RESUMO
Introduction: Endosymbiotic Wolbachia bacteria are widespread in nature, present in half of all insect species. The success of Wolbachia is supported by a commensal lifestyle. Unlike bacterial pathogens that overreplicate and harm host cells, Wolbachia infections have a relatively innocuous intracellular lifestyle. This raises important questions about how Wolbachia infection is regulated. Little is known about how Wolbachia abundance is controlled at an organismal scale. Methods: This study demonstrates methodology for rigorous identification of cellular processes that affect whole-body Wolbachia abundance, as indicated by absolute counts of the Wolbachia surface protein (wsp) gene. Results: Candidate pathways, associated with well-described infection scenarios, were identified. Wolbachia-infected fruit flies were exposed to small molecule inhibitors known for targeting those same pathways. Sequential tests in D. melanogaster and D. simulans yielded a subset of chemical inhibitors that significantly affected whole-body Wolbachia abundance, including the Wnt pathway disruptor, IWR-1 and the mTOR pathway inhibitor, Rapamycin. The implicated pathways were genetically retested for effects in D. melanogaster, using inducible RNAi expression driven by constitutive as well as chemically-induced somatic GAL4 expression. Genetic disruptions of armadillo, tor, and ATG6 significantly affected whole-body Wolbachia abundance. Discussion: As such, the data corroborate reagent targeting and pathway relevance to whole-body Wolbachia infection. The results also implicate Wnt and mTOR regulation of autophagy as important for regulation of Wolbachia titer.
RESUMO
BACKGROUND: Little is known about how bacterial endosymbionts colonize host tissues. Because many insect endosymbionts are maternally transmitted, egg colonization is critical for endosymbiont success. Wolbachia bacteria, carried by approximately half of all insect species, provide an excellent model for characterizing endosymbiont infection dynamics. To date, technical limitations have precluded stepwise analysis of germline colonization by Wolbachia. It is not clear to what extent titer-altering effects are primarily mediated by growth rates of Wolbachia within cell lineages or migration of Wolbachia between cells. RESULTS: The objective of this work is to inform mechanisms of germline colonization through use of optimized methodology. The approaches are framed in terms of nutritional impacts on Wolbachia. Yeast-rich diets in particular have been shown to suppress Wolbachia titer in the Drosophila melanogaster germline. To determine the extent of Wolbachia sensitivity to diet, we optimized 3-dimensional, multi-stage quantification of Wolbachia titer in maternal germline cells. Technical and statistical validation confirmed the identity of Wolbachia in vivo, the reproducibility of Wolbachia quantification and the statistical power to detect these effects. The data from adult feeding experiments demonstrated that germline Wolbachia titer is distinctly sensitive to yeast-rich host diets in late oogenesis. To investigate the physiological basis for these nutritional impacts, we optimized methodology for absolute Wolbachia quantification by real-time qPCR. We found that yeast-rich diets exerted no significant effect on bodywide Wolbachia titer, although ovarian titers were significantly reduced. This suggests that host diets affects Wolbachia distribution between the soma and late stage germline cells. Notably, relative qPCR methods distorted apparent wsp abundance, due to altered host DNA copy number in yeast-rich conditions. This highlights the importance of absolute quantification data for testing mechanistic hypotheses. CONCLUSIONS: We demonstrate that absolute quantification of Wolbachia, using well-controlled cytological and qPCR-based methods, creates new opportunities to determine how bacterial abundance within the germline relates to bacterial distribution within the body. This methodology can be applied to further test germline infection dynamics in response to chemical treatments, genetic conditions, new host/endosymbiont combinations, or potentially adapted to analyze other cell and tissue types.
