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

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.