title: Identification and Characterization of a Candidate Wolbachia pipientis Type IV Effector That Interacts with the Actin Cytoskeleton.
abstract: Many bacteria live as intracellular symbionts, causing persistent infections within insects. One extraordinarily common infection is that of Wolbachia pipientis, which infects 40% of insect species and induces reproductive effects. The bacteria are passed from generation to generation both vertically (through the oocyte) and horizontally (by environmental transmission). Maintenance of the infection within Drosophila melanogaster is sensitive to the regulation of actin, as Wolbachia inefficiently colonizes strains hemizygous for the profilin or villin genes. Therefore, we hypothesized that Wolbachia must depend on the host actin cytoskeleton. In this study, we identify and characterize a Wolbachia protein (WD0830) that is predicted to be secreted by the bacterial parasite. Expression of WD0830 in a model eukaryote (the yeast Saccharomyces cerevisiae) induces a growth defect associated with the appearance of aberrant, filamentous structures which colocalize with rhodamine-phalloidin-stained actin. Purified WD0830 bundles actin in vitro and cosediments with actin filaments, suggesting a direct interaction of the two proteins. We characterized the expression of WD0830 throughout Drosophila development and found it to be upregulated in third-instar larvae, peaking in early pupation, during the critical formation of adult tissues, including the reproductive system. In transgenic flies, heterologously expressed WD0830 localizes to the developing oocyte. Additionally, overexpression of WD0830 results in increased Wolbachia titers in whole flies, in stage 9 and 10 oocytes, and in embryos, compared to controls, suggesting that the protein may facilitate Wolbachia's replication or transmission. Therefore, this candidate secreted effector may play a role in Wolbachia's infection of and persistence within host niches.
  
  
  
  The obligate intracellular Wolbachia pipientis is a ubiquitous alphaproteobacterial symbiont of arthropods and nematodes and is related to the rickettsial pathogens Ehrlichia spp. and Anaplasma spp. Studies of Wolbachia cell biology suggest that this bacterium relies on host actin for efficient proliferation and transmission between generations. Here, we identified and characterized a Wolbachia protein that localizes to and manipulates the eukaryotic actin cytoskeleton, is expressed by Wolbachia during host development, and alters Wolbachia titers and localization in transgenic fruit flies. We hypothesize that WD0830 may be utilized by the bacterium to facilitate replication in or invasion of different niches during host development.
authors.affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA.
authors.forename: Kathy B
authors.initials: KB
authors.lastname: Sheehan
authors.affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA.
authors.forename: MaryAnn
authors.initials: M
authors.lastname: Martin
authors.affiliation: Department of Medicine, Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Cambridge, Massachusetts, USA.
authors.forename: Cammie F
authors.initials: CF
authors.lastname: Lesser
authors.affiliation: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA.
authors.forename: Ralph R
authors.initials: RR
authors.lastname: Isberg
authors.affiliation: Department of Biology, Indiana University, Bloomington, Indiana, USA irnewton@indiana.edu.
authors.forename: Irene L G
authors.initials: IL
authors.lastname: Newton
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000382:microbiology*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D020577:Wolbachia / Q000235:genetics* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins