Carbohydrate processing: an indispensable platform of pneumococcal virulence
Date
2021-04-30
Authors
Meier, Edward Peter William
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Abstract
Carbohydrate processing is fundamental for Streptococcus pneumoniae (pneumococcus) colonization, and it is also a major platform of its virulence. Many of the organism’s key carbohydrate processing enzymes target the glycans produced by the human host. However, pneumococcus can also utilize a limited number of plant derived carbohydrates known as dietary saccharides. This thesis focuses on characterizing structural and biochemical features related to four proteins involved in pneumococcal carbohydrate processing.
The first component of this thesis focuses on three proteins belonging to the pneumococcal raf locus. The pneumococcal raf locus refers to a cluster of genes involved in utilizing the dietary saccharides known as raffinose family oligosaccharides (RFOs). Intriguingly, several of the genes within the raf locus have been identified as virulence factors in signature-tagged mutagenesis- and directed knockout studies. However, our understanding of this locus is inhibited by a lack of biochemical and structural information regarding the encoded proteins. In this thesis, three crystal structures of the substrate binding protein RafE bound to the RFOs raffinose, stachyose and verbascose have been modelled. In addition, the binding affinity of RafE towards several different oligosaccharides has been determined. Furthermore, several substrate targets for the raf locus’ glycoside hydrolases, Aga and GtfA, are described and the kinetic parameters of Aga have been determined. Overall, this work shows that the raf locus contains all of the required biological machinery for RFO utilization. However, it was found that all of the proteins are biochemically inefficient towards their putative RFO targets. Thus, this work suggests that the raf locus may have an alternative role in vivo.
The second component of this thesis focuses on the S. pneumoniae Glycoside Hydrolase family 92 enzyme, better known as SpGH92. SpGH92 is a characterized exo-α1,2-mannosidase that is essential for High-Mannose N-glycans (HMNGs) degradation, and it was also a major virulence determinant in models of pneumonia and sepsis. Due to its profound impact on pathogenesis, SpGH92 has a high potential to become an alternative therapeutic target in future vaccine and drug trials. The work presented here describes a protocol utilizing Hydrogen-Deuterium-eXchange Mass-Spectrometry (HDX-MS) and Differential Scanning Fluorimetry (DSF) that can effectively detect changes in SpGH92 heat stability and structural dynamics upon interacting with different inhibitors. By utilizing a characterized α-mannosidase inhibitor we demonstrate the feasibility of detecting inhibitor induced changes in SpGH92 and we compare these changes with those of a novel inhibitor. Overall, this work has uncovered novel structural insights into the virulence factor controlling the deconstruction of HMNGs, and it has set the framework for future SpGH92 inhibitor screening assays using HDX-MS and DSF.
In summary, this thesis sheds light on the characteristics of several different proteins involved in S. pneumoniae carbohydrate processing, thereby contributing to the discovery and the development of future therapeutics targeting the prevention and treatment of pneumococcal infections.
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Keywords
Carbohydrate processing, Streptococcus pneumoniae, Pneumococcus, Virulence, Virulence factor, Pathogenesis, Obligate human pathogen, Pathobiont, Moonlighting proteins, CAZyme, Teichoic acid, Lipoteichoic acid, Dietary saccharide, N-glycan, High-mannose N-glycan, Host-pathogen interaction, Hydrogen-deuterium exchange mass-spectrometry, HDX-MS, X-ray crystallography, Enzyme kinetics, Isothermal titration calorimetry, ITC, Differential scanning fluorimetry, DSF