@phdthesis{Solger2021,
  author    = {Solger, Franziska},
  title     = {Central role of sphingolipids on the intracellular survival of \(Neisseria\) \(gonorrhoeae\) in epithelial cells},
  doi       = {10.25972/OPUS-24753},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-247534},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Neisseria gonorrhoeae are Gram-negative bacteria with diplococcal shape. As an obligate human pathogen, it is the causative agent of gonorrhoea, a sexually transmitted disease. Gonococci colonize a variety of mucosal tissues, mainly the urogenital tract in men and women. Occasionally N. gonorrhoeae invades the bloodstream, leading to disseminated gonococcal infection. These bacteria possess a repertoire of virulence factors, which expression patterns can be adapted to the environmental conditions of the host. Through the accumulation of antibiotic resistances and in absence of vaccines, some neisserial strains have the potential to spread globally and represent a major public health threat. Therefore, it is necessary to understand the exact molecular mechanisms underlying the successful infection and progression of gonococci within their host. This deeper understanding of neisserial infection and survival mechanisms is needed for the development of new therapeutic agents. In this work, the role of host-cell sphingolipids on the intracellular survival of N. gonorrhoeae was investigated. It was shown that different classes of sphingolipids strongly interact with invasive gonococci in epithelial cells. Therefore, novel and highly specific clickable sphingolipid analogues were applied to study these interactions with this pathogen. The formation of intra- and extracellular sphingosine vesicles, which were able to target gonococci, was observed. This direct interaction led to the uptake and incorporation of sphingosine into the neisserial membrane. Together with in vitro results, sphingosine was identified as a potential bactericidal reagent as part of the host cell defence. By using different classes of sphingolipids and their clickable analogues, essential structural features, which seem to trigger the bacterial uptake, were detected. Furthermore, effects of key enzymes of the sphingolipid signalling pathway were tested in a neutrophil infection model. In conclusion, the combination of click chemistry and infection biology made it possible to shed some light on the dynamic interplay between cellular sphingosine and N. gonorrhoeae. Thereby, a possible "catch-and-kill" mechanism could have been observed.},
  subject      = {Neisseria gonorrhoeae},
  language  = {en}
}
@phdthesis{Hagen2017,
  author    = {Hagen, Franziska},
  title     = {Sphingolipids in gonococcal infection},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-153852},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, has the potential to spread in the human host and cause a severe complication called disseminated gonococcal infection (DGI). The expression of the major outer membrane porin PorBIA is a characteristic of most gonococci associated with DGI. PorBIA binds to the scavenger receptor expressed on endothelial cells (SREC-I), which mediates the so-called low phosphate-dependent invasion (LPDI). This uptake mechanism enables N. gonorrhoeae to rapidly invade epithelial and endothelial cells in a phosphate-sensitive manner. We recently demonstrated that the neutral sphingomyelinase, which catalyses the hydrolysis of sphingomyelin to ceramide and phosphorylcholine, is required for the LPDI of gonococci in non-phagocytic cells. Neutral sphingomyelinase 2 (NSM2) plays a key role in the early PorBIA signaling by recruiting the PI3 kinase to caveolin. The following activation of the PI3 kinase-dependent downstream signaling leads to the engulfment of the bacteria. As a part of this work, I could confirm the involvement of the NSM2. The role of the enzyme was further elucidated by the generation of antibodies directed against NSM2 and the construction of an epithelium-based NSM2 knockout cell line using CRISPR/Cas9. The knockout of the NSM2 strongly inhibits the LPDI. The invasion could be, however, restored by the complementation of the knockout using an NSM2-GFP construct. However, the results could not be reproduced. In this work, I could show the involvement of further members of the sphingolipid pathway in the PorBIA-mediated invasion. Lipidome analysis revealed an increase of the bioactive molecules ceramide and sphingosine due to gonococcal infection. Both molecules do not only affect the host cell, but seem to influence the bacteria as well: while ceramide seems to be incorporated by the gonococci, sphingosine is toxic for the bacteria. Furthermore, the sphingosine kinase 2 (SPHK2) plays an important role in invasion, since the inhibition and knockdown of the enzyme revealed a negative effect on gonococcal invasion. To elucidate the role of the sphingosine kinases in invasion in more detail, an activity assay was established in this study. Additionally, the impact of the sphingosine-1-phosphate lyase (S1PL) on invasion was investigated. Inhibitor studies and infection experiments conducted with a CRISPR/Cas9 HeLa S1PL knockout cell line revealed a role of the enzyme not only in the PorBIA-mediated invasion, but also in the Opa50/HSPG-mediated gonococcal invasion. The signaling experiments allowed the categorization of the SPHK and S1PL activation in the context of infection. Like the NSM2, both enzymes play a role in the early PorBIA signaling events leading to the uptake of the bacteria. All those findings indicate an important role of sphingolipids in the invasion and survival of N. gonorrhoeae. In the last part of this work, the role of the NSM2 in the inhibition of apoptosis in neutrophils due to gonococcal infection was investigated. It could be demonstrated that the delayed onset of apoptosis is independent of neisserial porin and Opa proteins. Furthermore, the influence of neisserial peptidoglycan on PMN apoptosis was analysed using mutant strains, but no connection could be determined. Since the NSM2 is the most prominent sphingomyelinase in PMNs, fulfils manifold cell physiological functions and has already been connected to apoptosis, the impact of the enzyme on apoptosis inhibition due to gonococcal infection was investigated using inhibitors, with no positive results.},
  subject      = {gonococcal},
  language  = {en}
}