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Mechanically ventilated patients are at risk of ventilator-associated pneumonia, a serious infection of the lungs. Not every ventilated patient develops pneumonia due to a combination of the protective layer of mucus in the airways, the immune system and prophylactic antibiotic therapy. To date, only little was known about the antimicrobial factors produced by humans that protect the lungs against infection. Research described in this thesis was therefore aimed at investigating to what extent the lungs of ventilated patients can inhibit the growth of bacteria, the major causative agent of pneumonia Streptococcus pneumoniae in particular. To this end, the accumulated mucus in the patients’ lungs, sputum, was investigated. The most important conclusion was that sputum can indeed possess antimicrobial activity, explained either by a combination of antibiotics and S. pneumoniae-specific antibodies, or by the innate immune defenses. Thus, sputum may serve as a valuable source of information to unravel the complex interactions between the human host, antimicrobial factors and the microbiome of the lower respiratory tract. A possible consequence of pneumonia is the dissemination of bacteria from the lungs to the bloodstream and the brain, which may lead to meningitis. This thesis describes how this process takes place, and how the so-called choline-binding protein CbpL contributes to invasive pneumococcal infections. In addition, possible future approaches to prevent meningitis caused by this bacterium are proposed.
While ionizing radiation (IR) is a powerful tool in medical diagnostics, nuclear medicine,and radiology, it also is a serious threat to the integrity of genetic material. Mutagenic effects ofIR to the human genome have long been the subject of research, yet still comparatively little isknown about the genome-wide effects of IR exposure on the DNA-sequence level. In this study,we employed high throughput sequencing technologies to investigate IR-induced DNA alterationsin human gingiva fibroblasts (HGF) that were acutely exposed to 0.5, 2, and 10 Gy of 240 kVX-radiation followed by repair times of 16 h or 7 days before whole-genome sequencing (WGS).Our analysis of the obtained WGS datasets revealed patterns of IR-induced variant (SNV and InDel)accumulation across the genome, within chromosomes as well as around the borders of topologicallyassociating domains (TADs). Chromosome 19 consistently accumulated the highest SNVs andInDels events. Translocations showed variable patterns but with recurrent chromosomes of origin(e.g., Chr7 andChr16). IR-induced InDels showed a relative increase in number relative to SNVs anda characteristic signature with respect to the frequency of triplet deletions in areas without repetitiveor microhomology features. Overall experimental conditions and datasets the majority of SNVs pergenome had no or little predicted functional impact with a maximum of 62, showing damagingpotential. A dose-dependent effect of IR was surprisingly not apparent. We also observed a significantreduction in transition/transversion (Ti/Tv) ratios for IR-dependent SNVs, which could point to acontribution of the mismatch repair (MMR) system that strongly favors the repair of transitions overtransversions, to the IR-induced DNA-damage response in human cells. Taken together, our resultsshow the presence of distinguishable characteristic patterns of IR-induced DNA-alterations on agenome-wide level and implicate DNA-repair mechanisms in the formation of these signatures
Overall, the present thesis provides tools for virus characterization. Importantly, the application of the developed tools contributed to the fundamental knowledge of selected, veterinary relevant viruses in terms of their underlying biology and virus-host interaction.
By using in vitro models and full-genome sequencing, important new findings were gained that contributed to the deeper understanding of the selected viruses. Results show that in vitro models can be successfully modified to enable study of specific host factors that are important for viral entry. By genetically modifying a bovine cell line using CRISPR/CAS9 technology , a stable cell culture model was established that is now available to the research community, to study the virus-host interaction of pestiviruses. The model was further used to elucidate the adaptability of bovine viral diarrhea the virus and impact on infectivity and growth. By using deep sequencing, genetic changes that occurred during the adaption process of bovine viral diarrhea virus were identified and linked to the phenotype, allowing the characterization of genetic regions important for virus binding to the host cell.
Whole-genome analysis using deep-sequencing was further used to characterize circulating rabbit haemorrhagic disease virus (RHDV) strains from Germany. The study provides more than 50 full genomes of RHDV strains sampled between 2013 and 2020. Since the virus family is drastically under sampled, in particular in central Europe, these sequences represent a very valuable addition to the field. The investigation led further to the discovery of a novel recombinant virus strain in hares, that is likely still circulating today. This finding is of special interest, since it is the first detection of a recombination event between the genogroups RHDV and European brown hare syndrome virus (EBHSV) of Lagoviruses. It highlights the importance of full genome virus surveillance and the potential risk of virus variants that might evade diagnostic detection.
