date, the IgC domains of neutrophil CEACAMs have not been implicated in bacteria-CEACAM or CEACAM-CEACAM interactions. CEACAMs can be anchored to the membrane via a GPI anchor (CEACAMs 6 and 8) or a transmembrane domain with a cytosolic tail (CEACAMs 1, 3, and 4). The cytosolic tails of CEACAMs 1, 3, and 4 elicit intracellular signaling upon engagement and receptor clustering [1]. CEACAM1’s cytosolic tail contains an ITIM [2]. In contrast, CEACAM3 and CEACAM4 contain an ITAM that is characterized as a hemITAM (see below) [28].

Fig. 1.

CEACAM-dependent signaling events in response to Opa-expressing N. gonorrhoeae. ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif.

CEACAM1

CEACAM1 (CD66a, BGP-1) is an ITIM-containing receptor that is broadly expressed across human tissues. The N-terminal IgV domain of CEACAM1 contributes to cell-cell adhesion via homo-dimerization, or heterodimerization with CEACAMs 5, 6, and 8 on neighboring cells [29–31]. It can also form cis-homodimers, which elicit cell-intrinsic inhibitory signaling [32]. CEACAM1 on the membrane exists at an equilibrium between its monomeric and dimeric states [31–34]. Helicobacter pylori CEACAM-binding partner HopQ has been shown to directly disrupt CEACAM1 cis- and trans-homodimers by outcompeting binding with the N-domain [35, 36]. Whether Gc Opa proteins can directly disrupt CEACAM1 dimers and how this affects interactions with neutrophils are unknown.

CEACAM1 has 11 splice variants whose abundance differs among cell types. However, neutrophil CEACAM1 is limited to the CEACAM1-L isoform [37], which contains a full-length cytoplasmic motif, three IgC domains, and an IgV domain. In resting neutrophils, CEACAM1 resides in the membrane of secondary granules [7, 8]. Therefore, expression of CEACAM1 on the neutrophil plasma membrane is dynamic and responsive to various stimuli. When neutrophils are more activated, such as during infection, more CEACAM1 is expressed on the surface [38].

Although CEACAM1 is not canonically considered a phagocytic receptor, its engagement results in the internalization of Opa-bearing Gc [39–42]. Cellular internalization via CEACAM1 is endocytic in nature; it is actin independent, does not require an intracellular signaling domain, and requires cholesterol-containing plasma membrane microdomains where CEACAM1 is found [39–41, 43]. In HeLa cells transfected with human CEACAMs, internalization of Gc via CEACAM1 occurred less efficiently than internalization via CEACAM3 [39, 41]. However, the relative contribution of CEACAM1 to internalization kinetics of Gc by primary human neutrophils has not fully been defined.

Neutrophil CEACAM1 is phosphorylated via Src family kinases upon engagement and receptor clustering [44]. In epithelial and T cells, the phosphorylated ITIM recruits Src-homology region containing phosphatases (SHP-1 and 2) [45–48]. Likewise, antibody ligation of CEACAM1 induces SHP recruitment in mouse and rat neutrophils [49, 50]. However, CEACAM1-induced SHP recruitment has not yet been shown in human neutrophils. SH2 binding releases the autoinhibition of the SHP phosphatase active site [51, 52]. SHP-1 dephosphorylates spleen tyrosine kinase (Syk), a signaling molecule that is activated by several neutrophil-activating receptors, including FcγR, TLR4, and CEACAM3 [53]. Thus, SHP-1 acts as a negative regulator of neutrophil activation. Concordantly, SHP-1-deficient mouse neutrophils displayed aberrant activation in response to LPS, which could be counteracted by pharmacological Syk inhibition [49]. Vav, a guanine nucleotide exchange factor involved in Rac-dependent phagocytosis in neutrophils, has also been identified as a target of SHP-1 in natural killer cells [54]. In T cells, CEACAM1 engagement suppresses TCR-derived T-cell activation in a SHP-1 dependent manner [46, 55]. In contrast, CEACAM1 activation may not block the signaling of other receptors in human neutrophils and in fact might potentiate CEACAM3 signals (see [crosstalk with other neutrophil receptors] below).

