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Trait-dependent effects of biotic and abiotic filters on plant regeneration in Southern Ecuador
(2024)
Tropical forests have always fascinated scientists due to their unique biodiversity. However, our understanding of ecological processes shaping the complexity of tropical rainforests is still relatively poor. Plant regeneration is one of the processes that remain understudied in the tropics although this is a key process defining the structure, diversity and assembly of tropical plant communities. In my dissertation, I combine experimental, observational and trait-based approaches to identify processes shaping the assembly of seedling communities and compare associations between environmental conditions and plant traits across plant life stages. By working along a steep environmental gradient in the tropical mountains of Southern Ecuador, I was able to investigate how processes of plant regeneration vary in response to biotic and abiotic factors in tropical montane forests.
My dissertation comprises three complementary chapters, each addressing an individual research question. First, I studied how trait composition in plant communities varies in relation to the broad- and local-scale environmental conditions and across the plant life cycle. I measured key traits reflecting different ecological strategies of plants that correspond to three stages of the plant life cycle (i.e., adult trees, seed rain and recruiting seedlings). I worked on 81 subplots along an elevational gradient covering a large climatic gradient at three different elevations (1000, 2000 and 3000 m a.s.l.). In addition, I measured soil and light conditions at the local spatial scale within each subplot. My findings show that the trait composition of leaves, seeds and seedlings changed similarly across the elevational gradient, but that the different life stages responded differently to the local gradients in soil nutrients and light availability. Consequently, my findings highlight that trait-environment associations in plant communities differ between large and small spatial scales and across plant life stages.
Second, I investigated how seed size affects seedling recruitment in natural forests and in pastures in relation to abiotic and biotic factors. I set up a seed sowing experiment in both habitat types and sowed over 8,000 seeds belonging to seven tree species differing in seed size. I found that large-seeded species had higher proportions of recruitment in the forests compared to small-seeded species. However, small-seeded species tended to recruit better in pastures compared to large-seeded species. I showed that high surface temperature was the main driver of differences in seedling recruitment between habitats, because it limited seedling recruitment of large-seeded species. The results from this experiment show that pasture restoration requires seed addition of large-seeded species and active protection of recruiting seedlings in order to mitigate harmful conditions associated with high temperatures in deforested areas.
Third, I examined the associations between seedling beta-diversity and different abiotic and biotic factors between and within elevations. I applied beta-diversity partitioning to obtain two components of beta-diversity: species turnover and species richness differences. I associated these components of beta-diversity with biotic pressures by herbivores and fungal pathogens and environmental heterogeneity in light and soil conditions. I found that species turnover in seedling communities was positively associated with the dissimilarity in biotic pressures within elevations and with environmental heterogeneity between elevations. Further, I found that species richness differences increased primarily with increasing environmental heterogeneity within elevations. My findings show that the associations between beta-diversity of seedling communities and abiotic and biotic factors are scale-dependent, most likely due to differences in species sorting in response to biotic pressures and species coexistence in response to environmental heterogeneity.
My dissertation reveals that studying processes of community assembly at different plant life stages and spatial scales can yield new insights into patterns and processes of plant regeneration in tropical forests. I investigated how community assembly processes are governed by abiotic and biotic filtering across and within elevations. I also experimentally explored how the process of seedling recruitment depends on seed size-dependent interactions, and verified how these effects are associated with abiotic and biotic filtering. Identifying such processes is crucial to inform predictive models of environmental change on plant regeneration and successful forest restoration. Further exploration of plant functional traits and their associations with local-scale environmental conditions could effectively support local conservation efforts needed to enhance forest cover in the future and halt the accelerating loss of biodiversity.
Identification of new natural products from nematode-associated bacteria using mass spectrometry
(2023)
This work aims to find unknown natural products produced by bacteria, that live in close association with nematodes and to elucidate their structure by using mass spectrometry.
The first chapter of this work is dedicated to the detection of hitherto unknown natural products by using a metabolomics approach and subsequent structure elucidation of said compounds. This chapter includes metabolomics analysis of Xenorhabdus szentirmaii wild type and knockout mutants, overproduction of the target compound, identification of derivatives from other strains and MS based structure elucidation.
The second and third chapters are about natural products that protect C. elegans from B. thuringiensis infections.
