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Photorhabdus and Xenorhabdus are Gram-negative, entomopathogenic bacteria, living in endosymbiosis with the soil-dwelling nematode of the genera Steinernema and Heterorhabditis. The life cycle of these nematodes consists of non-feeding infective juvenile (IJ) stage, which actively searches for insects in the soil. After penetrating the insect prey, Photorhabdus and Xenorhabdus bacteria are released from the nematode gut. The bacteria proliferate and produce toxins to kill the insect. Photorhabdus and Xenorhabdus support nematode development throughout the life cycle and to get rid of food competitors by providing a wide variety of specialized metabolites (SMs). However, little is known about which SMs function as so called “food signals” to trigger the development process.
The IJs develop into adult, self-fertilizing hermaphrodites in a process called recovery, while feeding on cadaver and bacterial biomass. Heterorhabditis and Steinernema proceed to breed until nutrients are exhausted. Next generation IJs (NG-IJs) develop and leave the cadaver to search for another insect prey.
Photorhabdus and Xenorhabdus can be cultivated in defined medium under laboratory conditions. By placing IJs on a plate containing their respective bacterial symbiont, the complete life cycle of the nematodes can be observed in vitro. The in vitro nematode bioassay was used as a tool to investigate the development of the nematode.
The aim of this study was to find the food signals responsible for nematode development. Different Photorhabdus deletion strains unable to produce one or several SMs were co-cultivated with nematodes in the nematode bioassay. Subsequently, two aspects of the life cycle were investigated: recovery and NG-IJ development.
As isopropyl stilbene (IPS) is postulated to function as a food signal to support nematode recovery, it was used as a starting point for investigations. This study was focused on the biosynthetic pathway of IPS, including intermediates, side products and derivatives to investigate which one is in fact responsible for supporting nematode development.
The biosynthesis of IPS requires two precursors, phenylalanine and leucine (Figure 5). The first topic was focused on the phenylalanine derived pathway. Photorhabdus laumondii deletion mutants, defective in intermediate steps of this pathway, were created. The deletion of the genes coding for the phenylalanine ammonium lyase (stlA), converting phenylalanine into cinnamic acid (CA), the coenzyme A (CoA) ligase (stlB) and the operon coding for a ketosynthase and aromatase (stlCDE), were used. These strains were used for nematode bioassay including complementation of mutant phenotypes by feeding experiments. Recovery of nematodes grown on the deletion strains was always lower than recovery of nematodes grown on wild type bacteria. Feeding IPS to a deletion strain did not restore wild type level nematode recovery, thus IPS cannot be the food signal. Instead, the food signal must be another compound derived from this part of biosynthetic pathway. Lumiquinone and 2,5-dihydrostilbene are suggested to function as food signals and need to be investigated in future work.
The second part of this study was focused on the leucine derived pathway, which involved the Bkd complex forming the iso-branched part of IPS. A deletion of bkd was created and phenotypically analysed, subsequently performed with the nematode bioassay. Not only IPS but also other branched SMs, like photopyrones and phurealipids are synthetised by the Bkd complex. Deletions strains defective in producing photopyrones and phurealipids were also performed in nematode bioassays to investigate effects of these SMs individually. Branched SMs did not have an impact on nematode development, but nematodes grown on the ΔbkdABC strain showed a reduced nematode recovery and almost diminished NG-IJs development. As the Bkd complex also produces branched chain fatty acids (BCFAs), feeding experiments were performed with lipid extracts of wild type and mutant strain. All lipid extracts improved recovery, but only wild type lipids could complement NG-IJ development. This strongly indicates that BCFAs play an important role in NG-IJ development, which needs to be proven with purified BCFA feeding. This is an interesting finding, which could improve nematode production for biocontrol agent usage.
The role of IPS derived to epoxy stilbene (EPS) for nematode development, was another focus in the nematode life cycle. Recently it was demonstrated that EPS does not support nematode development. However, EPS forms adducts with amino acids. In my thesis, novel adducts containing the amino acid phenylalanine or a tetrapeptide were characterized. Another adduct, most likely being an EPS dimer, was also characterized. The biological role of such adducts was discussed to be potentially important for insect weakening and the structure of the novel compounds need to be structure elucidated and tested for bioactivity.
Es gibt viele Theorien, die sich mit der Auswirkung einer zunehmenden carnivoren Ernährung von Homininen auf Carnivorengilden beschäftigen. Aussterbeereignisse in der Carnivorengilde werden oft mit carnivoren Homininen in Verbindung gebracht. Um zu prüfen, ob solche Theorien überhaupt zutreffen, benötigt man zunächst ein Modell, das Effekte von Konkurrenzbeziehungen innerhalb von Carnivorengilden quantifiziert darstellt.
In dieser Arbeit ist daher ein Modell entwickelt worden, das die Konkurrenz um Beute innerhalb einer Carnivorengilde darstellt und ermöglicht Veränderungen durch das Eintreten neuer Mitglieder in die Gilde zu modellieren. Dieses Modell wurde zur Analyse der rezenten Großcarnivorengilden der Serengeti, des Krüger-National-Parks und des Bandipur-Biosphärenreservat verwendet. Ebenso ist es zur Analyse pleistozäner Großcarnivorengilden Javas eingesetzt worden.
