The complement-independent role of C4b-binding protein in Neisseria gonorrhoeae pathogenesis

Neisseria gonorrhoeae (Gc) is the human-specific pathogen that causes the sexually transmitted infection gonorrhea. Gonorrhea is a significant public health concern with no vaccine, increasing antibiotic resistance, and recurrent infections due to a lack of protective immunity. The hallmark immune response to Gc is an abundant neutrophilic influx. However, these neutrophils are unable to clear the infection, and viable, Opacity-associated (Opa) protein-expressing Gc (Opa+ Gc) are recovered in exudates from infected patients. Although Opa proteins promote colonization, they also engage carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) on the neutrophil surface, which can result in neutrophil activation and antigonococcal activities. How these two opposing outcomes are balanced to favor Opa expression during infection remains enigmatic.

In addition to neutrophils, serum is present in inflamed mucosal secretions. In this research, I unexpectedly found that normal human serum enhanced Opa+ Gc survival from primary human neutrophils from healthy donors. I hypothesized that soluble factor(s) in serum were affecting Gc-neutrophil interactions, and sought to determine the identity of the serum factor(s) responsible for enhanced Gc survival from neutrophils. Using mass spectrometry, I identified C4b-binding protein (C4BP), a canonical complement inhibitor protein, as a candidate protein of interest in serum and secretions relevant to gonorrhea. Using C4BP-depleted serum and purified C4BP, I confirmed that C4BP was necessary and sufficient for serum-mediated enhanced survival of Opa+ Gc.

Next, I characterized how C4BP modulated Gc-neutrophil interactions. C4BP suppressed Gc-induced neutrophil reactive oxygen species production and inhibited neutrophil association with and phagocytosis of Opa+ Gc. These effects required binding of the multimeric alpha chain form of C4BP to the surface of Gc. Though C4BP is a canonical complement inhibitor protein, the effects of C4BP on Gc-neutrophil interactions were complement-independent. To investigate whether C4BP might be disrupting Opa-CEACAM interactions, I developed a quantitative, imaging flow cytometry method to detect N-CEACAM binding to the surface of Opa+ Gc. Ultimately, using a panel of strains constitutively expressing single Opa proteins with unique CEACAM-binding profiles, I found that the role of C4BP in limiting neutrophil phagocytosis was specific to Gc expressing CEACAM-binding Opa proteins, and did not extend to bacteria that engaged neutrophils in other manners.

The results of this work underscore the importance of C4BP to Gc infection, not only as a contributor to serum resistance, but also as a modulator of neutrophil function. In the future, the effect of C4BP during in vivo infections and on therapeutic efficacy should be determined. Additionally, it is not known whether neutrophil signaling downstream of Opa-CEACAM engagement is modulated by C4BP, or if neutrophils process the C4BP bound to the Gc surface. The phenomena described here could extend to the numerous other microbes that bind C4BP or to other complement inhibitor proteins. This research identifies for the first time a complement-independent role for C4BP in enhancing the survival of a pathogenic bacterium from phagocytes, and contributes to our understanding of how Gc persists in neutrophil-rich conditions during infection.