579 Mikroorganismen, Pilze, Algen
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Stapylococcus aureus colonises the nose of healthy individuals but can also cause a wide range of infections. Amino acid (AA) synthesis and their availability is crucial to adapt to conditions encountered in vivo. Most S. aureus genomes comprise all genes required for AA biosynthesis. Nevertheless, different strains require specific sets of AAs for growth. In this study we show that regulation inactivates pathways under certain conditions which result in these observed auxotrophies. We analyzed in vitro and modeled in silico in a Boolean semiquantitative model (195 nodes, 320 edges) the regulatory impact of stringent response (SR) on AA requirement in S. aureus HG001 (wild-type) and in mutant strains lacking the metabolic regulators RSH, CodY and CcpA, respectively. Growth in medium lacking single AAs was analyzed. Results correlated qualitatively to the in silico predictions of the final model in 92% and quantitatively in 81%. Remaining gaps in our knowledge are evaluated and discussed. This in silico model is made fully available and explains how integration of different inputs is achieved in SR and AA metabolism of S. aureus. The in vitro data and in silico modeling stress the role of SR and central regulators such as CodY for AA metabolisms in S. aureus.
Cellular membranes form a boundary to shield the inside of a cell from the outside. This is of special importance for bacteria, unicellular organisms whose membranes are in direct contact with the environment. The membrane needs to allow the reception of information about beneficial and harmful environmental conditions for the cell to evoke an appropriate response. Information gathering is mediated by proteins that need to be correctly organized in the membrane to be able to transmit information. Several principles of membrane organization are known that show a heterogeneous distribution of membrane lipids and proteins. One of them is functional membrane microdomains (FMM) which are platforms with a distinct lipid and protein composition. FMM move within the membrane and their integrity is important for several cellular processes like signal transduction, membrane trafficking and cellular differentiation. FMM harbor the marker proteins flotillins which are scaffolding proteins that act as chaperones in tethering protein cargo to FMM. This enhances the efficiency of cargo protein oligomerization or complex formation which in turn is important for their functionality. The bacterium Bacillus subtilis contains two flotillin proteins, FloA and FloT. They form different FMM assemblies which are structurally similar, but differ in the protein cargo and thus in the specific function.
In this work, the mobility of FloA and FloT assemblies in the membrane was dissected using live-cell fluorescence microscopy techniques coupled to genetic, biochemical and molecular biological methods. A characteristic mobility pattern was observed which revealed that the mobility of both flotillins was spatially restricted. Restrictions were bigger for FloT resulting in a decreased diffusion coefficient compared to FloA. Flotillin mobility depends on the interplay of several factors. Firstly, the intrinsic properties of flotillins determine the binding of different protein interaction partners. These proteins directly affect the mobility of flotillins. Additionally, binding of interaction partners determines the assembly size of FloA and FloT. This indirectly affects the mobility, as the endo-cytoskeleton spatially restricts flotillin mobility in a size-dependent manner. Furthermore, the extracellular cell wall plays a dual role in flotillin mobility: its synthesis stimulates flotillin mobility, while at the same time its presence restricts flotillin mobility. As the intracellular flotillins do not have spatial access to the exo-cytoskeleton, this connection is likely mediated indirectly by their cell wall-associated protein interaction partners. Together the exo- and the endo-cytoskeleton restrict the mobility of FloA and FloT.
Similar structural restrictions of flotillin mobility have been reported for plant cells as well, where the actin cytoskeleton and the cell wall restrict flotillin mobility. These similarities between eukaryotic and prokaryotic cells indicate that the restriction of flotillin mobility might be a conserved mechanism.
The biosphere harbors a large quantity and diversity of microbial organisms that can thrive in all environments. Estimates of the total number of microbial species reach up to 1012, of which less than 15,000 have been characterized to date. It has been challenging to delineate phenotypically, evolutionary and ecologically meaningful lineages such as for example, species, subspecies and strains. Even within recognized species, gene content can vary considerably between sublineages (for example strains), a problem that can be addressed by analyzing pangenomes, defined as the non-redundant set of genes within a phylogenetic clade, as evolutionary units.
Species considered to be ecologically and evolutionary coherent units, however to date it is still not fully understood what are primary habitats and ecological niches of many prokaryotic species and how environmental preferences drive their genomic diversity. Majority of comparative genomics studies focused on a single prokaryotic species in context of clinical relevance and ecology. With accumulation of sequencing data due to genomics and metagenomics, it is now possible to investigate trends across many species, which will facilitate understanding of pangenome evolution, species and subspecies delineation.
