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The phage gene wmk is a candidate for male killing by a bacterial endosymbiont


Autoři: Jessamyn I. Perlmutter aff001;  Sarah R. Bordenstein aff001;  Robert L. Unckless aff003;  Daniel P. LePage aff001;  Jason A. Metcalf aff001;  Tom Hill aff003;  Julien Martinez aff005;  Francis M. Jiggins aff005;  Seth R. Bordenstein aff001
Působiště autorů: Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, United States of America aff001;  Vanderbilt Microbiome Initiative, Vanderbilt University, Nashville, Tennessee, United States of America aff002;  Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, United States of America aff003;  Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, United State of America aff004;  Department of Genetics, University of Cambridge, Cambridge, United Kingdom aff005;  Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, Tennessee, United States of America aff006;  Vanderbilt Institute for Infection, Immunology and Inflammation, Vanderbilt University, Nashville, Tennessee, United States of America aff007
Vyšlo v časopise: The phage gene wmk is a candidate for male killing by a bacterial endosymbiont. PLoS Pathog 15(9): e32767. doi:10.1371/journal.ppat.1007936
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.ppat.1007936

Souhrn

Wolbachia are the most widespread maternally-transmitted bacteria in the animal kingdom. Their global spread in arthropods and varied impacts on animal physiology, evolution, and vector control are in part due to parasitic drive systems that enhance the fitness of infected females, the transmitting sex of Wolbachia. Male killing is one common drive mechanism wherein the sons of infected females are selectively killed. Despite decades of research, the gene(s) underlying Wolbachia-induced male killing remain unknown. Here using comparative genomic, transgenic, and cytological approaches in fruit flies, we identify a candidate gene in the eukaryotic association module of Wolbachia prophage WO, termed WO-mediated killing (wmk), which transgenically causes male-specific lethality during early embryogenesis and cytological defects typical of the pathology of male killing. The discovery of wmk establishes new hypotheses for the potential role of phage genes in sex-specific lethality, including the control of arthropod pests and vectors.

Klíčová slova:

Biology and life sciences – Developmental biology – Embryology – Embryos – Organisms – Bacteria – Wolbachia – Eukaryota – Animals – Invertebrates – Arthropoda – Insects – Drosophila – Drosophila melanogaster – Viruses – Bacteriophages – Population biology – Population metrics – Sex ratio – Genetics – Phenotypes – Genomics – Comparative genomics – Gene expression – Computational biology – Research and analysis methods – Animal studies – Experimental organism systems – Model organisms – Animal models


Zdroje

1. Zug R, Hammerstein P. Still a host of hosts for Wolbachia: analysis of recent data suggests that 40% of terrestrial arthropod species are infected. PLoS One. 2012;7(6):e38544. Epub 2012/06/12. doi: 10.1371/journal.pone.0038544 22685581; PubMed Central PMCID: PMC3369835.

2. Weinert LA, Araujo-Jnr EV, Ahmed MZ, Welch JJ. The incidence of bacterial endosymbionts in terrestrial arthropods. Proceedings of the Royal Society B: Biological Sciences. 2015;282(1807):20150249. doi: 10.1098/rspb.2015.0249 25904667

3. Ferri E, Bain O, Barbuto M, Martin C, Lo N, Uni S, et al. New insights into the evolution of Wolbachia infections in filarial nematodes inferred from a large range of screened species. PLoS One. 2011;6(6):e20843. Epub 2011/07/07. doi: 10.1371/journal.pone.0020843 21731626; PubMed Central PMCID: PMC3120775.

4. Funkhouser-Jones LJ, van Opstal EJ, Sharma A, Bordenstein SR. The Maternal Effect Gene Wds Controls Wolbachia Titer in Nasonia. Curr Biol. 2018;28(11):1692–702 e6. Epub 2018/05/22. doi: 10.1016/j.cub.2018.04.010 29779872; PubMed Central PMCID: PMC5988964.

