Discovery and characterization of mechanisms that promote the intracellular growth of the pathogen Francisella tularensis

Abstract

The survival of pathogenic intracellular bacteria relies on their ability to establish and maintain a permissive niche. For Francisella tularensis, the causative agent of tularemia, this involves both escaping the degradative endocytic pathway and the acquisition of essential nutrients from the host cell once in the cytosol. In this work I investigate mechanisms employed by F. tularensis at both stages of intracellular infection. We and others have previously established that escape from endosomes is mediated via the action of a set of effector proteins secreted by the F. tularensis subsp. novicida pathogenicity island-encoded secretion system. I discovered that a substrate of this secretion system, OpiA, represents a previously undescribed, widespread family of bacterial phosphatidylinositide (PI) 3-kinase enzymes. Through biochemical and cell biology-based assays we were able to demonstrate OpiA is recruited to endocytic membranes and acts on the Francisella-containing phagosome to promote bacterial escape into the cytoplasm. Furthermore, I found that the phenotypic consequences of OpiA inactivation are mitigated by arresting endosomal maturation. Once in the cytoplasm, the ability of F. tularensis to grow is dependent upon nutrients derived from the host. Critical amongst these is glutathione (GSH), a tripeptide whose catabolism provides a source of cysteine that F. tularensis requires for growth. We performed a highly saturated transposon library screen which uncovered that F. tularensis encodes two distinct, yet essential pathways for the utilization of extracellular GSH. Additionally, my data demonstrates a unique role for each of these pathways during the course of infection. Finally, I provide evidence that one of these pathways, the ChaC-pathway, is involved in additional aspect of F. tularensis metabolism and stress defense. In total, this work highlights novel mechanisms utilized by F. tularensis to both manipulate and exploit host cell pathways to promote bacterial growth.