@phdthesis{Deutschmann2023,
  author    = {Deutschmann, Sally},
  title     = {Prävalenz und Epidemiologie von Infektionen mit \(Neisseria\) \(gonorrhoeae\) und deren Resistenzlage gegen{\"u}ber Cephalosporinen der dritten Generation bei tansanischen Patientinnen und Patienten mit bestehender HIV-Infektion eines Referenzkrankenhauses im Nordwesten Tansanias},
  doi       = {10.25972/OPUS-31773},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-317735},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Die Prävalenz von Infektionen mit Neisseria gonorrhoeae (NG) bei HIV-positiven Patientinnen und Patienten einer auf HIV-Infektionen spezialisierten Klinik in Mwanza im Nordwesten Tansani- as erwies sich mit 0,4\% bei Männern und 3,9\% bei Frauen als relativ niedrig. In dieser aktuellen Studie gab es unter Verwendung molekularer Antibiotikaresistenz(AMR)-Tests keine Hinweise auf eine Resistenz gegen Cephalosporine der dritten Generation. Weitere Kontrollen und Entwicklungen von NG- und AMR-Tests m{\"u}ssen implementiert werden. Molekulare Diagnoseverfahren auf der Basis von Urinproben als diagnostisches Material zum Nachweis von NG weisen wesentliche Vorteile f{\"u}r das Screening auf. Die Durchf{\"u}hrung eines Urinstreifentests hatte keinen positiven Vorhersagewert bez{\"u}glich einer Infektion mit NG oder einer AMR. K{\"u}nftige Untersuchungen sind anzuregen, um einerseits eine exaktere Angabe der Prävalenzraten und Risikofaktoren von Infektionen mit NG sowie deren Resistenzlage zu ermöglichen und andererseits eine effizientere Versorgung bzw. Behandlung der Gonorrhoe gewährleisten zu können.},
  subject      = {HIV},
  language  = {de}
}
@article{SteinerZacharyBaueretal.2023,
  author    = {Steiner, Thomas and Zachary, Marie and Bauer, Susanne and M{\"u}ller, Martin J. and Krischke, Markus and Radziej, Sandra and Klepsch, Maximilian and Huettel, Bruno and Eisenreich, Wolfgang and Rudel, Thomas and Beier, Dagmar},
  title     = {Central Role of Sibling Small RNAs NgncR_162 and NgncR_163 in Main Metabolic Pathways of Neisseria gonorrhoeae},
  series = {mBio},
  volume    = {14},
  journal   = {mBio},
  doi       = {10.1128/mbio.03093-22},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-313323},
  year      = {2023},
  abstract  = {Small bacterial regulatory RNAs (sRNAs) have been implicated in the regulation of numerous metabolic pathways. In most of these studies, sRNA-dependent regulation of mRNAs or proteins of enzymes in metabolic pathways has been predicted to affect the metabolism of these bacteria. However, only in a very few cases has the role in metabolism been demonstrated. Here, we performed a combined transcriptome and metabolome analysis to define the regulon of the sibling sRNAs NgncR_162 and NgncR_163 (NgncR_162/163) and their impact on the metabolism of Neisseria gonorrhoeae. These sRNAs have been reported to control genes of the citric acid and methylcitric acid cycles by posttranscriptional negative regulation. By transcriptome analysis, we now expand the NgncR_162/163 regulon by several new members and provide evidence that the sibling sRNAs act as both negative and positive regulators of target gene expression. Newly identified NgncR_162/163 targets are mostly involved in transport processes, especially in the uptake of glycine, phenylalanine, and branched-chain amino acids. NgncR_162/163 also play key roles in the control of serine-glycine metabolism and, hence, probably affect biosyntheses of nucleotides, vitamins, and other amino acids via the supply of one-carbon (C\(_1\)) units. Indeed, these roles were confirmed by metabolomics and metabolic flux analysis, which revealed a bipartite metabolic network with glucose degradation for the supply of anabolic pathways and the usage of amino acids via the citric acid cycle for energy metabolism. Thus, by combined deep RNA sequencing (RNA-seq) and metabolomics, we significantly extended the regulon of NgncR_162/163 and demonstrated the role of NgncR_162/163 in the regulation of central metabolic pathways of the gonococcus.},
  language  = {en}
}
@article{HeydarianRuehlRawaletal.2022,
  author    = {Heydarian, Motaharehsadat and R{\"u}hl, Eva and Rawal, Ravisha and Kozjak-Pavlovic, Vera},
  title     = {Tissue models for Neisseria gonorrhoeae research — from 2D to 3D},
  series = {Frontiers in Cellular and Infection Microbiology},
  volume    = {12},
  journal   = {Frontiers in Cellular and Infection Microbiology},
  issn      = {2235-2988},
  doi       = {10.3389/fcimb.2022.840122},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-263046},
  year      = {2022},
