Project offered for Ker Memorial PhD Studentship in Infectious Diseases
Burkholderia pseudomallei is the causative agent of melioidosis, a neglected tropical disease that can manifest as an acute infection with high mortality rates or lay dormant as a chronic infection with the potential to reactivate decades later. Infection can occur through inhalation or ingestion of the bacteria or through skin abrasions, resulting in a wide range of symptoms including localized skin ulcers, pneumonia, or systemic septicemia. B. pseudomallei demonstrates extraordinary antimicrobial resistance and there is no available vaccine, limiting our ability to prevent and treat this disease. To better understand this important human pathogen and aid in the development of new therapeutics, we have performed a large-scale genetic screen of B. pseudomallei mutants in a mouse model to identify genes required for infection. One of the genes identified and confirmed to be important for bacterial virulence is the putative RNA binding protein (RBP) Tex (bpls1527I). RBPs have recently emerged as important mediators of bacterial gene expression, regulating many processes important to pathogenesis and infection. Understanding the role of Tex in B. pseudomallei virulence could help shed important insight into the role of RNA binding proteins in melioidosis and help inform the development of new strategies to treat this deadly disease. This PhD project will address the following research questions:
1) What aspects of pathogenesis are regulated by Tex RPBs? The student will utilize three-dimensional tissue culture models of the human lung developed in the Moule lab to characterize the phenotypes of Tex mutants and determine the role of Tex in bacterial virulence
2) How does Tex influence the expression of bacterial virulence factors? – The RNA binding sites of Tex will be identified using a technique developed in the Granneman lab known as UV cross-linking and analysis of cDNA (CRAC) to determine what genes in the host and pathogen are regulated by this RBP. Highly enriched mRNA binders will be further characterised in vitro using biochemical and biophysical approaches. Network analysis of the data will inform us how gene expression is impacted by Tex and how this influences virulence.
3) How does Tex binding to individual RNAs impact infection? Target RNAs and RBP binding sites identified by CRAC will be mutated to characterize their infection phenotypes, confirming the role of this novel pathogenesis strategy infection
By identifying the mode of action of the Tex RBP, this project will identify key factors controlling the pathogenesis of this important human disease and help inform the development of new antimicrobials targeting bacterial RBPs to combat the global threat of AMR.
Training and skills development
This collaborative project will provide a student with the opportunity to develop interdisciplinary laboratory skills. Some of the techniques that the student will be trained in will include handling Category 2 bacterial pathogens, mammalian cell culture, construction and infection of three- dimensional models, confocal microscopy, molecular cloning, SDS page, PCR, RNA isolation, Illumina sequencing, in vitro analysis of protein-RNA interactions and bioinformatic data analysis of mass-spectrometry and high-throughput sequencing data
Furthermore, the student will also be given the opportunity to develop their writing and communication skills through the production of scientific manuscripts and the opportunity to regularly present their findings at lab meetings and academic conferences. The student will also be encouraged to participate in public engagement opportunities and networking events to develop their potential as a well-rounded scientist.