@phdthesis{Bauriedl2020,
  author    = {Bauriedl, Saskia Corinna},
  title     = {The influence of riboregulation on fitness and virulence in Neisseria meningitidis},
  doi       = {10.25972/OPUS-19297},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192978},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Neisseria meningitidis (N. meningitidis) is a human commensal that occasionally causes life-threatening infections such as bacterial meningitis and septicemia. Despite experi-mental evidence that the expression of small non-coding RNAs (sRNAs) as well as the RNA chaperone Hfq affect meningococcal physiology, the impact of RNA-based regula-tion (riboregulation) on fitness and virulence in N. meningitidis is only poorly understood. Therefore, this study addressed these issues using a combination of high-throughput tech-nologies. A differential RNA-sequencing (dRNA-seq) approach was applied to produce a single-nucleotide resolution map of the primary transcriptome of N. meningitidis strain 8013. The dRNA-seq analysis predicted 1,625 transcriptional start sites including 65 putative sRNAs, of which 20 were further validated by northern blot analysis. By Hfq RNA im-munopreci-pitation sequencing a large Hfq-centered post-transcriptional regulatory net-work comprising 23 sRNAs and 401 potential mRNA targets was identified. Rifampicin stability assays demonstrated that Hfq binding confers enhanced stability on its associat-ed sRNAs. Based on these data, the interactions of two paralogous sRNAs and their cog-nate target mRNA prpB were validated in vivo as well as in vitro. Both sRNAs directly repress prpB encoding a methylisocitrate lyse which was previously shown to be involved in meningococcal colonization of the human nasopharynx. Besides the well-described RNA chaperone Hfq, FinO-domain proteins have recently been recognized as a widespread family of RNA-binding proteins (RBPs) with regulatory roles in diverse bacteria. They display an intriguing bandwidth of target sites, ranging from a single RNA pair as recognized by plasmid-encoded FinO to the global RNA regu-lons of enterobacterial ProQ proteins. To better understand the intrinsic targeting mode of this RBP family, in vivo targets of the minimal ProQ protein of N. meningitidis were de-termined. In vivo UV crosslinking with RNA deep sequencing (UV-CLIP) identified as-sociations of ProQ with 16 sRNAs and 166 mRNAs encoding a variety of biological functions and thus revealed ProQ as another global RBP in meningococci. It could be shown that meningococcal ProQ predominantly binds to highly structured RNA regions including DNA uptake sequences (DUS) and rho-independent transcription terminators and stabilizes many of its RNA targets as proved by rifampicin stability experiments. As expected from the large suite of ProQ-bound RNAs, proQ deletion globally affects both gene and protein expression in N. meningitidis, changing the expression levels of at least 244 mRNAs and 80 proteins. Phenotypic analyses suggested that ProQ promotes oxida-tive stress tolerance and UV damage repair capacity, both of which are required for full virulence of N. meningitidis. Together, this work uncovers the co-existence of two major post-transcriptional regulons, one governed by ProQ, the other by Hfq, in N. meningitidis. It further highlights the role of these distinct RBPs and its associated sRNAs to bacterial virulence and indicates that riboregulation is likely to contribute to the way how meningococci adapt to different host niches.},
  subject      = {Neisseria meningitidis},
  language  = {en}
}
@phdthesis{Endres2024,
  author    = {Endres, Leo Maximilian},
  title     = {Development of multicellular \(in\) \(vitro\) models of the meningeal blood-CSF barrier to study \(Neisseria\) \(meningitidis\) infection},
  doi       = {10.25972/OPUS-34621},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-346216},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Neisseria meningitidis (the meningococcus) is one of the major causes of bacterial meningitis, a life-threatening inflammation of the meninges. Traversal of the meningeal blood-cerebrospinal fluid barrier (mBCSFB), which is composed of highly specialized brain endothelial cells (BECs), and subsequent interaction with leptomeningeal cells (LMCs) are critical for disease progression. Due to the human-exclusive tropism of N. meningitidis, research on this complex host-pathogen interaction is mostly limited to in vitro studies. Previous studies have primarily used peripheral or immortalized BECs alone, which do not retain relevant barrier phenotypes in culture. To study meningococcal interaction with the mBCSFB in a physiologically more accurate context, BEC-LMC co-culture models were developed in this project using BEC-like cells derived from induced pluripotent stem cells (iBECs) or hCMEC/D3 cells in combination with LMCs derived from tumor