Control of pathogen gene expression during symbiotic maintenance.

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Salmonella and Toxoplasma are widely spread intracellular pathogens. Salmonella typhi, the cause of typhoid fever, infects around 21.5 million people each year. Meanwhile around a third of the world's population is infected by Toxoplasma. Understanding host responses to pathogens is critical for developing effective intervention strategies. A major response of cells during pathogen infection is changes in gene expression, leading to changes in the proteins being produced in the cell. Proteins are essential biopolymers in all living organisms, playing roles as structural components of cells, enzymes, and immune response agents such as antibodies. Regulation of gene expression can occur at two levels: transcription (where mRNA is synthesised in the cell nucleus by the macromolecular machine RNA polymerase) and translation (where mRNA is decoded into proteins in the cell cytoplasm by the macromolecular machine known as the ribosome). When cells are stressed, specific gene expression pathways are activated (e.g. cytokines as part of the innate immune system). However, so far, very few studies have systematically studied these changes in gene expression at the level of translation. This is particularly true for bacterial and protozoan pathogens. The primary reason is due to technical difficulties with global monitoring of protein synthesis. Transcriptional regulation has been previously studied; however there is evidence that a significant amount of regulation also occurs at the translational level. This makes sense as direct modulation of protein synthesis provides a faster and more efficient response to pathogen infection, as it circumvents de novo mRNA transcription, processing and transport to the cell cytoplasm. Translational control is a highly dynamic process and global studies have only recently become possible with the advent of RiboSeq - a high-throughput technique that allows capturing the location and abundance of all ribosomes on mRNAs, allowing precise global measurement of real-time protein synthesis. I plan to carry out an integrated program of research aimed towards understanding the complex interplay of host and pathogen gene regulation, and its ultimate effect on the host proteome and host response to biotic stress. My research proposal will be executed in three stages. First, I will infect cells with either Salmonella or Toxoplasma and harvest samples at different time points for analysis of genome-wide responses at the level of RNA synthesis (using RNA sequencing, also known as RNASeq) as well as protein synthesis (using RiboSeq), permitting a sophisticated interrogation of host-pathogen interactions. By analysing global RNA and protein synthesis at different stages of infection, I will be able to distinguish genes that are activated at the early stage of infection at the level of protein synthesis, RNA synthesis, or both. Second, I hypothesise that there will be a number of genes activated at the level of protein synthesis during early infection that then go on to regulated the expression of other genes at the level of RNA synthesis (such genes are known as transcription factors and are common mediators of immune responses). This would provide the cell with a rapid response mechanism that initiates and shapes longer term anti-pathogen responses. I will use a technique known as chromatin-immunoprecipitation sequencing to capture and sequence the DNA bound by candidate transcription factors and compare the data with RNA expression profiles at later stages of infection to monitor their effects. Third, I will dissect the molecular mechanisms that drive switches in protein synthesis during early infection. Comparison between bacterial and eukaryotic pathogen infections will allow identification of common anti-pathogen pathways besides the responses that are specific for each pathogen. The results generated will be valuable for informing the development of intervention strategies.