Doctoral Thesis
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Metabolomics is the scientific study of metabolites of an organism, cell, or tissue. Metabolomics makes use of different analytical approaches. In this thesis, an analytical platform consisting of proton nuclear magnetic resonance spectroscopy (1H-NMR), gas chromatography-mass spectrometry (GC-MS, EI/quadrupol) and liquid chromatography-mass spectrometry (LC-MS, ESI/TOF) was used for metabolite analysis. Due to the high physicochemical diversity of metabolites, the usage of different analytics is profitable. Focusing on metabolome analysis of microorganisms, the development of viable protocols was prerequisite. To ensure metabolome samples of best possible quality, particularly the sampling procedure has to be optimized for each microorganism to be analyzed individually. In microbial metabolomics, the energy charge value is a commonly used parameter to assure high sample quality (Atkinson 1968). The pathogenic bacterium Staphylococcus aureus and the biotechnical relevant bacterium Bacillus subtilis were main target of research. The sampling protocol development “A protocol for the investigation of the intracellular Staphylococcus aureus metabolome” (Meyer et al. 2010) and “Methodological approaches to help unravel the intracellular metabolome of Bacillus subtilis”s (Meyer et al. 2013) confirmed the need for development and verification of viable protocols. It was observed, that minor differences in the sampling procedure can cause major differences in sample quality. Using the validated analytical platform and the optimized protocols, we were able to investigate the metabolome of S. aureus and B. subtilis under different conditions. Investigations of the pathogenic bacterium S. aureus are of major interest due to its increasing resistance to antibiotics. Methicillin (multi)-resistant S. aureus (MRSA) strains are responsible for several difficult-to-treat infections. The cell wall of bacteria is the target of an array of antibiotics, like the beta-lactam antibiotics. Our study “A metabolomic view of Staphylococcus aureus and Its Ser/Thr kinase and phosphatase deletion mutants: Involvement in cell wall biosynthesis” (Liebeke et al. 2010) revealed the influence of the serine-threonine kinase on cell wall biosynthesis of S. aureus. LC-MS based metabolome data uncovered prevalent wall teichoic acid precursors in the serine-threonine kinase deletion mutant (ΔpknB), and predominantly peptidoglycan precursors in the phosphatase deletion mutant (Δstp), compared to the S. aureus wild type strain 8325. This uncovered a so far undescribed importance of the serine-threonine kinase on the cell wall metabolism and provides new insights into its regulation. The nasopharynx and the human skin are often the ecological niche of S. aureus. Furthermore, S. aureus exists outside its host, for example on catheters. Depending on its niche, S. aureus is exposed to several stress factors and limitation conditions, such as carbon source limitation and starvation. To cope with the latter, a number of regulatory cellular processes take place. In “Life and death of proteins: a case study of glucose-starved Staphylococcus aureus” (Michalik et al. 2012) protein degradation during glucose starvation was monitored. An intriguing observation was that proteins involved in branch chain amino acid biosynthesis and purine nucleotide biosynthesis were distinctly down-regulated in the clpP mutant. This lead to the assumption of a stronger repression of CodY-dependent genes in the clpP mutant. Intracellular metabolome data revealed higher GTP concentrations in the clpP mutant. This may explain the higher CodY activity and thereby stronger repression of CodY-dependent genes in the clpP mutant. Since different S. aureus strains are known to colonize different niches, global carbon source (glucose, glucose 6-phosphate, glycerol, lactate, lactose and a mixture of all) and carbon source limitation dependent exo-metabolome analyses were performed using three different S. aureus strains (HG001: laboratory strain, EN493: human endocarditis isolate and RF122: bovine mastitis strain). The most apparent observation was that RF122 can utilize lactose best, while EN493 and HG001 are better at utilizing glucose-6-phosphate compared to the bovine RF122 strain. Bacillus subtilis is an extensively studied Gram-positive and non-pathogenic bacterium. In the functional genomics approach “System-wide temporal proteomics profiling in glucose-starved Bacillus subtilis” (Otto et al. 2010) growth phase dependent changes in the proteome, transcriptome and extracellular metabolome were monitored. By mass spectrometric analysis of five different cellular subfractions, ~ 52% of the predicted proteins could be identified. To confirm and complete the proteomic data transcriptome and extracellular metabolome analyses were performed. The extracellular metabolome data ensured that cells were glucose-starved and revealed growth phase dependent metabolic footprints. In “A time resolved metabolomics study: The influence of different carbon sources during growth and starvation of Bacillus subtilis” ((Meyer et al. 2013) submitted) four different compounded cultivation media were investigated as only glucose, glucose and malate, glucose and fumarate and glucose and citrate as carbon source. It could be shown, that B. subtilis is able to maintain an intracellular metabolite homeostasis independent of the available carbon source. On the other hand, in the exo-metabolome, carbon source as well as growth phase dependent differences were detected. Furthermore, in this study the influence of ATP and GTP on the activation of the alternative RNA polymerase sigma factor B (σB) was discussed. The concentration of ATP and GTP decreased for all conditions, as cells entered the stationary growth phase. While cell growth on solely glucose and during growth on glucose and additional malate, the ATP and GTP concentrations increased slightly when the consumption of the second carbon source was initiated. Only under these conditions, a considerable σB activity increase during the transition from exponential to stationary growth phase was observed. Furthermore, the developed sampling protocol for metabolome analysis of B. subtilis enabled us to be part of a “multi omics” system biological approach to study the physiological adjustment of B. subtilis to cope with osmotic stress under chemostat conditions.
