Defining the Roles of Human Carcinoembryonic Antigen-Related Cellular Adhesion Molecules during Neutrophil Responses to Neisseria gonorrhoeae

Abstract

Symptomatic infection of humans with Neisseria gonorrhoeae is characterized by a neutrophil-rich cervical or urethral exudate, suggesting that neutrophils are important both for the clearance of these bacteria and for the pathogenesis of gonorrhea. Neisseria interacts with neutrophils through ligation of human carcinoembryonic antigen related-cellular adhesion molecules (CEACAMs) by their surface-expressed Opa proteins, resulting in bacterial binding, engulfment, and neutrophil activation. Multiple CEACAMs are expressed by human neutrophils, and yet their coexpression has precluded understanding of the relative contribution of each CEACAM to functional responses of neutrophils during neisserial infection. In this work, we directly address the role of each CEACAM during infection by introducing individual human CEACAMs into a differentiated murine MPRO cell line-derived neutrophil model. Murine neutrophils cannot bind the human-restricted Neisseria; however, we show that introducing any of the Opa-binding CEACAMs of human neutrophils (CEACAM1, CEACAM3, and CEACAM6) allows binding and entry of Neisseria into murine neutrophils. While CEACAM1-and CEACAM6-expressing neutrophils bind more bacteria, neisserial uptake via these two receptors unexpectedly proceeds without appreciable neutrophil activation. In stark contrast, neisserial engulfment via CEACAM3 recapitulates the oxidative burst and intracellular granule release seen during human neutrophil infection. Finally, by coexpressing multiple CEACAMs in our model, we show that the expression of CEACAM1 and CEACAM6 potentiate, rather than hinder, CEACAM3-dependent responses of neutrophils, exposing a cooperative role for this family of proteins during neisserial infection of neutrophils. These observations illustrate a divergence in function of CEACAMs in neutrophils and implicate the human-restricted CEACAM3 in the neutrophil innate response to neisserial infection. Symptomatic gonococcal infection is caused by the humanrestricted bacterial pathogen Neisseria gonorrhoeae and involves a massive influx of polymorphonuclear neutrophils (PMNs) into the infected urogenital tract. This results in the characteristic PMN-filled urethral or cervical exudate, which is the hallmark of gonorrhea. PMNs are part of a "first line of defense" against bacterial infection through their prompt recruitment and activation at infected sites, where they internalize and neutralize invading pathogens via the production of reactive oxygen species and the release of antimicrobial agents (27). Recognition of bacteria by PMNs can involve specific binding to host-derived opsonins such as serum complement or immunoglobulins that coat the bacteria; however, the interaction between Neisseria and PMNs can also be opsonin independent (40). Specifically, these microorganisms can bind to and activate neutrophils directly via their colony opacity-associated (Opa) outer membrane proteins, the majority of which bind members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM)...

Innate Recognition by Neutrophil Granulocytes Differs between Neisseria gonorrhoeae Strains Causing Local or Disseminating Infections

“…6A). This is in agreement with previous results, showing that CEACAM3 engagement, but not CEACAM1 binding, by Opa CEA proteins is critical for efficient opsonin-independent uptake by human granulocytes (13,34,41). Furthermore, binding of MS11 Opa CEA-a proteins to CEACAM3 is known to trigger the production of reactive oxygen species by granulocytes and finally results in the destruction of the bacteria (34,42).…”

Selection for a CEACAM Receptor-Specific Binding Phenotype during Neisseria gonorrhoeae Infection of the Human Genital Tract

“…Characterization of individual CEACAM function in neutrophilic cell lines showed that Opa-mediated neutrophil activation is mediated by CEACAM3 signaling, while CEACAM1 and CEACAM6 allowed bacterial internalization without triggering production of reactive oxygen species (ROS) or release of toxic granule proteins (44). However, all of the studies focusing on neutrophil responses to Opa-CEACAM binding have been done exclusively with the commonly studied N. gonorrhoeae strains MS11 (44,45,47,61,62) and FA1090 (63,64).…”

“…We hypothesized that the high prevalence of Opa expression in our LPCIs suggests that Opa proteins facilitate infection. However, since each Opa variant may have a different spectrum of CEACAM binding, since the spectrum of binding has the potential to drastically affect the host cellular response to infection (28,30,32,38,43,44,47), and since the pattern of CEACAM expression varies between sexes and cell types, we sought to characterize the binding specificity of each N. gonorrhoeae isolate.…”

“…However, expression of human CEACAM1 in a mouse allowed persistent colonization by N. meningitidis (37), and a mouse model expressing human CEACAM5 showed increased gonococcal recovery from the lower genital tract (38). In contrast, Opa binding to neutrophil CEACAM3 drives inflammation and gonococcal clearance (39)(40)(41)(42)(43)(44)(45)(46)(47). The specific contribution of Opa binding to individual CEACAMs for N. gonorrhoeae colonization and pathogenesis in humans and how differences in CEACAM distribution between the sexes might affect the outcome of infection have not been addressed.…”

A novel factor H-FC chimeric immunotherapeutic molecule against Neisseria gonorrhoeae

“…To measure survival of gonococci in the presence of PMNs, an Opacity protein (Opa)-negative mutant of N. gonorrhoeae strain FA1090 that was grown in media containing 2 μg/ml CMP-Neu5Ac to sialylated LOS was added to 1 × 10 6 PMNs at a multiplicity of infection (MOI) of 10 (10 bacteria to 1 PMN). Opa-negative N. gonorrhoeae was used because select Opa proteins serve as ligands for human carcinoembryonic antigenrelated cell adhesion molecule 3 (CEACAM3) that is expressed by PMNs and results in phagocytosis (38). FHD1119G/HuFc was added at a concentration of 16.7 μg/ml, followed by 10% human complement (prepared as described above).…”