Variable Expression of Opa Proteins by Neisseria gonorrhoeae Influences Bacterial Association and Phagocytic Killing by Human Neutrophils.

Neisseria gonorrhoeae infection is characterized by local and abundant recruitment of neutrophils. Despite neutrophils' antimicrobial activities, viable N. gonorrhoeae is recovered from infected individuals, leading to the question of how N. gonorrhoeae survives neutrophil attack. One feature impacting N. gonorrhoeae-neutrophil interactions is the phase-variable opacity-associated (Opa) proteins. Most Opa proteins engage human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to facilitate bacterial binding and invasion. Neutrophils express two transmembrane CEACAMs, CEACAM1 and the granulocyte-specific CEACAM3. While N. gonorrhoeae isolated from infected individuals is frequently Opa+, expression of OpaD from strain FA1090, which interacts with CEACAMs 1 and 3, is associated with reduced N. gonorrhoeae survival after exposure to human neutrophils. In this study, we hypothesized that the receptor-binding capability of individual Opa proteins impacts bacterial survival in the presence of neutrophils. To test this hypothesis, we introduced opa genes that are constitutively expressed into a derivative of strain FA1090 with all 11 opa genes deleted. The engineered genes encode Opa proteins that bind CEACAM1 and -3, CEACAM1 but not CEACAM3, or neither CEACAM1 nor -3. N. gonorrhoeae expressing CEACAM3-binding Opa proteins survived significantly less well than bacteria expressing other Opa proteins when exposed to primary human neutrophils. The CEACAM3-binding N. gonorrhoeae had significantly greater association with and internalization by neutrophils. However, once internalized, bacteria were similarly killed inside neutrophils, regardless of Opa expression. Furthermore, Opa expression did not significantly impact neutrophil granule mobilization. Our findings indicate that the extent to which Opa proteins mediate nonopsonic binding is the predominant determinant of bacterial survival from neutrophils. IMPORTANCE Neisseria gonorrhoeae, the cause of gonorrhea, is an urgent-threat pathogen due to increasing numbers of infections and increased antibiotic resistance. Many surface components of N. gonorrhoeae are phase variable, including the Opa protein family of adhesins and invasins. While Opa protein expression is selected for in vivo, bacteria expressing some Opa proteins are readily killed by neutrophils, which are recruited to sites of infection. The reason for this discrepancy has remained unresolved. Our work shows that Opa-dependent differences in bacterial survival after exposure to primary human neutrophils correlates with Opa-dependent bacterial binding and phagocytosis. These findings underscore how the ability of N. gonorrhoeae to change Opa expression through phase variation contributes to bacterial resistance to neutrophil clearance.

Transcriptome-guided metabolic network analysis reveals rearrangements of carbon flux distribution in Neisseria gonorrhoeae during neutrophil co-culture.

The ability of bacterial pathogens to metabolically adapt to the environmental conditions of their hosts is critical to both colonization and invasive disease. Infection with Neisseria gonorrhoeae (the gonococcus, Gc) is characterized by the influx of neutrophils [polymorphonuclear leukocytes (PMNs)], which fail to clear the bacteria and make antimicrobial products that can exacerbate tissue damage. The inability of the human host to clear Gc infection is particularly concerning in light of the emergence of strains that are resistant to all clinically recommended antibiotics. Bacterial metabolism represents a promising target for the development of new therapeutics against Gc. Here, we generated a curated genome-scale metabolic network reconstruction (GENRE) of Gc strain FA1090. This GENRE links genetic information to metabolic phenotypes and predicts Gc biomass synthesis and energy consumption. We validated this model with published data and in new results reported here. Contextualization of this model using the transcriptional profile of Gc exposed to PMNs revealed substantial rearrangements of Gc central metabolism and induction of Gc nutrient acquisition strategies for alternate carbon source use. These features enhanced the growth of Gc in the presence of neutrophils. From these results, we conclude that the metabolic interplay between Gc and PMNs helps define infection outcomes. The use of transcriptional profiling and metabolic modeling to reveal new mechanisms by which Gc persists in the presence of PMNs uncovers unique aspects of metabolism in this fastidious bacterium, which could be targeted to block infection and thereby reduce the burden of gonorrhea in the human population. IMPORTANCE The World Health Organization designated Gc as a high-priority pathogen for research and development of new antimicrobials. Bacterial metabolism is a promising target for new antimicrobials, as metabolic enzymes are widely conserved among bacterial strains and are critical for nutrient acquisition and survival within the human host. Here we used genome-scale metabolic modeling to characterize the core metabolic pathways of this fastidious bacterium and to uncover the pathways used by Gc during culture with primary human immune cells. These analyses revealed that Gc relies on different metabolic pathways during co-culture with human neutrophils than in rich media. Conditionally essential genes emerging from these analyses were validated experimentally. These results show that metabolic adaptation in the context of innate immunity is important to Gc pathogenesis. Identifying the metabolic pathways used by Gc during infection can highlight new therapeutic targets for drug-resistant gonorrhea.