Role of host Arf GTPases and the scaffolding protein Tuba in the infection of human cells by bacterial pathogen Listeria monocytogenes
The gram-positive bacterium Listeria monocytogenes is an important cause of foodborne illnesses resulting in gastroenteritis, meningitis, or abortion. Listeria is a facultative intracellular pathogen that is able to induce its internalization into mammalian cells, replicate within those cells, and spread within host tissues using a process involving the host actin cytoskeleton. My Ph.D. work focused on molecular mechanisms employed by Listeria to establish infection in mammalian cells. In chapter 3 of my results section, I have investigated the molecular mechanism of internalisation ("entry") of Listeria into human epithelial cells. This entry process requires active participation of both the bacterium and host cell and is mediated by binding of the Listeria surface protein InlB to its human surface receptor Met. This interaction between InlB and Met ultimately results in localized polymerization of the host actin cytoskeleton and alterations in the plasma membrane of the host cell that engulf bacteria. How actin polymerization is stimulated during InlB-mediated entry of Listeria is not completely understood. In my PhD work, I demonstrated a role for the human GTPase Arf1 in actin polymerization during InlB-dependent entry into the human epithelial cell line HeLa. RNAi-mediated depletion of Arf1 inhibited entry of Listeria into HeLa cells and also reduced localized actin polymerization that normally accompanies entry. In cells, Arf1 activity is regulated by binding to GTP. Expression of a mutant form of Arf1 that is thought to decrease Arf1-GTP levels impaired Listeria entry and actin polymerization. Accumulation of Arf1 near bacterial sites indicated the active manipulation of Arf1 by Listeria. Arf1-GTP is known to regulate events such as membrane trafficking and actin polymerization through interaction with various effector proteins. Results from RNAi experiments indicate important roles for the Arf1 effectors AP-1, clathrin, PICK1 and PIP5K1A Listeria entry. Apart from Arf1, other members of the Arf family include Arf3, Arf4, Arf5, and Arf6. My RNAi results suggested minor roles for Arf4 in InlB-mediated entry of Listeria. The roles of Arf3, Arf5 and Arf6 in entry are currently unclear. Collectively, my results identify Arf1 as host factor exploited by Listeria to promote infection atleast in part by regulating the actin cytoskeleton downstream of Met receptor. In chapter 4 of my results section, I investigated aspects of the spread of Listeria from infected host cells to adjacent healthy cells. Cell-to-cell spread involves the formation of "protrusions", which are bacteria encased in projections of the host plasma membrane. Protrusion formation is mediated by the bacterial virulence protein InlC, which disrupts complexes between the human scaffolding protein Tuba and its mammalian ligands Sec31A and N-WASP. These two mammalian ligands interact with a carboxyl-terminal Src Homology 3 (SH3) domain in Tuba called 'SH36". In my Ph.D. work, I demonstrated that Sec31A binds selectively to SH36 and fails to interact with other SH3 domains in Tuba or SH3 domains in several other mammalian signaling proteins. In addition to my work with Sec31A, I also performed co-precipitation experiments that identified the cis-Golgi protein GM130 as a ligand of the amino-terminal region in Tuba that contains four tandem SH3 domains (SH31-4). In summary, the research in this thesis provides important insights on the mechanisms of entry and cell-to-cell spread of Listeria in human cells.
Advisor: Ireton, Keith
Degree Name: Doctor of Philosophy
Degree Discipline: Microbiology and Immunology
Publisher: University of Otago
Keywords: Arf; GTPases; Listeria; GM130
Research Type: Thesis