Hopkins, Adam P (2010) Molecular and Biochemical Characterisation of SiaP as a Sialic Acid Binding Protein Component of a TRAP Transporter of Sialic Acid. PhD thesis, University of York.
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Sialic acid utilisation plays an important role in the growth and persistence of the obligate human mucosal pathogen Haemophilus influenzae, which causes respiratory tract infections, septicaemia and meningitis. Like many other bacteria, H. influenzae can use host-derived sialic acids as carbon, nitrogen and energy sources, but also as a terminal modification on the LPS to better evade the human immune system. H. influenzae takes up exogenous sialic acid via a tripartite ATP-independent periplasmic (TRAP) transporter, SiaPQM. This possesses an extracytoplasmic substrate binding protein (SBP), SiaP, which binds the substrate in the periplasm and delivers it to the specific membrane permease, SiaQM. SiaP contains two globular domains, which close around the substrate upon binding. Here, the mechanism of sialic acid binding by SiaP is investigated using site-directed mutagenesis of residues in the ligand binding site and analogues of sialic acid. These, and several mutations on the surface of SiaP, were investigated for their effect on transport by SiaPQM in vitro, using SiaQM reconstituted into proteoliposomes, and in vivo, using expression of siaPQM in an E. coli strain lacking its native sialic acid transporter, NanT. It is demonstrated that stabilisation of the carboxylate group of sialic acid by the totally conserved Arginine-147 is important for high-affinity ligand binding, but is not essential for transport. Mutation of Aparagine-150 to Aspartate abolishes the function of the transporter without affecting ligand binding, suggesting the existence of a critical interaction between the components of the transporter. The catabolism of the sialic acid analogues was also examined in E. coli expressing different sialic acid transporters. This indicates that a wide variety of sialic acid analogues are potential carbon sources in many pathogenic bacteria.
|Item Type:||Thesis (PhD)|
|Academic Units:||The University of York > Biology (York)|
|Depositing User:||Mr Adam P Hopkins|
|Date Deposited:||14 Jun 2011 15:59|
|Last Modified:||08 Aug 2013 08:45|