The second chapter deals with natural products that protect the nematode host without killing the pathogen. I deployed molecular biology methods to generate deletion and overproduction strains of a target compound, identified it via LC-MS/MS analysis and used LC-MS/MS and lipidomics to analyse the chemical properties of the active compound.
The third chapter aims at finding natural products, which are produced by Pseudomonas strains MYb11 and MYb12, respectively. These natural products display the ability to protect C. elegans by killing B. thuringiensis. I identified said compounds via fractionation and subsequent bioactivity testing. After identification, I generated production strains of the target compounds and elucidated the structure of the bioactive derivative.
The last chapter deals with the structure elucidation of peptides produced by an unusual GameXPeptide synthetase in Xenorhabdus miraniensis. I analysed producer strains of GameXPeptides using LC-MS and elucidated the structural differences between the known GameXPeptides, produced by P. luminescens TT01, and the unusual ones produced by X. miraniensis.
Auf der Oberfläche von Erythrozyten, Thrombozyten und Neutrophilen befinden sich mehrere hundert verschiedene polymorphe, ungekoppelt vererbte Blutgruppenantigene. Dementsprechend birgt jede Bluttransfusion das Risiko einer Immunisierung gegen fremde Blutgruppenmerkmale. Auch während der Schwangerschaft können aufgrund väterlich vererbter Antigene Alloantikörper induziert werden. Deshalb muss das Blut vor jeder Transfusion oder während einer Schwangerschaft auf das Vorhandensein irregulärer erythrozytärer Antikörper untersucht werden. Dabei greifen die aktuellen diagnostischen Verfahren auf primäre, stabilisierte Testerythrozyten von Blutspendern zurück, deren relevante Blutgruppenantigene bekannt sind. Antikörperspezifitäten können anhand von Agglutinationsreaktionen der Testzellen mit dem zu untersuchenden Patientenplasma auf ein oder mehrere Antigene zurückgeführt werden. Ist jedoch ein Antikörper gegen ein häufiges, ein hochfrequentes oder ein nicht-polymorphes, ubiquitäres Antigen gerichtet, kann in Ermangelung Antigen-negativer Testzellen keine adäquate Diagnostik gewährleistet, die Verträglichkeit der Transfusion also nicht definitiv sichergestellt werden. Auch der medizinische Einsatz therapeutischer Antikörper, welche Antigene adressieren, die auch auf Erythrozyten exprimiert werden, führt zunehmend zu Problemen. Tests auf granulozytäre Antikörper sind mangelhaft bezüglich ihrer Robustheit, besitzen eine unzureichende Auflösung und sind zudem meist zeitaufwändig und daher teuer. Antikörper gegen humane Plättchenantigene spielen insbesondere in der Schwangerschaft eine Rolle; sie vermögen bei Neugeborenen thrombozytopenische Blutungen bis hin zu massiven Hirnblutungen zu verursachen, die zu schweren Entwicklungsstörungen führen können. Bisher erfolgt jedoch mangels geeigneter Reagenzien keine standardisierte pränatale Untersuchung auf thrombozytäre Antikörper. In dieser Arbeit wurde ein neuartiges Verfahren für die Identifikation und Differenzierung irregulärer Blutgruppenantikörper etabliert, welches auf gentechnisch hergestellten, xenogenen Testzellen basiert, die einzelne definierte humane Blutgruppenantigene auf ihrer Oberfläche präsentieren. Die nicht humanen Zellen co exprimieren Fluorochrome, anhand derer Antikörper-markierte Testzellen durchflusszytometrisch voneinander unterscheidbar sind. Weiterhin können die generierten Testzellen zur Depletion von Antikörpern aus polyagglutinierenden Plasmen unter Erhalt der anderen Antikörperspezifitäten verwendet werden. Diese Technologie könnte die konventionelle Diagnostik erheblich erleichtern und bietet zudem die Möglichkeit, therapeutische Antikörper (wie z. B. anti-CD38, anti CD47, etc.), die häufig zu Interferenzen mit der Routinediagnostik führen, spezifisch prädiagnostisch aus Patientenproben zu entfernen.