5. Ferree PM, Frydman HM, Li JM, Cao J, Wieschaus E, Sullivan W. Wolbachia utilizes host microtubules and Dynein for anterior localization in the Drosophila oocyte. PLoS pathogens. 2005;1(2):e14. Epub 2005/10/18. doi: 10.1371/journal.ppat.0010014 16228015; PubMed Central PMCID: PMC1253842.

6. Hurst GDD, Jiggins FM, Hinrich Graf von der Schulenburg J, Bertrand D, West SA, Goriacheva II, et al. Male–killing Wolbachia in two species of insect. Proceedings of the Royal Society of London Series B: Biological Sciences. 1999;266(1420):735–40. doi: 10.1098/rspb.1999.0698

7. Unckless RL, Jaenike J. Maintenance of a male-killing Wolbachia in Drosophila innubila by male-killing dependent and male-killing independent mechanisms. Evolution. 2012;66(3):678–89. Epub 2012/03/03. doi: 10.1111/j.1558-5646.2011.01485.x 22380432.

8. Hurst GD, Johnson AP, Schulenburg JH, Fuyama Y. Male-killing Wolbachia in Drosophila: a temperature-sensitive trait with a threshold bacterial density. Genetics. 2000;156(2):699–709. Epub 2000/10/03. 11014817; PubMed Central PMCID: PMC1461301.

9. Jaenike J, Dyer KA, Reed LK. Within-population structure of competition and the dynamics of male-killing Wolbachia. Evolutionary Ecology Research. 2003;5(7):1023–36.

10. Hurst GD, Graf von der Schulenburg JH, Majerus TM, Bertrand D, Zakharov IA, Baungaard J, et al. Invasion of one insect species, Adalia bipunctata, by two different male-killing bacteria. Insect Mol Biol. 1999;8(1):133–9. Epub 1999/02/02. 9927182.

11. Jiggins FM, Hurst GD, Majerus ME. Sex-ratio-distorting Wolbachia causes sex-role reversal in its butterfly host. Proceedings Biological sciences. 2000;267(1438):69–73. Epub 2000/02/12. doi: 10.1098/rspb.2000.0968 10670955; PubMed Central PMCID: PMC1690502.

12. Zeh DW, Zeh JA, Bonilla MM. Wolbachia, sex ratio bias and apparent male killing in the harlequin beetle riding pseudoscorpion. Heredity. 2005;95(1):41–9. Epub 2005/06/03. doi: 10.1038/sj.hdy.6800666 15931253.

13. Cheng B, Kuppanda N, Aldrich JC, Akbari OS, Ferree PM. Male-Killing Spiroplasma Alters Behavior of the Dosage Compensation Complex during Drosophila melanogaster Embryogenesis. Curr Biol. 2016;26(10):1339–45. Epub 2016/05/11. doi: 10.1016/j.cub.2016.03.050 27161498; PubMed Central PMCID: PMC4879104.

14. Skinner SW. Son-killer: a third extrachromosomal factor affecting the sex ratio in the parasitoid wasp, Nasonia (= Mormoniella) vitripennis. Genetics. 1985;109(4):745–59. Epub 1985/04/01. 3988039; PubMed Central PMCID: PMC1202505.

15. Brucker RM, Bordenstein SR. Speciation by symbiosis. Trends Ecol Evol. 2012;27(8):443–51. Epub 2012/05/01. doi: 10.1016/j.tree.2012.03.011 22541872.

16. Hornett EA, Charlat S, Duplouy AM, Davies N, Roderick GK, Wedell N, et al. Evolution of male-killer suppression in a natural population. PLoS Biol. 2006;4(9):e283. Epub 2006/08/29. doi: 10.1371/journal.pbio.0040283 16933972; PubMed Central PMCID: PMC1551922.

17. Bordenstein SR, O'Hara FP, Werren JH. Wolbachia-induced incompatibility precedes other hybrid incompatibilities in Nasonia. Nature. 2001;409(6821):707–10. Epub 2001/02/24. doi: 10.1038/35055543 11217858.

