Control of pathogen gene expression during symbiotic maintenance.

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.

Understanding the role of translational control in host response to pathogen infection is critical for developing intervention strategies as protein synthesis is a central process in all cells. However, currently most global studies are at the level of transcription (e.g. RNASeq and ChIP-Seq). Analysing genome-wide responses at the level of protein synthesis has only recently become possible due to the development of ribosome profiling (RiboSeq). Using a combination of RiboSeq, RNASeq and ChIP-Seq, I will investigate the translational responses to intracellular bacterial and protist infections and address the hypothesis that rapid translational responses following pathogen perturbation shape later transcriptional landscapes. I will test a key prediction of this hypothesis, namely that mRNAs with immediately enhanced translation include some that encode nuclear-localized proteins such as transcription factors.

My research program will focus initially on the prokaryote Salmonella enterica serovar Typhimurium (a mouse model for the typhoid fever pathogen) and the eukaryote Toxoplasma gondii. As intracellular pathogens, we can observe host responses unimpeded by cell lysis/autophagy. Both enter the host through ingestion, initially entering gut-epithelial cells, followed by macrophage infection. This will allow an interesting cross-Kingdom comparison of common host responses besides the elucidation of pathogen-specific responses, at both the translational and transcriptional levels. I will characterize the regulatory elements responsible for temporal switches in translational efficiencies - e.g. riboswitches within the mRNAs or trans-effectors that interact with the mRNAs. I will also investigate the anti-host mechanisms employed by each pathogen, and the molecular basis for pathogen adaptation to the highly acidic intra-macrophage environment.

Salmonella and Toxoplasma are important and widely spread intracellular pathogens of humans and animals. This is a 'basic-research' project that will advance fundamental understanding of several complex biological processes and lay the groundwork for future advances in the following areas.
(1) Salmonellosis: In human infections, Salmonella enterica (subspecies enterica) displays four different clinical manifestations: enteric fever, gastroenteritis, bacteraemia and other extraintestinal complications, and a chronic carrier state. The global burden of disease is substantial, with more than 90 million cases of gastroenteritis alone occurring each year. Salmonella typhi, the cause of typhoid fever, remains the predominant enteric fever worldwide, infecting annually around 21.5 million people (CDC:2017) with 12-30% mortality if untreated. Despite much research, the biology of this human-adapted bacterial pathogen is incompletely understood, as is the complexity of disease pathogenesis in endemic areas, particularly in Africa and Asia. Unfortunately, disease control is restricted by imperfect vaccines that are sub-immunogenic in young children and not cross-protective between different Salmonella spp serovars. Multi-drug-resistant (MDR) Salmonella serotypes are also emerging, in part due to a high mutation rate within its resistance genes, exacerbating the public health impact and leading to increased morbidity, mortality and treatment cost.
(2)Toxoplasmosis: Up to a third of the world's population have toxoplasmosis, a disease caused by Toxoplasma gondii, which infects all mammals. The pathogen is usually transmitted horizontally via the host ingesting sporulated oocysts from the environment or tissue cysts from undercooked meat. Infection can cause serious and occasionally fatal illness in immune-compromised individuals such as pregnant women and HIV/AIDS patients. Indeed, toxoplasmic encephalitis is a leading cause of death in HIV patients. The pathogen can also be transmitted vertically in pregnant women, resulting in severe ophthalmologic and neurologic defects in the foetus. Infections in healthy individuals are often symptomless, although parasites often lie dormant as cysts in the brain and the long-term effects are not clear. Currently, there is only one commercially available drug for treatment of toxoplasmosis - pyrimethamine, and, whilst effective in short-term treatment, the frequency of treatment failures associated with long-term use is high, particularly among HIV patients. Many determinants may contribute to treatment failures, including pre-existing differences in drug sensitivity between strains and drug resistance mutations. Therefore, there is a pragmatic need for further Toxoplasmosis drug development.
(3) Biotechnological tools: Non-canonical translation mechanisms have been and continue to be a source of valuable and broadly-applicable tools for molecular biological research (e.g. the EMCV IRES), with consequent economic benefits. Although such a use for the infection-triggered riboswitches for host mRNAs as biosensors appears relatively unlikely at the moment, it is a possibility that will be kept in mind as the research progresses.
Thus the potential beneficiaries are medical researchers in the public and private sector and through them the general public, besides the pharmaceutical and biotech industries.
The post doctoral research associate funded by this grant would acquire new expertise in molecular biology, pathology and bioinformatics which would be widely applicable in the UK biotech industry. In addition, I would expect to have a number of short- and long-term students pass through the lab in the same time period and acquire skills of broad relevance to the UK's economy and well-being.