CEACAM3

CEACAM3 (CD66d, CGM1) is a phagocytic, ITAM-containing receptor that is expressed only by granulocytes. Signaling downstream of CEACAM3 was first described in recombinant epithelial systems and has been further validated in neutrophil-like mouse cells transfected with human CEACAM3. When CEACAM3 clustering is engaged by bacterial ligands, Src family kinases phosphorylate the ITAM tyrosines Y230 and Y241 [56, 57]. Phosphorylated CEACAM3 ITAM recruits spleen tyrosine kinase (Syk), which is phosphorylated by Src family kinases [58]. Syk phosphorylates multiple downstream effectors to induce bactericidal activities, including degranulation, ROS production, and actin-dependent phagocytosis [58]. CEACAM3 can also function independently of Syk to elicit downstream signaling with similar ability to activate neutrophils. For instance, the ITAM of CEACAM3 can also recruit the Rac guanine nucleotide exchange factor Vav to directly induce phagocytosis independent of Syk; thus, CEACAM3 is distinct from neutrophil Fc receptors, whose ITAMs require Syk to initiate phagocytosis [59]. Nck1 and 2 adaptor proteins also directly bind CEACAM3 to recruit Rac effectors and assist in WAVE complex formation, another key step in the phagocytic pathway [60]. Similarly, phosphorylated CEACAM3 itself recruits the regulatory subunit of class 1 PI3Ks, which are phosphorylated by Src family kinases and activate the neutrophil oxidative burst [61]. Binding of these effectors requires only Y230 [59, 61]. Thus, CEACAM3 is described as containing a hemITAM, where only one of the two tyrosines is required for downstream signaling [28].

CEACAM3 engagement also induces a pro-inflammatory transcriptional profile and cytokine production. CEACAM3 engagement by Moraxella catarrhalis in human neutrophils results in Syk-dependent release of pro-inflammatory cytokines IL-8 and CCL3 [62]. This response is elicited by the Syk-Card9-NF-κB activation pathway, which has been previously described in mouse bone marrow-derived macrophages and dendritic cells [62, 63]. Similarly, bone marrow-derived neutrophils isolated from mice that are transgenic for CEACAMs including CEACAM3, but not wild-type mice, display a NF-κB transcriptional signature when infected with Opa-expressing Gc [64]. In this case, cytokine release requires PKCδ and Tak1-mediated activation of NF-κB, a pathway that has also been described as downstream of Syk in mouse dendritic cells [64, 65]. Activation of p38 MAPK, another Tak-1 activated effector, also plays a role in the CEACAM-dependent transcriptional response [64]. Together, CEACAM3-dependent signaling cascades result in neutrophil activation, phagocytic uptake, and enhanced inflammatory function.

CEACAM6

The GPI-anchored CEACAM6 resides in primary neutrophil granules and is translocated to the membrane upon degranulation. Once displayed, CEACAM6 resides on lipid rafts in the plasma membrane. Like CEACAM1, CEACAM6-mediated internalization occurs in an endocytosis-like pathway [39, 41]. CEACAM6-derived signaling has been most commonly described in cancer cells, where it promotes cancer progression by limiting apoptosis and facilitating invasion via c-Src and FAK-dependent signaling (reviewed in Ref. [66]). However, the role of CEACAM6 in neutrophils is less understood. CEACAM6 surface expression is increased in lung neutrophils of patients with severe asthma [67]. However, this was not shown to contribute to the severity of disease. During Candida albicans infection, CEACAM6 ligation with monoclonal antibodies results in increased production of IL-8 [68]. In addition to homodimerization, CEACAM6 can form heterodimers with other CEACAMs, including CEACAM1, and in doing so could facilitate cross-talk with them [29, 30]. The functional role of neutrophil CEACAM6 engagement by its bacterial binding partners remains largely undefined.

Opacity-Associated Proteins and Their Interaction with CEACAMs

Opacity-associated (Opa) proteins are outer-membrane proteins of Gc that function as adhesins, thereby facilitating intimate adherence and subsequent invasion of host cells (reviewed in Ref. [13]). A Gc isolate typically contains around 9–12 genes that encode Opa proteins [69–71]. Each gene is phase variable due to slipped-stranded mispairing of repetitive sequences in the opa signal sequence, allowing individual bacteria to express none, one, or multiple Opa proteins at a given time [72–76]. Extensive opa variation is thought to facilitate immune evasion while enhancing infectivity of certain bacteria in the Gc population. In a recent study assessing Opa diversity in 219 Gc isolates, the majority had 1–2 opa genes in-frame [69]. Opa protein expression is selected for in urethral infection, with production of some Opa proteins predominating over others, although the impact of the predominant Opa proteins on infection is unclear given the sequence variability among Opa proteins and between strains [77–83]. opa genes undergo extensive inter-gene recombination, with limited contribution of mutation and transformation of foreign DNA [69, 84–86]. Interestingly, different Gc isolates maintain identical copies of certain opa genes, suggesting a preference for retaining those specific Opa proteins [69].