  abstract  = {Neisseria gonorrhoeae is a human-specific pathogen that causes gonorrhea, the second most common sexually transmitted infection worldwide. Disease progression, drug discovery, and basic host-pathogen interactions are studied using different approaches, which rely on models ranging from 2D cell culture to complex 3D tissues and animals. In this review, we discuss the models used in N. gonorrhoeae research. We address both in vivo (animal) and in vitro cell culture models, discussing the pros and cons of each and outlining the recent advancements in the field of three-dimensional tissue models. From simple 2D monoculture to complex advanced 3D tissue models, we provide an overview of the relevant methodology and its application. Finally, we discuss future directions in the exciting field of 3D tissue models and how they can be applied for studying the interaction of N. gonorrhoeae with host cells under conditions closely resembling those found at the native sites of infection.},
  language  = {en}
}
@article{PetersFohmannRudeletal.2021,
  author    = {Peters, Simon and Fohmann, Ingo and Rudel, Thomas and Schubert-Unkmeir, Alexandra},
  title     = {A Comprehensive Review on the Interplay between Neisseria spp. and Host Sphingolipid Metabolites},
  series = {Cells},
  volume    = {10},
  journal   = {Cells},
  number    = {11},
  issn      = {2073-4409},
  doi       = {10.3390/cells10113201},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-250203},
  year      = {2021},
  abstract  = {Sphingolipids represent a class of structural related lipids involved in membrane biology and various cellular processes including cell growth, apoptosis, inflammation and migration. Over the past decade, sphingolipids have become the focus of intensive studies regarding their involvement in infectious diseases. Pathogens can manipulate the sphingolipid metabolism resulting in cell membrane reorganization and receptor recruitment to facilitate their entry. They may recruit specific host sphingolipid metabolites to establish a favorable niche for intracellular survival and proliferation. In contrast, some sphingolipid metabolites can also act as a first line defense against bacteria based on their antimicrobial activity. In this review, we will focus on the strategies employed by pathogenic Neisseria spp. to modulate the sphingolipid metabolism and hijack the sphingolipid balance in the host to promote cellular colonization, invasion and intracellular survival. Novel techniques and innovative approaches will be highlighted that allow imaging of sphingolipid derivatives in the host cell as well as in the pathogen.},
  language  = {en}
}
@phdthesis{Zachary2021,
  author    = {Zachary, Marie},
  title     = {Functional characterization of small non-coding RNAs of \(Neisseria\) \(gonorrhoeae\)},
  doi       = {10.25972/OPUS-24582},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-245826},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {During infection, bacteria need to adapt to a changing environment and have to endure various stress conditions. Small non-coding RNAs are considered as important regulators of bacterial gene expression and so allow quick adaptations by altering expression of specific target genes. Regulation of gene expression in the human-restricted pathogen Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhoea, is only poorly understood. The present study aims a better understanding of gene regulation in N. gonorrhoeae by studying small non-coding RNAs. The discovery of antisense RNAs for all opa genes led to the hypothesis of asRNA-mediated degradation of out-of-frame opa transcripts. Analysis of asRNA expression revealed a very low abundance of the transcripts and inclusion of another phase-variable gene in the study indicates that the asRNAs are not involved in degradation of out-of-frame transcripts. This doctoral thesis focuses on the analysis of trans-acting sRNAs. The sibling sRNAs NgncR_162 and NgncR_163 were discovered as post-transcriptional regulators altering expression of genes involved in metabolic processes, amino acid uptake and transcriptional regulation. A more detailed analysis by in silico and transcriptomic approaches showed that the sRNAs regulate a broad variety of genes coding for proteins of central metabolism, amino acid biosynthesis and degradation and several transport processes. Expression levels of the sibling sRNAs depend on the growth phase of the bacteria and on the growth medium. This indicates that NgncR_162 and NgncR_163 are involved in the adaptation of the gonococcal metabolism to specific growth conditions. This work further initiates characterisation of the sRNA NgncR_237. An in silico analysis showed details on sequence conservation and a possible secondary structure. A combination of in silico target prediction and differential RNA sequencing resulted in the identification of several target genes involved in type IV pilus biogenesis and DNA recombination. However, it was not successful to find induction conditions for sRNA expression. Interestingly, a possible sibling sRNA could be identified that shares the target interaction sequence with NgncR_237 and could therefore target the same mRNAs. In conclusion, this thesis provides further insights in gene regulation by non-coding RNAs in N. gonorrhoeae by analysing two pairs of sibling sRNAs modulating bacterial metabolism or possibly type IV pilus biogenesis.},
