Investigating the host determinants of Listeria monocytogenes cytosolic infection

Abstract

Listeria monocytogenes (Lm) is an intracellular foodborne pathogen which causes the severe disease listeriosis in immunocompromised, elderly, and pregnant individuals. Lm infects host cells using a well-defined intracellular lifecycle involving invasion, cytosolic replication, and cell-to-cell spread. While the bacterial virulence factors required for this lifecycle have been extensively studied, less is known about the host determinants of intracellular infection. The goal of this dissertation is to identify and characterize host proteins required for two stages of the Lm intracellular lifecycle: invasion of phagocytic cells through phagocytosis, and subsequent activation of virulence gene expression in the host cytosol. In this work, I developed and performed unbiased biochemical and genetic screens to identify host factors involved in these processes. First, the role of the host cytosol in activating the master virulence regulator PrfA was investigated using cytosolic cell-free extracts derived from Xenopus tropicalis eggs. These studies revealed that a host protein present in eukaryotic cytosol is required for activation of PrfA and expression of virulence factors. Fast protein liquid chromatography was used to separate Xenopus extracts and mass spectrometry identified candidate proteins involved in PrfA activation. As a complementary approach, a genome-wide CRISPR/Cas9 screen was performed and mutant macrophages with reduced levels of virulence factor expression were isolated. This screen also identified genes important for phagocytosis of Lm by macrophages. The role of one candidate gene, Pten, was extensively characterized. I discovered that the tumor suppressor PTEN promotes macrophage phagocytosis of Lm and L. ivanovii, but not other Gram-positive bacteria. Additionally, I found that PTEN enhances phagocytosis of Lm via its lipid phosphatase activity by promoting adherence to macrophages. Using conditional knockout mice lacking Pten in myeloid cells, PTEN-dependent phagocytosis was found to be important for host protection during oral Lm infection. Overall, this thesis provides a comprehensive identification of macrophage factors involved in regulating Lm virulence at the molecular level. In addition, the in vivo studies presented here demonstrate for the first time that macrophage phagocytosis is an important immune defense against invasive Lm during the foodborne route of infection.