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A Wolbachia deubiquitylating enzyme induces cytoplasmic incompatibility

Nature Microbiology volume 2, Article number: 17007 (2017) Cite this article

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Abstract

Wolbachia are obligate intracellular bacteria1 that infect arthropods, including approximately two-thirds of insect species2. Wolbachia manipulate insect reproduction by enhancing their inheritance through the female germline. The most common alteration is cytoplasmic incompatibility (CI)3–5, where eggs from uninfected females fail to develop when fertilized by sperm from Wolbachia-infected males. By contrast, if female and male partners are both infected, embryos are viable. CI is a gene-drive mechanism impacting population structure6 and causing reproductive isolation7, but its molecular mechanism has remained unknown. We show that a Wolbachia deubiquitylating enzyme (DUB) induces CI. The CI-inducing DUB, CidB, cleaves ubiquitin from substrates and is encoded in a two-gene operon, and the other protein, CidA, binds CidB. Binding is strongest between cognate partners in cidA-cidB homologues. In transgenic Drosophila, the cidA-cidB operon mimics CI when sperm introduce it into eggs, and a catalytically inactive DUB does not induce sterility. Toxicity is recapitulated in yeast by CidB alone; this requires DUB activity but is rescued by coexpressed CidA. A paralogous operon involves a putative nuclease (CinB) rather than a DUB. Analogous binding, toxicity and rescue in yeast were observed. These results identify a CI mechanism involving interacting proteins that are secreted into germline cells by Wolbachia, and suggest new methods for insect control.

Although Wolbachia infect both male and female germlines, the bacteria are removed in the later stages of spermatid differentiation. In a previous proteomic study we had therefore searched for Wolbachia (wPip strain) proteins associated with Wolbachia-modified mosquito sperm and identified the protein WPA0282 (ref. 16). The wPa_0282 gene is part of a two-gene operon (Fig. 1c). Given our identification (see below) of the second gene product, WPA0283, as a CI-inducing DUB (deubiquitylating enzyme), we renamed the genes cidA (wPa_0282) and cidB (wPa_0283). Ubiquitin is a small polypeptide that post-translationally modifies many proteins and has numerous functions. Protein ubiquitylation is highly dynamic and is reversed by cellular DUBs17. Genetic evidence from diverse Wolbachia strains suggests that the modification and rescue functionalities of CI arise from at least two independent genes, similar to bacterial toxin–antidote systems10. Most such toxin–antidote systems studied have simple two-gene operon structures. We therefore hypothesized that the cidA-cidB operon products might be the executers of CI.

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Figure 1: Modification–rescue hypothesis for CI.
Figure 2: Testing of the modification–rescue hypothesis in S. cerevisiae.
Figure 3: CidB is a DUB.
Figure 4: Induction of CI by transgenic cidA-cidB males.

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Acknowledgements

The authors thank A. Fallon, R. Tomko Jr and A. Chavez for critical review of the manuscript, L. Klasson for sharing preliminary data, H. Frydman and L. Cooley for fly lines, J. Carlson, K. Menuz, P. Graham and C. Yam for support with fly experimentation, C. Schlieker for use of instruments and C. Das and M. Sheedlo for HA-Ub-VME. Funding was provided by USDA-NIFA postdoctoral fellowship 2014-67012-22268 (to J.F.B.), the Marion Brooks-Wallace fellowship (to J.F.B.) and National Institutes of Health grant GM053756 (to M.H.).

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Author notes

  1. John F. Beckmann and Judith A. Ronau: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biophysics & Biochemistry, Yale University, 266 Whitney Avenue, New Haven, 06520, Connecticut, USA

    John F. Beckmann, Judith A. Ronau & Mark Hochstrasser

  2. Department of Molecular, Cellular, & Developmental Biology, Yale University, 266 Whitney Avenue, New Haven, 06520, Connecticut, USA

    Mark Hochstrasser

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J.F.B. and J.A.R. are equal contributors in the conception and performance of the experiments as well as writing of the manuscript. M.H. conceived experiments and wrote the manuscript.

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Correspondence to Mark Hochstrasser.

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Supplementary Information

Supplementary Figures 1–9, Supplementary Discussion, Supplementary References. (PDF 2407 kb)

Supplementary Table 1

Construct Database and Primer Database. (XLSX 29 kb)

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Beckmann, J., Ronau, J. & Hochstrasser, M. A Wolbachia deubiquitylating enzyme induces cytoplasmic incompatibility. Nat Microbiol 2, 17007 (2017). https://doi.org/10.1038/nmicrobiol.2017.7

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