Dataset Information

Transformation of the Drosophila Sex-Manipulative Endosymbiont Spiroplasma poulsonii and Persisting Hurdles for Functional Genetic Studies.

ABSTRACT: Insects are frequently infected by bacterial symbionts that greatly affect their physiology and ecology. Most of these endosymbionts are, however, barely tractable outside their native host, rendering functional genetics studies difficult or impossible. Spiroplasma poulsonii is a facultative bacterial endosymbiont of Drosophila melanogaster that manipulates the reproduction of its host by killing its male progeny at the embryonic stage. S. poulsonii, although a very fastidious bacterium, is closely related to pathogenic Spiroplasma species that are cultivable and genetically modifiable. In this work, we present the transformation of S. poulsonii with a plasmid bearing a fluorescence cassette, leveraging techniques adapted from those used to modify the pathogenic species Spiroplasma citri We demonstrate the feasibility of S. poulsonii transformation and discuss approaches for mutant selection and fly colonization, which are persisting hurdles that must be overcome to allow functional bacterial genetics studies of this endosymbiont in vivo IMPORTANCE Dozens of bacterial endosymbiont species have been described and estimated to infect about half of all insect species. However, only a few them are tractable in vitro, which hampers our understanding of the bacterial determinants of the host-symbiont interaction. Developing a transformation method for S. poulsonii is a major step toward genomic engineering of this symbiont, which will foster basic research on endosymbiosis. This could also open the way to practical uses of endosymbiont engineering through paratransgenesis of vector or pest insects.

SUBMITTER: Masson F

PROVIDER: S-EPMC7357483 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Transformation of the Drosophila Sex-Manipulative Endosymbiont Spiroplasma poulsonii and Persisting Hurdles for Functional Genetic Studies.

Masson Florent F Schüpfer Fanny F Jollivet Chloé C Lemaitre Bruno B

Applied and environmental microbiology 20200702 14

Insects are frequently infected by bacterial symbionts that greatly affect their physiology and ecology. Most of these endosymbionts are, however, barely tractable outside their native host, rendering functional genetics studies difficult or impossible. Spiroplasma poulsonii is a facultative bacterial endosymbiont of Drosophila melanogaster that manipulates the reproduction of its host by killing its male progeny at the embryonic stage. S. poulsonii, although a very fastidio ...[more]

PMID: 32444468

Similar Datasets

Project description:Spiroplasma bacteria are highly motile bacteria with no cell wall and a helical morphology. This clade includes many vertically transmitted insect endosymbionts, including Spiroplasma poulsonii, a natural endosymbiont of Drosophila melanogaster S. poulsonii bacteria are mainly found in the hemolymph of infected female flies and exhibit efficient vertical transmission from mother to offspring. As is the case for many facultative endosymbionts, S. poulsonii can manipulate the reproduction of its host; in particular, S. poulsonii induces male killing in Drosophila melanogaster Here, we analyze the morphology of S. poulsonii obtained from the hemolymph of infected Drosophila This endosymbiont was not only found as long helical filaments, as previously described, but was also found in a Y-shaped form. The use of electron microscopy, immunogold staining of the FtsZ protein, and antibiotic treatment unambiguously linked the Y shape of S. poulsonii to cell division. Observation of the Y shape in another Spiroplasma, S. citri, and anecdotic observations from the literature suggest that cell division by longitudinal scission might be prevalent in the Spiroplasma clade. Our study is the first to report the Y-shape mode of cell division in an endosymbiotic bacterium and adds Spiroplasma to the so far limited group of bacteria known to utilize this cell division mode. Most bacteria rely on binary fission, which involves elongation of the bacteria and DNA replication, followed by splitting into two parts. Examples of bacteria with a Y-shape longitudinal scission remain scarce. Here, we report that Spiroplasma poulsonii, an endosymbiotic bacterium living inside the fruit fly Drosophila melanogaster, divide with the longitudinal mode of cell division. Observations of the Y shape in another Spiroplasma, S. citri, suggest that this mode of scission might be prevalent in the Spiroplasma clade. Spiroplasma bacteria are wall-less bacteria with a distinctive helical shape, and these bacteria are always associated with arthropods, notably insects. Our study raises the hypothesis that this mode of cell division by longitudinal scission could be linked to the symbiotic mode of life of these bacteria.