Compaction and spheroid formation modulates stemness and differentiation of human pancreas organoids
(2023)
The incidence of diabetes type 1 (T1D) in children and young adults is increasing worldwide. T1D is well treated by insulin administration. However, there is currently no long-lasting cure for this ailment. The success rate of pancreatic islet transplantation to treat T1D is limited by the availability of patient-matched islets and the necessity of using life-long immunosuppressive medication. The difficulties caused by transplantation can be overcome by generating bio-engineered pancreatic islets from patient-derived progenitor cells. Aim of this thesis is to establish new strategies for the generation and analysis of pancreatic lineages derived from human progenitor cells. It reports on the optimization of a technique to form human pancreatic spheroids from hollow monolayered human pancreas organoids (hPOs) to investigate how cell-cell and cell-matrix interaction can be leveraged to induce endocrine differentiation of the pancreas progenitor cell organoids. We introduce cell aggregation protocols to generate endocrine pancreas cell lineages from ductal pancreatic cells. Next, we study the effect of co-culture with stromal and endothelial cells to promote cell differentiation toward a pancreatic fate enhancing β cells productivity.
This thesis has focused on identifying the differences in gene expression along with phenotypical transformation during differentiation of human pancreatic organoids (hPOs) towards human β cells to be used in the future of cellular therapeutics in treating T1D patients.
Xylose, an abundant sugar fraction of lignocellulosic biomass, is a five-carbon skeleton molecule. Since decades, utilization of this sugar has gained much attention and has been in particular focus as a substrate for production of biofuels like ethanol by microbial hosts, including Saccharomyces cerevisiae. In this yeast, xylose is naturally not used as a carbon source, but its utilization could be achieved by metabolic engineering either via the oxidoreductive route or through the isomerase pathway. Both pathways share xylulose as a common intermediate that must be phosphorylated before entering the endogenous metabolism via the non-oxidative pentose phosphate pathway (noxPPP). Besides this, in some bacteria a non-phosphorylating oxidative pathway for xylose degradation exists, known as Weimberg pathway, where a molecule of xylose is converted by a series of enzymes - xylose dehydrogenase (XylB), xylonate dehydratase (XylD), 3-keto-2-deoxy-xylonate dehydratase (XylX) and α-ketoglutarate semialdehyde dehydrogenase (KsaD) - to form α-ketoglutarate (AKG). Besides having several useful properties as a product, AKG could also be used for cell growth as an intermediate of the tricarboxylic acid (TCA) cycle. One target of the present study is to establish a functional Weimberg pathway in S. cerevisiae. Previous studies have shown that this task is not trivial, for instance due to the toxicity of xylonate (the first metabolite of the pathway) and the involvement of an iron-sulfur cluster dependent enzyme, the D-xylonate dehydratase. The assembly of iron-sulfur clusters on a heterologous protein in yeast is known to be challenging.
To establish the Weimberg pathway in yeast, the genes xylB, xylD, and xylX were obtained from Caulobacter cresentus and ksaD was from Corynebacterium glutamicum. In a variant, the dehydratase xylD was replaced with orf41 from Arthrobacter nicotinovorans, which is believed to be independent of iron-sulfur clusters. Growth of yeast cells on xylose as a sole carbon source was expected as an indicator of a functional Weimberg pathway. However, the heterologous expression of the codon optimized genes was not sufficient to reach this goal. Due to the complexity of the interactions of the heterologous pathway with the endogenous cellular processes, it was assumed that potential limitations could be overcome by adaptive laboratory evolution, using xylose as a sole source of carbon. Increasing selection pressure was applied on a strain with Weimberg pathway genes integrated into the genome over several generations. As a variant of the evolutionary engineering approach, mutator strains were generated. For this, RAD27 and MSH2 genes were deleted, which are involved in nucleotide excision and mismatch repair mechanisms, respectively. Some of the resulting strains PRY24, PRY25, PRY27 and PRY28 were able grow in xylose as a sole carbon source after evolutionary engineering. As a control, a non-mutator strain PRY19 was also included. Strikingly, only the mutator strains were able to consume xylose as a sole carbon source, which shows the feasibility of the approach.
