Background Wolbachia α-proteobacteria are essential for growth, reproduction and survival for many filarial nematode parasites of medical and veterinary importance. Endobacteria were discovered in filarial parasites by transmission electron microscopy in the 1970’s using chemically fixed specimens. Despite improvements of fixation and electron microscopy techniques during the last decades, methods to study the Wolbachia/filaria interaction on the ultrastructural level remained unchanged and the mechanisms for exchange of materials and for motility of endobacteria are not known. Methodology/Principal Finding We used high pressure freezing/freeze substitution to improve fixation of Brugia malayi and its endosymbiont, and this led to improved visualization of different morphological forms of Wolbachia. The three concentric, bilayer membranes that surround the endobacterial cytoplasm were well preserved. Vesicles with identical membrane structures were identified close to the endobacteria, and multiple bacteria were sometimes enclosed within a single outer membrane. Immunogold electron microscopy using a monoclonal antibody directed against Wolbachia surface protein-1 labeled the membranes that enclose Wolbachia and Wolbachia-associated vesicles. High densities of Wolbachia were observed in the lateral chords of L4 larvae, immature, and mature adult worms. Extracellular Wolbachia were sometimes present in the pseudocoelomic cavity near the developing female reproductive organs. Wolbachia-associated actin tails were not observed. Wolbachia motility may be explained by their residence within vacuoles, as they may co-opt the host cell’s secretory pathway to move within and between cells. Conclusions/Significance High pressure freezing/freeze substitution significantly improved the preservation of filarial tissues for electron microscopy to reveal membranes and sub cellular structures that could be crucial for exchange of materials between Wolbachia and its host.
References
[1]
Kozek WJ (1977) Transovarially-transmitted intracellular microorganisms in adult and larval stages of Brugia malayi. J Parasitol 63: 992–1000.
[2]
Kozek WJ, Marroquin HF (1977) Intracytoplasmic bacteria in Onchocerca volvulus. Am J Trop Med Hyg 26: 663–678.
[3]
McLaren DJ, Worms MJ, Laurence BR, Simpson MG (1975) Micro-organisms in filarial larvae (Nematoda). Trans R Soc Trop Med Hyg 69: 509–514.
[4]
Franz M, Buttner DW (1983) The fine structure of adult Onchocerca volvulus IV. The hypodermal chords of the female worm. Tropenmed Parasitol 34: 122–128.
[5]
Franz M, Copeman DB (1988) The fine structure of male and female Onchocerca gibsoni. Trop Med Parasitol 39 Suppl 4466–468.
[6]
Franz M, Schulz-Key H, Copeman DB (1987) Electron-microscopic observations on the female worms of six Onchocerca species from cattle and red deer. Parasitol Res 74: 73–83.
[7]
Kozek WJ, Raccurt C (1983) Ultrastructure of Mansonella ozzardi microfilaria, with a comparison of the South American (simuliid-transmitted) and the Caribbean (culicoid-transmitted) forms. Tropenmed Parasitol 34: 38–53.
[8]
Hoerauf A, Nissen-Pahle K, Schmetz C, Henkle-Duhrsen K, Blaxter ML, et al. (1999) Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility. J Clin Invest 103: 11–18.
[9]
Sironi M, Bandi C, Sacchi L, Di Sacco B, Damiani G, et al. (1995) Molecular evidence for a close relative of the arthropod endosymbiont Wolbachia in a filarial worm. Mol Biochem Parasitol 74: 223–227.
[10]
Taylor MJ, Bilo K, Cross HF, Archer JP, Underwood AP (1999) 16S rDNA phylogeny and ultrastructural characterization of Wolbachia intracellular bacteria of the filarial nematodes Brugia malayi, B. pahangi, and Wuchereria bancrofti. Exp Parasitol 91: 356–361.
[11]
Bandi C, Anderson TJ, Genchi C, Blaxter ML (1998) Phylogeny of Wolbachia in filarial nematodes. Proc Biol Sci 265: 2407–2413.
[12]
Fischer K, Beatty WL, Jiang D, Weil GJ, Fischer PU (2011) Tissue and stage-specific distribution of Wolbachia in Brugia malayi. PLoS Negl Trop Dis 5: e1174.
[13]
Frydman HM, Li JM, Robson DN, Wieschaus E (2006) Somatic stem cell niche tropism in Wolbachia. Nature 441: 509–512.
[14]
Hughes GL, Pike AD, Xue P, Rasgon JL (2012) Invasion of Wolbachia into Anopheles and other insect germlines in an ex vivo organ culture system. PLoS One 7: e36277.
[15]
Landmann F, Bain O, Martin C, Uni S, Taylor MJ, et al. (2012) Both asymmetric mitotic segregation and cell-to-cell invasion are required for stable germline transmission of Wolbachia in filarial nematodes. Biology Open 1: 536–547.
[16]
Fischer P, Schmetz C, Bandi C, Bonow I, Mand S, et al. (2002) Tunga penetrans: molecular identification of Wolbachia endobacteria and their recognition by antibodies against proteins of endobacteria from filarial parasites. Exp Parasitol 102: 201–211.
[17]
Melnikow E, Xu S, Liu J, Li L, Oksov Y, et al. (2011) Interaction of a Wolbachia WSP-like protein with a nuclear-encoded protein of Brugia malayi. Int J Parasitol 41: 1053–1061.
