@phdthesis{Yang2021,
  author    = {Yang, Tao},
  title     = {Functional insights into the role of a bacterial virulence factor and a host factor in Neisseria gonorrhoeae infection},
  doi       = {10.25972/OPUS-20895},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-208959},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Neisseria gonorrhoeae (GC) is a human specific pathogenic bacterium. Currently, N. gonorrhoeae developed resistance to virtually all the available antibiotics used for treatment. N. gonorrhoeae starts infection by colonizing the cell surface, followed by invasion of the host cell, intracellular persistence, transcytosis and exit into the subepithelial space. Subepithelial bacteria can reach the bloodstream and disseminate to other tissues causing systemic infections, which leads to serious conditions such as arthritis and pneumonia. A number of studies have well established the host-pathogen interactions during the initial adherence and invasion steps. However, the mechanism of intracellular survival and traversal is poorly understood so far. Hence, identification of novel bacterial virulence factors and host factors involved in the host-pathogen interaction is a crucial step in understanding disease development and uncovering novel therapeutic approaches. Besides, most of the previous studies about N. gonorrhoeae were performed in the conventional cell culture. Although they have provided insights into host-pathogen interactions, much information about the native infection microenvironment, such as cell polarization and barrier function, is still missing. This work focused on determining the function of novel bacterial virulence factor NGFG_01605 and host factor (FLCN) in gonococcal infection. NGFG_01605 was identified by Tn5 transposon library screening. It is a putative U32 protease. Unlike other proteins in this family, it is not secreted and has no ex vivo protease activity. NGFG_01605 knockout decreases gonococcal survival in the epithelial cell. 3D models based on T84 cell was developed for the bacterial transmigration assay. NGFG_01605 knockout does not affect gonococcal transmigration. The novel host factor FLCN was identified by shRNA library screening in search for factors that affected gonococcal adherence and/or internalization. We discovered that FLCN did not affect N. gonorrhoeae adherence and invasion but was essential for bacterial survival. Since programmed cell death is a host defence mechanism against intracellular pathogens, we further explored apoptosis and autophagy upon gonococcal infection and determined that FLCN did not affect apoptosis but inhibited autophagy. Moreover, we found that FLCN inhibited the expression of E-cadherin. Knockdown of E- cadherin decreased the autophagy flux and supported N. gonorrhoeae survival. Both non-polarized and polarized cells are present in the cervix, and additionally, E-cadherin represents different polarization properties on these different cells. Therefore, we established 3-D models to better understand the functions of FLCN. We discovered that FLCN was critical for N. gonorrhoeae survival in the 3-D environment as well, but not through inhibiting autophagy. Furthermore, FLCN inhibits the E-cadherin expression and disturbs its polarization in the 3-D models. Since N. gonorrhoeae can cross the epithelial cell barriers through both cell-cell junctions and transcellular migration, we further explored the roles FLCN and E-cadherin played in transmigration. FLCN delayed N. gonorrhoeae transmigration, whereas the knockdown of E-cadherin increased N. gonorrhoeae transmigration. In summary, we revealed roles of the NGFG_01605 and FLCN-E-cadherin axis play in N. gonorrhoeae infection, particularly in relation to intracellular survival and transmigration. This is also the first study that connects FLCN and human-specific pathogen infection.},
  language  = {en}
}
@phdthesis{Spannaus2015,
  author    = {Spannaus, Ralf},
  title     = {Regulation der foamyviralen Proteaseaktivit{\"a}t},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-113401},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Alle Retroviren prozessieren ihre Pol- und Strukturproteine mit Hilfe der viralen Protease. In dieser Arbeit wurden zentrale Mechanismen der Regulation der foamyviralen Protease untersucht und charakterisiert. Dazu wurde eine chromatographische Virusreinigungsmethode entwickelt und die relative Pol- und Env-Enkapsidierung bestimmt. Foamyviren enthalten weniger Pol als andere Retroviren aber deutlich mehr Env als humane Immunodefizienzviren. Die Pol-Inkorporation k{\"o}nnte durch die limitierte Prozessierung mit nur einer einzigen Schnittstelle in Gag und Pol kompensiert werden. Deshalb wurde untersucht, ob die foamyvirale Protease ein beschr{\"a}nktes Schnittstellenrepertoire aufweist. In Zellkulturen sind die Schnitt-stellenpositionen P2' und P2 auf die Aminos{\"a}urereste Valin und Valin/Asparagin beschr{\"a}nkt. Demnach hat die foamyvirale Protease ein eingeschr{\"a}nkteres Schnittstellenrepertoire als die Protease des humanen Immunodefizienzvirus. Weiterhin wurde hier gezeigt, dass die vollst{\"a}ndige reverse Transkription die Prozessierung von Gag voraussetzt und Proteaseaktivit{\"a}t-defiziente oder Gag-Schnittstellen-defiziente Viren keine vollst{\"a}ndige cDNA bilden k{\"o}nnen. Demnach kompensieren Foamyviren die niedrige Proteasekonzentration, indem sie sicherstellen, dass die reverse Transkription erst nach der Gag-Maturation vollendet werden kann. Weiterhin wird bei humanen Immunodefizienzviren durch die Gag-Maturation die essenzielle Mobilit{\"a}t der wenigen Env-Trimere auf der H{\"u}llmembran getriggert. Die erstmals in dieser Arbeit bei Foamyviren quantifizierte Env-Menge ergab, dass