The major aims of this thesis were 1) to annotate habitat preferences of prokaryotic species and strains; 2) investigate to what extent these environmental preferences drive genomic diversity of prokaryotes and to what extent phylogenetic constraints limit this diversification; 3) explore natural nucleotide identity thresholds to delineate species in bacteria in metagenomics gene catalogs; 4) explore species delineation for applications in subspecies and strain delineation in metagenomics.
The first part of the thesis describes methods to infer environmental preferences of microbial species. This data is a prerequisite for the analyses performed in the second part of the thesis which explores how the structure of bacterial pangenomes is predetermined by past evolutionary history and how is it linked to environmental preferences of the species. The main finding in this subchapter that habitat preferences explained up to 49% of the variance for pangenome structure, compared to 18% by phylogenetic inertia. In general, this trend indicates that phylogenetic inertia does not limit evolution of pangenome size and diversity, but that convergent evolution may overcome phylogenetic constraints. In this project we show that core genome size is associated with higher environmental ubiquity of species. It is likely this is due to the fact that species need to have more versatile genomes and most necessary genes need to be present in majority of genomes of that species to be highly prevalent. Taken together these findings may be useful for future predictive analyses of ecological niches in newly discovered species.
The third part of the thesis explores data-driven, operational species boundaries. I show that homologous genes from the same species from different genomes tend to share at least 95% of nucleotide identity, while different species within the same genus have lower nucleotide identity. This is in line with other studies showing that genome-wide natural species boundary might be in range of 90-95% of nucleotide identity. Finally, the fourth part of the thesis discusses how challenges in species delineation are relevant for the identification of meaningful within-species groups, followed by a discussion on how advancements in species delineation can be applied for classification of within-species genomic diversity in the age of metagenomics.
The putative attachment protein G of pneumonia virus of mice (PVM), a member of the Pneumoviruses, is an important virulence factor with so far ambiguous function in a virus-cell as well as in virus-host context. The sequence of the corresponding G gene is characterized by significant heterogeneity between and even within strains, affecting the gene and possibly the protein structure. This accounts in particular for the PVM strain J3666 for which two differing G gene organizations have been described: a polymorphism in nucleotide 65 of the G gene results in the presence of an upstream open reading frame (uORF) that precedes the main ORF in frame (GJ366665A) or extension of the major G ORF for 18 codons (GJ366665U). Therefore, this study was designed to analyse the impact of the sequence variations in the respective G genes of PVM strains J3666 and the reference strain 15 on protein expression, replication and virulence.
First, the controversy regarding the consensus sequence of PVM J3666 was resolved. The analysis of 45 distinct cloned fragments showed that the strain separated into two distinct virus populations defined by the sequence and structure of the G gene. This division was further supported by nucleotide polymorphisms in the neighbouring M and SH genes. Sequential passage of this mixed strain in the cell line standardly used for propagation of virus stocks resulted in selection for the GJ366665A-containing population in one of two experiments pointing towards a moderate replicative advantage. The replacement of the G gene of the recombinant PVM 15 with GJ366665A or GJ366665U, respectively, using a reverse genetic approach indicated that the presence of uORF within the GJ366665A significantly reduced the expression of the main G ORF on translational level while the potential extension of the ORF in GJ366665U increased G protein expression. In comparison, the effect of the G gene-structure on virus replication was inconsistent and dependent on cell line and type. While the presence of uORF correlated with a replication advantage in the standardly used BHK-21 cells and primary murine embryonic fibroblasts, replication in the murine macrophage cell line RAW 264.7 did not. In comparison, the GJ366665U variant was not associated with any effect on replication in cultured cells at all. Nonetheless, in-vivo analysis of the recombinant viruses associated the GJ366665U gene variant, and hence an increased G expression, with higher virulence whereas the GJ366665A gene, and therefore an impaired G expression, conferred an attenuated phenotype to the virus.
To extend the study to other G gene organizations, a recombinant PVM expressing a G protein without the cytoplasmic domain and for comparison a G-deletion mutant, both known to be attenuated in vivo, were studied. Not noticed before, this structure of the G gene was associated with a 75% reduction in G protein expression and a significant attenuation of replication in macrophage-like cells. This attenuation was even more prominent for the virus lacking G. Taking into consideration the higher reduction in G protein levels compared to the GJ366665A variant indicates that a threshold amount of G is required for efficient replication in these cells.
In conclusion, the results gathered indicated that the expression levels of the G protein were modulated by the sequence of the 5’ untranslated region of the gene. At the same time the G protein levels modulated the virulence of PVM.
