A cellular basis for Wolbachia recruitment to the host germline

Abstract

Wolbachia are among the most widespread intracellular bacteria, carried by thousands of metazoan species. The success of Wolbachia is due to efficient vertical transmission by the host maternal germline. Some Wolbachia strains concentrate at the posterior of host oocytes, which promotes Wolbachia incorporation into posterior germ cells during embryogenesis. The molecular basis for this localization strategy is unknown. Here we report that the wMel Wolbachia strain relies upon a two-step mechanism for its posterior localization in oogenesis. The microtubule motor protein kinesin-1 transports wMel toward the oocyte posterior, then pole plasm mediates wMel anchorage to the posterior cortex. Trans-infection tests demonstrate that factors intrinsic to Wolbachia are responsible for directing posterior Wolbachia localization in oogenesis. These findings indicate that Wolbachia can direct the cellular machinery of host oocytes to promote germline-based bacterial transmission. This study also suggests parallels between Wolbachia localization mechanisms and those used by other intracellular pathogens.

Figure 1. Localization of Wolbachia in Drosophila Oocytes

(A–L) Oocytes from D. melanogaster and D. simulans are shown, posterior end down. Phalloidin (cyan) indicates actin, while propidium iodide (yellow) labels Drosophila and Wolbachia DNA. (E'–L') Expanded views of the oocyte posterior show propidium iodide only. Arrows indicate posterior concentrations of wMel puncta. Panel rows, top to bottom: (A–D) stage 5, (E–H) stage 8, (E'–H') stage 8 posterior, (I–L) stage 10A, (I'–L') stage 10A posterior. Panel columns, left to right: (A, E, E', I, I') uninfected D. melanogaster, (B, F, F', J, J') wMel in D. melanogaster, (C, G, G', K, K') wRi in D. simulans, (D, H, H', L, L') wMel in D. simulans. (D) bar = 12.5 μm. (H, H', L, L') bars = 25 μm.

Figure 2. Effect of Microtubule, Kinesin-1, and osk Disruptions on wMel Posterior Localization

Stage 10A wMel-infected oocytes are shown with propidium iodide labeling. (A–I) Full-size images are accompanied by (A'–I') corresponding expanded views of the oocyte posterior pole. Conditions shown: (A, B) colchicine-DMSO-treated, (C) DMSO-treated, (D) Khc²⁷, (E) Khc²³, (F) Khc²⁷/+, (G) osk⁵⁴/oskDf^(3R)p-XT103, (H) osk⁵⁴/+, (I) oskDf^(3R)p-XT103/+. Arrows indicate enrichment of wMel at the (A) lateral and (A', C', E′, F', H', I') posterior cortex of the oocyte. Scale bars = 25 μm.

Figure 3. wMel in Oocytes That Exhibit Wild-Type and Ectopic Osk Localization

(A–F) Full-size oocytes and (A'–F') corresponding expanded views of the posterior cortex are shown. Rows: (A–C, A'–C') wMel in a wild-type oocyte, (D–F, D'–F') wMel in an osk⁵⁴/oskDf^(3R)p-XT103 oocyte carrying the osk-bicoid 3'UTR transgene. Columns: (A, A', D, D') propidium iodide stain, (B, B', E, E'), Osk antibody stain, (C, C', F, F') merged image showing propidium iodide (yellow) and Osk (cyan). Arrows indicate wMel and Osk co-localization. Scale bars = 25 μm.

Figure 4. Model for Strain-Specific Wolbachia Localization Strategies

wMel and wRi Wolbachia localize to the oocyte anterior from stages 3 to 6. Kinesin-1 transports Wolbachia away from the oocyte anterior during stages 7–9, carrying bacteria throughout the oocyte and toward the posterior pole. wRi remains evenly distributed into late oogenesis. In contrast, wMel Wolbachia near the posterior cortex interact with pole plasm to facilitate posterior wMel anchorage.