Project description:Facultative endosymbiotic bacteria, such as Wolbachia and Spiroplasma species, are commonly found in association with insects and can dramatically alter their host physiology. Many endosymbionts are defensive and protect their hosts against parasites or pathogens. Despite the widespread nature of defensive insect symbioses and their importance for the ecology and evolution of insects, the mechanisms of symbiont-mediated host protection remain poorly characterized. Here, we utilized the fruit fly Drosophila melanogaster and its facultative endosymbiont Spiroplasma poulsonii to characterize the mechanisms underlying symbiont-mediated host protection against bacterial and fungal pathogens. Our results indicate a variable effect of S. poulsonii on infection outcome, with endosymbiont-harboring flies being more resistant to Rhyzopus oryzae, Staphylococcus aureus, and Providencia alcalifaciens but more sensitive or as sensitive as endosymbiont-free flies to the infections with Pseudomonas species. Further focusing on the protective effect, we identified Transferrin-mediated iron sequestration induced by Spiroplasma as being crucial for the defense against R. oryzae and P. alcalifaciens. In the case of S. aureus, enhanced melanization in Spiroplasma-harboring flies plays a major role in protection. Both iron sequestration and melanization induced by Spiroplasma require the host immune sensor protease Persephone, suggesting a role of proteases secreted by the symbiont in the activation of host defense reactions. Hence, our work reveals a broader defensive range of Spiroplasma than previously appreciated and adds nutritional immunity and melanization to the defensive arsenal of symbionts.ImportanceDefensive endosymbiotic bacteria conferring protection to their hosts against parasites and pathogens are widespread in insect populations. However, the mechanisms by which most symbionts confer protection are not fully understood. Here, we studied the mechanisms of protection against bacterial and fungal pathogens mediated by the Drosophila melanogaster endosymbiont Spiroplasma poulsonii. We demonstrate that besides the previously described protection against wasps and nematodes, Spiroplasma also confers increased resistance to pathogenic bacteria and fungi. We identified Spiroplasma-induced iron sequestration and melanization as key defense mechanisms. Our work broadens the known defense spectrum of Spiroplasma and reveals a previously unappreciated role of melanization and iron sequestration in endosymbiont-mediated host protection. We propose that the mechanisms we have identified here may be of broader significance and could apply to other endosymbionts, particularly to Wolbachia, and potentially explain their protective properties.

Project description:Endosymbiotic bacteria associated with eukaryotic hosts are omnipresent in nature, particularly in insects. Studying the bacterial side of host-symbiont interactions is, however, often limited by the unculturability and genetic intractability of the symbionts. Spiroplasma poulsonii is a maternally transmitted bacterial endosymbiont that is naturally associated with several Drosophila species. S. poulsonii strongly affects its host's physiology, for example by causing male killing or by protecting it against various parasites. Despite intense work on this model since the 1950s, attempts to cultivate endosymbiotic Spiroplasma in vitro have failed so far. Here, we developed a method to sustain the in vitro culture of S. poulsonii by optimizing a commercially accessible medium. We also provide a complete genome assembly, including the first sequence of a natural plasmid of an endosymbiotic Spiroplasma species. Last, by comparing the transcriptome of the in vitro culture to the transcriptome of bacteria extracted from the host, we identified genes putatively involved in host-symbiont interactions. This work provides new opportunities to study the physiology of endosymbiotic Spiroplasma and paves the way to dissect insect-endosymbiont interactions with two genetically tractable partners.IMPORTANCE The discovery of insect bacterial endosymbionts (maternally transmitted bacteria) has revolutionized the study of insects, suggesting novel strategies for their control. Most endosymbionts are strongly dependent on their host to survive, making them uncultivable in artificial systems and genetically intractable. Spiroplasma poulsonii is an endosymbiont of Drosophila that affects host metabolism, reproduction, and defense against parasites. By providing the first reliable culture medium that allows a long-lasting in vitro culture of Spiroplasma and by elucidating its complete genome, this work lays the foundation for the development of genetic engineering tools to dissect endosymbiosis with two partners amenable to molecular study. Furthermore, the optimization method that we describe can be used on other yet uncultivable symbionts, opening new technical opportunities in the field of host-microbes interactions.

Dataset Information

Transformation of the Drosophila Sex-Manipulative Endosymbiont Spiroplasma poulsonii and Persisting Hurdles for Functional Genetic Studies.

Publications

Transformation of the <i>Drosophila</i> Sex-Manipulative Endosymbiont Spiroplasma poulsonii and Persisting Hurdles for Functional Genetic Studies.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets