570 Biowissenschaften; Biologie
Refine
Has Fulltext
- yes (3)
Is part of the Bibliography
- yes (3)
Year of publication
- 2021 (3) (remove)
Document Type
- Doctoral Thesis (3)
Language
- English (3) (remove)
Keywords
- Anthropocene (1)
- Aussterbedynamik (1)
- Ceramide (1)
- Chlamydia trachomatis (1)
- Expansion microscopy (1)
- Expansionsmikroskopie (1)
- Fluoreszenzmikroskopie (1)
- Folliculin (1)
- Gonococcal invasion (1)
- Hochauflösungsmikroskopie (1)
Institute
- Fakultät für Biologie (3) (remove)
Neisseria gonorrhoeae (GC) is a human specific pathogenic bacterium. Currently, N. gonorrhoeae developed resistance to virtually all the available antibiotics used for treatment. N. gonorrhoeae starts infection by colonizing the cell surface, followed by invasion of the host cell, intracellular persistence, transcytosis and exit into the subepithelial space. Subepithelial bacteria can reach the bloodstream and disseminate to other tissues causing systemic infections, which leads to serious conditions such as arthritis and pneumonia. A number of studies have well established the host-pathogen interactions during the initial adherence and invasion steps. However, the mechanism of intracellular survival and traversal is poorly understood so far. Hence, identification of novel bacterial virulence factors and host factors involved in the host-pathogen interaction is a crucial step in understanding disease development and uncovering novel therapeutic approaches. Besides, most of the previous studies about N. gonorrhoeae were performed in the conventional cell culture. Although they have provided insights into host-pathogen interactions, much information about the native infection microenvironment, such as cell polarization and barrier function, is still missing.
This work focused on determining the function of novel bacterial virulence factor NGFG_01605 and host factor (FLCN) in gonococcal infection. NGFG_01605 was identified by Tn5 transposon library screening. It is a putative U32 protease. Unlike other proteins in this family, it is not secreted and has no ex vivo protease activity. NGFG_01605 knockout decreases gonococcal survival in the epithelial cell. 3D models based on T84 cell was developed for the bacterial transmigration assay. NGFG_01605 knockout does not affect gonococcal transmigration.
The novel host factor FLCN was identified by shRNA library screening in search for factors that affected gonococcal adherence and/or internalization. We discovered that FLCN did not affect N. gonorrhoeae adherence and invasion but was essential for bacterial survival. Since programmed cell death is a host defence mechanism against intracellular pathogens, we further explored apoptosis and autophagy upon gonococcal infection and determined that FLCN did not affect apoptosis but inhibited autophagy. Moreover, we found that FLCN inhibited the expression of E-cadherin. Knockdown of E- cadherin decreased the autophagy flux and supported N. gonorrhoeae survival. Both non-polarized and polarized cells are present in the cervix, and additionally, E-cadherin represents different polarization properties on these different cells. Therefore, we established 3-D models to better understand the functions of FLCN. We discovered that FLCN was critical for N. gonorrhoeae survival in the 3-D environment as well, but not through inhibiting autophagy. Furthermore, FLCN inhibits the E-cadherin expression and disturbs its polarization in the 3-D models. Since N. gonorrhoeae can cross the epithelial cell barriers through both cell-cell junctions and transcellular migration, we further explored the roles FLCN and E-cadherin played in transmigration. FLCN delayed N. gonorrhoeae transmigration, whereas the knockdown of E-cadherin increased N. gonorrhoeae transmigration.
In summary, we revealed roles of the NGFG_01605 and FLCN-E-cadherin axis play in N. gonorrhoeae infection, particularly in relation to intracellular survival and transmigration. This is also the first study that connects FLCN and human-specific pathogen infection.
The importance of understanding species extinctions and its consequences for ecosystems and human life has been getting increasing public attention.
Nonetheless, regardless of how pressing the current biodiversity loss is, with rare exceptions, extinctions are actually not immediate.
Rather, they happen many generations after the disturbance that caused them.
This means that, at any point in time after a given disturbance, there is a number of extinctions that are expected to happen.
This number is the extinction debt.
As long as all the extinctions triggered by the disturbance have not happened, there is a debt to be paid.
This delay in extinctions can be interpreted as a window of opportunity, when conservation measures can be implemented.
In this thesis, I investigated the relative importance of ecological and evolutionary processes unfolding after different disturbances scenarios, to understand how this knowledge can be used to improve conservation practices aiming at controlling extinctions.
In the Introduction (chapter 1), I present the concept of extinction debts and the complicating factors behind its understanding.
Namely, I start by presenting i) the theoretical basis behind the definition of extinction debts, and how each theory informed different methodologies of study, ii) the complexity of understanding and predicting eco-evolutionary dynamics, and iii) the challenges to studying extinctions under a regime of widespread and varied disturbance of natural habitats.
I start the main body of the thesis (chapter 2) by summarizing the current state of empirical, theoretical, and methodological research on extinction debts.
In the last 10 years, extinction debts were detected all over the globe, for a variety of ecosystems and taxonomic groups.
When estimated - a rare occurrence, since quantifying debts requires often unavailable data - the sizes of these debts range from 9 to 90\% of current species richness and they have been sustained for periods ranging from 5 to 570 yr.
I identified two processes whose contributions to extinction debts have been studied more often, namely 1) life-history traits that prolong individual survival, and 2) population and metapopulation dynamics that maintain populations under deteriorated conditions.
Less studied are the microevolutionary dynamics happening during the payment of a debt, the delayed conjoint extinctions of interaction partners, and the extinction dynamics under different regimes of disturbances (e.g. habitat loss vs. climate change).