In addition to the mutator strain strategy, a further approach employed in the present study was the simultaneous expression of the Weimberg pathway in the cytosol and mitochondria. This was based on the reasoning that the iron-sulfur cluster biogenesis on XylD may be improved in the organelle and that the AKG is an intermediate of the TCA cycle. In the strain AHY02, all enzymes of the pathway were tagged with mitochondrial targeting signals in addition to a full cytosolically localized pathway. The localization of the mitochondrial variants was confirmed by fluorescence microscopy. Together with AHY02, CEN.PK2-1C wild type strain was also included as a control for evolution. When a selection pressure on xylose was applied, both strains - AHY02 and CEN.PK2-1C - were able to grow in the course of evolution. Deletion of the xylulokinase (XKS1) gene was found to be detrimental for both evolved strains in xylose-containing media. This suggests that the evolution of the endogenous oxidoreductive and noxPPP genes is responsible for growth of the evolved cells. For the evolved strain AHY02, it could also be possible that the Weimberg pathway genes supported to growth in addition to the oxidoreductive route. To elucidate the underlying molecular mechanisms, genome sequencing and reverse engineering approaches would be necessary in future.
In addition to screening for growth on xylose as a sole carbon source, a less stringent screening system was created to examine even a minor flux of xylose towards AKG. For this, all genes necessary for conversion of isocitrate to AKG where deleted, yielding a glutamate auxotrophic strain. In this system, the cells can grow on other carbon sources, whereas xylose is only provided as a source of AKG for the synthesis of glutamate...
Epithelial cells enable essential physiological functions, including absorption, morphogenesis, secretion, and transport. To execute these functions, epithelial cells often form three-dimensional shapes that include curved sheets of cells surrounding a pressurized fluid-filled lumen. These three-dimensional tissues (called domes) are essential for organ function, but when they are not working properly, developmental defects, inflammation, and cancer can ensue. Recently, it has been shown that the cells that form domes show active superelasticity on micropatterned plates.
We show here that the immortalized renal proximal tubule epithelial cell line, LLC-PK1, stereotypically forms tubules in 10 days. Tubule formation takes place in 4 stages. When cells are plated on a culture dish, they form a monolayer on the 1st day; on the 3rd day, three-dimensional structures are formed, called domes; and after the 4.5th day, these domes start fusing to begin the transition stage and transit to the tubule stage. At the end of the 10th day, differentiated, elongated, and matured tubes form (Figure 3.1). Therefore, tubule formation is a self-organized, stereotypic morphogenetic program under long-term, unperturbed tissue culture conditions.
We propose that tubulogenesis is a two-step process in proximal tubules by doming and wrapping. The process begins with dome formation, and as the cell layers come together in the transition stage at the edge of the dome, this leads to the formation of the lumen of the eventual tubule. We also found that F-actin provides the mechanical strength during the formation of these three-dimensional structures during tubule formation. To better understand this 4-step process on a molecular level, we performed proteomics of tubule formation to identify the different proteins that play a significant role in proximal tubule development. Importantly, we identified proximal tubule markers like synaptopondin, angiotensin 1-10, collectrin, polycystin 1, and polycystin 2. These proteins play an important role in renal tube formation and differentiation.
Cell division is carried out by highly conserved cyclin-CDK complexes, which phosphorylate various cellular components. Cyclin-CDKs act differently depending on the cell cycle phase and work cooperatively to create DNA replication and cytokinesis. Therefore, we identified that cyclin-B1, marker of proliferation Ki-67, the RAD51 recombinase, and proliferating cell nuclear antigen (PNCA) are upregulated in the monolayer stage, and the expression decreases as tubule formation takes place. The proximal tubule reabsorbs 60-65% of the glomerulus filtrate. Therefore, it requires a lot of energy generated by using the fatty acid oxidation (FAO) pathway. In our model, we found FAO expression is higher than that of the other metabolic pathways.
We found expression of an intricate protein network in mitochondria, which we interpret as a sign of mitochondrial homeostasis being vital for the FAO pathway to work. Furthermore, we also identified different types of transporters at each stage of proximal tubule formation, and we could recognize different cytoskeletal components playing a significant role in each stage of proximal tubule formation, for instance, at the monolayer stage, vimentin expression is high, and its expression is reduced as tubules form. Hence, this 2D system, at this step of characterization, seems suitable to use to study differential transport protein expression and how this might relate to physiological functions and syndromes.
