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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. / Neisseria gonorrhoeae, der Auslöser der sexuell übertragbaren Krankheit Gonorrhö, hat das Potenzial sich im menschlichen Wirt auszubreiten und eine schwere Komplikation, die disseminierende Gonokokkeninfektion (DGI), hervorzurufen. Die Expression des Porins PorBIA, das eines der häufigsten Proteine der äußeren Membran ist, stellt ein Charakteristikum der mit DGI assoziierten Gonokokken dar. PorBIA bindet an SREC-I (scavenger receptor expressed on endothelial cells), der die phosphatabhängige Invasion (low phosphate-dependent invasion LPDI) vermittelt. Dieser Aufnahmemechanismus erlaubt es N. gonorrhoeae Epithel- sowie Endothelzellen, schnell zu invadieren. Wir haben kürzlich gezeigt, dass die neutrale Sphingomyelinase 2 (NSM2), welche die Hydrolyse von Sphingomyelin zu Ceramid und Phosphorylcholin katalysiert, für die LPDI der Gonokokken in nicht-phagozytische Zellen benötigt wird. Dabei spielt die neutrale Sphingomyelinase 2 eine Schlüsselrolle in der frühen PorBIA Signalübertragung, indem sie die PI3 Kinase zu Caveolin rekrutiert. Die darauffolgende Aktivierung von nachgeschalteten Signalwegen, die von der PI3 Kinase abhängig sind, führt zur Aufnahme der Bakterien. Als Teil dieser Arbeit konnte ich die Beteiligung der NSM2 bestätigen. Die Rolle des Enzyms sollte durch die Herstellung von NSM2-spezifischen Antikörpern und einer auf Epithelzellen basierenden NSM2 knockout Zelllinie, die mit Hilfe des CRISPR/Cas9 Systems hergestellt wurde, aufgeklärt werden. Der knockout der NSM2 führte zu einer starken Inhibition der LPDI. Die Invasion konnte jedoch durch die Komplementation mit Hilfe eines NSM2-GFP Konstruktes wiederhergestellt werden. Wobei die Ergebnisse jedoch nicht reproduziert werden konnten. In dieser Arbeit konnte ich die Beteiligung weiterer Mitglieder des Sphingolipid Signalwegs an der PorBIA-vermittelten Invasion zeigen. Die Lipidomanalysen zeigten einen Anstieg der bioaktiven Moleküle Ceramide und Sphingosin aufgrund der Gonokokkeninfektion. Beide Moleküle beeinflussen nicht nur die Wirtszelle, sondern schienen auch Auswirkungen auf die Bakterien selbst zu haben: während Ceramid anscheinend von den Gonokokken aufgenommen wird, ist Sphingosin für die Bakterien toxisch. Weiterhin spielt die Sphingosinkinase 2 (SPHK2) eine wichtige Rolle in der Invasion, da die Inhibierung und der Knockdown des Enzyms die Gonokokkeninfektion negativ beeinflussen. Um die Rolle der Sphingosinkinasen in der Invasion im Detail zu erforschen, wurde in dieser Arbeit ein Aktivitätsassay etabliert. Außerdem wurde der Einfluss der Sphingosin-1-phosphat Lyase (S1PL) auf die Invasion erforscht. Inhibitorstudien und Infektionsexperimente, die mit einer CRISPR/Cas9 HeLa S1PL knockout Zelllinie durchgeführt wurden, zeigten, dass das Enzym nicht nur eine Rolle in der PorBIA-vermittelten, sondern auch in der Opa50/HSPG-vermittelten Gonokokkeninfektion spielt. Die Experimente, die bezüglich der zugrundeliegenden Signalwege durchgeführt wurden, erlaubten die Einordnung der Aktivierung der SPHK und der S1PL im Kontext der Invasion. Wie auch die NSM2, spielen beide Enzyme in der frühen PorBIA Signalübertragung eine Rolle, die schließlich zur Aufnahme der Bakterien führt. Alle diese Ergebnisse weisen auf eine wichtige Rolle der Sphingolipide für die Invasion und das Überleben von N. gonorrhoeae hin. Im letzten Teil dieser Arbeit, wurde die Inhibierung der Apoptose von Neutrophilen aufgrund der Gonokokkeninfektion untersucht. Es konnte gezeigt werden, dass das verspätete Einsetzen der Apoptose von neisseriellen Porinen und Opa Proteinen unabhängig ist. Weiterhin wurde der Einfluss von neisseriellem Peptidoglycan auf die Apoptose der Neutrophilen mit Hilfe von Mutanten untersucht, wobei eine Verbindung nicht bestätigt werden konnte. Da die NSM2 die bedeutendste Sphingomyelinase in Neutrophilen darstellt, sowie vielfältige zellphysiologische Funktionen erfüllt und im Vorfeld schon mit der Apoptose in Verbindung gebracht wurde, wurde der Einfluss des Enzymes auf die Inhibierung der Apoptose durch die Gonokokkeninfektion mit Hilfe von Inhibitoren überprüft.