Based on these observations, I proposed a roadmap for future research to focus on these less studies aspects.
In chapters 3 and 4, I started to follow this roadmap.
In chapter 3, I used a genomically-explicit, individual-based model of a plant community to study the microevolutionary processes happening after habitat loss and climate change, and potentially contributing to the settlement of a debt.
I showed that population demographic recovery through trait adaptation, i.e. evolutionary rescue, is possible.
In these cases, rather than directional selection, trait change involved increase in trait variation, which I interpreted as a sign of disruptive selection.
Moreover, I disentangled evolutionary rescue from demographic rescue and show that the two types of rescue were equally important for community resistance, indicating that community re-assembly plays an important role in maintaining diversity following disturbance.
The results demonstrated the importance of accounting for eco-evolutionary processes at the community level to understand and predict biodiversity change.
Furthermore, they indicate that evolutionary rescue has a limited potential to avoid extinctions under scenarios of habitat loss and climate change.
In chapter 4, I analysed the effects of habitat loss and disruption of pollination function on the extinction dynamics of plant communities.
To do it, I used an individual, trait-based eco-evolutionary model (Extinction Dynamics Model, EDM) parameterized according to real-world species of calcareous grasslands.
Specifically, I compared the effects of these disturbances on the magnitude of extinction debts and species extinction times, as well as how species functional traits affect species survival.
I showed that the loss of habitat area generates higher number of immediate extinctions, but the loss of pollination generates higher extinction debt, as species take longer to go extinct.
Moreover, reproductive traits (clonal ability, absence of selfing and insect pollination) were the traits that most influenced the occurrence of species extinction as payment of the debt.
Thus, the disruption of pollination functions arose as a major factor in the creation of extinction debts.
Thus, restoration policies should aim at monitoring the status of this and other ecological processes and functions in undisturbed systems, to inform its re-establishment in disturbed areas.
Finally, I discuss the implications of these findings to i) the theoretical understanding of extinction debts, notably via the niche, coexistence, and metabolic theories, ii) the planning conservation measures, including communicating the very notion of extinction debts to improve understanding of the dimension of the current biodiversity crisis, and iii) future research, which must improve the understanding of the interplay between extinction cascades and extinction debts.
The resolution of fluorescence light microscopy was long believed to be limited by the diffraction limit of light of around 200-250 nm described in 1873 by Ernst Abbe. Within the last decade, several approaches, such as structured illumination microscopy (SIM), stimulated emission depletion STED and (direct) stochastic optical reconstruction microscopy (d)STORM have been established to bypass the diffraction limit. However, such super-resolution techniques enabling a resolution <100 nm require specialized and expensive setups as well as expert knowledge in order to avoid artifacts. They are therefore limited to specialized laboratories. Recently, Boyden and colleagues introduced an alternate approach, termed expansion microscopy (ExM). The latter offers the possibility to perform superresolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded. Since its introduction in 2015, expansion microscopy has developed rapidly offering protocols for 4x, 10x and 20x expansion of proteins and RNA in cells, tissues and human clinical specimens.
Mitochondria are double membrane-bound organelles and crucial to the cell by performing numerous tasks, from ATP production through oxidative phosphorylation, production of many important metabolites, cell signaling to the regulation of apoptosis. The inner mitochondrial membrane is strongly folded forming so-called cristae. Besides being the location of the oxidative phosphorylation and therefore energy conversion and ATP production, cristae have been of great interest because changes in morphology have been linked to a plethora of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. However, cristae imaging remains challenging as the distance between two individual cristae is often below 100 nm. Within this work, we demonstrate that the mitochondrial creatine kinase MtCK linked to fluorescent protein GFP (MtCK-GFP) can be used as a cristae marker. Upon fourfold expansion, we illustrate that our novel marker enables visualization of cristae morphology and localization of mitochondrial proteins relative to cristae without the need for specialized setups. Furthermore, we show the applicability of expansion microscopy for several bacterial pathogens, such as Chlamydia trachomatis, Simkania negevensis, Neisseria gonorrhoeae and Staphylococcus aureus. Due to differences in bacterial cell walls, we reveal important aspects for the digestion of pathogens for isotropic expansion. We further show that expansion of the intracellular pathogens C. trachomatis and S. negevensis, enables the differentiation between the two distinct developmental forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We demonstrate the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside the chlamydial inclusion. Moreover, we show that expansion microscopy enables the investigation of bacteria, herein S. aureus, within LAMP1 and LC3-II vesicles. With the introduction of the unnatural α-NH2-ω-N3-C6-ceramide, we further present the first approach for the expansion of lipids that may also be suitable for far inaccessible molecule classes like carbohydrates. The efficient accumulation and high labeling density of our functionalized α-NH2-ω-N3-C6-ceramide in both cells and bacteria enables in combination with tenfold expansion nanoscale resolution (10-20 nm) of the interaction of proteins with the plasma membrane, membrane of organelles and bacteria. Ceramide is the central molecule of the sphingolipid metabolism, an important constituent of cellular membranes and regulates many important cellular processes such as differentiation, proliferation and apoptosis. Many studies report about the importance of sphingolipids during infection of various pathogens. While the transport of ceramide to Chlamydia has been reported earlier, one of the unanswered questions remaining was if ceramide forms parts of the outer or inner bacterial membrane. Expansion of α-NH2-ω-N3-C6-ceramide enabled the visualization of ceramide in the inner and outer membrane of C. trachomatis and their distance was determined to be 27.6 ± 7.7 nm.