Next, we inhibited different transporters using specific inhibitors and analyzed the effect on dome and tubule formation. We identified that Na+/K+ ATPase and vacuolar H+ ATPase play a significant role in the process of epithelial dynamics. Digoxin (a Na+/K+ ATPase inhibitor) treatment inhibits dome and tubule formation. Bafilomycin (a v-ATPase inhibitor) treatment demonstrated a delay in dome and tube formation. Therefore, this study shows that this 2D proximal tubule novel system can be used for screening of pharmacological leads in the context of specific aspects of kidney physiology.
Despite the recent success in growing kidney organoids, they are not well suited to investigate various pathophysiological conditions in vitro for several reasons: They grow in 3D and form a tissue that later needs to be dissected/cleared and stained to investigate pathophysiological changes. Moreover, organoids require complex and expensive protocols for generation and are challenging to use in screening approaches. Therefore, we set out to demonstrate feasibility for our 2D system using normal renal epithelial cells, which are the origin of various pathological conditions, to study pathophysiological conditions.
Bei den meisten erwachsenen Säugetieren führt ein Herzinfarkt zu Fibrose und Verlust von funktionellem Herzgewebe. Einige Wirbeltiere, wie der Zebrabärbling, besitzen jedoch die bemerkenswerte Fähigkeit, nach einer Schädigung ihres Herzgewebes verlorenes Gewebe zu regenerieren und so schädliche Folgen zu verhindern. Die lokale Immunantwort auf eine Verletzung wird zunehmend als eine wichtige Determinante für das regenerative Potential eines Gewebes gesehen. Das Komplementsystem ist Teil des humoralen Immunsystems. Historisch ist es als eine Sammlung von Protein bekannt, den Komplementkomponenten, die in der Leber synthetisiert werden und im Blutkreislauf zirkulieren. Bei Exposition gegenüber einem Auslöser, wie z. B. einem Pathogen, wird eine Komplementkomponentproteinspaltungskaskade initiiert, die dazu führen kann, dass Immunzellen rekrutiert werden, und, dass die Phagozytose erleichtert, ggf. die Zielzelle lysiert wird. Studien legen nahe, dass das Komplementsystem an zellulären Prozessen beteiligt sei, die für Entwicklungs- und Krankheitsprozesse entscheidend sind, wie etwa Proliferation und Dedifferenzierung. Es gibt Hinweise, dass das Komplementsystem eine Rolle bei Krebserkrankungen und bei regenerativen Prozessen spielen könnte. In verschiedenen Arten wurde eine lokale verletzungsinduzierte Expression von komplementkomponentkodierenden Genen in regenerierendem Gewebe beobachtet.
Einzelne Studien legen nahe, dass Funktionsverlust einzelner Komplementkomponenten regenerative Prozesse beeinträchtigt.
Offene Fragen bleiben jedoch: Ist die lokale Expression von mehreren komplementkomponentkodierenden Genen ein Merkmal von regenerierendem Gewebe, das sie von Geweben unterscheidet, welchem die Fähigkeit zur Regeneration fehlt? Und welche Rolle könnte das Komplementsystem und seine Komponenten während des regenerativen Prozesses spielen? Um diesen Fragen nachzugehen, wurde eine Expressionsanalyse von Zebrabärblingsgewebe nach Verletzung mittels RT-qPCR und in situ Hybridisierung durchgeführt: kardiale Kryoverletzung, Larvenrumpfamputation und Schwanzflossenamputation. Ich beobachtete, dass mehrere komplementkomponentkodierende Gene in diesen Geweben nach Verletzung induziert wurden. Die Interpretation veröffentlichter single cell RNAseq Datensätze legt nahe, dass diese komplementkomponentenkodierenden Gene von verschiedenen Zelltypen exprimiert werden, darunter Immunzellen, Epikardzellen und Fibroblasten. Um transkriptionelle Unterschiede zwischen regenerierendem und nicht regenerierendem Gewebe zu identifizieren, verwendete ich ein nicht regeneratives Zebrabärblingmodell, die il11ra- Mutante. Dieser Mutante fehlt die Fähigkeit, verschiedene Organe zu regenerieren, das ist der Fall beim Herzen, dem larvalen Rumpf, und der Schwanzflosse. Ich stellte fest, dass die Mehrheit der verletzungsinduzierten komplementkomponentkodierenden Gene il11ra nachgeschaltet war. Darüber hinaus zeigten Experimente unter Verwendung chemischer Inhibitoren, dass speziell die Expression der komplementkomponentkodierenden Gene c3a.1,
c4b und c7a im Larvenrumpfamputationsmodell durch den Il11-Stat3-Signalweg moduliert wird.