Structurally, Opa proteins are integral outer-membrane β-strand barrels with four extracellular loops. Most of the protein, around 60%, consists of four extracellular loops, of which two are hypervariable (HV1 and HV2). As implied by their name, the hypervariable loops are sequence-diverse and highly variable, while the membrane-inserted β-barrel domain is conserved [87, 88]. Most Opa proteins bind to one or more CEACAMs, while a subset bind to heparan sulfate proteoglycans, and some Opa proteins may bind both [24–27, 89–92]. HV1 and HV2 are essential for binding to CEACAMs [93]. Importantly, CEACAM-binding properties of Opa proteins cannot be inferred by sequence alone and require experimental determination, suggesting that the three-dimensional structure of these flexible loops dictates receptor specificity [88, 93]. Opa proteins specifically bind the GFFCC’ face of the N-terminal IgV domains of CEACAMs, with Y34 and I91 on human CEACAM1 being essential for binding [94–96].

Epithelial CEACAM1 was first identified as a receptor for Opa proteins in both Neisseria meningitidis and N. gonorrhoeae and was implicated in bacterial adherence and invasion [24, 27, 97]. CEACAMs 3, 5, and 6 were identified as additional receptors for Gc Opa proteins, with CEACAM3 acting as a phagocytic receptor on neutrophils [25, 26, 57, 98, 99]. Of the Opa proteins that bind CEACAM1, a subset also binds CEACAM3; to date, no Opa proteins that only bind CEACAM3 have been identified. For example, OpaF from strain FA1090 interacts with CEACAM1 but not CEACAM3, whereas FA1090 OpaD and OpaI bind both CEACAM1 and CEACAM3, demonstrating receptor-specific binding profiles among Opa variants [100]. The variability among Opa sequences and changes in CEACAM availability resulting from neutrophil degranulation allude to the array of responses that can occur as a result of Opa-CEACAM interaction on neutrophils.

CEACAM Engagement as a Defense against Gc

CEACAM as a Host Defense

Given the ubiquity of CEACAM1 and its capacity for inhibitory signaling, a variety of pathogens have been shown to co-opt CEACAM1 and other CEACAMs to colonize and invade epithelial sites. Beyond the Opa proteins of pathogenic Neisseria, H. pylori uses HopQ, M. catarrhalis uses UspA1, and Haemophilus influenzae (biovar aegyptius) uses OMP P1 to facilitate cellular attachment and invasion in their respective tissues [9, 10, 12]. Neutrophils counteract these pathogens bearing CEACAM-binding ligands by expressing the structurally similar but functionally distinct CEACAM3, which acts as a “decoy receptor” to trap and kill pathogens. The idea that CEACAM3 is a recently evolved immune defense against specific pathogens is reflected in its species specificity; primates are the only organisms possessing CEACAM3 [4, 101]. Mice, rabbits, and other species commonly used in laboratory studies lack CEACAM3 or any related homolog. Outside of primary human neutrophils, which are terminally differentiated and cannot be genetically manipulated, approaches used to study the function of human CEACAMs, including CEACAM3, include cells from CEACAM-transgenic mice and cell lines (human or non-human) that are engineered to ectopically express one or more human CEACAMs.

Phagocytic Killing and Neutrophil Activation

In alignment with the role of CEACAM3 in phagocytic clearance of other microbes, Gc producing Opa proteins that bind CEACAM3 (and other CEACAMs), such as FA1090 OpaD, exhibit rapid phagocytosis and killing by human neutrophils ex vivo, compared with Gc variants that do not express Opa proteins, or Opa variants that do not engage CEACAM3 [26, 42, 57, 100, 102–107]. After phagocytosis, primary and secondary granules are trafficked to deliver defensin and cathelicidin antimicrobial peptides, proteases, myeloperoxidase, and NADPH oxidase, along with other antimicrobial species to the bacteria-containing phagosome (reviewed in Ref. [108]). These defenses kill some, but not all, intracellular Gc [23, 104, 105, 109–120]; while CEACAM3 engagement by Gc induces robust ROS production, ROS activity is dispensable for killing Gc [105, 106, 115, 121–123]. Src family kinase activity is required for phagosome maturation and subsequent killing of Gc that had been phagocytosed via CEACAM3, while Syk and PI3K activity were dispensable for these activities [104]. Murine neutrophils expressing human CEACAM3 similarly exhibit an Opa-dependent phagocytic killing, oxidative burst, and granule mobilization in response to Gc infection [42, 64]. Intriguingly, Gc-containing phagosomes in patient exudates do not immunostain for primary granule components, suggesting additional contributors in vivo used by Gc to manipulate granule trafficking and promote intracellular bacterial survival in human neutrophils [110].