The human pathogen Aspergillus (A.) fumigatus is a fungal mold that can cause severe infections in immunocompromised hosts. Pathogen recognition and immune cell cross-talk are essential for clearing fungal infections efficiently. Immune cell interactions in particular may enhance individual cell activation and cytotoxicity towards invading pathogens.
This study analyzed the reciprocal cell activation of natural killer (NK) cells and monocyte-derived dendritic cells (moDCs) after stimulation with A. fumigatus cell wall fractions and whole-cell lysates. Furthermore, the impact of the on moDCs expressed fungal receptors Dectin-1 and TLR-2 on NK cell activation was analyzed. Stimulation of moDCs with ligands for Dectin-1 and TLR-2 and transfer of soluble factors on autologous NK cells showed that moDCs could induce NK cell activation solely by secreting factors. In summary, both cell types could induce reciprocal cell activation if the stimulated cell type recognized fungal morphologies and ligands. However, moDCs displayed a broader set of A. fumigatus receptors and, therefore, could induce NK cell activation when those were not activated by the stimulus directly.
Consequently, new fungal receptors should be identified on NK cells. The NK cell characterization marker CD56 was reduced detected in flow cytometry after fungal co-culture. Notably, this decreased detection was not associated with NK cell apoptosis, protein degradation, internalization, or secretion of CD56 molecules. CD56 was shown to tightly attach to hyphal structures, followed by its concentration at the NK-A. fumigatus interaction site. Actin polymerization was necessary for CD56 relocalization, as pre-treatment of NK cells with actin-inhibitory reagents abolished CD56 binding to the fungus. Blocking of CD56 suppressed fungal mediated NK cell activation and secretion of the immune-recruiting chemokines MIP-1α, MIP-1β, and RANTES, concluding that CD56 is functionally involved in fungal recognition by NK cells.
CD56 binding to fungal hyphae was inhibited in NK cells obtained from patients during immune-suppressing therapy after allogeneic stem cell transplantation (alloSCT). Additionally, reduced binding of CD56 correlated with decreased actin polymerization of reconstituting NK cells challenged with the fungus. The immune-suppressing therapy with corticosteroids negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES in NK cells after fungal stimulation ex vivo. Similar results were obtained when NK cells from healthy donors were treated with corticosteroids prior to fungal co-culture. Thus, corticosteroids were identified to have detrimental effects on NK cell function during infection with A. fumigatus.
Gonorrhea is the second most common sexually transmitted infection worldwide and is caused by Gram-negative, human-specific diplococcus Neisseria gonorrhoeae. It colonizes the mucosal surface of the female reproductive tract and the male urethra. A rapid increase in antibiotic resistance makes gonorrhea a serious threat to public health worldwide. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are not able to recapitulate all the features of infection. Therefore, a realistic in vitro cell culture model is urgently required for studying the gonorrhea infection. In this study, we established and characterized three independent 3D tissue models based on the porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. The histological, immunohistochemical, and ultra-structural analysis showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in the human host including the formation of epithelial monolayer, underlying connective tissue, mucus production, tight junction (TJ), and microvilli. In addition, functional analysis such as transepithelial electrical resistance (TEER) and barrier permeability indicated high barrier integrity of the cell layer. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results showed disruption of TJs and growing the interleukins production in response to the infection, which depends on the type of strain and cell. In addition, the 3D tissue models supported bacterial survival, which provided an appropriate in vitro model for long-term infection study. This could be mainly because of the high resilience of the 3D tissue models based on the SIS scaffold to the infection in terms of alteration in permeability, cell destruction, and bacterial transmigration.
During gonorrhea infection, a high level of neutrophils migrates to the site of infection. The studies also showed that N. gonorrhoeae can survive or even replicate inside the neutrophils. Therefore, studying the interaction between neutrophils and N. gonorrhoeae is substantially under scrutiny. For this purpose, we generated a 3D tissue model by triple co-culturing of human primary fibroblast cells, human colorectal carcinoma cells, and human umbilical vein endothelial cells. The tissue model was subsequently infected by N. gonorrhoeae. A perfusion-based bioreactor system was employed to recreate blood flow in the side of endothelial cells and consequently study human neutrophils transmigration to the site of infection. We observed neutrophils activation upon the infection. Furthermore, we demonstrated the uptake of N. gonorrhoeae by human neutrophils and reverse transmigration of neutrophils to the basal side carrying N. gonorrhoeae. In summary, the introduced 3D tissue models in this research represent a promising tool to investigate N. gonorrhoeae infections under close-to-natural conditions.