18. Jaenike J, Dyer KA, Cornish C, Minhas MS. Asymmetrical reinforcement and Wolbachia infection in Drosophila. PLoS Biol. 2006;4(10):e325. Epub 2006/10/13. doi: 10.1371/journal.pbio.0040325 17032063; PubMed Central PMCID: PMC1592313.

19. Engelstadter J, Hurst GD. The impact of male-killing bacteria on host evolutionary processes. Genetics. 2007;175(1):245–54. Epub 2006/12/08. doi: 10.1534/genetics.106.060921 17151259; PubMed Central PMCID: PMC1774985.

20. Telschow A, Engelstadter J, Yamamura N, Hammerstein P, Hurst GD. Asymmetric gene flow and constraints on adaptation caused by sex ratio distorters. Journal of evolutionary biology. 2006;19(3):869–78. Epub 2006/05/06. doi: 10.1111/j.1420-9101.2005.01049.x 16674583.

21. Hornett EA, Duplouy AM, Davies N, Roderick GK, Wedell N, Hurst GD, et al. You can't keep a good parasite down: evolution of a male-killer suppressor uncovers cytoplasmic incompatibility. Evolution. 2008;62(5):1258–63. Epub 2008/02/27. doi: 10.1111/j.1558-5646.2008.00353.x 18298644.

22. Jaenike J. Spontaneous emergence of a new Wolbachia phenotype. Evolution. 2007;61(9):2244–52. Epub 2007/09/05. doi: 10.1111/j.1558-5646.2007.00180.x 17767593.

23. Majerus TM, Majerus ME. Intergenomic arms races: detection of a nuclear rescue gene of male-killing in a ladybird. PLoS Pathog. 2010;6(7):e1000987. Epub 2010/07/16. doi: 10.1371/journal.ppat.1000987 20628578; PubMed Central PMCID: PMC2900309.

24. Hoffmann AA, Montgomery BL, Popovici J, Iturbe-Ormaetxe I, Johnson PH, Muzzi F, et al. Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission. Nature. 2011;476(7361):454–7. Epub 2011/08/26. doi: 10.1038/nature10356 21866160.

25. Dutra HL, Rocha MN, Dias FB, Mansur SB, Caragata EP, Moreira LA. Wolbachia Blocks Currently Circulating Zika Virus Isolates in Brazilian Aedes aegypti Mosquitoes. Cell host & microbe. 2016;19(6):771–4. Epub 2016/05/09. doi: 10.1016/j.chom.2016.04.021 27156023; PubMed Central PMCID: PMC4906366.

26. Berec L, Maxin D, Bernhauerova V. Male-killing bacteria as agents of insect pest control. Journal of Applied Ecology. 2016.

27. Magni GE. ‘Sex-Ratio’: a Non-Mendelian Character in Drosophila bifasciata. Nature. 1953;172:81. doi: 10.1038/172081a0 13072583

28. Harumoto T, Lemaitre B. Male-killing toxin in a bacterial symbiont of Drosophila. Nature. 2018;557(7704):252–5. Epub 2018/05/04. doi: 10.1038/s41586-018-0086-2 29720654; PubMed Central PMCID: PMC5969570.

29. LePage DP, Metcalf JA, Bordenstein SR, On J, Perlmutter JI, Shropshire JD, et al. Prophage WO genes recapitulate and enhance Wolbachia-induced cytoplasmic incompatibility. Nature. 2017;543(7644):243–7. Epub 2017/02/28. doi: 10.1038/nature21391 28241146; PubMed Central PMCID: PMC5358093.

30. Shropshire JD, On J, Layton EM, Zhou H, Bordenstein SR. One prophage WO gene rescues cytoplasmic incompatibility in Drosophila melanogaster. Proceedings of the National Academy of Sciences of the United States of America. 2018;115(19):4987–91. Epub 2018/04/25. doi: 10.1073/pnas.1800650115 29686091; PubMed Central PMCID: PMC5948995.