In dem Modell wird die verfügbare Beutemasse als limitierende Ressource für die Carnivorengilde betrachtet. Im ersten Schritt wird die Beute kategorisiert – in dieser Arbeit nach ihrer Körpermasse – und geprüft, welche Mitglieder dieselben Beutekategorien nutzen und welche für sie essentiell sind. Im zweiten Schritt wird die konkurrenzfreie Kapazität der Gildenmitglieder berechnet. Hierzu wird die für die gesamte Gilde verfügbare Beutemasse unter der Annahme verwendet, sie stehe einem Gildenmitglied allein zur Verfügung. Die konkurrenzfreie Kapazität ist daher die Populationsgröße, die ein Gildenmitglied mit dieser Beutemasse erreichen kann und stellt einen Referenzwert dar. Basierend auf diesem Referenzwert und der tatsächlichen Populationsgröße kann nun berechnet werden, zu welchem Anteil ein Mitglied diese Kapazität ausschöpft. Ist der Konsum an Beutemasse der übrigen Mitglieder in den essentiellen Beutekategorien bekannt, kann berechnet werden, zu welchem Anteil ein Mitglied durch ein anderes Mitglied von dieser Kapazität verliert. Dieser Verlust an Kapazität wird als Konkurrenzeffekt bezeichnet.
Dieses Modell ist sowohl auf rezente als auch fossile Gilden anwendbar. Um mit dem Modell die Konkurrenzeffekte zu berechnen, werden die Häufigkeit bzw. Populationsgröße, das Beutemassenspektrum sowie der tägliche Bedarf an Beutemasse benötigt.
Diese Größen können bei der Strukturanalyse von rezenten Gilden aus Freilandstudien entnommen werden. Im Falle fossiler Gilden müssen diese Größen erst rekonstruiert werden. Dafür sind in dieser Arbeit vorhandene Rekonstruktionsmethoden ergänzt, aber auch entwickelt worden, mit denen man basierend auf der Körpermasse fossiler Carnivora die benötigten Parameter rekonstruieren kann. Hierzu sind verschiedene Regressionen berechnet worden, die einen Zusammenhang zwischen verschiedenen Zahnparametern und der Körpermasse darstellen. Weiterhin sind Muster der Beutemassenspektren rezenter Carnivora untersucht worden und Regressionen berechnet worden, die zur Rekonstruktion der mittleren Beutemasse eines Carnivoren verwendet werden.
Die benötigten Daten der javanischen Gilden werden mit den eben genannten Regressionen rekonstruiert. Anschließend wird eine Strukturanalyse der genannten rezenten und fossilen Großcarnivorengilden durchgeführt.
Bei den drei rezenten Gilden ist eine generelle sich wiederholende Struktur erkennbar. Die erfolgreichsten Mitglieder schöpfen ihre Kapazität zu ca. 60 % aus und verfolgen eine soziale Lebensweise.
Dennoch werden die erfolgreichsten Mitglieder der Gilden von unterschiedlichen Arten repräsentiert. So sind dies der Löwe im Krüger-Nationalpark, die Tüpfelhyäne in der Serengeti oder der Rothund in Bandipur.
Bei den fossilen Gilden war diese Struktur allerdings nicht erkennbar. Hier schöpft der Tiger seine Kapazität in allen Gilden am stärksten aus und hat extrem hohe Konkurrenzeffekte (bis zu ca. 98 %) auf die übrigen Gildenmitglieder.
Diese Unterschiede können mit Isolationsbedingungen Javas als Insel zusammenhängen, die sich grundsätzlich auf Strukturen der Säugergemeinschaften auswirken.
Vermutlich konnte der Tiger durch Veränderungen der Körpermasse seine konkurrenzstarke Position in der Großcarnivorengilde Javas halten.
Das entwickelte Modell ermöglicht auch eine Modellierung von Szenarien, die verschiedene Möglichkeiten berücksichtigt. Diese sind vor allem Veränderungen der Populationsgrößen, aber auch Veränderungen der Körpermasse und daraus resultierende Verschiebungen der Beutemassenspektren.
In Beispielen der Trinil-Gilde wird gezeigt, dass die Rolle eines hyper- bzw. hypocarnivoren Homo erectus in der Gilde mit dem entwickelten Modell dargestellt werden kann. Auch lassen sich Szenarien modellieren, in denen ein hyper- bzw. hypocarnivorer Homo erectus in die Gilde eindringt und so die übrigen Mitglieder von bei ihrer Kapazitätsausschöpfung Einbuße hinnehmen müssen.
In dem Szenarium von Trinil wird erkennbar, dass nur ein hypercarnivorer Homo erectus einen starken Effekt auf die Gildenmitglieder hatte. Geht man von einem omnivoren Homo erectus aus, ist der Konkurrenzeffekt geringer und es sind keine Aussterbeereignisse zu erwarten.
Das Modell kann in weiteren Studien zur Testung von Hypothesen zu Aussterbeereignissen Aufklärung bieten. Durch Einbeziehung weiterer Faktoren wie Kleptoparasitismus und interspezifische Tötungen kann es noch erweitert werden. Auch eine Dynamisierung des Modells, die eine kontinuierlich zeitliche Veränderung der Gilden modellieren kann, ist in zukünftigen Studien möglich.
Xenorhabdus and Photorhabdus are bacterial genera that live in symbiosis with entomopathogenic nematodes of the genera Steinernema and Heterorhabditis, respectively. These nematodes infect insect larvae through the trachea and then enter the hemocoel. Once inside the hemocoel, the nematodes release the bacteria through their intestine. Thereafter, the bacteria become active and kill the larvae within 48 h. During this process, the immune system of the insect host is compromised by molecules produced and secreted by the bacteria. This illustrates that the bacteria possess not only a large arsenal of biological weaponry such as antibiotics and fungicides but also lipases, proteases, etc. Therefore, they are not only able to kill the insect but also protect the cadaver from other food competitors.
During the past decades, a large number of natural products have been identified from Xenorhabdus and Photorhabdus. However, the targets and functions for many of these biological molecules are still unknown. Therefore, the goal of the doctoral thesis is to elucidate the modes of action of these natural products from Xenorhabdus and Photorhabdus with the main focus on non-ribosomal peptides (NRPs). The work can be divided into two parts. Initially, it starts with the synthesis of natural compounds and various chemically modified derivatives. Besides that, a number of peptides were synthesized for other projects to either verify their structures or quantify the amount produced by the bacteria. Then, secondary analysis methods are applied and provide additional insight into the modes of action of these compounds.
