Complement, antimicrobial, and neutrophil responses to Neisseria gonorrhoeae 411 views

Neisseria gonorrhoeae (the gonococcus, Gc) is a Gram-negative bacterium and causative agent of the sexually transmitted infection gonorrhea. Gonorrhea is an urgent public health threat with rising incidence rates and detrimental effects on health and fertility. Gc colonizes its obligate human host at mucosal surfaces where it encounters a robust innate immune defense response including the complement system and neutrophils. However, Gc has evolved many mechanisms to evade killing by the human immune system. Moreover, Gc is rapidly becoming resistant to all classes of antibiotics used to combat this pathogen. Understanding the interplay between these opposing forces will be critical to developing new therapeutics and vaccines against this threat. A major function of the complement system is to generate 10-11nm membrane attack complex pores (MAC) comprised of components C5b-C9 which directly kill Gram-negative pathogens. Here, I found that MAC deposition disrupted both the gonococcal outer and inner membranes and broadly enhanced the activities of antibiotics as well as host-derived antimicrobials. MAC deposition re-sensitized a multidrug-resistant Gc isolate to clinically relevant antibiotics. I also found that complement complexes lacking C9 (2-4nm pores) were capable of anti-gonococcal bactericidal activity and increased activities of antimicrobials <2nm in diameter but that larger antimicrobials >4.5nm in diameter remained ineffectual. These findings shed further light on the biology and multifunctional capacity of the MAC to control bacterial pathogens. Immune control of Gc is also mediated by neutrophils, the first responders to infected mucosa. Interactions with pathogens or soluble factors at the neutrophil surface are critical for neutrophil activation and functions. To characterize neutrophil responses to inflammatory stimuli, I led the design of a designed a 22-color spectral flow cytometry panel profiling primary human neutrophil surface markers for phagocytosis, degranulation, migration, and chemotaxis. Care was taken to maximize panel adaptability to broad research questions in the field of neutrophil biology. This panel revealed that neutrophils exhibit conserved responses to known activating agents and Gc. However, the primary neutrophils demonstrated both intra- and inter-donor variability. Neutrophil activation correlated with infectious dose and number of Gc associated per neutrophil as determined by imaging flow cytometry. Multidimensional analyses identified neutrophil subpopulations in response to Gc infection and showed gonococci expressing opacity-associated adhesins more readily associated with neutrophils. Neutrophil interactions with mucosal pathogens occur at an epithelial surface which the neutrophils must traverse to engage the adhered bacteria. To this end, we examined a Transwell filter support system with an endocervical epithelial layer, infecting Gc, and migrating primary human neutrophils. Preliminary results showed transmigration across an infected epithelium in response to an as of yet unidentified bacterial stimulatory factor. Transepithelial migration was also observed to decrease neutrophil bactericidal activity against Gc compared to a non-transmigration model. Taken together, results from this work expand understanding on how cooperative innate immune effectors respond to and kill Gc. Complement complexes with and without C9 can both control Gc and enhance antimicrobial activity against this increasingly antibiotic-resistant pathogen. Gc induces heterogenic neutrophil activation and phagocytosis of the infecting bacteria with to be discovered impact on Gc viability. Understanding these phenomena can advance therapeutic developments that exploit complement-antimicrobial synergy to combat multidrug-resistant Gc and promote neutrophil subpopulations which better combat gonococci.

PHD (Doctor of Philosophy)

Neisseria gonorrhoeae; Complement; Neutrophil; Antibody

English

2025-04-25