The aims of this study were (i) to investigate the value of newly developed monoclonal antibody coagglutination reagents for serotyping gonococci according to Protein I variation between strains; (ii) to determine if these or similar monoclonal antibodies could be used in an immunological method for the non -cultural detection of N. gonorrhoeae; and (iii) to produce monoclonal antibodies directed against gonococcal beta- lactamase and to examine their value in a non - cultural diagnostic test for penicillinase- producing gonococci. A total of 1201 gonococcal isolates, obtained from 967 patients attending a sexually transmitted diseases clinic in Edinburgh, were classified serologically using two independently developed panels, each of 14 monoclonal antibodies. The distribution of serogroups WI and WII /III was shown to be 51% and 49% respectively. Using a combination of the two panels of reagents, WI strains could be subdivided into 19 serovars and WII /III strains into 53 serovars: The individual panels recognised 7 (Ph) and 12 (GS) WI serovars and 22 (Ph) and 24 (GS) WII /III serovars. Temporal and geographical variations in serovar patterns were observed, illustrating the dynamic nature of the circulating gonococcal population. A statistically significant correlation between the WII /III serovar combination Bropyt /Back and homosexually acquired infection was found (P < 0.001). This serovar combination accounted for 57% of homosexually acquired infections compared with 3% and 0.8% of infections in heterosexual men and women. A statistically significant correlation between the WII /III serovar Bajk and concomitant rectal infection in women was found (0.05 > P > 0.02). This serovar accounted for 28% of women with genital and rectal infection compared with 17% of women in whom infection was restricted to the genital site. Statistically significant associations between serogroup WII /III and certain serovars and penicillin susceptibility were also observed. In addition to their value in correlating certain strains with particular anatomical sites and in predicting the antibiotic susceptibility of an isolate, these serovar studies proved valuable in tracing infected contacts. A previously developed dot -blot immunoassay using polyclonal antisera raised against whole cell gonococci demonstrated poor sensitivity (26 %) in detecting gonococcal antigen in female cervical specimens: The specificity was 81.7 %. Replacement of polyclonal antisera with a panel of anti -Protein I monoclonal antibodies failed to improve the system, even though coagglutination reagents prepared with these antibodies had detected 99.7% of gonococcal strains. An immunoperoxidase staining technique was developed for the detection of gonococci directly in patient smears. An alternative panel of monoclonal antibodies developed for detection of gonococcal antigen was employed. Coagglutination reagents prepared using these antibodies had previously detected 97% of gonococcal strains. The sensitivity and specificity of this test when applied to 30 male urethral smears was 75% and 100% respectively. However, serovar analysis suggested that the incorporation of additional monoclonal antibodies to the panel would improve the sensitivity of this system. None of the hybridomas obtained from the fusions of myeloma cells and spleen cells from mice immunised with purified gonococcal beta -lactamase enzyme secreted antibodies specific for gonococcal beta -lactamase. It was concluded from this study that (i) the resolution achieved using two panels of monoclonal antibody coagglutination reagents was better than that achieved with either panel alone and increases the value of serotyping for the study of gonococcal epidemiology; (ii) a large variety of antigenic types of gonococci are circulating in the population at any one time and this antigenic pattern does not remain static, therefore constant surveillance of gonococcal strains will be necessary to ensure that immunological detection techniques remain effective; and (iii) the immun peroxidase assay using monoclonal antibodies against PrI has potential for use as an 'on- the -spot' test for the diagnosis of gonococcal infection.