Naturwissenschaften
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Auflistung Naturwissenschaften nach Autor:in "Alshaar, Belal"
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Item Lipidomic and structural characterization of the cell wall of Streptococcus pneumoniae(2025-05-13) Alshaar, BelalStreptococcus pneumoniae (Spn) is a Gram-positive bacterium that naturally colonizes various niches within the human body, but can also lead to infections with varying degrees of symptoms. Despite the availability of vaccines, Spn infections remain a significant health concern, exacerbated by the rising threat of antibiotic resistance. This growing challenge underscores the urgent need to explore new drug targets that are common across different strains. A particularly promising target is the pneumococcal cell wall, which has become a central point of structure-focused research. The cellular membrane, a key component of this wall, plays a crucial role in host-pathogen interactions. Nevertheless, the precise quantities of its components are still not well understood. This thesis presents a first-of-its-kind quantitative analysis of the pneumococcal lipidome, conducted in close collaboration with the group of Prof. Dr. Sven Hammerschmidt from the University of Greifswald. The implementation of a standardized pipeline applicable for in-depth microbial lipidomics on pneumococcal samples is described. Further, I examined how the knockout of selected genes influenced the membrane lipid composition. As a proof of concept, the wild-type serotype 2 strain S. pneumoniae D39 was compared to three mutant strains with single gene knockouts. The targeted mutant strains investigated here are: 1) tacL: TacL is responsible for the attachment of the polymeric teichoic acid chain on the glycolipid anchor, thus forming the lipoteichoic acid (LTA); 2) lgt: Lgt (diacylglyceryl transferase) catalyzes the diacylation of preprolipoproteins via a thioester bond by transferring a diacylglycerol moiety from a phosphatidylglycerol (PG) precursor; 3) cps: enzymes of the cps cluster are responsible for capsule polysaccharide formation. To date, such quantitative lipidome studies have not been performed for Spn. Methodically, shotgun lipidomics was employed and the development of a customized mix of internal standards ensured accurate quantitative analysis. The analysis allowed quantification of more than 100 lipids across the following six classes: diacylglycerol, glucosyldiacylglycerol (GlcDAG), galactosylglucosyldiacylglycerol (GalGlcDAG), cardiolipin, phosphatidylcholine and PG, with GlcDAG and GalGlcDAG being the predominant classes. Gas Chromatography-Mass Spectrometry (GC-MS) and Thin Layer Chromatography in conjunction with high-resolution tandem mass spectrometry were further utilized for the validation of identified lipid species. The lipid composition of tacL and lgt strains showed no significant changes compared to the wild type, while the non-encapsulated strain exhibited significant adaptations. Another aim of this thesis was to optimize a GC-MS method for the accurate quantification of pneumococcal wall glycopolymers, particularly wall teichoic acid and lipoteichoic acid. This is a challenging task for researchers in the field since decades. This quantification technique is anticipated to provide a basis for studying enzymes that affect teichoic acid levels without using radioactive materials in cultures and may also be used for capsule polysaccharide quantification, advancing our understanding of pneumococcal cell wall regulation. Preliminary results from this optimized method have already provided insights into how LytR, which plays an essential role in attaching the wall teichoic acid to the peptidoglycan, influences the teichoic acids ratio. In summary, a robust quantitative method for lipidome analysis has been established, along with a developed method which delivered the first successful proof of concept for teichoic acids quantification in Spn. These approaches enable future investigations into membrane homeostasis and biosynthesis regulation in other mutants or under various growth conditions.