Foamyviren 28 mal mehr Env- pro Gag-Molek{\"u}l als humane Immunodefizienzviren besitzen. Wahrscheinlich dient dieser hohe Env-Gehalt der Kompensation der eingeschr{\"a}nkten Env-Mobilit{\"a}t, die durch die limitierte Gag-Prozessierung an nur einer carboxyterminalen Schnittstelle verursacht wird. Da f{\"u}r die Aktivierung der foamyviralen Protease virale Ribonukleins{\"a}ure ben{\"o}tigt wird, wurde untersucht, welche Pol-Dom{\"a}nen f{\"u}r die Aktivierung der Protease ben{\"o}tigt werden. Im Gegensatz zur Integrase, deren Deletion in reduzierter Proteaseaktivit{\"a}t resultierte, war die funktionelle RNaseH-Dom{\"a}ne essenziell f{\"u}r die Gag-Prozessierung. Die Substitution der foamyviralen RNaseH durch RNaseH-Dom{\"a}nen von anderen Retroviren resultierte in genomunabh{\"a}ngiger Proteaseaktivit{\"a}t in Zellen und genomabh{\"a}ngiger Proteaseaktivit{\"a}t in den rekombinanten Viren. Demnach scheint die dimerstabilisierende Funktion der RNaseH durch direkte Protein-Protein-Interaktion oder durch unspezifische RNA-Bindung verursacht zu werden.},
  subject      = {Spumaviren},
  language  = {de}
}
@phdthesis{Dunkel2013,
  author    = {Dunkel, Nico},
  title     = {Regulation of virulence-associated traits of the human fungal pathogen Candida albicans by nitrogen availability},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83076},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Nitrogen-regulated pathogenesis describes the expression of virulence attributes as direct response to the quantity and quality of an available nitrogen source. As consequence of nitrogen availability, the opportunistic human fungal pathogen Candida albicans changes its morphology and secretes aspartic proteases [SAPs], both well characterized virulence attributes. C. albicans, contrarily to its normally non-pathogenic relative Saccharomyces cerevisiae, is able to utilize proteins, which are considered as abundant and important nitrogen source within the human host. To assimilate complex proteinaceous matter, extracellular proteolysis is followed by uptake of the degradation products through dedicated peptide transporters (di-/tripeptide transporters [PTRs] and oligopeptide transporters [OPTs]). The expression of both traits is transcriptionally controlled by Stp1 - the global regulator of protein utilization - in C. albicans. The aim of the present study was to elucidate the regulation of virulence attributes of the pathogenic fungus C. albicans by nitrogen availability in more detail. Within a genome wide binding profile of Stp1, during growth with proteins, more than 600 Stp1 target genes were identified, thereby confirming its role in the usage of proteins, but also other nitrogenous compounds as nitrogen source. Moreover, the revealed targets suggest an involvement of Stp1 in the general adaption to nutrient availability as well as in the environmental stress response. With the focus on protein utilization and nitrogen-regulated pathogenesis, the regulation of the major secreted aspartic protease Sap2 - additionally one of the prime examples of allelic heterogeneity in C. albicans - was investigated in detail. Thereby, the heterogezygous SAP2 promoter helped to identify an unintended genomic alteration as the true cause of a growth defect of a C. albicans mutant. Additionally, the promoter region, which was responsible for the differential activation of the SAP2 alleles, was delimited. Furthermore, general Sap2 induction was demonstrated to be mediated by distinct cis-acting elements that are required for a high or a low activity of SAP2 expression. For the utilization of proteins as nitrogen source it is also crucial to take up the peptides that are produced by extracellular proteolysis. Therefore, the function and importance of specific peptide transporters was investigated in C. albicans mutants, unable to use peptides as nitrogen source (opt1Δ/Δ opt2Δ/Δ opt3Δ/Δ opt4Δ/Δ opt5Δ/Δ ptr2Δ/Δ ptr22Δ/Δ septuple null mutants). The overexpression of individual transporters in these mutants revealed differential substrate specificities and expanded the specificity of the OPTs to dipeptides, a completely new facet of these transporters. The peptide-uptake deficient mutants were further used to elucidate, whether indeed proteins and peptides are an important in vivo nitrogen source for C. albicans. It was found that during competitive colonization of the mouse intestine these mutants exhibited wild-type fitness, indicating that neither proteins nor peptides are primary nitrogen sources required to efficiently support growth of C. albicans in the mouse gut. Adequate availability of the preferred nitrogen source ammonium represses the utilization of proteins and other alternative nitrogen sources, but also the expression of virulence attributes, like Sap secretion and nitrogen-starvation induced filamentation. In order to discriminate, whether ammonium availability is externally sensed or determined inside the cell by C. albicans, the response to exterior ammonium concentrations of ammonium-uptake deficient mutants (mep1Δ/Δ mep2Δ/Δ null mutants) was investigated. This study showed that presence of an otherwise suppressing ammonium concentration did not inhibit Sap2 proteases secretion and arginine-induced filamentation in these mutants. Conclusively, ammonium availability is primarily determined inside the cell in order to control the expression of virulence traits. In sum, the present work contributes to the current understanding of how C. albicans regulates expression of virulence-associated traits in response to the presence of available nitrogen sources - especially proteins and peptides - in order to adapt its lifestyle within a human host.},
  subject      = {Candida albicans},
  language  = {en}
}