31. Beckmann JF, Ronau JA, Hochstrasser M. A Wolbachia deubiquitylating enzyme induces cytoplasmic incompatibility. Nature microbiology. 2017;2:17007. Epub 2017/03/02. doi: 10.1038/nmicrobiol.2017.7 28248294; PubMed Central PMCID: PMC5336136.

32. Bordenstein SR, Bordenstein SR. Eukaryotic association module in phage WO genomes from Wolbachia. Nature communications. 2016;7:13155. Epub 2016/10/12. doi: 10.1038/ncomms13155 27727237; PubMed Central PMCID: PMC5062602.

33. Sasaki T, Kubo T, Ishikawa H. Interspecific transfer of Wolbachia between two lepidopteran insects expressing cytoplasmic incompatibility: a Wolbachia variant naturally infecting Cadra cautella causes male killing in Ephestia kuehniella. Genetics. 2002;162(3):1313–9. Epub 2002/11/28. 12454075; PubMed Central PMCID: PMC1462327.

34. Richardson KM, Schiffer M, Griffin PC, Lee SF, Hoffmann AA. Tropical Drosophila pandora carry Wolbachia infections causing cytoplasmic incompatibility or male killing. Evolution. 2016;70(8):1791–802. Epub 2016/06/11. doi: 10.1111/evo.12981 27282489; PubMed Central PMCID: PMC4980230.

35. Metcalf JA, Jo M, Bordenstein SR, Jaenike J, Bordenstein SR. Recent genome reduction of Wolbachia in Drosophila recens targets phage WO and narrows candidates for reproductive parasitism. PeerJ. 2014;2:e529. Epub 2014/08/29. doi: 10.7717/peerj.529 25165636; PubMed Central PMCID: PMC4137656.

36. Riparbelli MG, Giordano R, Ueyama M, Callaini G. Wolbachia-mediated male killing is associated with defective chromatin remodeling. PLoS One. 2012;7(1):e30045. Epub 2012/02/01. doi: 10.1371/journal.pone.0030045 22291901; PubMed Central PMCID: PMC3264553.

37. Mitsuhashi W, Ikeda H, Muraji M. Fifty-year trend towards suppression of Wolbachia-induced male-killing by its butterfly host, Hypolimnas bolina. Journal of insect science (Online). 2011;11:92. Epub 2011/08/30. doi: 10.1673/031.011.9201 21870980; PubMed Central PMCID: PMC3281488.

38. Charlat S, Hornett EA, Fullard JH, Davies N, Roderick GK, Wedell N, et al. Extraordinary flux in sex ratio. Science (New York, NY). 2007;317(5835):214. Epub 2007/07/14. doi: 10.1126/science.1143369 17626876.

39. Sheeley SL, McAllister BF. Mobile male-killer: similar Wolbachia strains kill males of divergent Drosophila hosts. Heredity. 2009;102(3):286–92. Epub 2009/01/15. doi: 10.1038/hdy.2008.126 19142204.

40. Bordenstein SR, Wernegreen JJ. Bacteriophage flux in endosymbionts (Wolbachia): infection frequency, lateral transfer, and recombination rates. Molecular biology and evolution. 2004;21(10):1981–91. Epub 2004/07/16. doi: 10.1093/molbev/msh211 15254259.

41. Lindsey ARI, Rice DW, Bordenstein SR, Brooks AW, Bordenstein SR, Newton ILG. Evolutionary Genetics of Cytoplasmic Incompatibility Genes cifA and cifB in Prophage WO of Wolbachia. Genome biology and evolution. 2018;10(2):434–51. Epub 2018/01/20. doi: 10.1093/gbe/evy012 29351633; PubMed Central PMCID: PMC5793819.

42. Sampson BJ, Stafne ET, Marshall-Shaw DA, Stringer SJ, Mallette T, Werle CT, et al., editors. Environmental ethanol as a reproductive constraint on spotted wing Drosophila and implications for control in Rubus and other fruits2016: International Society for Horticultural Science (ISHS), Leuven, Belgium.

