Bacterial sialic acid degradation: membrane transport and enzymology
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
The overarching aim of this thesis is to understand how sialic acids are transported into the bacterial cell and then degraded by bacterial pathogens. In heavily sialylated environments, bacterial pathogens utilise host-derived sialic acid as a nutrient source, and this pathway constitutes a novel and unexploited target for antibiotic drug design. Three sialic acid transporters, from two distinct gene families were investigated to probe how sialic acid is transported across the cytoplasmic membrane. The Yersinia pestis sugar proton symporter (NanT) exists as a single species in solution, while the Staphylococcus aureus sodium solute symporter (SSS) and Proteus mirabilis SSS appear to be in a selfassociation. It is likely that the SSS sialic acid transporters exist in a monomer-dimer equilibrium, although higher oligomeric structures cannot be ruled out. The structure of the P. mirabilis SSS sialic acid transporter was solved, which is the first known structure of a sialic acid transporter to be presented. It was solved in complex with sialic acid and two sodium ions, providing insight into how this transporter mediates the movement of sialic acid across the membrane. In addition, the structure presents a novel conformation among sodium symporters and the structural basis for the movement of sialic acid across the membrane is elucidated. Following the import of sialic acid into the bacterial cell, N-acetylneuraminate lyase is the first enzyme involved in its degradation. The structure, function and inhibition of methicillin-resistant S. aureus (MRSA) N-acetylneuraminate lyase were investigated. Solution and structural studies confirmed that this enzyme is tetrameric. Kinetic analysis was employed to test an inhibitor of N-acetylneuraminate lyase enzymes. This molecule demonstrated strong species-specific inhibition against MRSA N-acetylneuraminate lyase. The structure of MRSA N-acetylneuraminate lyase in complex with this inhibitor demonstrated that altered binding modes between N-acetylneuraminate lyase enzymes may account for variable inhibition between species. In addition, evidence for a protein-protein interaction between the S. aureus SSS sialic acid transporter and MRSA Nacetylneuraminate lyase is presented. Thus, these proteins may interact at the cytoplasmic membrane, allowing the degradation of sialic acid to be initiated upon entering the cell. The structure, function and catalytic mechanism of the third enzyme involved in sialic acid degradation, N-acetylmannosamine-6-phosphate 2-epimerase, were explored from MRSA. This enzyme was demonstrated to adopt a dimeric architecture in solution, consistent with the crystal structure that was solved and presented. A multi-enzyme coupled assay was developed to assess N-acetylmannosamine-6-phosphate 2-epimerase activity in real time. Residues were probed that may be important for catalysis and tested by mutagenesis and kinetic analysis. A novel mechanism of carbohydrate epimerisation is proposed for this enzyme, whereby catalysis occurs via a proton displacement mechanism mediated by the substrate. Overall, this work provides new data that enriches our understanding of the import and degradation of sialic acid in clinically important human bacterial pathogens.