Serological assay were used to study the persistency of antibodies developed during a natural infection with Schmallenberg virus. It could be shown that these antibodies are long lasting and therefore, re-emergence of this virus in Europe is likely favoured by introduction of naïve animals into a herd and not by decreasing antibody-titers over time.
Overall, the discoveries described in this thesis underline the importance of adequate tools for virus characterization and they give valuable answers to fundamental questions regarding the biology of the different viruses.
New World arenaviruses represent an important group of zoonotic pathogens that pose a serious threat to human health. While some virus species cause severe disease, resulting in hemorrhagic fever and neurological symptoms, other closely related family members exhibit little or no pathogenicity. For instance, Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, while the closely related Tacaribe virus (TCRV) is avirulent in humans. Little is known about host cell responses to infection, or how they contribute to virulence; however, TCRV strongly induces caspase-dependent apoptosis (i.e. non-inflammatory programmed cell death) in infected cells, whereas JUNV does not.
In order to better understand the connection between apoptosis and pathogenesis, we sought to unravel the regulation of pro- and anti-apoptotic signaling in response to arenavirus infection. We demonstrated that apoptosis induced by TCRV proceeds over the mitochondrial-regulated intrinsic pathway and involves activation of p53 (accumulation and phosphorylation), activation of the pro-apoptotic BH3-only factors Puma and Noxa (accumulation), as well as inactivation of another pro-apoptotic factor called Bad (phosphorylation). The regulation of these factors in response to TCRV infection is accompanied by other classical hallmarks of intrinsic apoptosis, such as disorganization of the mitochondrial network, cytochrome c release, PS flipping, caspase cleavage and nuclear condensation. The involvement of the BH3-only factors as key players in regulating TCRV-induced apoptosis could also be validated in knockout cells, which showed either suppressed or increased apoptosis depending on the respective activation (i.e. Puma and Noxa) or inactivation (i.e. Bad) status of the respective BH3 protein. Interestingly, while JUNV does not trigger late stages of apoptosis induction (i.e. caspase activation, nuclear condensation and cell death), we could show that it activates similar upstream pro-apoptotic signaling events including activation of p53, Puma and Noxa. This supports the current hypothesis that JUNV actively evades the induction of apoptosis through the involvement of a mechanism targeting late steps in the apoptotic cascade. Specifically, this model proposes that intrinsic activation is suppressed at the level of caspase activation by JUNV NP, which serves as an alternative substrate for caspase cleavage.
Additionally, in order to identify viral factors associated with the induction of apoptosis, a full genome sequencing of TCRV was performed and contributed to the validation and correction of substantial errors reported in existing sequences for TCRV. With the help of this sequence, correct expression plasmids containing the viral genes for NP, GP and Z were constructed and tested for their ability to induce apoptosis in vitro. This revealed that both TCRV and JUNV Z are triggers for apoptosis, which further supports our finding that JUNV also induces activation of pro-apoptotic factors. Again, consistent with a model where JUNV NP blocks caspase activation directly, co-expression of JUNV Z and NP abrogated caspase activation, while simultaneous expression of TCRV NP and Z still resulted in cell death.
Finally, identification of the specific apoptotic factors involved in regulating TCRV-induced apoptosis (i.e. Bad, Puma and Noxa) and the generation of the respective knockout cell lines allowed us to investigate what influence apoptosis induction has on virus infection. Interestingly, knockout of these factors showed no direct impact on virus growth in Vero cells. However, TCRV particles produced in cells with the individual pro-apoptotic (i.e. Puma and Noxa) or anti-apoptotic (i.e. Bad) factors knocked out showed altered infectivity in primary human monocytes and macrophages, which represent important target cells for arenaviruses. Since TCRV particles that originate from the different knockout cells would be expected to contain different amounts of PS in their envelope (depending on the level of apoptosis taking place), this suggests a role of apoptosis in facilitating PS-receptor-mediated entry and/or PS-receptor signaling through downstream kinases, either of which could be contributing to successful infection in professional phagocytic cells. In particular, phosphorylation of some of the identified factors involved in regulating TCRV-induced apoptosis indicates the involvement of upstream kinases from diverse signaling pathways, some of which also play a role in regulating cytokine production – another host cell reaction that differs significantly between TCRV- and JUNV-infected monocytes and macrophages. As such, these findings represent an exciting basis for a possible connection between apoptotic responses and the regulation of pro- and anti-inflammatory cytokine responses via their associated upstream signaling processes and provide a starting point for future studies that will help us to better understand how these processes contribute to arenavirus pathogenicity.
