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Permanent link (DOI): https://doi.org/10.7939/R3163C

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Unique cellular interactions between the obligate intracellular bacteria Wolbachia pipientis and its insect host Open Access

Descriptions

Other title
Subject/Keyword
Proteomics
cytoplasmic incompatibility
DNA damage
Antioxidant
Reactive oxygen species
Wolbachia
Symbiosis
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Brennan, Lesley Jean
Supervisor and department
Keddie, B. Andrew (Biological Sciences)
Harris, Harriet L. (Biological Sciences)
Examining committee member and department
Perlman, Steve (Department of Biology, University of Victoria)
Evans, David (Medical Microbiology and Immunology, University of Alberta)
Gallin, Warren (Biological Sciences, University of Alberta)
Department
Department of Biological Sciences
Specialization

Date accepted
2011-08-31T15:30:11Z
Graduation date
2011-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Wolbachia are maternally inherited obligate intracellular bacteria found in arthropods, where they induce feminization, male-killing, parthenogenesis, and cytoplasmic incompatibility (CI). CI is conditional male sterility, in which Wolbachia-infected males successfully mate with infected females, but crosses between infected males and uninfected females result in embryonic death. How sperm are modified and how the Wolbachia-infected egg rescues them, resulting in normal embryonic development, is unknown. The objective of this thesis is to contribute to an understanding of the cellular biology of Wolbachia-host interactions, including the mechanism of CI. Protein expression in Wolbachia-infected and uninfected Aedes albopictus cells was evaluated by 2D PAGE. Proteins expressed exclusively in the presence of Wolbachia were identified, and included host actin depolymerizing factor and bacterial single-strand binding protein, GroES, 3,4-dihydroxy-2-butanone 4-phosphate synthase, nucleoside diphosphate kinase, and proteins involved in bacterial protein synthesis. Three host proteins (copper zinc superoxide dismutase, glutathione peroxidase, and peroxiredoxin) and two bacterial proteins (iron superoxide dismutase and bacterioferritin) having antioxidant activity were also identified. Antioxidants neutralize reactive oxygen species (ROS) generated by aerobic respiration or an immune response and induce cellular damage. Flow cytometric and microscopic analysis confirmed that ROS is elevated in infected cells and is associated with Wolbachia-containing vacuoles in the host cell cytoplasm. In Drosophila simulans flies, antioxidant assays showed that ROS is elevated in infected reproductive tissues, particularly the testes. To evaluate the effect of ROS accumulation, DNA damage was measured in Ae. albopictus cell lines by DNA dot blotting for the oxidative lesion 8-oxo-dG, which revealed an 8% increase in damage in DNA from infected cells. In D. simulans flies, analysis of 8-oxo-dG in DNA from whole males by mass spectrometry showed a slight increase in the lesion in infected flies, while single cell gel electrophoresis of spermatocytes revealed a 20% increase in single and double-stranded breaks as a result of Wolbachia infection. The conclusion from these results is that redox homeostasis is maintained in Wolbachia-infected insects as a whole. However in the densely infected testes Wolbachia-mediated ROS production exceeds antioxidant capacity resulting in oxidative DNA damage. The potential role of this damage in cytoplasmic incompatibility is discussed.
Language
English
DOI
doi:10.7939/R3163C
Rights
License granted by Lesley Brennan (lbrennan@ualberta.ca) on 2011-08-30T21:11:37Z (GMT): Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of the above terms. The author reserves all other publication and other rights in association with the copyright in the thesis, and except as herein provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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