Characterisation of Neisseria gonorrhoeae secreted outer membrane vesicles and their role in immune evasion

Neisseria gonorrhoeae is a strict human pathogen that causes the sexually transmitted disease gonorrhoea, which affects approximately 106 million people worldwide. It has evolved a diverse range of strategies to establish long-term colonisation of the human mucosal surfaces. Importantly, N. gonorrhoeae resists clearance by innate immune cells, such as macrophages, which can result in sustained inflammation, tissue damage, and bacterial dissemination. The underlying molecular mechanism of innate immune evasion remains largely unknown, given that there is little evidence that N. gonorrhoeae secrete effector proteins to modulate host cell responses. The major focus of the work described in this thesis was to determine how N. gonorrhoeae controls macrophage immune responses. In particular, I focused on the role of outer membrane vesicles (OMVs) that are shed by an increasing number of bacteria, enabling potential communication with host cells. In chapter 3 of this thesis, I was able to establish a method that allowed me to isolate highly purified OMVs from N. gonorrhoeae. Using quantitative proteomic approaches, I determined that the protein content of purified N. gonorrhoeae OMVs is enriched for outer-membrane proteins, whereby the porin PorB is the most abundant protein, constituting 35 % of the total OMV proteome. Purified OMVs also contained proteins known to be important for N. gonorrhoeae virulence, such as opacity proteins, but also proteins that may affect vesicle formation (NlpD, AmiC, and RmpM) as well as several uncharacterised proteins. In contrast, traditional purification of OMVs resulted in crude preparations that contained numerous cytosolic proteins. PorB has previously been shown to damage the mitochondria of host cells during infection, but the trafficking route from the outer-membrane of N. gonorrhoeae has never been characterised. In Chapter 3, I have now described the cellular route of PorB, which includes the uptake of OMVs by bone marrow-derived macrophages (BMDMs) and trafficking of PorB to mitochondria. In confocal and single molecule localisation microscopy, PorB co-localised to the mitochondrial outer-membrane marker, Tom20. Furthermore, N. gonorrhoeae secreted OMVs induce apoptotic cell death in macrophages, as evidenced by the release of mitochondrial cytochrome c and caspase activation, which followed localisation of PorB to mitochondria. In addition to PorB, host factors also regulate macrophage apoptosis. In this context, I have now shown for the first time that OMVs can induce rapid extrinsic apoptosis dependent on caspase-8 (Chapter 3). In addition, the pro-survival protein BCL-XL prevented OMV-induced intrinsic apoptosis (Chapter 3). Finally, I have uncovered a novel of the pro-apoptotic MCL-1S as an essential host factor that regulates macrophage apoptosis in response to N. gonorrhoeae OMVs (Chapter 3).
Little is known about how Gram-negative bacteria regulate OMV biogenesis. The proteome of N. gonorrhoeae OMVs contains several proteins that may directly, or indirectly, affect OMV biogenesis. In Chapter 4, I have focused on the proteins TsaP, NlpD, FtsN, AmiC. HldA and GmhB, which are thought to function in peptidoglycan (PG) binding, cell division, and lipid chain biosynthesis, and may thus regulate OMV abundance. To directly test this, defined genetic mutants of N. gonorrhoeae were generated and OMV biogenesis was determined. By electron microscopy, I identified that loss of AmiC and NlpD induced hyper-blebbing cellular morphology, resulting in increased levels of OMVs compared to wild-type. Given that there is great interest in developing a vaccine against N. gonorrhoeae based on naturally produced OMVs, these genetically-engineered hyper-blebbing mutants may enable more effective vaccine approaches. Thus, the molecular characterisation of OMVs coupled with the identification of their roles in N. gonorrhoeae infection may open up new strategies to improve immune clearance of Neisseria, as well as dampen inflammatory responses, both of which are of interest in the treatment of gonococcal-induced sexually transmitted infections.