UCSF

The intracellular bacterial pathogen Legionella pneumophila (L.p.) secretes ~330 bacterial effector proteins into the host cell that interfere with numerous cellular pathways and often regulate host cell proteins through post-translational modifications. Many aspects of L.p.-mediated pathogenesis, including the functions and targets of most effectors, remain elusive. To obtain a global overview of host cell rewiring and potential targets of these effectors, we analyzed the host cell proteome for changes in protein abundance, phosphorylation, and ubiquitination during L.p. infection. Our analysis reveals dramatic spatiotemporal changes in the host cell proteome that are dependent on the secretion of bacterial effectors. In Chapter 2, we show that L.p. substantially reshapes the mitochondrial proteome and induces a mitochondrial stress response with many similarities to the mitochondrial unfolded protein response (UPRmt). L.p. modulates downstream adaptive responses to mitochondrial stress by blocking the translation of transcription factors ATF4 and CHOP while allowing for the translation of ATF3. To our knowledge, this is the first evidence of manipulation of the UPRmt by a bacterial pathogen in mammalian cells. In Chapter 3, we show that L.p. infection results in increased ubiquitination of host proteins regulating subcellular trafficking and membrane dynamics, most notably 63 of ~160 mammalian Ras superfamily small GTPases. We determine that these small GTPases undergo non-degradative ubiquitination, and link their ubiquitination to recruitment to the Legionella-containing vacuole membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central role in cross-family small GTPase ubiquitination, and that these effectors function upstream of SidE-family ligases in the poly-ubiquitination and retention of GTPases in the LCV membrane. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. Overall, the findings presented in this dissertation position L.p. as a tool to better understand the regulation of mitochondria and small GTPases in uninfected contexts.