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Cardiovascular diseases are the most common cause of death in industrial nations. The basis of these diseases is a dysfunction in the interaction between the cells the heart is composed of. The main types of cells making up the human heart are cardiomyocytes that build the myocardium and provide the contraction properties, endothelial cells that delimit the blood flowing through the inner chambers and coronary arteries from the myocardial tissue, and fibroblasts, which build the connective tissue. A common process in the development of cardiovascular diseases is the formation of fibrosis due to injury of the endothelium and subsequent infiltration of the cardiac tissue by immune cells, and inflammatory agents like cytokines. Cytokines exert different functions in cardiac cells. Tumor necrosis factor α (TNFα) is an inducer of apoptosis. Transforming growth factor ß (TGFß) is known for activation of proliferation. Other cytokines like C-X-C motif chemokine 11 (CXCL11), interleukin-6 (IL-6), or brain-derived neurotrophic factor (BDNF) have not yet been investigated or their impact on such cells is unknown. Eventually, however, fibrotic scar tissue arises from the transition from fibroblasts to myofibroblasts leading to a stiffening of the cardiac muscle and impaired pump function. In order to prevent the occurrence of these events the balance of proliferation, migration, and differentiation of cardiac cells needs to be controlled very delicately.
The mechanisms controlling these interactions are still not well understood, which is why this work aimed at the elucidation of molecular mechanisms within the three main cell types that might play a role in the regulation of cardiac function. A proteomic approach using mass spectrometry was used to identify alterations in protein levels that could provide hints about the involved pathways and find new players as candidates for more detailed investigation. Initially, the proteomic composition of HL-1 cardiomyocytes, L929 fibroblasts, and human umbilical vein endothelial cells (HUVECs) that were cultivated in standard growth conditions without stress was investigated. Half of the total protein intensity was made up by only 42 to 53 proteins, depending on the cell type. More than a third of all proteins were identified in all three cell types, which may be proteins performing common cell functions. Indeed, the proteins displaying the highest abundance seem to be predominantly involved in such common cellular functions as the regulation of glucose metabolism or the cytoskeleton. More specific functions like heart development and muscle contraction were found enriched in cardiomyocytes as were mitochondrial proteins. The proportion of proteins with extracellular localization and function was higher in fibroblasts and endothelial cells.
Secondly, the impact of cytokines on the proliferative behavior and the proteomic composition of cardiomyocytes and fibroblasts was analyzed. HL-1 cardiomyocytes and L929 fibroblasts were treated with different concentrations of cytokines with a cytotoxic, proliferative, or yet unknown effect on these cells. While HL-1 cells exhibited no macroscopic reaction to any of the cytokines used, cytotoxic/growth inhibitory (TNFα, CXCL11) and proliferative (TGFß, IL6, BDNF) effects were observed for L929 cells. The latter also showed CXCL11-induced upregulated EIF2 signaling, pointing to a higher need of protein synthesis.
The third aim was the examination of proteome adaptations in endothelial cells due to different kinds of stress, as these cells are the first line of defense against inflammatory agents or injury and therefore prone to wounding. The role of the growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in wounding and starvation was another object of this study as they are known for their angiogenic and cell survival supporting properties. Additionally, the impact of the cellular sex on the response to stress and growth factors was examined, because a person’s sex plays an important role in susceptibility, risk factors, and outcome of cardiovascular diseases. This has mainly been attributed to the different hormone levels, especially the higher levels of estrogen in premenopausal women, which exerts cardioprotective properties, but also genetic background was reported to play an important role. Only few studies that examined the molecular properties of HUVECs considered the cellular sex and if so, the genetic bias of unrelated samples was not taken into account. This is why Lorenz and colleagues at the Charité in Berlin collected HUVECs from newborn twins of opposite sex, cultivated them without stress in standard growth medium, exposed them to wounding and serum starvation, and investigated the impact of the growth factors and the sex on migrational behavior and metabolic issues. The current work focused on the alterations of not only the intra- but also the extracellular proteome, because paracrine signaling is crucial for intercellular communication in order to cope with stress. General differences between male and female cells were observed for proteins encoded on the X chromosome with higher levels in females (DDX3X, UBA1, EIF1AX, RPS4X, HDHD1), except for one protein with higher levels in male cells (G6PD). A Y-chromosomal protein was, for the first time, identified in endothelial cells (DDX3Y). Wounding, starvation, and growth factor treatment led to alterations and sex-specific different levels in an unexpectedly high number of proteins, with VEGF showing a stronger impact than bFGF. Many proteins with alterations observed without taking the sex into account, were actually only changed in male or female cells. Some proteins were regulated in opposite directions, or growth factors inhibited their secretion in a sex-specific way by unknown mechanisms. Tissue factor pathway inhibitor 2 (TFPI2) should be emphasized as a protein with sex-specific differences, especially in the extracellular space and with increased levels after starvation and VEGF treatment. These observations suggest a temporal lack in TFPI2 synthesis and secretion in male cells, which might explain the enhanced adaptation of females to wounding.
