Morphological and functional characterization of host proteins during infections by actin-hijacking bacterial pathogens

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Listeria monocytogenes
Shigella flexneri
Salmonella enterica serovar Typhimurium

Cells, much like mammals, possess an internal skeleton. This cellular skeleton (called the cytoskeleton) provides structure to cells, enables their movement within the environment and promotes the internalization of extracellular cargo (endocytosis). Many pathogens have devised strategies to hijack the cytoskeleton and other crucial sub-cellular processes for their disease processes. The bacterium Listeria monocytogenes (L. monocytogenes) utilizes the clathrin endocytic machinery to invade cells, and later, the actin polymerization machinery to generate actin-rich comet/rocket tails to move within and amongst host cells. Salmonella enterica serovar Typhimurium (S. Typhimurium) and Shigella flexneri (S. flexneri) generate actin-rich membrane ruffles at the cell surface to enter cells. Once inside, S. Typhimurium occupies a long-lived vacuole, whereas S. flexneri generates comet/rocket tails. Enteropathogenic Escherichia coli (EPEC) on the other hand remain extracellular and co-opt clathrin and actin to form motile pedestals directly beneath the site of bacterial adherence. In this thesis, I explored the involvement of several host actin- and/or endocytic-associated proteins during bacterial infections and simultaneously used these infections to gain insight into novel roles of the proteins studied. In chapter 2, I discovered that L. monocytogenes co-opts the actin-associated protein palladin during its entry and intracellular motility. Importantly, I revealed that palladin can functionally replace the Arp2/3 complex during bacterial actin-based motility. In chapter 3, I uncovered that the internalization strategy used by L. monocytogenes to transfer between host cells exploits caveolin-mediated endocytosis. In chapter 4, I investigated the host enzyme cyclophilin A (CypA) and found that it is crucial for maintaining the structural integrity of L. monocytogenes membrane protrusions generated during bacterial dissemination events. In chapter 5, I determined that CypA restricts S. Typhimurium invasion but is dispensable for EPEC pedestal formation. Finally, in chapters 6 and 7, I examined the receptor of CypA, CD147, and found that this membrane protein, like CypA, is crucial for the proper formation and function of L. monocytogenes membrane protrusions. In conclusion, my research has 2 major implications: 1) I have uncovered new insight into the mechanisms behind how actin-hijacking pathogens cause disease and 2) I have demonstrated novel cellular functions for host actin-associated proteins.

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This thesis may be printed or downloaded for non-commercial research and scholarly purposes. Copyright remains with the author.
Julian Guttman
Science: Biological Sciences Department
Thesis type: 
(Thesis) Ph.D.