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dc.contributor.advisorIreton, Keith
dc.contributor.authorBhalla, Manmeet Pal Singh
dc.date.available2018-02-12T21:58:49Z
dc.date.copyright2018
dc.identifier.citationBhalla, M. P. S. (2018). The human kinases mTOR, PKC-α, and their substrate Filamin A promote infection of human cells by Listeria monocytogenes (Thesis, Doctor of Philosophy). University of Otago. Retrieved from http://hdl.handle.net/10523/7847en
dc.identifier.urihttp://hdl.handle.net/10523/7847
dc.description.abstractThe bacterium Listeria monocytogenes is a food borne intracellular pathogen capable of causing gastroenteritis, abortions, or meningitis with a high mortality rate. Listeria is a facultative intracellular pathogen that is internalized into human cells, replicates inside cells, and spreads within host tissues. Listeria actively induces its internalisation (also known as “entry”) into cell types generally thought as non-phagocytic, such as intestinal epithelial cells, hepatocytes, trophoblast cells, and endothelial cells. One of the major pathways of Listeria entry into human cells is mediated by interaction of the bacterial surface protein Internalin B (InlB) with its host receptor, the Met tyrosine kinase. At least two host physiological processes are stimulated downstream of Met and contribute to internalisation of Listeria. One of these processes is actin polymerization, which is thought to provide a protrusive force that remodels the host plasma membrane, allowing the membrane to enwrap bacteria during entry. The second host process involved in InlB-mediated entry is exocytosis, the fusion of intracellular vesicles with the plasma membrane. Exocytosis controls internalisation of Listeria by delivering the human GTPase Dynamin2, and perhaps other host signalling proteins, to sites in the plasma membrane interacting with bacteria. The host type IA phosphoinositide 3-kinase (PI3-K) pathway is activated downstream of the Met receptor and plays a critical role in actin polymerization during entry of Listeria. Recent RNA interference (RNAi)-based screens targeting components of the PI3-K pathway identified the serine/threonine kinase Mammalian target of Rapamycin (mTOR) as a host signalling protein needed for efficient internalisation of Listeria. mTOR is present in two distinct complexes, termed mTORC1 or mTORC2. These complexes have different substrates and biological functions. The molecular mechanism by which mTORC1 or mTORC2 might control Listeria entry was not known and thus, formed the starting point of this dissertation. In Chapter 3 of this thesis, I present evidence indicating that mTOR acts together with PKC-α to promote exocytosis during InlB-mediated entry of Listeria into the human epithelial cell line HeLa. The specific findings in this chapter are as follows. First, Listeria was demonstrated to activate both mTORC1 and mTORC2, as assessed by phosphorylation of the substrates p70S6K or Akt, respectively. Activation of mTORC1 or mTORC2 was also caused by purified soluble InlB protein or InlB coupled to inert particles (latex beads). These InlB-coated beads were used as a model for Listeria entry in several experiments in this thesis. I also found that RNAi-mediated depletion of components specific to mTORC1 or mTORC2 (Raptor or Rictor) inhibited internalisation of Listeria. These results demonstrated important roles for both mTOR complexes in InlB-mediated entry. Further RNAi studies indicated that entry of Listeria is controlled by the mTORC1 effectors 4E-BP1 and HIF-1α, and the mTORC2 substrate PKC-α. The remaining studies in this chapter 3 focused on the mechanisms by which mTOR and PKC-α promote InlB-dependent internalisation. Infection of host cells with Listeria or treatment with InlB protein stimulated mTOR-dependent phosphorylation of PKC-α, suggesting that mTOR and PKC-α act together to control bacteria entry. Work with chemical inhibitors or a kinase dead form of PKC-α indicated that InlB-mediated entry requires the kinase activity of PKC-α. Laser scanning confocal microscopy was used to investigate the roles of mTOR and PKC-α in actin polymerization and exocytosis during InlB-mediated internalisation. Actin polymerization was assessed using fluorescently labeled phalloidin, whereas exocytosis was detected using a probe consisting of the vesicular SNARE protein VAMP3 fused to a GFP tag. The GFP moiety becomes surface-exposed upon fusion of exocytic vesicles with the plasma membrane. The effect of RNAi-mediated depletion of mTOR or PKC-α on exocytosis or actin polymerization during InlB-dependent entry was examined. My results indicate that both mTOR and PKC-α are required for efficient exocytosis during InlB-dependent entry. PKC-α also impacted actin polymerization during entry, whereas mTOR had no measurable effect on this process. In Chapter 4, I show that the human scaffolding protein Filamin A (FlnA) is regulated by mTOR and has an important role in InlB-dependent entry of Listeria into HeLa cells. Evidence is also presented suggesting that mTOR, PKC-α, FlnA, and a GTPase known as RalA may form a pathway that controls exocytosis during InlB-mediate uptake. The specific results are as follows. Results involving RNAi-mediated depletion of FlnA indicated an important role for this host protein in InlB-mediated entry and exocytosis in HeLa cells. In addition, phosphorylation of FlnA on Ser-2152 was stimulated during entry of InlB-coated beads into HeLa cells. Experiments with FlnA mutant protein unable to be phosphorylated on Ser-2152 indicated that phosphorylation at this site contributes to InlB-dependent entry. The GTPase RalA is a known binding partner of FlnA. Experiments involving RNAi-mediated depletion of RalA indicated an important role for this host protein in InlB-dependent entry and exocytosis. Using RNAi and confocal microscopy analysis, it was found that both RalA and mTOR are needed for recruitment of FlnA to areas in the plasma membrane surrounding InlB-coated beads. These latter results suggest that mTOR and RalA might control exocytosis, in part, by localizing FlnA to plasma membrane sites where InlB-dependent entry occurs. Collectively, my findings identify mTOR, PKC-α, FlnA and RalA as host signalling factors exploited by Listeria to promote its internalisation into host cells. These results also suggest that mTOR, PKC-α, FlnA and RalA may act together in a pathway to control host exocytosis during InlB-mediated entry.
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectListeria
dc.subjectmTOR
dc.subjectPKC-alpha
dc.subjectRNA-i
dc.subjectRalA
dc.subjectexocytosis
dc.titleThe human kinases mTOR, PKC-α, and their substrate Filamin A promote infection of human cells by Listeria monocytogenes
dc.typeThesis
dc.date.updated2018-02-12T07:54:50Z
dc.language.rfc3066en
thesis.degree.disciplineMicrobiology and Immunology
thesis.degree.nameDoctor of Philosophy
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.interloanno
otago.openaccessAbstract Only
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