High Pressure Freezing/Freeze Substitution Fixation Improves the Ultrastructural Assessment of Wolbachia Endosymbiont – Filarial Nematode Host Interaction

BackgroundWolbachia 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 FindingWe 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/SignificanceHigh 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.背景：沃尔巴克氏体（Wolbachia，α-proteobacteria）对诸多具有医学与兽医学重要性的丝虫线虫的生长、繁殖及存活均不可或缺。1970年代，研究人员通过化学固定标本结合透射电子显微镜（transmission electron microscopy），在丝虫体内发现了这类内共生细菌。尽管近数十年来固定技术与电子显微镜技术均有进步，但在超微结构层面研究沃尔巴克氏体与丝虫互作的方法仍未更新，且内共生细菌的物质交换机制与运动模式至今尚不明确。 方法/主要发现：本研究采用高压冷冻/冷冻替代（high pressure freezing/freeze substitution）技术优化马来布鲁丝虫（Brugia malayi）及其内共生体的固定效果，显著提升了沃尔巴克氏体不同形态的可视化质量。环绕内共生菌细胞质的三层同心双层膜结构得以完好保存。研究人员在靠近内共生菌的区域发现了具有相同膜结构的囊泡，且有时可见多个细菌被包裹在单个外膜之内。使用针对沃尔巴克氏体表面蛋白1（Wolbachia surface protein-1）的单克隆抗体开展的免疫金电子显微镜（immunogold electron microscopy）实验，成功标记了包裹沃尔巴克氏体及沃尔巴克氏体相关囊泡的膜结构。在第四期幼虫（L4幼虫）、未成熟与成熟成虫的侧索中，均观察到高密度的沃尔巴克氏体。细胞外的沃尔巴克氏体有时会出现在发育中的雌性生殖器官附近的假体腔（pseudocoelomic cavity）内。本研究未观察到与沃尔巴克氏体相关的肌动蛋白尾（actin tails）。沃尔巴克氏体的运动可通过其在液泡内的驻留加以解释：这类细菌或许会利用宿主细胞的分泌通路，在细胞内部及细胞之间移动。 结论/意义：高压冷冻/冷冻替代技术显著改善了用于电子显微镜研究的丝虫组织保存效果，揭示了可能对沃尔巴克氏体与其宿主之间的物质交换至关重要的膜结构与亚细胞结构。