Summary
Streptococcus pneumoniae (the pneumococcus), a bacterium belonging to the normal flora in the human respiratory tract, continues to be an important pathogen due to its contribution to morbidity and mortality among children, the elderly, and immunocompromised persons. Global estimates of pneumococcal deaths among children declined by 51% between 2000 and 2015. This achievement was mainly due to the introduction of pneumococcal conjugate vaccines (PCVs) in countries with the highest pneumococcal burden. Since May 2012, children in Ghana have been receiving PCV vaccination as part of routine immunization. The continuous monitoring of the pneumococcus after PCV introduction is essential to understand the changing epidemiology of the pathogen in the population.
This study therefore, aims to determine the (1) prevalence, serotypes, and sequence types of pneumococcal isolates, (2) antibiotic susceptibility patterns and the genetic basis for the antibiotic resistance among these pneumococcal isolates, and (3) prevalence of selected virulence genes that have been identified as potential vaccine candidates. Nasopharyngeal swabs were obtained from vaccinated children under five years of age in Cape Coast, Ghana. Six years after PCV implementation, we provide data on the epidemiology of pneumococcal strains circulating among children in Cape Coast Ghana. Standard microbiological and molecular techniques were used to identify and characterize the pneumococcal strains.
Overall, pneumococcal carriage prevalence was 29.4% (151/513). All participating children were fully vaccinated. Of the 26 different serotypes identified, the top five PCV13 serotypes (VT) were 6B, 23F, 19F, 3, 6A and non-PCV13 vaccine serotypes (NVT) were 23B, 13, 11A, 15B, and 34. PCV13 coverage was 38.4%, however, more than half of the isolates were NVT with a coverage rate of 61.6%. The isolates were highly susceptible to levofloxacin, ceftriaxone, vancomycin, and erythromycin. However, marked resistance to cotrimoxazole and tetracycline was observed. The reduction in penicillin resistance (35.8%) as compared to pre-vaccination data (45% - 63%) suggests an attributable effect from PCV13 vaccination. However, penicillin resistance was also detected in some NVT serotypes. Overall, 28.5% of the isolates resistant to three or more different classes of antibiotics were classified as multidrug-resistant (MDR). To analyze the genetic basis for resistance to penicillin, erythromycin and tetracycline, pbp2b, ermB, mefA, and tetM genes were amplified.
Thirty-eight (70%) out of the 54 penicillin-resistant isolates contained the pbp2b resistance gene. Out of the 11 erythromycin-resistant isolates, 7 (63.6) and 4 (36.4%) were positive for the ermB and mefA genes, respectively. The tetM gene was detected in 85 (98.8%) of the 86 tetracycline resistance isolates.
To determine the extent to which potential protein-based vaccines could be protective in Ghanaian children, we sought to determine the prevalence of selected virulence genes among the isolates. The lytA, pavB, and cpsA genes were present in all the carrier isolates. However, psrP, pcpA, pilus islet (PI) PI-1, and PI-2 were present in 62.7%, 87.5%, 11.8%, and 6.5% of the strains, respectively. The psrP and pcpA virulence genes were evenly distributed among all the serotypes. However, the pilus islets were detected in only seven serotypes namely 19F, 6B, 9V, 6A, 13, 11A, and 23B. Five serotype 19F isolates possessed both PI-1 and PI-2. Furthermore, the pilus islets were associated with multidrug resistance.
The predominant NVT serotype 23B and isolates resistant to ≥ 4 antibiotics were analysed by multilocus sequence typing (MLST). Nine known sequence types (STs) and 10 novel STs were identified. Seven out of the 10 new STs belonged to serotype 23B, while the remaining 3 were VTs 6B and 19F. A capsular switch was identified among isolates of ST802, which comprised of both serotype 23F and 19F. The majority of serotype 23B strains belonged to ST172. The ST172 is associated with serotype 23F and a single locus variant (SLV) of internationally disseminated clone ST338 (Colombia23F-26). Consequently, ST172 was characterised with marked antibiotic resistance and with traits of capsular switching. One serotype 6B strain was identified as a SLV of ST273 (Greece6B-22) while two serotype 9V strains belonged to the internationally disseminated clone ST156 (Spain9V-3).
In conclusion, this study showed a marginal decline in overall pneumococcal carriage prevalence, persistence of VTs despite the increase in NVTs, and the occurrence of serotype replacement and capsular switching. In addition, sequence types related to internationally disseminated clones are circulating in Ghana. With the high pcpA and psrP coverage detected,including these genes in protein-based vaccines could provide adequate protection for Ghanaian Children.
