Investigation of the relationship between the human proteins Sec31A and Tuba in infection by the bacterial pathogen Listeria monocytogenes.
Le, Phuong Huynh Bao

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Le, P. H. B. (2013). Investigation of the relationship between the human proteins Sec31A and Tuba in infection by the bacterial pathogen Listeria monocytogenes. (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/4413
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http://hdl.handle.net/10523/4413
Abstract:
Listeria monocytogenesis causes food-borne disease leading to meningitis or abortion. A critical aspect of Listeria virulence is the ability of the pathogen to spread between mammalian cells using an F-actin-dependent motility process. During cell-cell spread, Listeria is propelled through the host cytoplasm by bacterial-induced actin ‘comet tails’. Contact of motile bacteria with the host plasma membrane leads to the formation of pathogen-containing membrane extensions or ‘protrusions’, which are then engulfed by neighbouring cells. It has been demonstrated in previously published work that the Listeria protein InlC promotes the formation of protrusions by binding as well as inhibiting a human cytoplasmic adaptor protein called Tuba. Apart from Tuba, the human trafficking protein Sec31A also controls Listeria spread. This project is motivated by the hypothesis that human Tuba and Sec31A might cooperate to control Listeria spread by forming a complex in human cells. To test this hypothesis, experiments were performed to determine if Tuba and Sec31A interact and/or co-localize in vivo. Using co-precipitation assays, it was found that a purified Src Homology 3 (SH3) domain from Tuba associates with Sec31A. Interestingly, the Listeria InlC protein binds to the same SH3 domain in Tuba and displaces Sec31A from this domain. However, attempts to verify interaction between endogenous Tuba and Sec31A in co-immunoprecipitation experiments were unsuccessful. Confocal microscopy analysis was performed to determine the subcellular localization of endogenous Tuba and Sec31A. Depending on the antibodies used, Tuba localized pre-dominantly either to the Golgi apparatus or near cell-cell junctions. Sec31A localized in vesicular structures thought to be endoplasmic reticulum exit sites. Experiments with one of the anti-Tuba antibodies indicated that ~ 10% of the total cellular pool of Tuba co-localizes with Sec31A. However, when two other Tuba antibodies were used little or no co-localization between Tuba and Sec31A was detected. Altogether, the results from this project indicate that host Tuba and Sec31A have the potential to interact and suggest that the Listeria InlC protein might control spreading by disrupting Tuba/Sec31A complexes. However, firm conclusions cannot be presently made because of the inability to detect Tuba and Sec31A interaction by co-immunoprecipitation and variable data on co-localization of Tuba and Sec31A. Future experiments should focus on immunoprecipation approaches capable of detecting complexes of low affinity and/or abundance, and confirming specificity of Tuba antibodies.
Date:
2013
Advisor:
Ireton, Keith
Degree Name:
Master of Science
Degree Discipline:
Microbiology and Immunology
Publisher:
University of Otago
Keywords:
Microbiology; foodborne; pathogen; Listeria; Sec31A; Tuba; NewZealand; Otago
Research Type:
Thesis
Languages:
English
Collections
- Microbiology and Immunology [180]
- Thesis - Masters [3378]