Investigating substrate specificity in SiaT mutants. (2020)
Type of ContentTheses / Dissertations
Degree NameMaster of Science
PublisherUniversity of Canterbury
AuthorsNewton-Vesty, Michael Charlesshow all
Staphylococcus aureus and Proteus mirabilis are pathogenic bacteria that are resistant to many clinical antibiotics and the identification of new inhibitory compounds is a priority for human health. Sialic acids are scavenged by S. aureus and P. mirabilis from mammalian hosts and can be used as a source of carbon and nitrogen. In a range of a range of pathogenic bacteria, colonization and persistence is dependent on the utilization of sialic acids. Therefore, a potential target for the development of novel antibiotics in these organisms is the sodium sialic acid symporters (SiaT), termed SaSiaT and PmSiaT respectively.
N-Acetylneuraminic acid and N-glycolylneuraminic acid are the most abundant sialic acids. Although the SiaT proteins from these organisms are homologous, SaSiaT has a higher affinity for N-glycolylneuraminic acid and PmSiaT has a higher affinity for N- acetylneuraminic acid. The substrate binding site of these SiaT transporters differs by only three amino acid residues. Examination of the previously determined X-ray crystal structure of PmSiaT, and the subsequently modelled SaSiaT structure, suggests that these three residues may be responsible for the opposite substrate specificity observed for these two SiaT homologues.
This thesis focuses on further investigating the substrate preference of SaSiaT and PmSiaT by engineering two SiaT triple mutants with the three binding site residues swapped to each other’s binding site. In PmSiaT, three residues were mutated to the respective residues in SaSiaT producing a mutant referred to as PmSiaT YNN (Phe78Tyr, Gln82Asn and Phe243Asn). For SaSiaT, these three residues were mutated producing a mutant referred to SaSiaT FQF (Tyr79Phe, Asn83Gln and Phe244Asn). Microscale thermophoresis and isothermal titration calorimetry were used to test whether these mutations would reverse the observed sialic acid preference. The results show that the PmSiaT mutant, PmSiaT YNN, could no longer bind sialic acids. A bacterial growth assay showed that the SaSiaT mutant, SaSiaT FQF, displayed reversed sialic acid preference in vivo, however overall activity was significantly reduced.
These results suggest that while the binding site substitutions of PmSiaT and SaSiaT play a role in determining sialic acid preference, it is likely to be influenced by additional factors that help to specify which type of sialic acid is preferred by these transporters. Overall the work completed in this thesis will help underpin novel drug design to target sialic acid transporters, particularly those of P. mirabilis and S. aureus.