A Wolbachia deubiquitylating enzyme induces cytoplasmic incompatibility

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.

Figure 1

Modification-Rescue hypothesis for CI. a. Crossing Wolbachia-infected males (red) with uninfected females (black) yields nonviable embryos due to a sperm-derived modification. b. Crossing infected males and similarly infected females rescues viability due to a rescue factor in the infected egg. c. Operon from Wolbachia (wPip strain) proposed to induce CI; the wPa_0282 and wPa_0283 genes encode CidA and the deubiquitylating enzyme CidB, respectively. d. Paralogous operon from wPip in which a putative DUF1703 nuclease, CinB (wPa_0295) might also induce CI. e. Orthologous cidA-cidB operon from wMel, a Wolbachia strain isolated from D. melanogaster. f. Pull-down assays of operon partners reveal interaction specificity (6 replicates). His6-tagged β-galactosidase (LacZ) is a negative control.

Figure 2

Testing of the Modification-Rescue hypothesis in Saccharomyces cerevisiae. a. Expression of Wolbachia proteins from a galactose-inducible GAL1 promoter on minimal medium lacking uracil and containing galactose or glucose (3 replicates). Control plasmids pYES2 (empty vector) and LacZ (negative control) cause no defects. Both CidB and CinB expression blocks yeast growth at high temperature. Inactivation of the Ulp1-like protease by a C1025A mutation (CidB*) or the putative DUF1703 nuclease by mutation of the D-E-K triad to A-A-A (CinB*) eliminates toxicity. b. Coexpression of CidB or CinB with different upstream operon components on minimal media lacking uracil and leucine shows growth rescue only with cognate partners (3 replicates). Vector is pRS425.

Figure 3

CidB is a DUB. a. DUB reactivity with the N-terminally HA-tagged suicide inhibitor, UbVME (3 replicates). Shown is an anti-HA immunoblot analysis of 30-min reactions performed at room temperature. UbVME reacts with the wild-type CidB protein but not the C1025A catalytic mutant (CidB*). TsUCH37 is a positive control. CidA at 100-fold molar excess does not inhibit UbVME reactivity. b. Cleavage by CidB of K48- and K63-linked ubiquitin chains assayed by anti-ubiquitin immunoblotting (3 replicates). Usp2 is a positive control. Enzyme and polyubiquitin chains were at 50 nM and 500 nM, respectively, and reactions were at 37°C for 1 h. c. CidB has a ~4-fold preference for K63-ubiquitin dimers compared to K48-linked dimers. Shown is a plot of initial velocity (divided by total enzyme concentration) as a function of substrate concentration from three independent experiments. Error bars are standard deviations.

Figure 4

Induction of CI by transgenic cidA-cidB males. a. D. melanogaster males carrying transgenic cidA-cidB are sterile when mated to wild-type (WT) females (n=30 mating vials). Males with transgenic cidA-cidB* harboring the CidB active-site mutation C1025A (operon*) are fully fertile as are females with the active transgenic operon. CidA by itself has no effect on fertility; no strain singly transgenic for cidB could be isolated. EGFP is a negative control. Error bars are standard deviations. b. CI-like defects in the male pronucleus initially appear in late prophase, during the first division of the apposed female and male pronuclei, and accrue through mitosis. Abnormal cytology was observed in 56 transgenic CI embryos fixed after 18 min of development. c. Quantification of transgenic cidA-cidB (CI) embryos’ mitotic defects including uncondensed paternal chromosomes, delayed segregation of paternal chromosomes, or chromosomal bridging during the first zygotic cell cycle. Sample sizes of observed transgenic and WT embryos were 63 and 29, respectively. d. Quantification of developmental progress in transgenic (“CI”) embryos. At 24 h after egg laying, embryos were classified into three categories. Early, pre-blastoderm formation; Mid, blastoderm until segmentation stages; and Late, segmented stages. Quantification is based on three samples of approximately 200 embryos each. 60% of CI embryos arrested development in the early stage compared to 12% from the wild-type (WT) control. Significant p values (< 0.005) are indicated by (*). Error bars are standard deviations.