43. Curtsinger JW. Components of selection in X chromosome lines of Drosophila melanogaster: sex ratio modification by meiotic drive and viability selection. Genetics. 1984;108(4):941–52. Epub 1984/12/01. 6439600; PubMed Central PMCID: PMC1224275.

44. Montenegro H, Souza WN, da Silva Leite D, Klaczko LB. Male-killing selfish cytoplasmic element causes sex-ratio distortion in Drosophila melanogaster. Heredity. 2000;85 Pt 5:465–70. Epub 2000/12/21. doi: 10.1046/j.1365-2540.2000.00785.x 11122425.

45. Dyer KA, Jaenike J. Evolutionarily stable infection by a male-killing endosymbiont in Drosophila innubila: molecular evidence from the host and parasite genomes. Genetics. 2004;168(3):1443–55. Epub 2004/12/08. doi: 10.1534/genetics.104.027854 15579697; PubMed Central PMCID: PMC1448788.

46. Dyer KA, Jaenike J. Evolutionary dynamics of a spatially structured host-parasite association: Drosophila innubila and male-killing Wolbachia. Evolution; international journal of organic evolution. 2005;59(7):1518–28. Epub 2005/09/13. 16153037.

47. Petrella LN, Smith-Leiker T, Cooley L. The Ovhts polyprotein is cleaved to produce fusome and ring canal proteins required for Drosophila oogenesis. Development (Cambridge, England). 2007;134(4):703–12. Epub 2007/01/12. doi: 10.1242/dev.02766 17215303.

48. Landmann F, Orsi GA, Loppin B, Sullivan W. Wolbachia-mediated cytoplasmic incompatibility is associated with impaired histone deposition in the male pronucleus. PLoS pathogens. 2009;5(3):e1000343. Epub 2009/03/21. doi: 10.1371/journal.ppat.1000343 19300496; PubMed Central PMCID: PMC2652114.

49. Harumoto T, Fukatsu T, Lemaitre B. Common and unique strategies of male killing evolved in two distinct Drosophila symbionts. Proceedings Biological sciences. 2018;285(1875). Epub 2018/03/23. doi: 10.1098/rspb.2017.2167 29563258; PubMed Central PMCID: PMC5897628.

50. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. The Phyre2 web portal for protein modeling, prediction and analysis. Nature protocols. 2015;10(6):845–58. Epub 2015/05/08. doi: 10.1038/nprot.2015.053 25950237; PubMed Central PMCID: PMC5298202.

51. Kim M, Kim HJ, Son SH, Yoon HJ, Lim Y, Lee JW, et al. Noncanonical DNA-binding mode of repressor and its disassembly by antirepressor. Proceedings of the National Academy of Sciences of the United States of America. 2016;113(18):E2480–8. Epub 2016/04/22. doi: 10.1073/pnas.1602618113 27099293; PubMed Central PMCID: PMC4983836.

52. Luscombe NM, Austin SE, Berman HM, Thornton JM. An overview of the structures of protein-DNA complexes. Genome biology. 2000;1(1):Reviews001. Epub 2000/12/06. doi: 10.1186/gb-2000-1-1-reviews001 11104519; PubMed Central PMCID: PMC138832.

53. Harumoto T, Anbutsu H, Lemaitre B, Fukatsu T. Male-killing symbiont damages host's dosage-compensated sex chromosome to induce embryonic apoptosis. Nature communications. 2016;7:12781. Epub 2016/09/22. doi: 10.1038/ncomms12781 27650264; PubMed Central PMCID: PMC5036004.

54. Veneti Z, Bentley JK, Koana T, Braig HR, Hurst GD. A functional dosage compensation complex required for male killing in Drosophila. Science (New York, NY). 2005;307(5714):1461–3. Epub 2005/03/05. doi: 10.1126/science.1107182 15746426.

55. Hurst GD, Jiggins FM. Male-killing bacteria in insects: mechanisms, incidence, and implications. Emerg Infect Dis. 2000;6(4):329–36. Epub 2000/07/25. doi: 10.3201/eid0604.000402 10905965; PubMed Central PMCID: PMC2640894.