In neutrophils from CEACAM3-transgenic mice, CEACAM3 engagement by Gc initiates an intracellular signaling cascade resulting in induction of an NF-κB-mediated pro-inflammatory transcriptional program and consequent cytokine production [64]. Engagement of CEACAM3 by M. catarrhalis in primary human neutrophils elicits a similar CEACAM3-driven NF-κB pro-inflammatory response [62]. In neutrophil-like HL-60 cells, NF-κB activation delays neutrophil apoptosis during Gc infection [124], in keeping with reports that Gc infection prolongs neutrophil lifespan [125, 126]. However, CEACAM ligation, NF- κB activity, and neutrophil lifespan during Gc infection have not yet been directly linked.

Cross-Talk with Other Signaling Pathways Induced by N. gonorrhoeae

CEACAM3 can cross-talk with and amplify other activating signals during Gc infection. For instance, Gc engages TLR2 via outer membrane protein porin and other lipoproteins and TLR4 via its lipooligosaccharide (LOS) [127–132]. TLR4 signaling intersects with CEACAM3 signaling at the CARD9-BCL10-MALT1 complex, which is activated downstream of both Syk in the case of CEACAM3 and MyD88 in the case of TLR4 [133]. This complex is a key player in NF-κB activation downstream of both receptors, and its dual activation by CEACAM3 and TLR4 promotes the pro-inflammatory cytokine response to Gc infection [133]. TLR2 signals similarly through the MyD88 pathway to amplify CEACAM3-derived signals [133].

Activation of neutrophils, including by CEACAM3 engagement, triggers degranulation of both primary and secondary granules, increasing levels of CEACAM6 and CEACAM1 on the neutrophil surface, respectively [8, 38]. The consequences of this increase in surface CEACAM1 and 6 remain to be fully explored. While they are expected to increase the potential for interaction with Opa-expressing Gc, the differential signaling downstream of each CEACAM may alter phagocytosis and neutrophil activation.

CEACAM1 canonically elicits inhibitory signaling via its ITIM in a SHP1/2-dependent manner, as described in T cells, where Opa-expressing Gc suppresses TCR-derived signaling in a CEACAM1-dependent manner [47]. SHP-1 can dephosphorylate both Syk and Vav, and CEACAM1 has been shown to counteract TLR-derived Syk activation in neutrophils [49, 53, 54]. In theory, CEACAM1-derived SHP activation would directly counteract CEACAM3-derived signaling, as well. However, the ability of CEACAM1 to inhibit CEACAM3-derived signals has not yet been directly shown in neutrophils. In fact, there is evidence of the opposite: Gc expressing Opa proteins that bind CEACAM1, but not CEACAM3, were found to augment CEACAM3-dependent signals in neutrophil-like mouse MPRO cells expressing both receptors [42]. Interpretation of these results is complicated by the overexpression of these receptors, which may not reflect the signaling events in human neutrophils. Thus, the molecular basis of CEACAM1 and 3 cooperation or competition in neutrophils during Gc infection remains unresolved.

Regardless, it is likely that other ITIM-containing receptors that can also be engaged during Gc infection elicit signaling to counteract CEACAM3-derived signals (see [engaging inhibitory receptors to impede CEACAM3-driven signals] below). From this perspective, it is intriguing that two negative regulators that directly interact with CEACAM3 have been identified: GRB14 and PTPRJ [134, 135]. GRB14 is an SH2-containing adaptor protein that can bind the 230 phosphorylation site of CEACAM3 [135]. While GRB14 lacks enzymatic activity, its association with CEACAM3 prevents phagocytic activity, presumably by preventing binding of signaling-competent adaptors. Conversely, PTPRJ is a receptor-type protein tyrosine phosphatase that directly dephosphorylates both Y230 and Y241 of CEACAM3 to inhibit its downstream functions [134]. The deletion of PTPRJ in HL-60 cells overexpressing CEACAM3 results in increased CEACAM3 phagocytic activity, and overexpression of PTPRJ in the same cells diminishes CEACAM3 phagocytic activity. It is an open question if and how pathogens including Gc take advantage of these proteins to dampen CEACAM3 signals.