During the thesis, I carried out peptide synthesis either manually or with an automatic synthesizer system from Biotage. Here, the Fmoc-protecting group strategy was preferred in most cases. Natural products, such as silathride, xenoautoxin, phenylethylamide, tryptamide, rhabdopeptide, 3-hydroxyoctanoic acid, and PAX, were produced during this process. Furthermore, new peptide derivatives derived from synthetic NRPS approaches using the XU concept or SYNZIP were generated as standards.
Most of these natural compounds were experimentally verified by MIC tests (broth microdilution, plate diffusion) to be biologically active. For example, silathride, phenylethylamide, and tryptamide showed quorum quenching effects when tested against Chromobacterium violaceum. Initial results from collaborators (PD Dr. Nadja Hellmann/Mainz) showed that tryptamide and phenylethylamide interact with membrane or membrane proteins.
(R)-3-hydroxyoctanoic acid was synthesized to verify the molecule structure of phototemtide A, a cyclic lipopeptide with antiprotozoal activity. The rhabdopeptides are another class, which showed remarkable antiprotozoal effects. However, their mode of action was unknown. These compounds are relatively short peptide sequences, which contain hydrophobic residues, such as valine, leucine, or phenylalanine. Moreover, they possess N methylation, resulting in a rod-shaped highly hydrophobic structure. In this work, I synthesized eight new derivatives of rhabdopeptides for photo-affinity labeling (PAL). These molecules should react covalently under UV-light irradiation with the biological target of the peptides. In addition, these derivatives can be enriched in a pull-down assay using click chemistry. Afterward, analytic methods such as mass detection (proteome analysis) can be applied to elucidate the protein targets.
The PAX peptides derivatives are well-known to have anti-microbial activities and believed to be secreted into the environment by the producing bacteria. However, I found that the majority of these peptides are located in the cell pellet fraction and not in the supernatant. This has been shown through quantification using HPLC MS. New PAX derivatives were synthesized, which carry a moiety suitable for covalent modification using click-chemistry, therefore being functionalizable with a fluorescence dye. In collaboration with Dr. Christoph Spahn (Prof. Dr. Mike Heilemann group), we used confocal, as well as super-resolution microscopy, in particular, single-molecule localization microscopy (SMLM) to investigate the spatial distribution of clickable PAX molecules and revealed that they localize at the bacterial membrane. Furthermore, bioactivity assays revealed that the promotor exchanged X. doucetiae PAX mutants, which do not produce PAX molecules without chemical induction (hereby termed as pax-), were more susceptible to several insect AMPs tested. Based on these findings, a new dual mechanism of action for PAX was proposed. Besides the previously shown antimicrobial activity, these molecules with a positive net charge of +5 (pH = 7) would bind to the negatively charged bacterial surface. Hereby, the surface charge (typically negative) would be inversed resulting in a protective effect for Xenorhabdus against other positively charged AMPs. Furthermore, PAX was investigated as AMP against E. coli to study its antimicrobial mechanism of action. Here, the results show that PAX can disrupt the E. coli membrane at higher concentrations (> 30 µg/ml), enter the cytosol, and lead to reorganization of subcellular structures, such as the nucleoid during this process.
Another aspect of secondary analysis is the application of proteomic analysis. Therefore, I induced X. nematophila, X. szentirmaii, and P. luminescens with insect lysate. These samples were analyzed using HPLC-MS/MS (Q Exactive) together with a database approach (Maxquant/Andromeda). The results showed that in all strains the lipid degradation and the glyoxylate pathway were induced. This is in line with the given insect lysate diet, which mostly contained lipids. Moreover, several interesting unknown peptides and proteins were also upregulated and might get into the focus of future research.
Colorectal cancer (CRC) has the third highest incidence and the fourth highest mortality rate worldwide and represents a substantial health care burden and affects the life of millions of people. CRC is a genetic disease caused by the stepwise accumulation of genetic alterations. The initiating event in colorectal carcinogenesis is the aberrant activation of the WNT pathway, but other pathways are also commonly deregulated, including the PI3K/AKT pathway. A number of previous studies using genetically engineered mouse models aimed at dissecting the exact role of PI3K/AKT pathway in CRC, but have yielded in rather conflicting results. Despite the inconsistent results, these studies already put forward the idea that PI3K/AKT signaling in combination with other genetic events might substantially contribute to tumor progression.
Since the PI3K/AKT pathway is frequently activated in CRC, it represents an ideal candidate for therapeutic intervention. Although extensive efforts had led to the development of numerous inhibitors targeting the PI3K/AKT pathway, the diversity of genetic alterations can challenge the identification of the most effective therapeutic targets. Therefore, the discovery of shared tumor-promoting mechanisms downstream of these genetic alterations might unravel new biomarkers and druggable targets. The aim of this study was to elucidate the precise role of PI3K/AKT pathway during the course of colorectal carcinogenesis and to decipher novel pro-tumorigenic molecular mechanisms downstream of PI3K/AKT activation that can be used for therapeutic intervention.
To obtain a better insight into the role of the PI3K/AKT pathway during colorectal carcinogenesis, mice expressing an oncogenic variant of AKT1 (AktE17K) specifically in the intestinal epithelial cells (IEC) were used. At the age of 6 months untreated AktE17K mice showed clearly perturbed intestinal homeostasis, but no tumor formation. To induce colonic tumorigenesis, AktE17K mice were subjected to treatment with the colonic carcinogen azoxymethane (AOM). In response to AOM, AktE17K mice developed invasive but nonmetastatic tumors, which showed strong nuclear accumulation of TP53. To investigate the role of PI3K/AKT signaling specifically in CRC progression, AktE17K mice were crossed to TP53- deficient mice (Tp53ΔIEC). Unlike AktE17K mice, untreated Tp53ΔIECAktE17K, developed highly invasive small intestinal tumors by the age of 6 months. To investigate the role of AKT hyperactivation in colonic tumor progression, Tp53ΔIECAktE17K mice were subjected to AOM treatment. AKT hyperactivation significantly enhanced tumor progression and induced metastatic dissemination.
