@phdthesis{Weiss2021, author = {Weiß, Esther}, title = {Host-pathogen interactions of natural killer cells and Aspergillus fumigatus: Relevance of immune cell cross-talk and fungal recognition receptors}, doi = {10.25972/OPUS-20607}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-206077}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {The human pathogen Aspergillus (A.) fumigatus is a fungal mold that can cause severe infections in immunocompromised hosts. Pathogen recognition and immune cell cross-talk are essential for clearing fungal infections efficiently. Immune cell interactions in particular may enhance individual cell activation and cytotoxicity towards invading pathogens. This study analyzed the reciprocal cell activation of natural killer (NK) cells and monocyte-derived dendritic cells (moDCs) after stimulation with A. fumigatus cell wall fractions and whole-cell lysates. Furthermore, the impact of the on moDCs expressed fungal receptors Dectin-1 and TLR-2 on NK cell activation was analyzed. Stimulation of moDCs with ligands for Dectin-1 and TLR-2 and transfer of soluble factors on autologous NK cells showed that moDCs could induce NK cell activation solely by secreting factors. In summary, both cell types could induce reciprocal cell activation if the stimulated cell type recognized fungal morphologies and ligands. However, moDCs displayed a broader set of A. fumigatus receptors and, therefore, could induce NK cell activation when those were not activated by the stimulus directly. Consequently, new fungal receptors should be identified on NK cells. The NK cell characterization marker CD56 was reduced detected in flow cytometry after fungal co-culture. Notably, this decreased detection was not associated with NK cell apoptosis, protein degradation, internalization, or secretion of CD56 molecules. CD56 was shown to tightly attach to hyphal structures, followed by its concentration at the NK-A. fumigatus interaction site. Actin polymerization was necessary for CD56 relocalization, as pre-treatment of NK cells with actin-inhibitory reagents abolished CD56 binding to the fungus. Blocking of CD56 suppressed fungal mediated NK cell activation and secretion of the immune-recruiting chemokines MIP-1α, MIP-1β, and RANTES, concluding that CD56 is functionally involved in fungal recognition by NK cells. CD56 binding to fungal hyphae was inhibited in NK cells obtained from patients during immune-suppressing therapy after allogeneic stem cell transplantation (alloSCT). Additionally, reduced binding of CD56 correlated with decreased actin polymerization of reconstituting NK cells challenged with the fungus. The immune-suppressing therapy with corticosteroids negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES in NK cells after fungal stimulation ex vivo. Similar results were obtained when NK cells from healthy donors were treated with corticosteroids prior to fungal co-culture. Thus, corticosteroids were identified to have detrimental effects on NK cell function during infection with A. fumigatus.}, subject = {Nat{\"u}rliche Killerzelle}, language = {en} } @phdthesis{Peters2021, author = {Peters, Simon}, title = {The impact of sphingolipids on \(Neisseria\) \(meningitidis\) and their role in meningococcal pathogenicity}, doi = {10.25972/OPUS-22623}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-226233}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {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.}, subject = {Neisseria meningitidis}, language = {en} } @article{MachataSreekantapuramHuennigeretal.2021, author = {Machata, Silke and Sreekantapuram, Sravya and H{\"u}nniger, Kerstin and Kurzai, Oliver and Dunker, Christine and Schubert, Katja and Kr{\"u}ger, Wibke and Schulze-Richter, Bianca and Speth, Cornelia and Rambach, G{\"u}nter and Jacobsen, Ilse D.}, title = {Significant Differences in Host-Pathogen Interactions Between Murine and Human Whole Blood}, series = {Frontiers in Immunology}, volume = {11}, journal = {Frontiers in Immunology}, issn = {1664-3224}, doi = {10.3389/fimmu.2020.565869}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-222575}, year = {2021}, abstract = {Murine infection models are widely used to study systemic candidiasis caused by C. albicans. Whole-blood models can help to elucidate host-pathogens interactions and have been used for several Candida species in human blood. We adapted the human whole-blood model to murine blood. Unlike human blood, murine blood was unable to reduce fungal burden and more substantial filamentation of C. albicans was observed. This coincided with less fungal association with leukocytes, especially neutrophils. The lower neutrophil number in murine blood only partially explains insufficient infection and filamentation control, as spiking with murine neutrophils had only limited effects on fungal killing. Furthermore, increased fungal survival is not mediated by enhanced filamentation, as a filament-deficient mutant was likewise not eliminated. We also observed host-dependent differences for interaction of platelets with C. albicans, showing enhanced platelet aggregation, adhesion and activation in murine blood. For human blood, opsonization was shown to decrease platelet interaction suggesting that complement factors interfere with fungus-to-platelet binding. Our results reveal substantial differences between murine and human whole-blood models infected with C. albicans and thereby demonstrate limitations in the translatability of this ex vivo model between hosts.