Gonococci Manipulate and Evade CEACAM-Dependent Killing by Human Neutrophils

Although neutrophils possess multiple bactericidal mechanisms, Gc has evolved ways to evade killing by neutrophils, specifically the pathways elicited by CEACAM3. These include skewing the population toward an Opa-negative phenotype, selection against Opa proteins that bind CEACAM3, recruiting soluble host factors, and simultaneously engaging inhibitory neutrophil receptors (Fig. 2).

Fig. 2.

N. gonorrhoeae evasion of CEACAM3-mediated killing in neutrophils. ITAM, immunoreceptor tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; C4BP, C4b-binding protein; CR3, complement receptor 3; CMP-NANA, cytidine 5′-monophospho-N-acetylneuraminic acid.

Phase Variation to an Opa-Negative State

Non-opsonized Opa-negative Gc is less efficiently internalized by neutrophils, shows better survival, and does not elicit an oxidative burst when encountering human neutrophils [26, 99, 100, 103, 104, 107, 115, 136–139]. Despite the selection for Opa expressors observed in vivo [77, 78, 80–82, 140, 141], a fraction of progeny will undergo phase variation and not express Opa proteins. These Opa-negative bacteria are internalized via complement receptor 3 (CR3, CD11b/CD18). CR3 is a highly expressed integrin that is critical for the chemotaxis and phagocytic activity of neutrophils and macrophages [142]. In comparison to CEACAM3-mediated phagocytosis, CR3-mediated phagocytosis of Gc is “silent”: it does not elicit kinase-dependent signaling events, the oxidative burst, or mobilization of primary granules [143]. As a result, the bacteria reside in immature phagosomes that lack full bactericidal activity [110]. The CR3 canonical ligand is the inactive complement fragment iC3b, and complement-opsonized Gc interacts with primary human neutrophils similarly to Opa-negative Gc, except phagocytosis is more rapid [110, 144]. In contrast, IgG-opsonized Gc, phagocytosed via Fcγ receptors, triggers ROS production and formation of primary granule-containing phagolysosomes, implicating the route of phagocytosis as a driver of survival outcomes for Gc [104, 115, 145].

Phase variation to avoid CEACAM3 engagement: just as Gc can phase-vary off Opa expression, the fact that each opa gene undergoes slipped-strand mispairing during DNA replication means that progeny expressing different Opa repertoires continually arise. In male volunteers infected with predominantly Opa-negative Gc, there is in vivo selection for particular Opa proteins that persist throughout the course of the infection [77, 78, 146]. For strain FA1090, the Opa proteins predominating over the course of infection are not strong CEACAM3 binders [77, 147]. In a study using clinical specimens for Opa expression and assessment of CEACAM binding profiles, there was in vivo selection for expression of phase-variable Opa proteins that bind CEACAM1 and CEACAM5 but selection against expression of Opa variants that bind CEACAM3 [102]. Furthermore, some clinical isolates from disseminated Gc infection express Opa variants that bind epithelial CEACAMs (like CEACAM1) but do not bind CEACAM3 [148]. These in vivo investigations raise the question as to why Gc maintains Opa variants that bind to CEACAM3 at all. Given that Opa structural and not sequence features of its extracellular loops affect CEACAM engagement (see above), the ability of any variant to engage different CEACAMs will vary in unpredictable ways. However, there are additional ways beyond the Opa sequence or expression that Gc uses to avoid CEACAM3 engagement.

Recruiting soluble host factors to impede CEACAM3-mediated phagocytosis: our group made the unexpected observation that serum-opsonized Opa-expressing Gc was less, not more, phagocytosed by human neutrophils. We identified C4b-binding protein (C4BP) as the serum component mediating this effect. C4BP is a ∼570 kDa protein complex that binds directly to the gonococcal surface via outer membrane protein porin, where it inhibits complement activation on Gc [149–151]. C4BP is both necessary and sufficient to suppress the non-opsonic phagocytosis and oxidative burst elicited by Opa-expressing Gc [152]. However, C4BP did not affect phagocytosis of Opa-negative bacteria, bacteria expressing a non-CEACAM-binding Opa protein, or IgG-opsonized bacteria. In a recent study, we used Chinese hamster ovary cells transfected to express human CEACAMs to show that C4BP directly inhibits Opa-CEACAM interactions, with a more potent inhibitory effect on CEACAM3 compared to CEACAM1 [153]. Using chimeric CEACAM constructs, this degree of inhibition was attributed to the shorter extracellular domain of CEACAM3. Conversely, smaller, nonmultimeric versions of C4BP poorly inhibited Opa-CEACAM interactions on CHO cells or human neutrophils. These results are consistent with the hypothesis that C4BP sterically hinders Opa-CEACAM3 interactions. It remains to be explored if and how Gc recruits other soluble factors to its surface that impede CEACAM-mediated phagocytic killing.