To get a better insight how AKT signaling can promote tumor progression, whole tumor tissues from AOM-treated Tp53ΔIEC and Tp53ΔIECAktE17K mice were subjected to next generation mRNA sequencing and phospho-proteomic analysis by mass spectrometry. Both analyses indicated that AKT hyperactivation expands the inflammatory tumor microenvironment and upregulates pathways associated with invasion and metastasis. Importantly, Gene Set Enrichment Analysis revealed that AOM-induced colon tumors of Tp53ΔIECAktE17K animals, are highly similar in their gene expression profile to the CMS4 subtype of human CRC, which is associated with worse overall- and relapse-free survival7 . Gene expression analysis also suggested elevated NOTCH signaling in the Tp53ΔIECAktE17K tumors. Interestingly, while the expression of Notch3 mRNA was increased in the tumors of Tp53ΔIECAktE17K mice, the expression of the other NOTCH receptors was unaffected by AKT hyperactivation. In vitro experiments using TP53-deficient mouse tumor organoids with hyperactive AKT signaling confirmed the direct, tumor cell-intrinsic link between AKT activation and increased Notch3 expression. Moreover, inhibition of EZH2 mimicked the effect of AKT hyperactivation on Notch3 expression, suggesting that AKT regulates Notch3 via an epigenetic mechanism.
Knock-down of Notch3 in TP53-deficient mouse tumor organoids with hyperactive AKT signaling resulted in differential regulation of several pathways with potential role in invasion and metastasis and in cell death and survival. Subsequent in vivo experiments confirmed the role of NOTCH3 signaling in CRC progression. Treatment of AOM-induced Tp53ΔIECAkt E17K mice with a NOTCH3 antagonistic antibody or the γ-secretase inhibitor DAPT significantly reduced invasion and metastasis. Importantly, NOTCH3 expression was also found to be associated with human CRC progression, suggesting that NOTCH3 represent a valid target for the treatment of CRC. This work, using genetically engineered mouse models and advanced in vitro techniques, has demonstrated a strong tumor promoting role for PI3K/AKT signaling in CRC progression and has identified NOTCH3 signaling as a potential therapeutic target downstream of the PI3K/AKT pathway.
Colorectal cancer (CRC) has the third highest incidence and the fourth highest mortality rate worldwide and represents a substantial health care burden and affects the life of millions of people. CRC is a genetic disease caused by the stepwise accumulation of genetic alterations. The initiating event in colorectal carcinogenesis is the aberrant activation of the WNT pathway, but other pathways are also commonly deregulated, including the PI3K/AKT pathway. A number of previous studies using genetically engineered mouse models aimed at dissecting the exact role of PI3K/AKT pathway in CRC, but have yielded in rather conflicting results. Despite the inconsistent results, these studies already put forward the idea that PI3K/AKT signaling in combination with other genetic events might substantially contribute to tumor progression. Since the PI3K/AKT pathway is frequently activated in CRC, it represents an ideal candidate for therapeutic intervention. Although extensive efforts had led to the development of numerous inhibitors targeting the PI3K/AKT pathway, the diversity of genetic alterations can challenge the identification of the most effective therapeutic targets. Therefore, the discovery of shared tumor-promoting mechanisms downstream of these genetic alterations might unravel new biomarkers and druggable targets. The aim of this study was to elucidate the precise role of PI3K/AKT pathway during the course of colorectal carcinogenesis and to decipher novel protumorigenic molecular mechanisms downstream of PI3K/AKT activation that can be used for therapeutic intervention.
To obtain a better insight into the role of the PI3K/AKT pathway during colorectal carcinogenesis, mice expressing an oncogenic variant of AKT1 (AktE17K) specifically in the intestinal epithelial cells (IEC) were used. At the age of 6 months untreated AktE17K mice showed clearly perturbed intestinal homeostasis, but no tumor formation. To induce colonic tumorigenesis, AktE17K mice were subjected to treatment with the colonic carcinogen azoxymethane (AOM). In response to AOM, AktE17K mice developed invasive but non-metastatic tumors, which showed strong nuclear accumulation of TP53. To investigate the role of PI3K/AKT signaling specifically in CRC progression, AktE17K mice were crossed to TP53-deficient mice (Tp53ΔIEC). Unlike AktE17K mice, untreated Tp53ΔIEC; AktE17K, developed highly invasive small
intestinal tumors by the age of 6 months. To investigate the role of AKT hyperactivation in colonic tumor progression, Tp53ΔIEC; AktE17K mice were subjected to AOM treatment. AKT hyperactivation significantly enhanced tumor progression and induced metastatic dissemination.
To get a better insight how AKT signaling can promote tumor progression, whole tumor tissues from AOM-treated Tp53ΔIEC and Tp53ΔIEC; AktE17K mice were subjected to next generation mRNA sequencing and phospho-proteomic analysis by mass spectrometry. Both analyses indicated that AKT hyperactivation expands the inflammatory tumor microenvironment and upregulates pathways associated with invasion and metastasis. Importantly, Gene Set Enrichment Analysis revealed that AOM-induced colon tumors of Tp53ΔIEC; AktE17K animals, are highly similar in their gene expression profile to the CMS4 subtype of human CRC, which is associated with worse overall- and relapse-free survival. Gene expression analysis also suggested elevated NOTCH signaling in the Tp53ΔIEC; AktE17K tumors. Interestingly, while the expression of Notch3 mRNA was increased in the tumors of Tp53ΔIEC; AktE17K mice, the expression of the other NOTCH receptors was unaffected by AKT hyperactivation. In vitro experiments using TP53-deficient mouse tumor organoids with hyperactive AKT signaling confirmed the direct, tumor cell-intrinsic link between AKT activation and increased Notch3 expression. Moreover, inhibition of EZH2 mimicked the effect of AKT hyperactivation on Notch3 expression, suggesting that AKT regulates Notch3 via an epigenetic mechanism.