56. Pinto SB, Stainton K, Harris S, Kambris Z, Sutton ER, Bonsall MB, et al. Transcriptional regulation of Culex pipiens mosquitoes by Wolbachia influences cytoplasmic incompatibility. PLoS pathogens. 2013;9(10):e1003647. doi: 10.1371/journal.ppat.1003647 24204251

57. Zhang G, Hussain M, O’Neill SL, Asgari S. Wolbachia uses a host microRNA to regulate transcripts of a methyltransferase, contributing to dengue virus inhibition in Aedes aegypti. Proceedings of the National Academy of Sciences. 2013;110(25):10276–81.

58. Bhattacharya T, Newton IL, Hardy RW. Wolbachia elevates host methyltransferase expression to block an RNA virus early during infection. PLoS pathogens. 2017;13(6):e1006427. doi: 10.1371/journal.ppat.1006427 28617844

59. Lux SA, Vilardi JC, Liedo P, Gaggl K, Calcagno GE, Munyiri FN, et al. Effects of Irradiation on the Courtship Behavior of Medfly (Diptera, Tephritidae) Mass Reared for the Sterile Insect Technique. The Florida Entomologist. 2002;85(1):102–12.

60. Barclay HJ. Modeling incomplete sterility in a sterile release program: interactions with other factors. Population Ecology. 2001;43(3):197–206. doi: 10.1007/s10144-001-8183-7

61. Telschow A, Hammerstein P, Werren JH. The effect of Wolbachia versus genetic incompatibilities on reinforcement and speciation. Evolution. 2005;59(8):1607–19. Epub 2005/12/07. 16329235.

62. Hurst GD, McVean GAT. Parasitic male-killing bacteria and the evolution of clutch size. Ecological entomology. 1998;23(3):350–3.

63. Baym M, Kryazhimskiy S, Lieberman TD, Chung H, Desai MM, Kishony R. Inexpensive multiplexed library preparation for megabase-sized genomes. PloS one. 2015;10(5):e0128036. Epub 2015/05/23. doi: 10.1371/journal.pone.0128036 26000737; PubMed Central PMCID: PMC4441430.

64. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Journal of computational biology: a journal of computational molecular cell biology. 2012;19(5):455–77. Epub 2012/04/18. doi: 10.1089/cmb.2012.0021 22506599; PubMed Central PMCID: PMC3342519.

65. Longdon B, Fabian DK, Hurst GD, Jiggins FM. Male-killing Wolbachia do not protect Drosophila bifasciata against viral infection. BMC microbiology. 2012;12 Suppl 1:S8. Epub 2012/03/02. doi: 10.1186/1471-2180-12-s1-s8 22376177; PubMed Central PMCID: PMC3287519.

66. Ellegaard KM, Klasson L, Naslund K, Bourtzis K, Andersson SG. Comparative genomics of Wolbachia and the bacterial species concept. PLoS genetics. 2013;9(4):e1003381. Epub 2013/04/18. doi: 10.1371/journal.pgen.1003381 23593012; PubMed Central PMCID: PMC3616963.

67. Boetzer M, Pirovano W. Toward almost closed genomes with GapFiller. Genome biology. 2012;13(6):R56. Epub 2012/06/27. doi: 10.1186/gb-2012-13-6-r56 22731987; PubMed Central PMCID: PMC3446322.

68. Sullivan W, Ashburner M, Hawley RS. Drosophila protocols. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2000. xiv + 697 pp. p.

69. Hall S, Ward REt. Septate Junction Proteins Play Essential Roles in Morphogenesis Throughout Embryonic Development in Drosophila. G3 (Bethesda, Md). 2016;6(8):2375–84. Epub 2016/06/05. doi: 10.1534/g3.116.031427 27261004; PubMed Central PMCID: PMC4978892.

70. Capra JA, Singh M. Predicting functionally important residues from sequence conservation. Bioinformatics (Oxford, England). 2007;23(15):1875–82. Epub 2007/05/24. doi: 10.1093/bioinformatics/btm270 17519246.

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