The metabolomic approach is one part of the "-omics" cascade further comprising genomic, transcriptomic, and proteomic investigations. Since information about the metabolome of the important human pathogenic bacterium Staphylococcus aureus is scarce, the aim of this thesis is the characterization of the exo- and endometabolome of this bacterium on a most global scale. For this, the metabolomic platform consisting of the analytical instruments used for 1H-NMR spectroscopy, HPLC-MS, and GC-MS analysis was applied. First, the requirements for an accurate sampling procedure for the analysis of intracellular metabolites are presented, explaining important pitfalls during the sampling and the subsequent metabolome analysis via HPLC-MS and GC-MS (book chapter I). The challenging task of the metabolite identification is demonstrated, as well as the requirements for absolute quantification of intracellular metabolites. In order to enhance the knowledge about the staphylococcal physiology and the biochemical network, the impact of different stresses and varying cultivation media on the bacterial metabolite pool was investigated in several studies. In article I, a first description of the primary metabolism of growing S. aureus COL cells cultivated aerobically in CDM is provided. This study also monitored the adaptation to glucose starvation on the level of metabolites and proteins. The uptake of all amino acids and the secretion and reuse of overflow metabolites were analyzed in a time-dependent manner. During the switch to a non-growing state, a drastic rearrangement of the amino acid pool in the bacterial cells was detected, and intracellular amounts of glycolytic intermediates were found to decrease in parallel to extracellular glucose exhaustion. During infection processes, S. aureus has to cope with varying levels of oxygen supply, including anaerobic conditions. A global metabolomic approach investigated the adaptation of S. aureus COL to strict anaerobic conditions using CDM as the culture medium. Thereby only linear growth was possible despite the higher uptake rate of glucose compared to aerobically, logarithmically growing cells. In an anoxic environment, S. aureus mainly switched on the less reliable lactic acid fermentation. Only serine and threonine but no alanine were significantly taken up. Subsequent glucose limitation led to energy starvation indicated by a drop in the adenylate energy charge. This was accompanied with an arrest of the fermentative metabolism and declining numbers of colony-forming units without taking advantage of the energy supplying arginine deiminase pathway. Compared to the established CDM, the eukaryotic cell culture medium RPMI 1640 provides more in vivo-like growth conditions. In article II, the growth behavior and the metabolic footprint of the S. aureus strains COL and HG001 were investigated during the aerobic cultivation in RPMI 1640 medium. Both strains are commonly used in laboratory research. The observed uptake and secretion pattern of extracellular metabolites provides important information for infection studies in which this medium is used for the precultivation of S. aureus. The extracellular accumulation of the noncanonical D-amino acid D-isoleucine was an interesting outcome. The strain specific metabolic footprint points to noteworthy differences in the biochemical system of both strains. Moreover, this study demonstrates the impact of the cultivation medium on the metabolic status of bacterial cells. Due to increasing resistance against a large number of antibiotics, community- and hospital- acquired infections with S. aureus are of major concern in medical therapy. Thus, greater knowledge about adaptive mechanisms after antibiotic treatment is required. In article III, the response of S. aureus HG001 to antibiotics with varying target sides, such as ciprofloxacin, erythromycin, fosfomycin, vancomycin, and ampicillin, was investigated on the metabolite level. Thereby, the abundances of 176 intracellular metabolites were observed in a time-dependent manner, thus providing the most comprehensive experimental metabolite dataset so far available for S. aureus. None of the antibiotic compounds led to alterations of single metabolite amounts, but mostly entire metabolic pathways were affected. The intermediates of the cell wall biosynthesis were affected by each antibiotic, confirming this pathway as the most potential target for new antibacterial compounds. The metabolite composition of human nasal secretions and human sweat was analyzed, since such secretions present natural habitats of S. aureus during the colonization of typical host sides. The results confirm that the bacteria has to cope with low concentrations of most of the amino acids but large amounts of urea and lactate during host colonization. Considering the supply of amino acids, the results support the usage of the RPMI 1640 medium as a step to more in vivo-like cultivation experiments. Moreover, essential information for future studies about the adaptation of S. aureus to more in vivo growth conditions is provided. Altogether, the metabolomic approach was proven to be an important tool for helping unravel the complex bacterial metabolism and the environmental factors that also play a role in the virulence of Staphylococcus aureus.