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Neisseria gonorrhoeae (Gc) is a human-specific pathogen that causes the sexually transmitted infection gonorrhea. Gc survives in neutrophil-rich gonorrheal secretions, and recovered bacteria predominantly express phase-variable, surface-expressed opacity-associated (Opa) proteins (Opa+). However, expression of Opa proteins like OpaD decreases Gc survival when exposed to human neutrophils ex vivo. Here, we made the unexpected observation that incubation with normal human serum, which is found in inflamed mucosal secretions, enhances survival of Opa+ Gc from primary human neutrophils. We directly linked this phenomenon to a novel complement-independent function for C4b-binding protein (C4BP). When bound to the bacteria, C4BP was necessary and sufficient to suppress Gc-induced neutrophil reactive oxygen species production and prevent neutrophil phagocytosis of Opa+ Gc. This research identifies for the first time a complement-independent role for C4BP in enhancing the survival of a pathogenic bacterium from phagocytes, thereby revealing how Gc exploits inflammatory conditions to persist at human mucosal surfaces., Competing Interests: The authors have no competing interests to declare., (Copyright: © 2023 Werner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Neisseria gonorrhoeae (Gc) must overcome the limitation of metals such as zinc to colonize mucosal surfaces in its obligate human host. While the zinc-binding nutritional immunity proteins calprotectin (S100A8/A9) and psoriasin (S100A7) are abundant in human cervicovaginal lavage fluid, Gc possesses TonB-dependent transporters TdfH and TdfJ that bind and extract zinc from the human version of these proteins, respectively. Here we investigated the contribution of zinc acquisition to Gc infection of epithelial cells of the female genital tract. We found that TdfH and TdfJ were dispensable for survival of strain FA1090 Gc that was associated with Ect1 human immortalized epithelial cells, when zinc was limited by calprotectin and psoriasin. In contrast, suspension-grown bacteria declined in viability under the same conditions. Exposure to murine calprotectin, which Gc cannot use as a zinc source, similarly reduced survival of suspension-grown Gc, but not Ect1-associated Gc. We ruled out epithelial cells as a contributor to the enhanced growth of cell-associated Gc under zinc limitation. Instead, we found that attachment to glass was sufficient to enhance bacterial growth when zinc was sequestered. We compared the transcriptional profiles of WT Gc adherent to glass coverslips or in suspension, when zinc was sequestered with murine calprotectin or provided in excess, from which we identified open reading frames that were increased by zinc sequestration in adherent Gc. One of these, ZnuA, was necessary but not sufficient for survival of Gc under zinc-limiting conditions. These results show that adherence protects Gc from zinc-dependent growth restriction by host nutritional immunity proteins.

Human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are a family of receptors that mediate intercellular interactions. Pathogenic bacteria have ligands that bind CEACAMs on human cells. Neisseria gonorrhoeae (Gc) encodes numerous unique outer membrane opacity-associated (Opa) proteins that are ligands for one or more CEACAMs. CEACAMs that are expressed on epithelial cells facilitate Gc colonization, while those expressed on neutrophils affect phagocytosis and consequent intracellular survival of Gc. Since Opa protein expression is phase-variable, variations in receptor tropism affect how individual bacteria within a population interact with host cells. Here we report the development of a rapid, quantitative method for collecting and analyzing fluorescence intensity data from thousands of cells in a population using imaging flow cytometry to detect N-CEACAM bound to the surface of Opa-expressing Gc. We use this method to confirm previous findings regarding Opa-CEACAM interactions and to examine the receptor-ligand interactions of Gc expressing other Opa proteins, as well as for other N-CEACAM proteins. © 2020 International Society for Advancement of Cytometry., (© 2020 International Society for Advancement of Cytometry.)

Neisseria gonorrhoeae (the gonococcus, Gc) triggers a potent inflammatory response and recruitment of neutrophils to the site of infection. Gc survives exposure to neutrophils despite these cells' antimicrobial products, such as reactive oxygen species (ROS). ROS production in neutrophils is initiated by NADPH oxidase, which converts oxygen into superoxide. The subunits of NADPH oxidase are spatially separated between granules (gp91(phox)/p22(phox)) and the cytoplasm (p47(phox), p67(phox), and p40(phox)). Activation of neutrophils promotes the coassembly of NADPH oxidase subunits at phagosome and/or plasma membranes. While Gc-expressing opacity-associated (Opa) proteins can induce neutrophils to produce ROS, Opa-negative (Opa-) Gc does not stimulate neutrophil ROS production. Using constitutively Opa- and OpaD-positive (OpaD+) Gc bacteria in strain FA1090, we now show that the difference in ROS production levels in primary human neutrophils between these backgrounds can be attributed to differential assembly of NADPH oxidase. Neutrophils infected with Opa- Gc showed limited translocation of NADPH oxidase cytoplasmic subunits to cellular membranes, including the bacterial phagosome. In contrast, these subunits rapidly translocated to neutrophil membranes following infection with OpaD+ Gc. gp91(phox) and p22(phox) were recruited to Gc phagosomes regardless of bacterial Opa expression. These results suggest that Opa- Gc interferes with the recruitment of neutrophil NADPH oxidase cytoplasmic subunits to membranes, in particular, the p47(phox) "organizing" subunit, to prevent assembly of the holoenzyme, resulting in an absence of the oxidative burst.

CR3 (CD11b/CD18; αmβ2 integrin) is a conserved phagocytic receptor. The active conformation of CR3 binds the iC3b fragment of complement C3 as well as many host and microbial ligands, leading to actin-dependent phagocytosis. There are conflicting reports about how CR3 engagement affects the fate of phagocytosed substrates. Using imaging flow cytometry, we confirmed that binding and internalization of iC3b-opsonized polystyrene beads by primary human neutrophils was CR3-dependent. iC3b-opsonized beads did not stimulate neutrophil reactive oxygen species, and most beads were found in primary granule-negative phagosomes. Similarly, Neisseria gonorrhoeae that does not express phase-variable Opa proteins suppresses neutrophil reactive oxygen species and delays phagolysosome formation. Here, binding and internalization of Opa-deleted (Δopa) N. gonorrhoeae by adherent human neutrophils was inhibited using blocking antibodies against CR3 and by adding neutrophil inhibitory factor, which targets the CD11b I-domain. No detectable C3 was deposited on N. gonorrhoeae in the presence of neutrophils alone. Conversely, overexpressing CD11b in HL-60 promyelocytes enhanced Δopa N. gonorrhoeae phagocytosis, which required the CD11b I-domain. Phagocytosis of N. gonorrhoeae was also inhibited in mouse neutrophils that were CD11b-deficient or treated with anti-CD11b. Phorbol ester treatment upregulated surface CR3 on neutrophils in suspension, enabling CR3-dependent phagocytosis of Δopa N. gonorrhoeae. Neutrophils exposed to Δopa N. gonorrhoeae had limited phosphorylation of Erk1/2, p38, and JNK. Neutrophil phagocytosis of unopsonized Mycobacterium smegmatis , which also resides in immature phagosomes, was CR3-dependent and did not elicit reactive oxygen species. We suggest that CR3-mediated phagocytosis is a silent mode of entry into neutrophils, which is appropriated by diverse pathogens to subvert phagocytic killing. [ABSTRACT FROM AUTHOR]