  subject      = {Neisseria gonorrhoeae},
  language  = {en}
}
@phdthesis{Heydarian2021,
  author    = {Heydarian, Motaharehsadat},
  title     = {Development of human 3D tissue models for studying \(Neisseria\) \(gonorrhoeae\) infection},
  doi       = {10.25972/OPUS-20496},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204967},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Gonorrhea is the second most common sexually transmitted infection worldwide and is caused by Gram-negative, human-specific diplococcus Neisseria gonorrhoeae. It colonizes the mucosal surface of the female reproductive tract and the male urethra. A rapid increase in antibiotic resistance makes gonorrhea a serious threat to public health worldwide. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are not able to recapitulate all the features of infection. Therefore, a realistic in vitro cell culture model is urgently required for studying the gonorrhea infection. In this study, we established and characterized three independent 3D tissue models based on the porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. The histological, immunohistochemical, and ultra-structural analysis showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in the human host including the formation of epithelial monolayer, underlying connective tissue, mucus production, tight junction (TJ), and microvilli. In addition, functional analysis such as transepithelial electrical resistance (TEER) and barrier permeability indicated high barrier integrity of the cell layer. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results showed disruption of TJs and growing the interleukins production in response to the infection, which depends on the type of strain and cell. In addition, the 3D tissue models supported bacterial survival, which provided an appropriate in vitro model for long-term infection study. This could be mainly because of the high resilience of the 3D tissue models based on the SIS scaffold to the infection in terms of alteration in permeability, cell destruction, and bacterial transmigration. During gonorrhea infection, a high level of neutrophils migrates to the site of infection. The studies also showed that N. gonorrhoeae can survive or even replicate inside the neutrophils. Therefore, studying the interaction between neutrophils and N. gonorrhoeae is substantially under scrutiny. For this purpose, we generated a 3D tissue model by triple co-culturing of human primary fibroblast cells, human colorectal carcinoma cells, and human umbilical vein endothelial cells. The tissue model was subsequently infected by N. gonorrhoeae. A perfusion-based bioreactor system was employed to recreate blood flow in the side of endothelial cells and consequently study human neutrophils transmigration to the site of infection. We observed neutrophils activation upon the infection. Furthermore, we demonstrated the uptake of N. gonorrhoeae by human neutrophils and reverse transmigration of neutrophils to the basal side carrying N. gonorrhoeae. In summary, the introduced 3D tissue models in this research represent a promising tool to investigate N. gonorrhoeae infections under close-to-natural conditions.},
  subject      = {3D-Gewebemodell},
  language  = {en}
}
@phdthesis{Solger2021,
  author    = {Solger, Franziska},
  title     = {Central role of sphingolipids on the intracellular survival of \(Neisseria\) \(gonorrhoeae\) in epithelial cells},
  doi       = {10.25972/OPUS-24753},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-247534},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Neisseria gonorrhoeae are Gram-negative bacteria with diplococcal shape. As an obligate human pathogen, it is the causative agent of gonorrhoea, a sexually transmitted disease. Gonococci colonize a variety of mucosal tissues, mainly the urogenital tract in men and women. Occasionally N. gonorrhoeae invades the bloodstream, leading to disseminated gonococcal infection. These bacteria possess a repertoire of virulence factors, which expression patterns can be adapted to the environmental conditions of the host. Through the accumulation of antibiotic resistances and in absence of vaccines, some neisserial strains have the potential to spread globally and represent a major public health threat. Therefore, it is necessary to understand the exact molecular mechanisms underlying the successful infection and progression of gonococci within their host. This deeper understanding of neisserial infection and survival mechanisms is needed for the development of new therapeutic agents. In this work, the role of host-cell sphingolipids on the intracellular survival of N. gonorrhoeae was investigated. It was shown that different classes of sphingolipids strongly interact with invasive gonococci in epithelial cells. Therefore, novel and highly specific clickable sphingolipid analogues were applied to study these interactions with this pathogen. The formation of intra- and extracellular sphingosine vesicles, which