The results of this work lay the basis for future investigation by providing a database of intra- and extracellular proteome changes due to different environmental circumstances. It strongly suggests the investigation of male and female HUVECs, and other cells, separately to avoid the impact of the sex observed in this work. Essentially, the observations suggest a number of candidate proteins for more detailed investigations of endothelial and cardiovascular diseases.
Symbiotic interactions are a key element of biological systems. One powerful strategy to gain insight into these interactions, and into biological systems in general, is the analysis of proteins expressed in situ using metaproteomics. In this thesis, host-microbe interactions in two mutualistic associations between chemosynthetic sulfur-oxidizing endosymbionts and marine invertebrates, the deep-sea tubeworm Riftia pachyptila and the shallow-water clam Codakia orbicularis, were studied by adapted and optimized metaproteomics methods.
The Riftia symbiosis, which inhabits hydrothermal vents in the deep sea, and in which the host completely depends on its symbiont for nutrition, has fascinated researchers for about four decades. Yet, the interaction mechanisms between both partners have been understudied so far. Additionally, while different aspects of the host’s biology have been described, a comprehensive analysis has been lacking. Moreover, although only one symbiont 16S rRNA phylotype is present in Riftia, the symbiont population of the same host expresses proteins of various redundant or opposed metabolic pathways at the same time. As the symbionts also exhibit a wide variety in size and shape, symbionts of different size might have dissimilar physiological functions, which remained as of now to be elucidated. In this thesis, we addressed both, the host-symbiont interaction mechanisms, and physiological roles of symbiont subpopulations. A comprehensive Riftia host and symbiont protein database was generated as prerequisite for metaproteomics studies by de novo sequencing the host’s transcriptome and combining it with existing symbiont protein databases. This database was then used for metaproteomics comparisons of symbiont-containing and symbiont-free Riftia tissues, to gain insights into host-symbiont interactions on the protein level. The impact of energy availability on host-symbiont interactions was studied by comparing specimens with stored sulfur (i.e., high energy availability) with specimens in which sulfur storages were depleted. We employed optimized liquid chromatography peptide separation to increase metaproteome coverage. With this analysis, we identified proteins and mechanisms likely involved in maintaining the symbiosis, under varying environmental conditions. We unraveled key interaction mechanisms, i.e.: (i) the host likely digests its symbionts using abundant digestive enzymes, and, at the same time, (ii) a considerable part of the worm’s proteome is involved in creating stable internal conditions, thus maintaining the symbiont population. Furthermore, (iii) the symbionts probably employ eukaryote-like proteins to communicate with the host. (iv) Under conditions of restricted energy availability, the host apparently increases digestion pressure on the symbiotic population to sustain itself.
Riftia symbionts of different size apparently have dissimilar metabolic roles, as revealed in this thesis. We enriched symbionts of different sizes using gradient centrifugation. These enrichments were subjected to protein extraction using a protocol optimized for the small sample amount available. Metaproteomics analysis included a gel-based workflow and evaluation of the complex dataset with machine learning techniques. Based on our metaproteomics study, we propose that Riftia symbionts of different cell size correspond to dissimilar physiological differentiation stages. Smaller cells are apparently engaged in cell differentiation and host interactions. Larger cells, on the other hand, seem to be more involved in synthesis of various organic compounds. Supposedly, in large symbionts endoreduplication cycles lead to polyploidy. Our results indicate that the Riftia symbiont employs a large part of its metabolic repertoire at the same time in the stable host environment.
The symbiont of the shallow-water clam Codakia orbicularis, which, like the Riftia symbiont, relies on reduced sulfur compounds as energy source and fixes inorganic carbon, is, unexpectedly, also able to fix atmospheric nitrogen, as shown by metaproteomic, genomic and biochemical analysis. Potentially, this benefits the host, as Codakia digests its symbiont and might thus supplement its diet with organic nitrogen fixed by the symbionts in addition to organic carbon in its nitrogen-poor seagrass habitat.