Zur Klärung der Frage, ob das Komplementsystem und/ oder seine Komponenten eine Rolle während der Regeneration spielen, wurden verschiede Funktionsverlustmodelle generiert und im larvalen Rumpfamputationsmodell auf mögliche Aberrationen getestet. Zum einen generierte ich Überexpressionslinien von endogenen Inhibitoren der Komplementproteinspaltungskaskade. Überexpression eines etablierten Komplementsysteminhibitors rca2.1/ tecrem führte zu einer im Vergleich zu Wildtyp- Geschwistern verringerten Regeneration des larvalen Rumpfs. Zum anderen generierte ich Funktionsverlustmutanten von individuellen Komplementkomponenten durch CRISPR/Cas9 vermittelter Mutagenese, und zwar für masp1, masp2, cfd, c1s, c4b, c5 und c9. Die larvale Rumpfregeneration war in diesen Mutanten unauffällig. Allerdings zeigten c4b Mutanten eine verringerte Kardiomyozytenproliferation und eine differenzielle Expression von einigen Markergenen, einschließlich einer erhöhten Expression von inflammatorischen Zytokinen.
Meine Studien führten zu neuen Einblicken in das Komplementsystem im Kontext der Regeneration. Ich fand heraus, dass mehrere komplementkomponentenkodierenden Gene in regenerierendem Zebrabärblinggewebe exprimiert werden, und zwar im Herzgewebe, im larvalen Rumpf und in der adulten Flosse. Darüber hinaus zeige ich, dass die verletzungsinduzierte Expression von komplementkodierenden Genen in regenerierendem Gewebe dem Regenerationsmasterregulator il11ra nachgeschaltet ist. Speziell c3a.1, c4b und c7a wurden durch il11/ stat3 reguliert...
The capacity of pathogenic bacteria to adhere to host cells and to avoid subsequent clearance by the host´s immune response is the initial and most decisive step leading to infections. Human pathogenic bacteria circulating in the bloodstream need to find ways to interact with endothelial cells (ECs) lining the blood vessels to infect and colonise the host. The extracellular matrix (ECM) of ECs might represent an attractive initial target for bacterial interaction, as many bacterial adhesins have reported affinities to ECM proteins, particularly fibronectin (Fn). Trimeric autotransporter adhesins (TAA) have been described as important pathogenicity factors of Gram-negative bacteria. The TAA from human pathogenic Bartonella henselae, Bartonella adhesin A (BadA), is one of the longest and best characterised adhesin and represents a prototypic TAA due to its domain architecture. B. henselae, the causative agent of cat scratch disease, endocarditis, and bacillary angiomatosis, adheres to ECs and ECM proteins via BadA interaction.
In this research, it was determined that the interaction between BadA and Fn is essential for B. henselae host cell adhesion. BadA interactions were identified within the heparin-binding domains of Fn, and the exact binding sites were revealed by mass spectrometry analysis of chemically crosslinked whole-cell bacteria and Fn. It turned out that specific BadA interactions with defined Fn regions represent the molecular basis for bacterial adhesion to ECs. These data were confirmed by using BadA-deficient bacteria and CRISPR-Cas FN1 knockout ECs. It was also identified that BadA binds to Fn from both cellular and plasma origin, suggesting that B. henselae binding to Fn might possibly take part in other infection processes apart from bacterial adherence, e.g. evasion from the host cell immune system.
Interactions between TAAs and Fn represent a key step for adherence of B. henselae to ECs. Still, Fn-mediated binding is of more significant importance for pathogenic bacteria than broadly recognised. Fn removal from the ECM environment of ECs, also reduced adherence of Staphylococcus aureus, Borrelia burgdorferi, and Acinetobacter baumannii to host cells Interactions between adhesins and Fn might therefore represent a crucial step for the adhesion of human-pathogenic Gram-negative and Gram-positive bacteria targeting the ECs as a niche of infection or as means for persistence.