were able to target gonococci, was observed. This direct interaction led to the uptake and incorporation of sphingosine into the neisserial membrane. Together with in vitro results, sphingosine was identified as a potential bactericidal reagent as part of the host cell defence. By using different classes of sphingolipids and their clickable analogues, essential structural features, which seem to trigger the bacterial uptake, were detected. Furthermore, effects of key enzymes of the sphingolipid signalling pathway were tested in a neutrophil infection model. In conclusion, the combination of click chemistry and infection biology made it possible to shed some light on the dynamic interplay between cellular sphingosine and N. gonorrhoeae. Thereby, a possible "catch-and-kill" mechanism could have been observed.},
  subject      = {Neisseria gonorrhoeae},
  language  = {en}
}
@article{HeydarianSchweinlinSchwarzetal.2021,
  author    = {Heydarian, Motaharehsadat and Schweinlin, Matthias and Schwarz, Thomas and Rawal, Ravisha and Walles, Heike and Metzger, Marco and Rudel, Thomas and Kozjak-Pavlovic, Vera},
  title     = {Triple co-culture and perfusion bioreactor for studying the interaction between Neisseria gonorrhoeae and neutrophils: A novel 3D tissue model for bacterial infection and immunity},
  series = {Journal of Tissue Engineering},
  volume    = {12},
  journal   = {Journal of Tissue Engineering},
  doi       = {10.1177/2041731420988802},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259032},
  pages     = {2041731420988802},
  year      = {2021},
  abstract  = {Gonorrhea, a sexually transmitted disease caused by the bacteria Neisseria gonorrhoeae, is characterized by a large number of neutrophils recruited to the site of infection. Therefore, proper modeling of the N. gonorrhoeae interaction with neutrophils is very important for investigating and understanding the mechanisms that gonococci use to evade the immune response. We have used a combination of a unique human 3D tissue model together with a dynamic culture system to study neutrophil transmigration to the site of N. gonorrhoeae infection. The triple co-culture model consisted of epithelial cells (T84 human colorectal carcinoma cells), human primary dermal fibroblasts, and human umbilical vein endothelial cells on a biological scaffold (SIS). After the infection of the tissue model with N. gonorrhoeae, we introduced primary human neutrophils to the endothelial side of the model using a perfusion-based bioreactor system. By this approach, we were able to demonstrate the activation and transmigration of neutrophils across the 3D tissue model and their recruitment to the site of infection. In summary, the triple co-culture model supplemented by neutrophils represents a promising tool for investigating N. gonorrhoeae and other bacterial infections and interactions with the innate immunity cells under conditions closely resembling the native tissue environment.},
  language  = {en}
}
@phdthesis{Yang2020,
  author    = {Yang, Manli},
  title     = {\(Chlamydia\) \(trachomatis\) metabolism during infection and metatranscriptome analysis in \(Neisseria\) \(gonorrhoeae\) coinfected STD patients},
  doi       = {10.25972/OPUS-18499},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-184993},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Chlamydia trachomatis (Ct) is an obligate intracellular human pathogen. It causes blinding trachoma and sexually transmitted disease such as chlamydia, pelvic inflammatory disease and lymphogranuloma venereum. Ct has a unique biphasic development cycle and replicates in an intracellular vacuole called inclusion. Normally it has two forms: the infectious form, elementary body (EB); and the non-infectious form, reticulate body (RB). Ct is not easily amenable to genetic manipulation. Hence, to understand the infection process, it is crucial to study how the metabolic activity of Ct exactly evolves in the host cell and what roles of EB and RB play differentially in Ct metabolism during infection. In addition, Ct was found regularly coinfected with other pathogens in patients who got sexually transmitted diseases (STDs). A lack of powerful methods to culture Ct outside of the host cell makes the detailed molecular mechanisms of coinfection difficult to study. In this work, a genome-scale metabolic model with 321 metabolites and 277 reactions was first reconstructed by me to study Ct metabolic adaptation in the host cell during infection. This model was calculated to yield 84 extreme pathways, and metabolic flux strength was then modelled regarding 20hpi, 40hpi and later based on a published proteomics dataset. Activities of key enzymes involved in target pathways were further validated by RT-qPCR in both HeLa229 and HUVEC cell lines. This study suggests that Ct's major active pathways involve glycolysis, gluconeogenesis, glycerolphospholipid biosynthesis and pentose phosphate pathway, while Ct's incomplete tricarboxylic acid cycle and fatty acid