Engaging inhibitory receptors to impede CEACAM3-driven signals: sialic acid-binding Ig-type lectins (Siglecs) are a family of proteins on neutrophils and other cells that are important for self-recognition and preventing cellular complement deposition (reviewed in Ref. [154]). In line with these activities, many Siglecs, including Siglecs 5 and 9 on human neutrophils, contain ITIMs to dampen inflammatory signals [155–158]. Under homeostatic conditions, Siglecs engage terminal sialic acid residues displayed on host cell glycoproteins and glycolipids (reviewed in Refs. [154, 159]). Conversely, many pathogens decorate their surface with sialic acid to exploit these inhibitory receptors and evade immune clearance [160–163]. Gc modifies its LOS to incorporate sialic acid by scavenging the sialic acid precursor cytidine-5′-monophosphate-N-acetylneuraminic acid from the host, using bacterial enzyme LOS sialyltransferase (Lst) [164–173]. Sialylated Gc is found in urethral secretions and is more resistant to lysis by human complement [166, 174–180]. Independent of complement, sialylated, Opa-expressing Gc elicits less ROS production from human neutrophils and less degranulation [161, 181]. We found that antibody-mediated blockade of Siglecs restored neutrophil ROS production by sialylated Opa-expressing bacteria, implicating Siglecs as the transducer of a signal to dampen CEACAM3-dependent neutrophil activation [161]. In contrast to the effect of C4BP, sialylation does not strongly affect phagocytosis of Opa-expressing Gc, and bacterial survival is slightly enhanced [161, 178, 181], though the impact of sialylation was variable depending on the study. These results imply that rather than impeding CEACAM3 engagement, sialylation stimulates Siglecs to send inhibitory signals to counteract neutrophil activation; how this is achieved is a topic of ongoing study. It is also an open question if sialylation similarly affects other ways in which Gc interacts with neutrophils or with epithelial cells, which have their own array of Siglecs.

Conclusion

In the 30 years since CEACAMs were identified as receptors for Opa-expressing Gc, much has been learned about how CEACAMs promote neutrophil responsiveness to Gc and how Gc attempts to avoid CEACAM-mediated clearance by neutrophils. It still remains to be uncovered how CEACAM signaling changes in response to neutrophil activation state, when different CEACAMs and other receptors are surface-exposed, and the consequences of engaging different CEACAMs on both Gc and neutrophil viability over time. These questions can be addressed with reductionist cell culture systems as well as more complex models, including human CEACAM transgenic mice that can be genitally infected with Opa-expressing Gc, and ex vivo models of human epithelial monolayers where neutrophils transmigrate in response to Gc. It is also important to investigate Opa-CEACAM interactions using currently circulating bacterial isolates in addition to well-defined laboratory strains of Gc. Findings with these systems will shed light on a key aspect of the pathogenesis of gonorrhea and by extension infection by other microbes that engage human CEACAMs.

Acknowledgment

Figures were created in BioRender. https://BioRender.com/yxnvvm5.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This work was supported by the National Institutes of Health (R37AI097312, U19AI189178) (A.K.C.). M.W.B. was supported in part by the National Institutes of Health (T32 AI007046, F31AI188753) and the Robert R. Wagner Fellowship from the University of Virginia. The funders had no role in the design, data collection, data analysis, and reporting of this work.

Author Contributions

M.W.B.: conceptualization, funding acquisition, visualization, writing – original draft, and writing – review and editing. A.M.T.M.: conceptualization, visualization, writing – original draft, and writing – review and editing. A.K.C.: conceptualization, funding acquisition, project administration, supervision, writing – original draft, and writing – review and editing.

Funding Statement

This work was supported by the National Institutes of Health (R37AI097312, U19AI189178) (A.K.C.). M.W.B. was supported in part by the National Institutes of Health (T32 AI007046, F31AI188753) and the Robert R. Wagner Fellowship from the University of Virginia. The funders had no role in the design, data collection, data analysis, and reporting of this work.