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Male-killing toxin in a bacterial symbiont of Drosophila

Naturevolume 557pages252255 (2018) | Download Citation

Abstract

Several lineages of symbiotic bacteria in insects selfishly manipulate host reproduction to spread in a population1, often by distorting host sex ratios. Spiroplasma poulsonii2,3 is a helical and motile, Gram-positive symbiotic bacterium that resides in a wide range of Drosophila species4. A notable feature of S. poulsonii is male killing, whereby the sons of infected female hosts are selectively killed during development1,2. Although male killing caused by S. poulsonii has been studied since the 1950s, its underlying mechanism is unknown. Here we identify an S. poulsonii protein, designated Spaid, whose expression induces male killing. Overexpression of Spaid in D. melanogaster kills males but not females, and induces massive apoptosis and neural defects, recapitulating the pathology observed in S. poulsonii-infected male embryos5,6,7,8,9,10,11. Our data suggest that Spaid targets the dosage compensation machinery on the male X chromosome to mediate its effects. Spaid contains ankyrin repeats and a deubiquitinase domain, which are required for its subcellular localization and activity. Moreover, we found a laboratory mutant strain of S. poulsonii with reduced male-killing ability and a large deletion in the spaid locus. Our study has uncovered a bacterial protein that affects host cellular machinery in a sex-specific way, which is likely to be the long-searched-for factor responsible for S. poulsonii-induced male killing.

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Acknowledgements

We thank the Bloomington Stock Center in the USA and the Department of Drosophila Genomics and Genetic Resources at Kyoto Institute of Technology in Japan (DGGR) for fly stocks; the Developmental Studies Hybridoma Bank at the University of Iowa for monoclonal antibodies; the Drosophila Genomics Resource Center in the USA for DNA vectors; J. Jaenike, T. Murata, and M. Kuroda for providing fly strains; T. Fukatsu for the S. poulsonii-infected fly stocks that were originally established in his laboratory by T.H; J. Lucchesi for providing antibodies; the BioImaging & Optics Platform (BIOP) in EPFL for confocal microscopy; K. Harshman and the Lausanne Genomic Technologies Facility (GTF) at the University of Lausanne (UNIL) for whole-genome sequencing and draft genome assemblies; and C. Vorburger and S. Perlman for comments and suggestions on the manuscript; and the members of the laboratory for discussion and support. This work was supported by the European Research Council (ERC) Advanced Grant 339970 and the Swiss National Science Foundation (SNSF) Sinergia Grant CRSII3_154396. The purchase of the PacBio RS II system in UNIL was supported in part by the Loterie Romande through the Fondation pour la Recherche en Médecine Génétique.

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Nature thanks P. Ferree and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Affiliations

  1. Global Health Institute, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

    • Toshiyuki Harumoto
    •  & Bruno Lemaitre

Authors

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  2. Search for Bruno Lemaitre in:

Contributions

T.H. conceived of and designed the study, performed the experiments, analysed the data and wrote and edited the manuscript. B.L. conceived and administered the study and wrote and edited the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Toshiyuki Harumoto or Bruno Lemaitre.

Extended data figures and tables

  1. Extended Data Fig. 1 Identification and characterization of the partial male-killing S. poulsonii strain.

    a, An illustration showing the origin of the S. poulsonii strains analysed in this study. Pictures show male-killing (MK) S. poulsonii of D. melanogaster. MSRO-Ug is the original male-killing strain maintained in the Oregon-R wild-type fly. Fly stocks (Df(3L)H99 and Sxl-eGFP) artificially infected with this original strain showed complete male killing (100% MK) for the first 20 generations. Afterwards, one strain (MSRO-SE) started to show the partial male-killing phenotype, whereas the other (MSRO-H99) kept the ability to induce complete male killing. See Methods section ‘Identification and characterization of the partial male-killing S. poulsonii strain’ for more detail. b, c, Sex-ratio analysis of the adult progeny obtained from Oregon-R flies infected with MSRO-Ug, MSRO-H99, and MSRO-SE. We repeated experiments three times on the fourth, fifth and sixth generations (G4–6) after the establishment of infection. In c, the relative number of male offspring (percentage of females) obtained from Oregon-R female flies infected with MSRO-SE are plotted. Data points were excluded if the total count of flies was below 10. d, Relative titre of S. poulsonii within individual female flies. Adult females infected with three MSRO strains were kept for 0, 7 and 14 days after eclosion and analysed by qPCR. Data were normalized with respect to females from day 0 that were infected with MSRO-Ug. Different letters indicate statistically significant different groups (P < 0.01; N.S., not significant, P > 0.05; Steel–Dwass test; see Supplementary Table 2). Please note that the titres of the three strains are comparable and even the higher titre in old females (see 14 days in d) fails to induce complete male killing in MSRO-SE (c). Box and dot plots are as in Fig. 1c and sample sizes (n, number of adult flies) are shown at the bottom. Source Data