Knock-down of Notch3 in TP53-deficient mouse tumor organoids with hyperactive AKT signaling resulted in differential regulation of several pathways with potential role in invasion and metastasis and in cell death and survival. Subsequent in vivo experiments confirmed the role of NOTCH3 signaling in CRC progression. Treatment of AOM-induced Tp53ΔIEC; AktE17K mice with a NOTCH3 antagonistic antibody or the γ-secretase inhibitor DAPT significantly reduced invasion and metastasis. Importantly, NOTCH3 expression was also found to be associated with human CRC progression, suggesting that NOTCH3 represent a valid target for the treatment of CRC. This work, using genetically engineered mouse models and advanced in vitro techniques, has demonstrated a strong tumor promoting role for PI3K/AKT signaling in CRC progression and has identified NOTCH3 signaling as a potential therapeutic target downstream of the PI3K/AKT pathway.
Investigating the influence of truffle´s microbiome and genotype on the aroma of truffle fungi
(2019)
Truffles (Tuber spp.) are belowground forming fungi that develop in association with roots of various host trees and shrubs. Their fruiting bodies are renowned for their enticing aromas which vary considerably, even within truffles of the same species. This aroma variability might be attributed to factors such as geographical origin, degree of fruiting body maturation, truffle genotype and microbiome (microbial communities that colonise truffle fruiting bodies) which often co-vary. Although the influence of specific factors is highlighted by several studies, discerning the contribution of each factor remains a challenge since it requires an appropriate experimental design. The primary purpose of this thesis was to gain insight into the influence of truffle’s genotype and microbiome on truffle aroma.
This doctoral thesis is comprised of four chapters. Chapter1 (Vahdatzadeh et al., 2018) aimed to exclusively elucidate the influence of truffle genotype on truffle aroma by investigating the aroma of nine mycelial strains of the white truffle Tuber borchii. We also assessed whether strain selection could be employed to improve the human- perceived truffle aroma. Quantitative differences in aroma profiles among strains could be observed upon feeding of amino acids. Considerable aroma variabilities among strains were attributed to important truffle volatiles, many of which might be derived from amino acid catabolism through the Ehrlich pathway. 13 C-labelling experiments confirmed the existence of the Ehrlich pathway in truffles for leucine, isoleucine, methionine, and phenylalanine. Sensory analyses further demonstrated that the human nose can differentiate among strains. Our results illustrated the influence of truffle genotype on truffle aroma and showed how strain selection could be used to improve the human-perceived truffle aroma.
In chapter 2 the existing knowledge on the composition of bacterial community of four truffle species was compiled using meta-analysis approach (Vahdatzadeh et al., 2015). We highlighted the endemic microbiome of truffle as well as similarities and differences in the composition of microbial community within species at various phases of their life cycle. Furthermore, the potential contribution of truffle microbiome in the formation of truffle odorants was studied. Our findings showed that truffle fruiting bodies harbour complex microbial community composed of bacteria, yeasts, filamentous fungi, and viruses with bacteria being the dominant group. Regardless of truffle species, the composition of endemic microbiome of fruiting bodies appeared very similar and was dominated by α-Proteobacteria class. However, striking differences were observed in the bacterial community composition at various stages of the life cycle of truffle.Our analyses further suggested that odorants common to many truffle species might be produced by both truffle fungi and microbes, whereas specific truffle odorants might be derived from microbes only. Nevertheless, disentangling the origin of truffle odorants is very challenging, since acquiring microbe-free fruiting bodies are currently not possible.
Chapter 3 (Splivallo et al., 2019) further characterises truffle-associated bacterial communities of fruiting bodies of the black truffle T. aestivum from two different orchards. It aimed at defining the native microbiome in this truffle species, evaluating the variability of their microbiome across orchards, and assessing factors that shape assemblages of the bacterial communities. The dominant bacterial communities in T. aestivum revealed to be similar in both orchards: although a large portion of fruiting bodies were dominated by the α-Proteobacteria class (Bradyrhizobium genus) similar to other so far-assessed truffle species, in few cases β-Proteobacteria (Polaromonas genus), or Sphingobacteria (Pedobacter genus) were found to be predominant classes. Moreover, factors shaping bacterial communities influenced the two orchards differently, with spatial location within the orchard being the main driver in Swiss orchard and collection season in the French one. Surprisingly, in contrast to other fungi, truffle genotype and the degree of fruiting body maturity seemed not to contribute in shaping the assembly of truffle microbiome. Altogether, our data highlighted the existence of heterogeneous bacterial communities in T. aestivum fruiting bodies which are dominated by either of the three bacterial classes and mainly by the α-Proteobacteria class, irrespective of geographical origin. They further illustrated that determinants driving the assembly of various bacterial communities within truffle fruiting bodies are site-specific. Truffles are highly perishable delicacies with a short shelf life (1-2 weeks), and their aroma changes profoundly upon storage. Since truffle aroma might be at least partially produced by the truffle microbiome, chapter 4 (Vahdatzadeh et al., 2019) focuses on assessing the influence of the truffle microbiome on aroma deterioration of T.aestivum during post harvest storage. Specifically, volatile profile and bacterial communities of fruiting bodies collected from four different regions (three in France and one in Switzerland) were studied over nine days of storage. Our findings demonstrated the gradual replacement of dominant bacterial classes in fresh truffles (α-Proteobacteria, β-Proteobacteria, and Sphingobacteria) by food spoilage bacteria (members of γ- Proteobacteria and Bacilli classes), regardless of the initial diversity of the bacterial classes. This shift in the bacterial community also correlated with changes in volatile profiles, and markers for truffle freshness and spoilage could be identified. Ultimately, network analysis illustrated possible links among those volatile markers and specific bacterial classes. Our data showed that storage deeply influenced the composition of bacterial community as well as aroma of truffle fruiting bodies. They also illustrated the correlation between the shift in truffle microbiome, from commensal to detrimental, and the change of aroma profile, possibly leading to the loss of fresh truffle aroma. Overall, the work undertaken in this thesis demonstrated that truffle genotype and microbiome had a stronger influence on truffle aroma than previously believed.