}, language = {en} } @article{KunzRuehlingMoldovanetal.2021, author = {Kunz, Tobias C. and R{\"u}hling, Marcel and Moldovan, Adriana and Paprotka, Kerstin and Kozjak-Pavlovic, Vera and Rudel, Thomas and Fraunholz, Martin}, title = {The Expandables: Cracking the Staphylococcal Cell Wall for Expansion Microscopy}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {11}, journal = {Frontiers in Cellular and Infection Microbiology}, issn = {2235-2988}, doi = {10.3389/fcimb.2021.644750}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-232292}, year = {2021}, abstract = {Expansion Microscopy (ExM) is a novel tool improving the resolution of fluorescence microscopy by linking the sample into a hydrogel that gets physically expanded in water. Previously, we have used ExM to visualize the intracellular Gram-negative pathogens Chlamydia trachomatis, Simkania negevensis, and Neisseria gonorrhoeae. Gram-positive bacteria have a rigid and thick cell wall that impedes classic expansion strategies. Here we developed an approach, which included a series of enzymatic treatments resulting in isotropic 4× expansion of the Gram-positive pathogen Staphylococcus aureus. We further demonstrate the suitability of the technique for imaging of planktonic bacteria as well as endocytosed, intracellular bacteria at a spatial resolution of approximately 60 nm with conventional confocal laser scanning microscopy.}, language = {en} } @article{HempelmannHartlebvanStraatenetal.2021, author = {Hempelmann, Alexander and Hartleb, Laura and van Straaten, Monique and Hashemi, Hamidreza and Zeelen, Johan P. and Bongers, Kevin and Papavasiliou, F. Nina and Engstler, Markus and Stebbins, C. Erec and Jones, Nicola G.}, title = {Nanobody-mediated macromolecular crowding induces membrane fission and remodeling in the African trypanosome}, series = {Cell Reports}, volume = {37}, journal = {Cell Reports}, number = {5}, doi = {10.1016/j.celrep.2021.109923}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-270285}, year = {2021}, abstract = {The dense variant surface glycoprotein (VSG) coat of African trypanosomes represents the primary host-pathogen interface. Antigenic variation prevents clearing of the pathogen by employing a large repertoire of antigenically distinct VSG genes, thus neutralizing the host's antibody response. To explore the epitope space of VSGs, we generate anti-VSG nanobodies and combine high-resolution structural analysis of VSG-nanobody complexes with binding assays on living cells, revealing that these camelid antibodies bind deeply inside the coat. One nanobody causes rapid loss of cellular motility, possibly due to blockage of VSG mobility on the coat, whose rapid endocytosis and exocytosis are mechanistically linked to Trypanosoma brucei propulsion and whose density is required for survival. Electron microscopy studies demonstrate that this loss of motility is accompanied by rapid formation and shedding of nanovesicles and nanotubes, suggesting that increased protein crowding on the dense membrane can be a driving force for membrane fission in living cells.}, language = {en} } @article{DuehringGermerodtSkerkaetal.2015, author = {D{\"u}hring, Sybille and Germerodt, Sebastian and Skerka, Christine and Zipfel, Peter F. and Dandekar, Thomas and Schuster, Stefan}, title = {Host-pathogen interactions between the human innate immune system and Candida albicans - understanding and modeling defense and evasion strategies}, series = {Frontiers in Microbiology}, volume = {6}, journal = {Frontiers in Microbiology}, number = {625}, doi = {10.3389/fmicb.2015.00625}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151621}, year = {2015}, abstract = {The diploid, polymorphic yeast Candida albicans is one of the most important human pathogenic fungi. C. albicans can grow, proliferate and coexist as a commensal on or within the human host for a long time. However, alterations in the host environment can render C. albicans virulent. In this review, we describe the immunological cross-talk between C. albicans and the human innate immune system. We give an overview in form of pairs of human defense strategies including immunological mechanisms as well as general stressors such as nutrient limitation, pH, fever etc. and the corresponding fungal response and evasion mechanisms. Furthermore, Computational Systems Biology approaches to model and investigate these complex interactions are highlighted with a special focus on game-theoretical methods and agent-based models. An outlook on interesting questions to be tackled by Systems Biology regarding entangled defense and evasion mechanisms is given.}, language = {en} }