biopsies. Distinct BEC and LMC layers as well as characteristic expression of cellular markers were observed using transmission electron microscopy (TEM) and immunofluorescence staining. Clear junctional expression of brain endothelial tight and adherens junction proteins was detected in the iBEC layer. LMC co-culture increased iBEC barrier tightness and stability over a period of seven days, as determined by sodium fluorescein (NaF) permeability and transendothelial electrical resistance (TEER). Infection experiments demonstrated comparable meningococcal adhesion and invasion of the BEC layer in all models tested, consistent with previously published data. While only few bacteria crossed the iBEC-LMC barrier initially, transmigration rates increased substantially over 24 hours, despite constant high TEER. After 24 hours of infection, deterioration of the barrier properties was observed including loss of TEER and altered expression of tight and adherens junction components. Reduced mRNA levels of ZO-1, claudin-5, and VE-cadherin were detected in BECs from all models. qPCR and siRNA knockdown data suggested that transcriptional downregulation of these genes was potentially but not solely mediated by Snail1. Immunofluorescence staining showed reduced junctional coverage of occludin, indicating N. meningitidis-induced post-transcriptional modulation of this protein, as previous studies have suggested. Together, these results suggest a potential combination of transcellular and paracellular meningococcal traversal of the mBCSFB, with the more accessible paracellular route becoming available upon barrier disruption after prolonged N. meningitidis infection. Finally, N. meningitidis induced cellular expression of pro-inflammatory cytokines and chemokines such as IL-8 in all mBCSFB models. Overall, the work described in this thesis highlights the usefulness of advanced in vitro models of the mBCSFB that mimic native physiology and exhibit relevant barrier properties to study infection with meningeal pathogens such as N. meningitidis.},
  subject      = {Bakterielle Hirnhautentz{\"u}ndung},
  language  = {en}
}
@phdthesis{HagmanngebKischkies2016,
  author    = {Hagmann [geb. Kischkies], Laura Violetta},
  title     = {Stringent response regulation and its impact on ex vivo survival in the commensal pathogen \(Neisseria\) \(meningitidis\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144352},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Neisseria meningitidis is a commensal bacterium which sometimes causes serious disease in humans. Recent studies in numerous human pathogenic bacteria have shown that the stringent response contributes to bacterial virulence. Therefore, this study analyzed the regulation of the stringent response in meningococci and in particular of RelA as well as its contribution to ex vivo fitness in a strain- and condition- dependent manner by using the carriage strain α522 and the hyperinvasive strain MC58 in different in vitro and ex vivo conditions. Growth experiments revealed that both wild-type strains were almost indistinguishable in their ex vivo phenotypes. However, quantitative real time PCR (qRT-PCR) found differences in the gene expression of relA between both strains. Furthermore, in contrast to the MC58 RelA mutant strain α522 deficient in RelA was unable to survive in human whole blood, although both strains showed the same ex vivo phenotypes in saliva and cerebrospinal fluid. Moreover, strain α522 was depended on a short non-coding AT-rich repeat element (ATRrelA) in the promoter region of relA to survive in human blood. Furthermore, cell culture experiments with human epithelial cells revealed that in both strains the deletion of relA resulted in a significantly decreased invasion rate while not significantly affecting adhesion. In order to better understand the conditional lethality of the relA deletion, computational and experimental analyses were carried out to unravel differences in amino acid biosynthetic pathways between both strains. Whereas strain MC58 is able to synthesize all 20 amino acids, strain α522 has an auxotrophy for cysteine and glutamine. In addition, the in vitro growth experiments found that RelA is required for growth in the absence of external amino acids in both strains. Furthermore, the mutant strain MC58 harboring an ATRrelA in its relA promoter region showed improved growth in minimal medium supplemented with L-cysteine and/or L-glutamine compared to the wild-type strain. Contrary, in strain α522 no differences between the wild-type and the ATRrelA deletion mutant were observed. Together this indicates that ATRrelA interferes with the complex regulatory interplay between the stringent response pathway and L-cysteine as well as L-glutamine metabolism. It further suggests that meningococcal virulence is linked to relA in a strain- and condition- depended manner. In conclusion, this work highlighted the role of the stringent response and of non-coding regulatory elements for bacterial virulence and indicates that virulence might be related to the way how meningococci accomplish growth within the host environments.},