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.
Characterizing the hologenome of Lasallia pustulata and tracing genomic footprints of lichenization
(2017)
The lichen symbiosis – consisting of fungal mycobionts and photoautotroph photobionts (green algae or cyanobacteria) – is globally successful. It covers an estimated 6% of the global surface with habitats ranging from deserts to the arctic. This success is reflected in the diversity of the mycobionts, with around 21% of all fungal species participating in lichen symbioses that can be facultative or obligate. Lichenization is furthermore evolutionary old, with fossil evidence for lichens reaching back 415 million years. For an individual fungal lineage, the Lecanoromycetes, the lichenization happened around 300 million years ago. This longstanding symbiotic relationship and the diversity of observed symbiotic dependency make them promising models to study the genomic consequences that follow the establishment of symbioses. Despite this, only little is known about the genomic effects of lichenization and extreme symbiotic dependency. To fill this gap we sequenced the hologenome of the lichen Lasallia pustulata, where the mycobiont could so far not been cultivated, suggesting that it might be more dependent on its symbionts.
As the poor culturability of lichen symbionts renders their genomes inaccessible to standard sequencing practices, we evaluated the extent to which different metagenome sequencing- and de novo assembly-strategies can be used to sequence and reconstruct the genomes of the individual symbionts. We find that the abundances of individual genomes present in the L. pustulata hologenome vary substantially, with the mycobiont being most abundant. Using in silico generated data sets and real Illumina sequencing data for L. pustulata we observe that the skewed abundances prevent a contiguous assembly of the underrepresented genomes when using only short-read sequencing. We conclude that short-read sequencing can offer first insights into lichen hologenomes. The fragmentation of the reconstructions hinders downstream analyses into the genomic consequences of lichenization though, as these are focused on identifying the gain and loss of genes.
We thus demonstrate a hybrid genome assembly strategy that is based on both short- and long-read sequencing. We show that this strategy is capable of creating highly contiguous genome reconstructions, not only for the L. pustulata mycobiont but also its photobiont Trebouxia sp., along with substantial amounts of the bacterial microbiome. A subsequent analysis of the microbiome of L. pustulata – performed over nine different samples collected in Germany and Italy – showed a stable taxonomic composition across the geographic range. We find that Acidobacteriaceae, which are known to thrive in nutrient poor habitats, are the dominant taxa. These would make them well adapted for the co-habitation with L. pustulata, which largely grows on rocks. Whether the Acidobacteriaceae are functionally involved in the lichen symbiosis is unclear so far.
As further comparative genomic studies rely on comprehensive genome annotations, we evaluate the completeness and fidelity of the gene annotations for the mycobiont L. pustulata as well as four further Lecanoromycetes. This reveals that un- and mis-annotated genes impact all evaluated genomes, with artificially joined genes and unannotated genes having the largest impact. In addition to these factors we find that the sequence composition – especially G/C-rich inverted repeats – lead to sequencing errors that interfere with the gene prediction. We minimize the effects of these artifacts through a rigorous curation.
Given the extremely sparse taxon sampling of available green alga genomes, we focus our search for the genomic footprints of lichenization on the mycobionts. We compare the genomes of the Lecanoromycetes to their closest relatives, the Eurotiomycetes and Dothideomycetes. This reveals that the last common ancestor of the Lecanoromycetes has lost around 10% of its genes after they split from the non-lichenized ancestor they share with the Eurotiomycetes. These losses are furthermore enriched, showing an excessive loss of genes involved with the degradation of polysaccharides. The loss of these genes fits a change from an ancestral saprotrophic lifestyle that depends on degrading complex plant matter, to the symbiotic lifestyle that relies on simpler nutrients provided by the photobionts. While the last common ancestor of the Lecanoromycetes additionally gained around 400 genes these could so far not be further characterized due to a lack of functionally annotated reference data.
As the mycobiont L. pustulata could so far not been grown in axenic culture, we initially expected to find an extensive genomic remodeling compared to the other mycobionts that easily grow in culture. We do not find evidence for this. Analyzing both the contraction of gene families and the loss of genes, we observe that L. pustulata and Umbilicaria muehlenbergii – its close relative that is easily grown in culture – share most of these. Furthermore, L. pustulata does not show an excessive loss of evolutionary old and well-conserved genes. These effects are mirrored on the functional level, as neither gene family contractions nor gene losses show a functional enrichment. This is partially due to the lack of functional reference data, analogous to the genes gained in the Lecanoromycetes, rendering their characterization hard. Thus, further studies on the genomic consequences of lichenization and differences in symbiotic dependence will have to be conducted, including larger taxon sets. This will be even more important for the photobionts, as the Chlorophyta are even more sparsely sampled today, hindering an effective functional and evolutionary study.
Subject of this thesis was the investigation of the actin-interacting and glucocorticoid-sensitive Protein DRR1 (or Fam107a) and its role in promoting stress resilience in the murine hippocampus.
