Date of Award

Spring 2022

Document Type


Degree Name

Doctor of Philosophy (PhD)



First Advisor

Roy, Craig


Legionella pneumophila is a natural pathogen of freshwater protozoa and an opportunistic pathogen of humans. Legionella is the causative agent of Legionnaires’ disease, a potentially fatal atypical pneumonia. This bacteria lives a unique intracellular lifestyle that involves internalization via phagocytosis, rapid avoidance of endocytic degradation, and subversion of host pathways to decorate its compartment into an ER-like, replication-permissive vacuole called the Legionella-Containing Vacuole (LCV). This process is largely mediated by a special secretion machinery called the Dot/Icm type IVb secretion system. This system is predicted to secrete over 300 effector proteins into the host cytosol during intracellular infection and is essential for virulence. Studies over the past two decades have focused on identifying effectors important for Legionella virulence and their specific interactions with the host. Although multiple effectors have been characterized and multiple pathways of Legionella subversion of host pathways have been described, the majority of effectors encoded by L. pneumophila remain uncharacterized. Here, I describe the establishment of insertion sequencing (INSeq) as a valuable tool to comprehensively study the virulence of effector proteins during infections of multiple hosts. Using INSeq in combination with a liquid handling robot, a clonally arrayed pool of Legionella pneumophila mutants was generated, and an effector mutant sublibrary was established and used to perform a high-throughput screen in multiple hosts. I verified effector mutants with virulence defects in this screen and further demonstrated the function of the effector RavY in promoting bacterial intracellular replication after the formation of a mature and protective vacuole. Additionally, I generated a Nlrc4-/- Myd88-/- mouse line that supports sustained replication of flagellated Legionella. Using the effector mutant pool, I performed another INSeq screen in this new mouse line and comprehensively examined the virulence of effector mutants in a 7-day infection. The enhanced resolution of this screen allowed the discovery of novel growth phenotypes, including a large number of effector mutants with fitness advantages during infection. This includes Lpg1354, an effector with predicted GDSL hydrolase activity that may be associated with the growth advantage of the mutant strain and its molecular activity. Together, the establishment of the L. pneumophila effector mutant pool, the Nlrc4-/- Myd88-/- mouse model for sustained in vivo infection, and the identification of novel effector mutants with virulence phenotypes in multiple hosts provide valuable insight on the importance of a large number of previously unstudied effector proteins during Legionella infection. These results may facilitate further characterization of Legionella effectors and shed light on their implications in Legionella pathogenesis and disease.