[18]
Voronin D, Cook DA, Steven A, Taylor MJ (2012) Autophagy regulates Wolbachia populations across diverse symbiotic associations. Proc Natl Acad Sci U S A 109: E1638–1646.
[19]
Ghedin E, Hailemariam T, DePasse JV, Zhang X, Oksov Y, et al. (2009) Brugia malayi gene expression in response to the targeting of the Wolbachia endosymbiont by tetracycline treatment. PLoS Negl Trop Dis 3: e525.
[20]
Hawes P, Netherton CL, Mueller M, Wileman T, Monaghan P (2007) Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells. J Microsc 226: 182–189.
[21]
Salvenmoser W, Egger B, Achatz JG, Ladurner P, Hess MW (2010) Electron microscopy of flatworms standard and cryo-preparation methods. Methods Cell Biol 96: 307–330.
[22]
Weimer RM (2006) Preservation of C. elegans tissue via high-pressure freezing and freeze-substitution for ultrastructural analysis and immunocytochemistry. Methods Mol Biol 351: 203–221.
Bazzocchi C, Mortarino M, Grandi G, Kramer LH, Genchi C, et al. (2008) Combined ivermectin and doxycycline treatment has microfilaricidal and adulticidal activity against Dirofilaria immitis in experimentally infected dogs. Int J Parasitol 38: 1401–1410.
[25]
Sacchi L, Corona S, Kramer L, Calvi L, Casiraghi M, et al. (2003) Ultrastructural evidence of the degenerative events occurring during embryogenesis of the filarial nematode Brugia pahangi after tetracycline treatment. Parassitologia 45: 89–96.
[26]
Melnikow E, Xu S, Liu J, Bell AJ, Ghedin E, et al. (2013) A Potential Role for the Interaction of Wolbachia Surface Proteins with the Brugia malayi Glycolytic Enzymes and Cytoskeleton in Maintenance of Endosymbiosis. PLoS Negl Trop Dis 7: e2151.
[27]
Beveridge TJ (1999) Structures of gram-negative cell walls and their derived membrane vesicles. J Bacteriol 181: 4725–4733.
[28]
Foster J, Ganatra M, Kamal I, Ware J, Makarova K, et al. (2005) The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode. PLoS Biol 3: e121.
[29]
Kawafune K, Sato M, Toyooka K, Nozaki H (2012) Ultrastructure of the rickettsial endosymbiont "MIDORIKO" in the green alga Carteria cerasiformis as revealed by high-pressure freezing and freeze-substitution fixation. Protoplasma 250: 949–953.
[30]
Serio AW, Jeng RL, Haglund CM, Reed SC, Welch MD (2010) Defining a core set of actin cytoskeletal proteins critical for actin-based motility of Rickettsia. Cell Host Microbe 7: 388–398.
[31]
Van Kirk LS, Hayes SF, Heinzen RA (2000) Ultrastructure of Rickettsia rickettsii actin tails and localization of cytoskeletal proteins. Infect Immun 68: 4706–4713.
[32]
Albertson R, Casper-Lindley C, Cao J, Tram U, Sullivan W (2009) Symmetric and asymmetric mitotic segregation patterns influence Wolbachia distribution in host somatic tissue. J Cell Sci 122: 4570–4583.
[33]
Kulp A, Kuehn MJ (2010) Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu Rev Microbiol 64: 163–184.
[34]
Kuehn MJ, Kesty NC (2005) Bacterial outer membrane vesicles and the host-pathogen interaction. Genes Dev 19: 2645–2655.
[35]
Amano A, Takeuchi H, Furuta N (2010) Outer membrane vesicles function as offensive weapons in host-parasite interactions. Microbes Infect 12: 791–798.
[36]
Frohlich K, Hua Z, Wang J, Shen L (2012) Isolation of Chlamydia trachomatis and membrane vesicles derived from host and bacteria. J Microbiol Methods 91: 222–230.
[37]
McMahon KJ, Castelli ME, Garcia Vescovi E, Feldman MF (2012) Biogenesis of outer membrane vesicles in Serratia marcescens is thermoregulated and can be induced by activation of the Rcs phosphorelay system. J Bacteriol 194: 3241–3249.
[38]
Canton J, Kima PE (2012) Interactions of pathogen-containing compartments with the secretory pathway. Cell Microbiol 14: 1676–1686.
[39]
Cho KO, Kim GW, Lee OK (2011) Wolbachia bacteria reside in host Golgi-related vesicles whose position is regulated by polarity proteins. PLoS One 6: e22703.
[40]
Heuer D, Rejman Lipinski A, Machuy N, Karlas A, Wehrens A, et al. (2009) Chlamydia causes fragmentation of the Golgi compartment to ensure reproduction. Nature 457: 731–735.
[41]
Voronin DA, Dudkina NV, Kiseleva EV (2004) A new form of symbiotic bacteria Wolbachia found in the endoplasmic reticulum of early embryos of Drosophila melanogaster. Dokl Biol Sci 396: 227–229.
[42]
Darby AC, Armstrong SD, Bah GS, Kaur G, Hughes MA, et al. (2012) Analysis of gene expression from the Wolbachia genome of a filarial nematode supports both metabolic and defensive roles within the symbiosis. Genome Res 22: 2467–2477.