This research demonstrated that combining large-scale analysis approaches to describe protein-protein interactions with supportive functional readouts (binding assays) allows for the discrimination of crucial interactions involved in bacterial adhesion to the host. The herein-described experimental approaches and tools might guide future research for other pathogenic bacteria and represent an initial point for the future generation of anti-virulence strategies to inhibit bacterial binding to host cells.
Brain development is a complex and highly organized process that relies on the coordinated interaction between neurons and vessels. These cell systems form a neurovascular link that involves the exchange of oxygen, ions, and other physiological components necessary for proper neuronal and vascular function. This physiologically coupled process is executed through analogous structural and molecular signaling mechanisms shared by both cell types. At the neurovascular interface, the cellular crosstalk via these shared signaling mechanisms allows for the synchronized expansion and integration of neurons and vessels into complex cellular networks. This study investigated the role of VEGFR2, a receptor for vascular endothelial growth factor (VEGF), during postnatal neuronal development in the mouse hippocampus. Prior studies have revealed physiological roles of VEGF, a pro-angiogenic morphogen, in nervous system development. However, it was unclear if VEGF signaling had a direct effect on neuronal physiology and function through neuronal-expressing receptors. In this investigative work, we identified a previously unknown function of VEGFR2, whereby VEGF-induced signaling coordinates the development and circuitry integration of CA3 pyramidal neurons in the early postnatal mouse hippocampus. Mechanistically, we found that VEGFR2 signaling requires receptor endocytosis, a process mediated by ephrinB2. We also found that VEGF-induced cooperative signaling between VEGFR2 and ephrinB2 is functionally required for the dendritic arborization and spine maturation of developing CA3 neurons during the first few postnatal weeks. Moreover, in a collaborative effort with the research group of Carmen Ruiz de Almodovar, formerly at the University of Heidelberg, we simultaneously studied VEGF-induced VEGFR2 signaling in CA3 axonal development. Together, we aimed to gain a comprehensive understanding of the complex interplay between VEGF and VEGFR2 signaling during the early postnatal development of CA3 neurons. Ruiz de Almodovar’s research group found that, unlike the branch and spine development of CA3 dendrites, VEGF-VEGFR2 signaling promotes axonal development through mechanisms that are independent of ephrinB2 function. Our findings on CA3 dendritic development are reported in the published manuscript, Harde et al. (2019), and the complementary work on CA3 axonal development from Ruiz de Almodovar's group is presented in the co-published manuscript, Luck et al. (2019). Although the totality of Ruiz de Almodovar's group's work on CA3 axons is not fully discussed here, it is referenced where noted to provide biological context for our findings on CA3 dendritic development.
VEGFR2 signaling within neurovascular niches is known to play a role in the neurogenesis of neural progenitor cells during embryonic development and within the adult brain. However, the precise localization of neuronal VEGFR2 expression and functional role within the nervous system during postnatal brain development was unknown. To investigate this, we used immunohistochemistry to identify the spatial expression of VEGFR2 within the mouse hippocampus during the first few weeks after birth. Our results showed that VEGFR2 was predominantly expressed within the hippocampal vasculature, consistent with prior studies. However, we also observed localized VEGFR2 expression in pyramidal cell neurons of the hippocampal CA3 region by postnatal day 10 (P10). This spatially restricted postnatal expression of VEGFR2 in CA3 neurons suggested a potential role in the development of these neurons during this developmental stage.
The first two weeks after birth in the mouse hippocampus is a critical period for the development of neuronal circuits, as neurons undergo extensive dendritic arborization and spine formation. To explore the role of VEGFR2 in the postnatal nervous system, we used a Nes-cre VEGFR2lox/- mouse line to target the deletion of VEGFR2 expression within the nervous system while preserving normal receptor expression in all other cell types. We also generated corresponding control mice that were negative for Nes-cre. By breeding these mice with Thy1-GFP reporter mice, we could analyze the functional consequences of VEGFR2 by assessing the morphologies of CA3 dendritic trees and spine density and maturation at P10 and P15, respectively. Our analysis showed that CA3 neurons in Nes-cre VEGFR2lox/- mice had less complex dendritic arbors compared to control mice. There were significant reductions in total length and branch points, particularly in areas located 100-250 μm from the cell soma within the stratum radiatum layer. Additionally, Nes-cre VEGFR2lox/- mice exhibited a significant decrease in spine density accompanied by an increased proportion of immature spines. These findings suggest that VEGFR2 plays a crucial role in the proper development of CA3 dendrites and spines during the early postnatal weeks.
