Translational control of a bacterial micro-injection system

Recent sequencing technologies have allowed insights into the complexity of post-transcriptional regulation in bacteria, in particular the importance of non-coding small RNAs (sRNA). Our research has focused on the zoonotic pathogen, enterohaemorrhagic Escherichia coli (EHEC) O157 that is present in cattle as a primary host and infects humans as an incidental host, often through food and drink. Human EHEC infection can be life threatening due bacterial expression of Shiga toxins during colonisation of the gastrointestinal tract. To promote attachment and persistence in the gut, EHEC strains produce a type 3 secretion (T3S) system. This amazing micro-syringe injects a cocktail of proteins into host cells in order to supress innate responses and prolong attachment of the bacteria. If you knock out the injection system, EHEC O157 can no longer colonise cattle and T3S is considered to be equally important for human infection. Moreover, similar injection systems are produced by Salmonella, Shigella, Chlamydia, Yersinia and Pseudomonas and T3S has been shown to be critical for virulence in these bacterial pathogens. Not surprisingly, these injection systems have received a lot of attention as targets for interventions. For example our group has developed a vaccine for use in cattle that includes the main injection filament antigen (EspA) and we are working on small molecule inhibitors of the system. The above is to make the case that the system is important and the more we can understand about its regulation and assembly, the more we should be able to exploit this information to prevent its production and/or function.
A T3S system is a complex nano-machine that is related to the rotating bacterial flagella system. It has a basal apparatus that is composed of about 15 proteins that span the inner and outer membranes and then a short needle which is further elongated into a filament (EspA). At the tip of this a pore forms into the host eukaryotic cell with the aid of two further proteins (EspB/D). Once the secretion system is build ATP hydrolysis provides the energy to pump 'effector' proteins from the bacterium into the host cell where they manipulate cellular pathways to promote bacterial infection.
The project: We have evidence that the assembly is staged, with a checkpoint after building the basal apparatus and before EspA filament production. We know that this control involves a translational switch in the mRNA encoding the filament. We have evidence that the conformation of this switch is critical for expression of the injection filament. Our hypothesis is that the mRNA is only translated and key proteins produced for filament assembly (SepL and EspA) when the mRNA can interact with a complex of proteins produced earlier in the assembly process. So you will not get filament production unless the basal apparatus is in position. Furthermore, we propose that this translation will occur at the basal apparatus as a result of these interactions, placing he proteins ready for use at the apparatus. We hypothesize that this localised translation at the apparatus could explain the increased sensitivity of T3S production to specific antibiotics (). The project will use three separate approaches to investigate the process:
(1) Mutagenesis of translational fusions to investigate sequences required for translation and interacting partners (Gally Lab). (2) Mapping of the mRNA structure both in the bacterium and in vitro using chemical techniques (Granneman Lab). (3) Imaging the localisation of the wild type and mutated transcripts in the bacterial cell and in the context of EspA filaments (El Karoui Lab). Throughout the process we will aim to apply our understanding to develop inhibitors of T3S assembly which may have broad applicability across multiple bacterial pathogens.