Characterisation of the function and mechanism of a binding-protein dependent secondary transporter

Many bacteria rely almost completely on being able to acquire nutrients from their environment and in bacteria that can only live inside our bodies these chemicals often come from us. One bacterium, Haemophilus influenzae, uses a chemical called sialic acid as a nutrient but also covers its surface in this molecule allowing it to become invisible to components of our immune system. This bacterium is able to acquire the sialic acid using a particular collection of proteins in its membrane called a TRAP transporter. This project aims to study in detail the TRAP transporter that is essential for sialic acid uptake by H. influenzae. If we can determine exactly how this transporter works, we have a better chance at being able to develop novel antimicrobials against this bacterium as we have already demonstrated that if this system is inactive then H. influenzae cannot causes disease. In addition, this TRAP transporter is very amenable to study in the laboratory and we wish to use this particular system to learn a lot of general things about how these transporters work. They are not just limited to functioning in sialic acid uptake, but different TRAP transporters have evolved to transport a wide range of compounds and are present in a wide range of environment, for example, many bacteria that live in the sea (in salty environments) appears to have many TRAP transporters. One of the objectives of this project to try and find out if the presence of salt (sodium) is important for the mechanism of TRAP transporters, which would correlate with them being used preferentially by marine organisms. The results of the project will tell us much more about how one human pathogen is able to capture sialic acid, but also the basic mechanims used by thousands of other bacteria to capture chemicals from the environment using TRAP transporters.

All free-living prokaryotes rely heavily on nutrient acquisition from their environments and many years of study of the routes by which these compounds are taken up into bacterial cells has revealed two very common types of transporters. One of these families is the ATP-binding cassette (ABC) transporters that use an extracytoplasmic solute receptor (ESR) to confer high affinity to an ATP powered (primary) transporter. The second type are known as secondary transporters as they use electrochemical gradients to energize transport and are most well represented by the major facilitator superfamily (MFS), which includes the lactose permease (LacY). This project studies a third class of transporter that is a hybrid between ABC and MFS transporters in that they are ESR-dependent secondary transporters. They are widespread in both bacteria and archaea but are poorly studied and this project focuses on a particular member of the tripartite ATP-independent periplasmic (TRAP) transporter family that is a virulence factor in Haemophilus influenzae due to its essential role in sialic acid transport in this pathogen. In the project we wish to study the properties of this TRAP transporter in vitro using purified components reconstituted into proteoliposomes to characterize the energetics and kinetics of the sialic acid uptake process. Also, we wish to discover how an ESR protein can interact with a secondary transporter and by using the structure of the ESR from this system, SiaP, will test hypotheses as to regions in the protein that we propose to be important for ligand binding and recognition by the membrane subunits of the transporter. This will be the first ever in vitro characterization of this important family of prokaryotic solute transporters.