In view of a growing world population and the finite nature of fossil resources, the development of eco-friendly production processes is essential for the transition towards a sustainable industry. Methanol, which can be produced both petrochemically and from renewable resources, offers itself as bridging technology and attractive alternative raw material for biotechnological processes. This work describes developments for the progress of the well-studied methylotrophic α proteobacterium Methylorubrum extorquens AM1 towards an efficient methylotrophic cell factory. Although many homologous and heterologous production routes have already been described and realized for M. extorquens in a laboratory scale, no industrial process has yet been realized. Three major reasons can be identified for this: (1) A limited choice of tools for genetic modifications, (2) a lack of understanding of carbon fluxes and side reactions occurring in modified strains, such as product reimports, and (3) the lack of tailored production strains for profitable target products and optimized bioprocessing protocols. The aim of the present work was to achieve developments for the mentioned areas. As a model application, the high-level production of chiral dicarboxylic acids from the substrate methanol was chosen. Enantiomerically pure chiral compounds are of great interest, e.g., as building blocks for chiral drugs. The ethylmalonyl CoA metabolic pathway (EMCP) which is part of the primary metabolism of M. extorquens, harbors unique chiral CoA-ester intermediates. Their acid derivatives can be released by cleavage of the CoA-moiety using heterologous enzymes. The dicarboxylic acids 2 methylsuccinic acid and mesaconic acid were produced in a previous study by introducing the heterologous thioesterase YciA into M. extorquens. In the said study, a combined product titer of 0.65 g/L was obtained in shake flask experiments. These results serve as the basis for the developments in the present work.
First, the previously described reuptake of products was thoroughly investigated and dctA2, a gene encoding for an acid transporter, was identified as target for reducing the product reuptake. In addition, reuptake of mesaconic acid was prevented by converting it to (S)-citramalic acid, a product not metabolizable by M. extorquens, by the introduction of a heterologous mesaconase. Together with 2-methylsuccinic acid, for which a high enantiomeric excess of (S)-2-methylsuccinic acid was determined, a second chiral molecule was thus added to the product spectrum. For the release of dicarboxylic acid products, YciA, a broad-range thioesterase that accepts a variety of CoA-esters with different chain lengths as substrates, was chosen. The enzyme should theoretically be able to hydrolyze all CoA-esters of interest present in the EMCP. However, in culture supernatants of M. extorquens strains that were overexpressing the corresponding yciA gene, only mesaconic acid and 2 methylsuccinic acid could be detected. To expand the substrate spectrum of YciA thioesterase with respect to other EMCP intermediates, semi-rational enzyme engineering was attempted. Screening of the corresponding strains carrying the respective YciA variants did not result in strains capable of producing new dicarboxylic acid products. However, the experiments revealed an amino acid position that strongly affected the production of mesaconic acid and 2-methylsuccinic acid in vivo. By substituting the according amino acid in YciA, the maximum titers of mesaconic acid and 2-methylsuccinic acid could be increased substantially. Application of an improved thioesterase variant in a second E. coli-based process confirmed the enhanced activity of the enzyme. The desired extension of the product spectrum by another chiral molecule (2-hydroxy-3-methylsuccinic acid, presumably the (2S,3R)-form) was finally achieved by using an alternative thioesterase. Tailored fermentation strategies were developed for the high-level production of the above-mentioned products.
As second part of the work, two novel genetic tools for M. extorquens were developed and characterized. The pBBR1-derived plasmid pMis1_1B was shown to be stably maintained in M. extorquens cells. In addition, its suitability for co-transformations with other plasmids was demonstrated. The second tool, the cumate-inducible promoter Ps6, is tailored for expression of pathways with toxic products, as the transcription of genes controlled by Ps6 is strongly repressed in the absence of an inducer.
Overall, the present work demonstrates the enormous potential of using M. extorquens as a methylotrophic cell factory. In the applications shown, the biotechnological production of high-priced chiral molecules is combined with the use of an attractive alternative substrate. In addition, new achievements and approaches are presented to facilitate the development of future M. extorquens production strains.