The human specific gram-negative bacterium Neisseria meningitidis (Nme, meningococci) is a common colonizer of the upper respiratory tract. Upon becoming invasive, Nme can cause meningitis and life-threatening sepsis. The most important immune defense mechanism in invasive meningococcal disease (IMD) is the complement mediated killing of bacteria. The complement cascade is activated through different pathogen associated patterns and finally leads to the lysis of the bacteria by the membrane attack complex. In addition to the direct bacterial killing, the complement system is also an important player in different inflammatory processes. A hallmark of IMD is an overreaction of the immune system and the release of the potent anaphylatoxins C3a and C5a by the complement system is an important factor hereby. There are three anaphylatoxin receptors (ATRs), the C3aR, the C5aR1 and the C5aR2, capable of detecting these anaphylatoxins. It has already been shown that blocking the ATR C5aR1 strongly benefitted the outcome of IMD in a murine sepsis model. However, the roles of ATRs C3aR and C5aR2 in IMD are still unclear. This work aims to analyze the role of these ATRs in meningococcal sepsis and to identify possible underlying mechanisms. Furthermore, a possible involvement of the complement system, the ATRs and the type II CRISPR/Cas system on nasopharyngeal colonization is analyzed. In vivo depletion experiments showed that without neutrophils or monocytes/macrophages the complement system alone was not able to clear a low dose Nme infection, which highlights the importance of cellular components in IMD. Analyzing the role of the ATRs in knock-out mice with high dose Nme infections, revealed that the lack of C5aR2, like the lack of C5aR1, was beneficial for the outcome of meningococcal induced sepsis. In contrast, the lack of C3aR in knock-out mice was detrimental. The positive outcome associated with the C5aRs could be reproduced by using an antagonist against both C5aRs or an antagonist specifically against C5aR1 in WT mice. These findings are giving hope to future therapeutic applications. Next, a possible contribution of neutrophils to this positive outcome was analyzed. Absence of C5aR1 led to a decrease of degranulation by neutrophils in a murine whole blood model, while the other ATRs showed no effect. Neutrophil analysis in human whole blood, on the other hand, revealed a reduced oxidative burst and IL-8 secretion upon inhibition of all three ATRs. A functional difference between the C5aRs and the C3aR in neutrophils was observed in phagocytosis, which was reduced upon C3aR inhibition, but was unaltered with C5aR1 or C5aR2 inhibition. Possible underlying mechanisms in the phosphorylation of ERK1/2 were analyzed in bone marrow derived macrophages isolated from ATR knock-out mice. The later phosphorylation of ERK1/2 in macrophages without C5aR1 or C5aR2 expression might explain, why blocking the C5aRs is beneficial for the outcome of IMD in mice. In contrast to these findings, the colonization of the nasopharynx in huCEACAM 1 expressing mice by Nme did not seem to depend on the Complement system factors C3 and C5 nor the ATRs. Additionally, no difference in the colonization could be observed in this model using Nme mutants lacking different parts of the type 2 CRISPR/Cas system. Conclusively, this work highlights the importance of the complement system, the ATRs and the cellular components in IMD. Contrariwise, these factors did not play a role in the analyzed nasopharyngeal infection model. The beneficial effects of C5aR1 and C5aR2 lack/inhibition in IMD might have medicinal applications, which could support the standard therapies of IMD in the future.

The obligate human pathogen Neisseria meningitidis is a major cause of sepsis and meningitis worldwide. It affects mainly toddlers and infants and is responsible for thousands of deaths each year. In this study, different aspects of the importance of sphingolipids in meningococcal pathogenicity were investigated. In a first step, the acid sphingomyelinase (ASM), which degrades membrane sphingomyelin to ceramide, was studied in the context of meningococcal infection. A requirement for ASM surface activity is its translocation from the lysosomal compartment to the cell surface, a process that is currently poorly understood. This study used various approaches, including classical invasion and adherence assays, flow cytometry, and classical and super resolution immunofluorescence microscopy (dSTORM). The results showed that the live, highly piliated N. meningitidis strain 8013/12 induced calcium-dependent ASM translocation in human brain microvascular endothelial cells (HBMEC). Furthermore, it promoted the formation of ceramide-rich platforms (CRPs). In addition, ASM translocation and CRP formation were observed after treating the cells with pili-enriched fractions derived from the same strain. The importance for N. meningitidis to utilize this pathway was shown by the inhibition of the calcium-dependent ASM translocation, which greatly decreased the number of invasive bacteria. I also investigated the importance of the glycosphingolipids GM1 and Gb3. The results showed that GM1, but not Gb3, plays an important role in the ability of N. meningitidis to invade HBMEC. By combining dSTORM imaging and microbiological approaches, we demonstrated that GM1 accumulated prolifically around bacteria during the infection, and that this interaction seemed essential for meningococcal invasion. Sphingolipids are not only known for their beneficial effect on pathogens. Sphingoid bases, including sphingosine, are known for their antimicrobial activity. In the last part of this study, a novel correlative light and electron microscopy approach was established in the combination with click chemistry to precisely localize azido-functionalized sphingolipids in N. meningitidis. The result showed a distinct concentration-dependent localization in either the outer membrane (low concentration) or accumulated in the cytosol (high concentration). This pattern was confirmed by mass spectrometry on separated membrane fractions. Our data provide a first insight into the underlying mechanism of antimicrobial sphingolipids.