Donnie Pickel
Published: 2021
Total Pages: 103
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Antimicrobial resistant Neisseria gonorrhoeae (Ng) is an urgent threat to public health worldwide. Ng causes gonorrhea, the second most prevalent sexually transmitted bacterial infection. Left untreated, gonorrhea can lead to pelvic inflammatory disease, ectopic pregnancy, sterility, and in men can increase susceptibility to HIV. The current recommended treatment is a dual therapy with two antibiotics azithromycin and ceftriaxone. However, many countries are reporting cases with reduced effectiveness of this treatment method. This has placed a greater urgency on the development of preventative therapeutics. There is currently no commercial vaccine available for Ng infection. The development of preventative treatment strategies has long been impeded by Ng’s ability to evade the immune system, particularly the complement system. Ng is an obligate human pathogen and has evolved numerous mechanisms to interact with host-derived molecules that negatively regulate the activation of the immune system. Previous characterization of these mechanisms has focused on one class of complement inhibitors, fluid-phase inhibitors. To fully understand Ng immune evasion and the host molecules that contribute to it, investigating the role of the other class of complement inhibitors, membrane-associated complement inhibitors (mCIs) is needed. mCIs like CD46, CD55, and CD59 are expressed by human epithelial cells to prevent formation of the membrane attack complex on the surface of human cells. I hypothesized that these proteins may be sequestered by Ng during infection and create localized regions of immune activation allowing Ng to persist. To test this, we used a co-culture serum bactericidal assay (ccSBA) where human epithelial cells were infected with Ng, treated with normal human serum, a vehicle for the components of the complement system, and monitored bacterial viability over time. Using the ccSBA we demonstrated that increasing host expression of mCIs resulted in higher levels of Ng survival, while decreasing mCI expression resulted in lower Ng survival. We then used CRISPR to knock out all three of CD46, CD55, and CD59 simultaneously. Surprisingly, this had the opposite effect of what we expected, resulting in a substantial increase in Ng survival. We observed that the lack of mCIs rendered host cells susceptible to complement and speculated that this could result in the release of host-derived molecules that stimulated an alternative mechanism of Ng immune evasion. We used a strain of Ng deficient in this alternative mechanism and found that the absence of mCI expression resulted in a significant reduction in Ng survival. Finally, we expressed each of CD46, CD55, and CD59 individually and found that all three could support Ng survival alone. In summary, this dissertation shows that mCIs are involved in Ng immune evasion. Further it demonstrates the value of the ccSBA in investigating the role of host-derived molecules in Ng immune evasion.
