RESUMO
Wolbachia are common bacteria among terrestrial arthropods. These endosymbionts transmitted through the female germline manipulate their host reproduction through several mechanisms whose most prevalent form called Cytoplasmic Incompatibility -CI- is a conditional sterility syndrome eventually favoring the infected progeny. Upon fertilization, the sperm derived from an infected male is only compatible with an egg harboring a compatible Wolbachia strain, this sperm leading otherwise to embryonic death. The Wolbachia Cif factors CidA and CidB responsible for CI and its neutralization function as a Toxin-Antitoxin system in the mosquito host Culex pipiens. However, the mechanism of CidB toxicity and its neutralization by the CidA antitoxin remain unexplored. Using transfected insect cell lines to perform a structure-function analysis of these effectors, we show that both CidA and CidB are chromatin interactors and CidA anchors CidB to the chromatin in a cell-cycle dependent-manner. In absence of CidA, the CidB toxin localizes to its own chromatin microenvironment and acts by preventing S-phase completion, independently of its deubiquitylase -DUB- domain. Experiments with transgenic Drosophila show that CidB DUB domain is required together with CidA during spermatogenesis to stabilize the CidA-CidB complex. Our study defines CidB functional regions and paves the way to elucidate the mechanism of its toxicity.
Assuntos
Proteínas de Drosophila , Wolbachia , Animais , Masculino , Cromatina/metabolismo , Wolbachia/fisiologia , Sêmen/metabolismo , Animais Geneticamente Modificados , Drosophila/metabolismo , Citoplasma/metabolismo , Proteína Centromérica A/metabolismo , Proteínas de Drosophila/metabolismoRESUMO
Wolbachia are widespread endosymbiotic bacteria that manipulate the reproduction of arthropods through a diversity of cellular mechanisms. In cytoplasmic incompatibility (CI), a sterility syndrome originally discovered in the mosquito Culex pipiens, uninfected eggs fertilized by sperm from infected males are selectively killed during embryo development following the abortive segregation of paternal chromosomes in the zygote. Despite the recent discovery of Wolbachia CI factor (cif) genes, the mechanism by which they control the fate of paternal chromosomes at fertilization remains unknown. Here, we have analyzed the cytological distribution and cellular impact of CidA and CidB, a pair of Cif proteins from the Culex-infecting Wolbachia strain wPip. We show that expression of CidB in Drosophila S2R+ cells induces apoptosis unless CidA is co-expressed and associated with its partner. In transgenic Drosophila testes, both effectors colocalize in germ cells until the histone-to-protamine transition in which only CidB is retained in maturing spermatid nuclei. We further show that CidB is similarly targeted to maturing sperm of naturally infected Culex mosquitoes. At fertilization, CidB associates with paternal DNA regions exhibiting DNA replication stress, as a likely cause of incomplete replication of paternal chromosomes at the onset of the first mitosis. Importantly, we demonstrate that inactivation of the deubiquitylase activity of CidB does not abolish its cell toxicity or its ability to induce CI in Drosophila. Our study thus demonstrates that CI functions as a transgenerational toxin-antidote system and suggests that CidB acts by poisoning paternal DNA replication in incompatible crosses.