We proposed the hypothesis that DRR1 through its actin-binding properties specifically modulates neuronal actin dynamics and promotes resilience through synaptic plasticity leading to subsequently improvement of cognitive performance and social behavior. The accompanied AMPA-receptor transport could create an efficient way regulating neural function and complex behavior during stress episodes.
By utilizing fluorescent immunohistochemistry, we showed basal expression of DRR1 primarily in the murine cerebellum and hippocampal CA3 and CA1 area. Co-staining with different cell marker proteins showed DRR1 expression in neurons, microglia and especially in astrocytic end-feet, which create contact to the brain vasculature.
To test whether DRR1 and AMPA receptor function correlate to modulate stress-associated consequences, primary hippocampal neuron cultures were transduced with adeno-associated virus (AAV) for overexpression or suppression of the protein. Western Blot analysis showed a positive correlation between the AMPA-receptor subunit GluR2 and DRR1 amounts. Further the application of the proximity ligation assay (PLA) in untreated neural cultures indicated interaction between DRR1 and the AMPA receptor subunit GluR2. To address whether DRR1 even affects AMPAR trafficking we performed the “newly inserted assay” after AAV-treatment of primary hippocampal neuron cultures. Suppression of DRR1 revealed less newly inserted GluR2 subunits as compared to controls. Inconclusive were the results upon DRR1 overexpression, however they point to no changes.
In the second part we correlated behavioral phenotypes originating from in vivo overexpression and suppression of DRR1 in the murine hippocampus with potential alterations in neuronal morphology. Therefore, in vitro analysis was performed utilizing AAV transduced primary hippocampal cultures overexpressing or suppressing DRR1. Synchronously the viral vector included a green fluorescent protein (GFP) being expressed throughout the complete neural cell. GFP staining was used to verify successful transfection and for reconstruction of dendritic arbors and dendritic stretches for spine classification. DRR1 suppression showed reduced total spine numbers especially evoked by reduced numbers of immature spine classes – namely long thin spines and filopodia. Whereas mature mushroom spines and stubby spines were unaffected. By overexpressing DRR1, tendencies inclined against higher total dendritic lengths, branch points and increased dendritic arbors in comparison to controls. In regard of spines, total numbers were unaffected. However, mature mushroom spines were significantly declined in numbers, but compensated by increased numbers of immature long thin spines and filopodia.
Chronic social defeat stress (CSDS) is widely used in mouse models to study the effects of stress and resilience. We exposed C57Bl/6J mice expressing GFP under the Thy1 promoter CSDS and categorized them into resilient (R+/-), susceptible (R-/-) and non-learning (R+/+) mice following a modified social interaction test (MSIT). We found alterations in CA1 spine compositions with resilient animals resembling the untreated phenotype. Stress susceptible and non-learning animals displayed reduced numbers in stubby spines with simultaneous increases in mature mushroom spines. In addition, we could detect a tendency towards more immature spines in susceptible animals and non-learners, mirroring our in vitro results.
Finally, we present a different investigative approach in this thesis. Sequenced acute stress was previously found to compromise cognition including spine loss.
We aimed to investigate the implication of acute stress on DRR1 levels and its occurrence in diverse cell types of the brain. We subjected one group of C57Bl/6J mice to acute stress and injected another group with the artificial glucocorticoid DEX. Six hours post stress, animals were perfused and brains were subsequently immunobiologically analyzed. We found DRR1 protein levels elevated in the hippocampus of stressed and DEX-treated animals compared to controls. Interestingly, DRR1 seemed was especially elevated in endothelial cells. This coincides with our investigations finding DRR1 present in astrocytic end-feet under basal conditions and might claim a participation of DRR1 in the blood-brain-barrier integrity.
Our results show DRR1 as actin-interacting and glucocorticoid-sensitive gene affecting structural plasticity of hippocampal spines. Moreover, DRR1 directly interacts with AMPA glutamate receptors and presumably is involved in AMPA trafficking to the postsynaptic membrane. In addition, this study could demonstrate that DRR1 is expressed by other cell types of the brain. Of special interest is DRR1’s occurrence in astrocytic end-feet and endothelial cells suggesting a role as integrator of cell-cell communication and to this end also acting as modifier of stress-induced consequences at the neurovascular unit.
In vivo data of chronically stressed mice displayed no phenotypic differences in hippocampal pyramidal neurons of resilient animals as compared to unstressed mice. Morphological alterations of spine structures were particularly visible in stress susceptible and non-learning animals. Integrating our findings with existing behavioral data, we can conclude that DRR1 plays a role in stress resilience whereby it needs to be expressed in a tightly managed homeostatic equilibrium.
Microsporidia are a group of parasites that infect a wide range of species, many of which play important roles in agriculture and human disease. At least 14 microsporidian species have been confirmed to cause potentially lifethreatening infectious diseases in both immunocompromised and immunocompetent humans. Approximately 1,400 species of microsporidia have been described. Depending on their host and habitat they are classified into three groups, the aquasporidia, the terresporidia and the marinosporidia.
Microsporidia were originally classified as fungi by Naegeli (1857). However, their lack of typical eukaryotic components – such as mitochondria, Golgi bodies or peroxisomes – suggested to place the microsporidia together with other amitochondriate protists within the Archezoa kingdom. This "microsporidia-early" hypothesis was further supported by molecular phylogenies inferred from individual genes. Despite this evidence, the placement of microsporidia as an early branching eukaryote remained a topic for debate. The phylogeny of microsporidia is prone to suffer from biases in their reconstruction. The high evolutionary rate of microsporidian proteins tends to place these proteins together with other fast evolving lineages, a phenomenon known as long-branch attraction. In 1996, the first molecular phylogenetic studies placed the microsporidia inside the fungi.
Subsequently, several further studies located the microsporidia at different positions inside the fungal clade. Since then, microsporidia have been considered as members of the Ascomycota, Zygomycota, Cryptomycota, or as a sister group to the Ascomycota and Basidiomycota, or even as the sister group of all fungi.