  subject      = {Neisseria meningitidis},
  language  = {en}
}
@phdthesis{Muenstermann2022,
  author    = {M{\"u}nstermann, Marcel},
  title     = {The roles of the anaphylatoxin receptors during invasive disease as well as mucosal colonization caused by \(Neisseria\) \(meningitidis\)},
  doi       = {10.25972/OPUS-26975},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-269759},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {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.},
  subject      = {Anaphylatoxine},
  language  = {en}
}
@phdthesis{Pawlik2013,
  author    = {Pawlik, Marie-Christin},
  title     = {Gene expression in the human pathogen Neisseria meningitidis: Adaptation to serum exposure and zinc limitation},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-78758},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {Neisseria meningitidis is a facultative human pathogen that occasionally shows strong resistance against serum complement exposure. Previously described factors that mediate meningococcal serum resistance are for example the capsule, LPS sialylation, and expression of the factor H binding protein. I aimed for identification of novel serum resistance factors, thereby following two approaches, i) the analysis of the impact of global regulators of gene expression on serum resistance; and ii) a comparative analysis of closely related strains differing in serum resistance. (i) Of six meningococcal global regulators of gene expression studied, only mutation of the zinc uptake regulator Zur reduced complement deposition on meningococci. Little was known about meningococcal Zur and regulatory processes in response to zinc. I therefore elucidated the yet unidentified meningococcal Zur regulon comparing the transcriptional response of the N. meningitidis strain MC58 under zinc-rich and zinc-deficient conditions using a common reference design of microarray analysis. The meningococcal Zur regulon comprises 17 genes, of which 15 genes were repressed and two genes were activated at high zinc condition. Amongst the Zur-repressed genes were genes involved in zinc uptake, tRNA modification, and ribosomal assembly. A 23 bp meningococcal consensus Zur binding motif (Zur box) with a conserved central palindrome was established (TGTTATDNHATAACA) and detected in the promoter region of all regulated transcriptional units (genes/operons). In vitro binding of meningococcal Zur to the Zur box of three selected genes was shown for the first time using EMSAs. Binding of meningococcal Zur to DNA depended specifically on zinc, and mutations in the palindromic sequence constrained Zur binding to the DNA motif. ii) Three closely related strains of ST-41/44 cc from invasive disease and carriage which differed in their resistance to serum complement exposure were analysed to identify novel mediators of serum resistance. I compared the strains' gene content by microarray analysis which revealed six genes being present in both carrier isolates, but absent in the invasive isolate. Four of them are part of two Islands of horizontally transferred DNA, i.e. IHT-B and -C. The working group furthermore applied a comprehensive screening assay, a transcriptome and a proteome analysis leading to identification of three target proteins. I contributed to establish the role of these three proteins in serum resistance: The adhesin Opc mediates serum resistance by binding of vitronectin, a negative regulator of the complement system; the hypothetical protein NMB0865 slightly contributes to serum resistance by a yet unknown mechanism; and NspA, recently identified to bind the negative complement regulator factor H, led to considerable reduced complement-mediated killing.},
  subject      = {Komplement <Immunologie>},
  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}
}
@phdthesis{Schielke2010,
  author    = {Schielke, Stephanie},
  title     = {Functional and molecular characterization of FarR - a transcriptional regulator of the MarR family in Neisseria meningitidis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-48550},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2010},
