Identification and characterization of proteins involved in the cytoskeletal rearrangements caused by bacterial pathogens

Date created: 
Enteropathogenic Escherichia coli
Listeria monocytogenes
Salmonella enterica serovar Typhimurium
Klebsiella pneumoniae

Bacterial pathogens have evolved to alter the cytoskeleton of their hosts during their respective infection processes. The extracellular bacterium, enteropathogenic Escherichia coli (EPEC), generates an actin-rich pedestal to “surf” along the host cell surface. In contrast, L. monocytogenes (L. monocytogenes) invades its host and polymerizes actin filaments to generate a comet tail for movement within and among host cells of epithelia. Salmonella enterica serovar Typhimurium (S. Typhimurium) induces actin-rich membrane-ruffles to invade its host cell. These bacteria have evolved to generate their respective actin-rich structures to colonize the intestinal epithelia. To further characterize the actin-rich structures generated by these bacteria, I selected four proteins from a mass spectrometry analysis of EPEC pedestals previously conducted in our laboratory. I found that the known actin-bundling proteins calponin 1 and calponin 2 decorated all the actin-rich structures formed by these three bacteria. Another actin-stabilizing protein transgelin (SM22) also decorated EPEC pedestals and L. monocytogenes comet tails. Moreover, the formation of pedestals and comet tails were dependent on SM22 protein levels. Aside from these three members of the calponin family, I found that a ubiquitin conjugating enzyme Ube2N was enriched at the invasion events and at the plasma membrane-bound comet tails formed by L. monocytogenes. This novel association of Ube2N with actin structures at the plasma membrane led to my discovering that Ube2N binds directly to actin, and that Ube2N function influences actin-based whole cell motility. Another bacterial pathogen, Klebsiella pneumoniae (K. pneumoniae), has been shown by others to alter the host actin cytoskeleton. I have found that the disassembly of the host microtubule networks precedes these actin cytoskeletal alterations in lung epithelial cells, and show that the Klebsiella pneumoniae gene ytfL (Kp ytfL) initiates this microtubule disassembly and that the katanin catalytic subunit A like 1 protein (KATNAL1) as well as the katanin regulatory subunit B1 protein (KATNB1) are activated to cause microtubule severing. Through this, I identified the bacterial initiator and the host cell effector proteins responsible for K. pneumoniae-induced microtubule disassembly. From these, I identified proteins that are novel to the actin structures of EPEC, L. monocytogenes and S. Typhimurium as well as effector proteins that are crucial for the novel host microtubule alterations of K. pneumoniae.

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Senior supervisor: 
Julian Guttman
Science: Biological Sciences Department
Thesis type: 
(Thesis) Ph.D.