Human donor milk (HDM) provides appropriate nutrition and offers protective functionsin preterm infants. The aim of the study is to examine the impact of different storage conditions onthe stability of the human breast milk peptidome. HDM was directly frozen at−80◦C or stored at−20◦C (120 h), 4◦C (6 h), or room temperature (RT for 6 or 24 h). The milk peptidome was profiledby mass spectrometry after peptide collection by ultrafiltration. Profiling of the peptidome covered3587 peptides corresponding to 212 proteins. The variance of the peptidome increased with storagetemperature and time and varied for different peptides. The highest impact was observed whensamples were stored at RT. Smaller but significant effects were still observed in samples stored at4◦C, while samples showed highest similarity to those immediately frozen at−80◦C when storedat−20◦C. Peptide structures after storage at RT for 24 h point to the increased activity of thrombinand other proteases cleaving proteins at lysine/arginine. The results point to an ongoing proteindegradation/peptide production by milk-derived proteases. They underline the need for immediatefreezing of HDM at−20◦C or−80◦C to prevent degradation of peptides and enable reproducibleinvestigation of prospectively collected samples.
The transcriptome of non-coding RNA (ncRNA) species is increasingly focused in Alzheimer’s disease (AD) research. NcRNAs comprise, among others, transfer RNAs, long non-coding RNAs and microRNAs (miRs), each with their own specific biological function. We used smallRNASeq to assess miR expression in the hippocampus of young (3 month old) and aged (8 month old) Tg4-42 mice, a model system for sporadic AD, as well as age-matched wildtype controls. Tg4-42 mice express N-truncated Aβ4–42, develop age-related neuron loss, reduced neurogenesis and behavioral deficits. Our results do not only confirm known miR-AD associations in Tg4-42 mice, but more importantly pinpoint 22 additional miRs associated to the disease. Twenty-five miRs were differentially expressed in both aged Tg4-42 and aged wildtype mice while eight miRs were differentially expressed only in aged wildtype mice, and 33 only in aged Tg4-42 mice. No significant alteration in the miRNome was detected in young mice, which indicates that the changes observed in aged mice are down-stream effects of Aβ-induced pathology in the Tg4-42 mouse model for AD. Targets of those miRs were predicted using miRWalk. For miRs that were differentially expressed only in the Tg4-42 model, 128 targets could be identified, whereas 18 genes were targeted by miRs only differentially expressed in wildtype mice and 85 genes were targeted by miRs differentially expressed in both mouse models. Genes targeted by differentially expressed miRs in the Tg4-42 model were enriched for negative regulation of long-term synaptic potentiation, learning or memory, regulation of trans-synaptic signaling and modulation of chemical synaptic transmission obtained. This untargeted miR sequencing approach supports previous reports on the Tg4-42 mice as a valuable model for AD. Furthermore, it revealed miRs involved in AD, which can serve as biomarkers or therapeutic targets.
The Membrane Transporter OAT7 (SLC22A9) Is Not a Susceptibility Factor for Osteoporosis in Europeans
(2020)
Bone production, maintenance, and modeling are a well-balanced process involving mineralization by osteoblasts and resorption by osteoclasts. Sex steroid hormones, including their conjugated forms, contribute majorly to maintaining this balance. Recently, variants in the SLC22A9 gene have been associated with osteoporosis in Korean females. We had recently shown that SLC22A9, encoding organic anion transporter 7 (OAT7), is an uptake transporter of estrone sulfate and identified several genetic variants in Europeans leading to functional consequences in vitro. We therefore hypothesized that SLC22A9 genetic variants may contribute to the pathophysiology of osteoporosis in Europeans. To test this hypothesis, we examined the associations of SLC22A9 variants with bone quality, fractures, and bone turnover markers. We genotyped SLC22A9 variants in 5,701 (2,930 female) subjects (age range, 20–93 years) extracted from the population-based Study of Health in Pomerania (SHIP and SHIP-TREND) covered by the Illumina Infinium HumanExome BeadChip version v1.0 (Exome Chip). Descriptive data (e.g., history of fractures), ultrasonography of the calcaneus, as well as serum concentrations of carboxy-terminal telopeptide of type I collagen, amino-terminal propeptide of type I procollagen, and vitamin D were determined. Comprehensive statistical analyses revealed no association between low-frequency and rare SLC22A9 variants and bone quality, fractures, and bone turnover markers. Our results indicate that single genetic SLC22A9 variants do not have a major impact on osteoporosis risk prediction in Europeans, yet findings need to be replicated in larger-scale studies.