  abstract  = {Neisseria meningitidis is a facultatively pathogenic human commensal and strictly adapted to its niche within the human host, the nasopharynx. Not much is known about the regulatory processes required for adaptation to this environment. Therefore the role of the transcriptional regulator NMB1843, one of the two predicted regulators of the MarR family in the meningococcal genome, was investigated. As this gene displayed a high sequence homology to FarR, the Fatty acid resistance Regulator in N. gonorrhoeae, we designated the meningococcal protein FarR (NmFarR). Homology modeling of this protein revealed a dimeric structure with the characteristic winged helix-turn-helix DNA binding motif of the MarR family. NmFarR is highly conserved among meningococcal strains and expression of farR during exponential growth is controlled post-transcriptionally, being highest in the late exponential phase. By means of electrophoretic mobility shift assays (EMSAs) the direct and specific binding of FarR to the farAB promoter region was shown, comparable to its homologue in gonococci. As FarR is involved in fatty acid resistance in N. gonorrhoeae, susceptibility assays with the medium chain lauric acid (C12:0), the long chain saturated palmitic acid (C16:0) and the long chain unsaturated linoleic acid (C18:2) were performed, testing a wide variety of strains of both species. In contrast to the unusually susceptible gonococci, a high intrinsic fatty acid resistance was detected in almost all meningococcal isolates. The molecular basis for this intrinsic resistance in N. meningitidis was elucidated, showing that both a functional FarAB efflux pump system as well as an intact lipopolysaccharide (LPS) are responsible for palmitic acid resistance. However, even despite circumvention of the intrinsic resistance, FarR could not be connected with fatty acid resistance in meningococci. Instead, FarR was shown to directly and specifically repress expression of the Neisseria adhesin A (nadA), a promising vaccine candidate absent in N. gonorrhoeae. Microarray analyses verified these results and disclosed no further similarly regulated genes, rendering the FarR regulon the smallest regulon in meningococci reported until now. The exact FarR binding site within the nadA promoter region was identified as a 16 bp palindromic repeat and its influence on nadA transcription was proved by reporter gene fusion assays. This repression was also shown to be relevant for infection as farR deficient mutant strains displayed an increased attachment to epithelial cells. Furthermore, farR transcription was attested to be repressed upon contact with active complement components within human serum. Concluding, it is shown that FarR adopted a role in meningococcal host niche adaptation, holding the balance between immune evasion by repressing the highly antigenic nadA and host cell attachment via this same adhesin.},
  subject      = {Transkription <Genetik>},
  language  = {en}
}
@phdthesis{Swiderek2005,
  author    = {Swiderek, Halina},
  title     = {Typing and genome comparison of Neisseria meningitidis by DNA-microarrays},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-13374},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2005},
  abstract  = {In the present thesis, two projects on the use of microarray technology for molecular epidemiology of Neisseria meningitidis have been followed. The first one evaluated microarrays based on polymorphism-directed oligonucleotide design for typing of N. meningitidis adopting the multilocus sequence typing (MLST) concept. The number of oligonucleotides needed to cover all known polymorphisms was much lower compared to the number needed if a tiling strategy would have been chosen. Initial experiments using oligonucleotides 28-32 nucleotides in length, revealed that the applied hybridisation protocols were highly specific. However, despite of several optimisation steps, the rate of misidentification of oligonucleotides remained >1.8\% in consecutive validation experiments using arrays representing the genetic diversity at three MLST loci. This finding led to the assumption that the high density of polymorphic sites and extensive GC-content variations at N. meningitidis MLST loci hindered the successful implementation of MLST microarrays based on polymorphism-directed oligonucleotide design. In the 1980s, the ET-15 clone emerged within the ST-11 complex of N. meningitidis. This new clone was associated with severe meningococcal disease and outbreaks world-wide. Therefore, the goal of the second project was to identify genetic differences between ET-15 strains and other ST-11 strains using whole genome microarray technology. Three genes encoding hypothetical proteins were identified to be present in all ET-15 strains but absent in other ST-11 strains. This finding together with unpublished observation from our group suggested that several genome alterations occurred before the clonal expansion of the ET-15 clone started. The role that these three genes play in the pathogenicity of the ET-15 clone is unclear. The genome comparisons revealed furthermore that studies of the ET-15 clone displayed approximately two-fold less gene content variation than ST-11 strains not belonging to the ET-15 clone. This finding is in accordance with the recent emergence and clonal expansion of the ET-15 variant.},
  subject      = {Neisseria meningitis},
  language  = {en}
}