Characterisation of Neisseria gonorrhoeae secreted outer membrane vesicles and their role in immune evasion
Creators: name: Deo, Pankaj
Publisher: name: Figshare
Year: 2017
Subjects: Uncategorized

description: 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). 
  <br>   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.