The difficulties in determining the evolutionary origin of microsporidia are not only caused by their lack of several cellular components but also by their reduced genomes and metabolism. Being obligate intracellular parasites, microsporidia successfully reduced their genome sizes, down to the range of bacteria. As the smallest eukaryotic genome described so far, the genome of Encephalitozoon intestinalis is just 2.3 Mbp, about half the size of the one of Escherichia coli. Due to their low number of protein coding genes (less than 4,000), microsporidia are thought to retain only genes essential for their survival and development. Furthermore, several key metabolic pathways are missing in the microsporidia, such as the citric acid cycle, oxidative phosphorylation, or the de novo biosynthesis of nucleotides. As a result they are in an obligatory dependence on many primary metabolites from the hosts. However, the presence of hsp70 protein suggests a more complex genome of the microsporidian ancestor. Consequently, the small microsporidian genomes and the reduced metabolism would be consequences of a secondary loss process that molded the contemporary microsporidia from a functionally more complex ancestral species. However, it remains unclear whether the last common ancestor (LCA) of the microsporidia was already reduced, or whether the genome compaction was lineage-specific and started from a more complex LCA.
We investigated the evolutionary history of the contemporary microsporidia through the reconstruction and analysis of their LCA. As a first step in our analysis, we have developed and implemented a software facilitating an intuitive data analysis of the large presence absence-patterns resulting from the tracing of microsporidian proteins in gene sets of many different species. These so called phylogenetic profiles can now be dynamically visualized and explored with PhyloProfile. The software allows the integration of other additional information layers into the phylogenetic profile, such as the similarity of feature architecture (FAS) between the protein under study and its orthologs. The FAS score can be displayed along the presence-absence pattern, which can help to identify orthologs that have likely diverged in function. PhyloProfile closes the methodological gap that existed between tools to generate large phylogenetic profiles to delineate the evolutionary history and the contemporary distribution of large – and ultimately complete – gene sets, and the more function-oriented analysis of individual protein. In the next step we tackled the problem of how to transfer functional annotation from one protein to another. We have developed HamFAS that integrates a targeted ortholog search based on the HaMStR algorithm with a weighted assessment of feature architecture similarities (FAS) between orthologs. In brief, for a seed protein we identify orthologs in reference species in which proteins have been functionally annotated based on manually curated assignments to KEGG Ortholog (KO) groups. The FAS scores between the orthologs and seed proteins are calculated. Subsequently, we compute pairwise FAS scores for all reference proteins within a KO group. A group's mean FAS score serves then as cutoff that must be exceeded to warrant transfer of its KO identifier to the seed. A benchmark using a manually curated yeast protein set showed that HamFAS yields the best precision (98.5%) when compared with two state-of-the-art annotation tools, KAAS and BlastKOALA. Furthermore, HamFAS achieves a higher sensitivity. On average HamFAS annotates almost 50% more proteins than KAAS or BlastKOALA.
With this extended bioinformatics toolbox at hand, we aimed at reconstructing the evolutionary history of the microsporidia. We generated a robust phylogeny of microsporidia using a phylogenomics approach. As a data basis, we identified a set of microsporidian proteins encoded by 80 core genes with one-to-one orthologs. A maximum likelihood analysis of this data
with 48 fungi and additionally in 13 species from more distantly related such as animals and plants combined in a supermatrix strongly supported the hypothesis that microsporidia form the sister group of the fungi. We confirmed that the data explains this microsporidia-fungi relationship significantly better than any other of the previously proposed phylogenetic hypotheses.
On the basis of this phylogeny, and of the phylogenetic profiles of microsporidian proteins, we then focused on reconstructing the dynamics microsporidian genome evolution. Between 2% of the proteins in the compact microsporidia Encephalitozoon intestinalis and up to 49% of the proteins of Edhazardia aedis are private for individual microsporidian species. A comparison of the sequence characteristics of these proteins to that of proteins with orthologs in other microsporidian species revealed individual differences. Yet, without further evidences it remains unclear whether these private genes are indeed lineage-specific innovations contributing to the adaptation of each microsporidium to its host, or whether these are artifacts introduced in the process of gene annotation. A total of 14,410 microsporidian proteins could then be grouped into 1605 orthologous groups that can be traced back to the last common ancestor of the microsporidia (LCA set). We found that 94% of the microsporidian LCA proteins could be tracked back to the last eukaryotic common ancestor. The high evolutionary age of these proteins, together with the resistance against gene loss in the microsporidia suggests that the corresponding functions are essential for eukaryotic life. Further 3% of the LCA proteins could be dated to the common ancestor microsporidia share with the fungi. Only 3% of the LCA proteins appear as microsporidia specific inventions. These proteins are potentially of importance for the evolutionary of the obligate parasitic lifestyle nowadays shared by all microsporidia.
The functional annotation and metabolic pathway analysis of the microsporidian LCA protein set gave us more insight into the adaptation of the microsporidia to their parasitic lifestyle and the origin of the microsporidian genome reduction. The presence of E1 and E3 components of the pyruvate dehydrogenase complex and the mitochondrial hsp70 protein support an ancestral presence of mitochondria in the ancestral microsporidia. In addition, several ancient proteins that complement gapped metabolic pathways were found in the microsporidian LCA. They suggested a more complex genome and metabolism in the LCA. However, our reconstruction of the metabolic network of the microsporidian LCA still lacks many main pathways. For example, the TCA cycle for effective energy production, and key enzymes that are required for in vivo synthesis of critical metabolites like purines and pyrimidines appear absent. We therefore find that the parasitic lifestyle and the genome reduction already occurred in the microsporidian LCA. This ancestral state was followed by further losses and gains during the evolution of each individual microsporidian lineage.