biosynthesis are less active. EB is more activated in almost all these carbohydrate pathways than RB. Result suggests the survival of Ct generally requires a lot of acetyl-CoA from the host. Besides, both EB and RB can utilize folate biosynthesis to generate NAD(P)H but may use different pathways depending on the demands of ATP. When more ATP is available from both host cell and Ct itself, RB is more activated by utilizing energy providing chemicals generated by enzymes associated in the nucleic acid metabolism. The forming of folate also suggests large glutamate consumption, which is supposed to be converted from glutamine by the glutamine-fructose-6-phosphate transaminase (glmS) and CTP synthase (pyrG). Then, RNA sequencing (RNA-seq) data analysis was performed by me in a coinfection study. Metatranscriptome from patient RNA-seq data provides a realistic overview. Thirteen patient samples were collected and sequenced by our collaborators. Six male samples were obtained by urethral swab, and seven female samples were collected by cervicovaginal lavage. All the samples were Neisseria gonorrhoeae (GC) positive, and half of them had coinfection with Ct. HISAT2 and Stringtie were used for transcriptomic mapping and assembly respectively, and differential expression analysis by DESeq2, Ballgown and Cuffdiff2 are parallelly processed for comparison. Although the measured transcripts were not sufficient to assemble Ct's transcriptome, the differential expression of genes in both the host and GC were analyzed by comparing Ct positive group (Ct+) against Ct-uninfected group. The results show that in the Ct+ group, the host MHC class II immune response was highly induced. Ct infection is associated with the regulation of DNA methylation, DNA double-strand damage and ubiquitination. The analysis also shows Ct infection enhances host fatty acid beta oxidation, thereby inducing mROS, and the host responds to reduce ceramide production and glycolysis. The coinfection upregulates GC's own ion transporters and amino acid uptake, while it downregulates GC's restriction and modification systems. Meanwhile, GC has the nitrosative and oxidative stress response and also increases the ability for ferric uptake especially in the Ct+ group compared to Ct-uninfected group. In conclusion, methods in bioinformatics were used here in analyzing the metabolism of Ct itself, and the responses of the host and GC respectively in a coinfection study with and without Ct. These methods provide metabolic and metatranscriptomic details to study Ct metabolism during infection and Ct associated coinfection in the human microbiota.},
  subject      = {chlamydia trachomatis},
  language  = {en}
}
@article{HeydarianYangSchweinlinetal.2019,
  author    = {Heydarian, Motaharehsadat and Yang, Tao and Schweinlin, Matthias and Steinke, Maria and Walles, Heike and Rudel, Thomas and Kozjak-Pavlovic, Vera},
  title     = {Biomimetic human tissue model for long-term study of Neisseria gonorrhoeae infection},
  series = {Frontiers in Microbiology},
  volume    = {10},
  journal   = {Frontiers in Microbiology},
  number    = {1740},
  issn      = {1664-302X},
  doi       = {10.3389/fmicb.2019.01740},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-197912},
  year      = {2019},
  abstract  = {Gonorrhea is the second most common sexually transmitted infection in the world and is caused by Gram-negative diplococcus Neisseria gonorrhoeae. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are only of limited use. Therefore, a suitable in vitro cell culture model for studying the complete infection including adhesion, transmigration and transport to deeper tissue layers is required. In the present study, we generated three independent 3D tissue models based on porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. Functional analyses such as transepithelial electrical resistance (TEER) and FITC-dextran assay indicated the high barrier integrity of the created monolayer. The histological, immunohistochemical, and ultra-structural analyses showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in human host including the epithelial monolayer, the underlying connective tissue, mucus production, tight junction, and microvilli formation. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results indicated that the disruption of tight junctions and increase in interleukin production in response to the infection is strain and cell type-dependent. In addition, the models supported bacterial survival and proved to be better suitable for studying infection over the course of several days in comparison to commonly used Transwell® models. This was primarily due to increased resilience of the SIS scaffold models to infection in terms of changes in permeability, cell destruction and bacterial transmigration. In summary, the SIS scaffold-based 3D tissue models of human mucosal tissues represent promising tools for investigating N. gonorrhoeae infections under close-to-natural conditions.},
  language  = {en}
}