Mechanically ventilated patients are at risk of ventilator-associated pneumonia, a serious infection of the lungs. Not every ventilated patient develops pneumonia due to a combination of the protective layer of mucus in the airways, the immune system and prophylactic antibiotic therapy. To date, only little was known about the antimicrobial factors produced by humans that protect the lungs against infection. Research described in this thesis was therefore aimed at investigating to what extent the lungs of ventilated patients can inhibit the growth of bacteria, the major causative agent of pneumonia Streptococcus pneumoniae in particular. To this end, the accumulated mucus in the patients’ lungs, sputum, was investigated. The most important conclusion was that sputum can indeed possess antimicrobial activity, explained either by a combination of antibiotics and S. pneumoniae-specific antibodies, or by the innate immune defenses. Thus, sputum may serve as a valuable source of information to unravel the complex interactions between the human host, antimicrobial factors and the microbiome of the lower respiratory tract. A possible consequence of pneumonia is the dissemination of bacteria from the lungs to the bloodstream and the brain, which may lead to meningitis. This thesis describes how this process takes place, and how the so-called choline-binding protein CbpL contributes to invasive pneumococcal infections. In addition, possible future approaches to prevent meningitis caused by this bacterium are proposed.
Lipoproteins of Staphylococcus aureus represent a major class of surface proteins, which are anchored to the outer leaflet of the cell membrane. Although they play a key role in the immune response and virulence, the majority of lipoproteins in this organism is still of unknown function. The aim of our study was to investigate the function of so far poorly or uncharacterized lipoproteins in S. aureus strain Newman. To this end, an integrated bioinformatical approach was applied to define the pan-lipoproteome of 123 completely sequenced S. aureus strains. In total, this analysis predicted 192 different potential lipoproteins, with a core lipoproteome of 39 and a variable lipoproteome of 153 lipoproteins. Out of those 192 lipoproteins, 141 are so far functionally uncharacterized. Primarily focusing on members of the core-lipoproteome with unknown or poorly characterized function, 24 lipoproteins or co-encoded neighbor proteins were selected for further characterization. Of those 24 proteins, 20 S. aureus markerless deletion mutants were constructed (S. aureus delta l01 - delta l20) and screened for an altered growth behavior under various conditions. Here, three mutants showed a temperature-sensitive phenotype, two mutants formed aggregates in the TSB of the manufacturer Merck (TSBMerck), and four mutants showed reduced growth under osmotic stress with 8% NaCl. An altered aggregation behavior was observed for four mutants in the presence of Triton X-100 and for eleven mutants in the presence of SDS. Furthermore, ten mutants revealed an impaired biofilm formation capacity as well as reduced hemolytic activity. Interestingly, S. aureus deletion mutants delta l14 (delta NWMN_1435) and delta l16 (delta NWMN_0646) showed an altered phenotype under nearly all tested growth and stress conditions. Most strikingly, both deletion mutants demonstrated dramatic defects in cell morphology and cell division during the transient growth phase in TSBMerck and were therefore selected for further detailed characterization. Electron microscopy imaging of the two mutants revealed an irregular cell shape, increased cell size, multiple displaced division septa, and incomplete separation of daughter cells resulting in the formation of cell aggregates in TSBMerck. Complementarily, microarray-based transcriptome analysis and whole-genome sequencing of S. aureus delta l14 and delta l16 suppressor mutants strongly point to a functional association of both lipoproteins with cell envelope- or cell division-related processes. Specifically, multiple hints suggest a functional connection of both lipoproteins with lipo- or wall teichoic acids. Of note, the phenotypes of S. aureus delta l14 and delta l16 are conditional and appear under some, but not all growth conditions. Thus, it is conceivable that the function of L14 and L16 is modulated by metabolic processes, or that the proteins might be part of a “backup system” becoming important only under certain conditions. Collectively, we propose that L14 and L16 fulfill a basic role in cell envelope- or cell division-related processes under specific growth conditions. Particularly, the activity of L14 and L16 might be necessary for the function or localization of lipo- or wall teichoic acids, and thus, might be linked to the regulation of autolysins. In conclusion, this study reveals important insights into the function of two so far uncharacterized but highly conserved lipoproteins in S. aureus.