Tuberculosis (TB), caused by the respiratory pathogen Mycobacterium tuberculosis (Mtb) is one of the leading causes of death worldwide. Upon infection, Mtb is taken up into phagosomes of alveolar macrophages. Mtb is known to hinder phagosome maturation and thereby evade degradation. Growing evidence suggests that damaging the phagosomal membrane is an additional critical mechanism for the survival of Mtb within macrophages and its intracellular pathogenesis. Two key Mtb virulence factors, the virulence lipid PDIM and the type VII protein secretion system EsxA contribute to phagosomel membrane damage. However, the host factors involved in repairing Mtb-induced phagosomal membrane damage and thereby constrain the bacterium are less well known. The host ESCRT system is one factor known to promote repair of Mtb-damaged membranes, but additional host factors likely also contribute. To identify such factors, the lab previously performed a genome wide CRISPR screen to identify candidate genes required to repair Mtb-mediated phagosomal membrane damage. Identified hits included the phosphoinositide kinase PIKFYVE, the cystic fibrosis transmembrane conductance regulator CFTR, and the solute carrier family member SLC11A1. In order to investigate these candidate genes, we propose the following aims:
- Aim 1: Generate and validate monoclonal CRISPR knockouts for each of these genes.
- Aim 2: Evaluate the role of these genes in Mtb infection and membrane damage repair using both the generated monoclonal knockouts and chemical inhibitors as an orthogonal approach.
- Aim 3: Gain insights into the mechanisms involved in membrane repair mediated by these candidate genes.
Through this project we hope to elucidate key host factors involved in repair mechanisms during Mtb induced membrane damage.