A Tale of TonBs: Exploring the role of multiple TonB transporters in oral bacterial physiology

Transport of small molecules and ions is key to the physiology and virulence of a range of human pathogens and is facilitated by several membrane traversing transport systems. One of the most important, and least understood is the TonB-dependent transport system- which is widespread among Gram-negative pathogens and often involved in key virulence traits such as iron-uptake and virulence gene regulation. This inner membrane protein acts (in concert with companion proteins) to energize uptake of a variety of small molecules across the Outer membrane into the periplasm via interactions with outer membrane TonB-dependent Transporters (TBDT). In oral and gut Bacteroidetes these transporters have recently been shown to be responsible for the ability of these bacteria to access host and dietary carbohydrates in systems known as Polysaccharide Utilisation Loci (PULs) and are key to survival in humans. Our work uncovered a novel PUL responsible for sialic acid uptake in the periodontal pathogen Tannerella forsythia and showed it is central to biofilm formation and cellular infection in vitro. However, in T. forsythia and the gut commensal B. fragilis and other Bacteroidetes there are over 50 TBDTs that seem to be energized by at least 3 TonB inner membrane complexes. There is to date no evidence as to whether these TonB proteins function in a specific or redundant fashion to energize all of or specific subsets of these TBDTs that may relate to the ability of the bacteria to access nutrients or respond to their environment via sensing extracellular signals- a question that is key to understanding how bacteria live within and colonise human mouths and guts and designing methods to modulate their expression and potentially suppress bacterial growth.

Our team has established an ex vivo system in E.coli to test and assess function of the 3 putative TonB systems of T. forsythia and B. fragilis that will be utilized in this project to assess function of these TonBs to transport sialic acid but also Iron containing hemin. In addition you will utilize gene deletion techniques to remove the TonBs from these organisms and assess their function in vitro in biofilm and cellular interaction studies using epithelial cell models (oral and gut) and also assess the ability of bespoke TonB-inhibitory peptides that preliminary data suggest inhibit TBDT transport as a potential route to novel antimicrobials. Furthermore the project will examine gene expression profiles of these transporters during biofilm formation and in infection models using qPCR to decipher their regulation and potential function in these processes.

The student will gain a broad training in a range of techniques including molecular microbiology, eukaryotic cell infection models, biofilm growth and gene expression profiling. This project thus provides an excellent framework on which to build a successful career.