  2. Extended Data Fig. 2 Whole-genome sequencing studies of the male-killing S. poulsonii variants.

    a, Comparison of the genomic features of three S. poulsonii strains. MSRO-H99 and MSRO-SE are newly obtained variants isolated in this study (Extended Data Fig. 1a). As a control, data from the previously reported male-killing S. poulsonii genome16 are also indicated. b, Whole-genome alignment of the three S. poulsonii strains. To start the alignment from the dnaA gene, contig 1 of MSRO-H99 was split into two fragments (contig 1.1 and 1.2). The locations of the contigs corresponding to extra chromosomes (putative plasmids; see Methods section ‘Genomic data analyses and protein domain searches’) are shown as ‘extra’. spaid (gene ID from GenBank BioProject PRJNA416288: SMH99_26490) and spaid ΔC (gene ID: SMSE_25110) are located on these extra chromosomes in MSRO-H99 (contig 4) and MSRO-SE (contig 2), respectively.

  3. Extended Data Fig. 3 Genetic alterations of the spaid locus in the partial male-killing S. poulsonii strain.

    The genome structures around the spaid loci in the male-killing (a, MSRO-Ug and MSRO-H99) and the partial male-killing (b, MSRO-SE) S. poulsonii strains. Genes encoded on opposite strands are shown in different colours (red and blue, respectively). An 828-bp deletion and nucleotide substitutions (coloured in red; corresponding amino acid sequences are presented in one-letter code) in the 3′ region of the spaid gene are indicated. These sequence alterations were confirmed using Sanger sequencing (see Methods).

  4. Extended Data Fig. 4 Neural defects of Spaid-expressing embryos.

    Representative images of stage 13–14 female (a, n = 14) and male (bn = 16) embryos maternally expressing Spaid and stained for TUNEL (green) and neural cells (Elav, magenta). Single-channel images of Elav are also shown. The outlined region in b is magnified in c with single-channel images of Elav and TUNEL. Embryos were co-stained for Elav, TUNEL, Sxl and DNA, and selected channels are shown in a–c and Fig. 2a, b, respectively.

  5. Extended Data Fig. 5 Spaid acts through the dosage compensation machinery.

    a–c, Representative images of stage 13–14 embryos ectopically expressing the MSL complex (H83M2 transgene), stained for TUNEL (green) and Sxl (magenta). GFP-expressing control female (a, n = 15), Spaid-expressing female (b, n = 20), and male (c, n = 19) embryos are shown. d, Quantification of TUNEL signals in H83M2 embryos at stages 13–14. Different characters indicate significantly different groups (P < 0.001; Steel–Dwass test; see Supplementary Table 2). The box and dot plot (females, red; males, blue) is as in Fig. 1c and sample sizes (n, number of embryos) are shown at the bottom. e, f, Representative images of epithelial cells of stage 8–10 male embryos expressing GFP (e, n = 25) and Spaid (f, n = 25), stained for DNA (green) and MSL1 (magenta) from the datasets analysed in Fig. 3. All UAS transgenes were expressed maternally. Source Data

  6. Extended Data Fig. 6 Expression of Spaid using a weak GAL4 driver.

    The number of adult progeny (females, red; males, blue) obtained from crosses between the armadillo-GAL4 driver line (weak and ubiquitous expression) and four UAS transgenic lines (GFP, Spaid, ΔANK, and ΔOTU; n = 6 independent crosses for GFP and Spaid, n = 8 independent crosses for ΔANK and ΔOTU). The UAS-GFP line was used as a negative control. With this weak GAL4 driver, Spaid still eliminated all male progeny, while both ΔANK and ΔOTU had no impact on male viability. An asterisk indicates the statistically significant difference (P < 0.01; N.S., not significant, P > 0.05; two-tailed Mann–Whitney U test; see Supplementary Table 2). Box and dot plots are as in Fig. 1c. The total numbers of adult counts for each genotype and sex are shown at the bottom. Source Data

  7. Extended Data Fig. 7 A proposed model for Spaid-induced male-killing phenotypes.

    Spaid utilizes the OTU domain and ankyrin repeats (ANK) to target the host nucleus and the male X chromosome. ‘MSL’ and ‘Ac’ indicate the dosage compensation complex and resultant histone acetylation, respectively. See text for other explanations.

Supplementary information

  1. Supplementary Information

    This file contains Supplementary Tables 1-2. Supplementary Table 1 contains a list of PCR primers (the names and sequences of PCR primers are indicated with the names of the target organisms) and Supplementary Table 2 contains exact P values (statistically significant pairs with an alpha level of 0.05 are indicated in red characters).

  2. Reporting Summary

  3. Supplementary Data

    Summary of conserved domain database search results for the proteins annotated in the Spiroplasma strains MSRO-H99 (Data 1), MSRO-SE (Data 2), and in the previously published genome (Data 3; MSRO, 2015). Spaid in MSRO-H99 (SMH99_26490) and Spaid ∆C in MSRO-SE (SMSE_25110) are shown in red characters.

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