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Open Access

Peer-reviewed

Research Article

A Cellular Basis for Wolbachia Recruitment to the Host Germline

  • Laura R Serbus,

    Affiliation Molecular, Cell, and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America

    ⨯
  • William Sullivan

    To whom correspondence should be addressed. E-mail: sullivan@biology.ucsc.edu

    Affiliation Molecular, Cell, and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America

    ⨯

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 machintery 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.

Author Summary

This study focuses on Wolbachia, a genus of intracellular bacteria carried by insect and nematode host species. It was recently shown that Wolbachia carried into the human body by the host nematode Onchocerca volvulus trigger an immune response that leads to African river blindness. Findings like these raise fundamental questions of how Wolbachia interact with host cells to perpetuate Wolbachia infection. Distinct from many pathogenic bacteria, Wolbachia are transmitted throughout host populations primarily from females to their offspring, similar to mitochondrial inheritance. The molecular basis for this transmission strategy is unclear. Here we show that Wolbachia transmission is aided by a complex mechanism in egg development. Our study suggests that Wolbachia are transported inside the egg as cargo of molecular motors that walk along microtubule filaments. This directs Wolbachia to the posterior of maturing eggs, thus placing Wolbachia at the site where reproductive cells form during embryogenesis and ensuring Wolbachia integration into those cells. Furthermore, both factors intrinsic to Wolbachia and host molecules specifying reproductive cell fates are necessary to maximize posterior concentration of Wolbachia in the egg. This suggests that Wolbachia manipulate conserved cellular machinery in egg development to direct their transmission to the next host generation.

Citation: Serbus LR, Sullivan W (2007) A Cellular Basis for Wolbachia Recruitment to the Host Germline. PLoS Pathog 3(12): e190. https://doi.org/10.1371/journal.ppat.0030190

Editor: David S. Schneider, Stanford University, United States of America

Received: July 19, 2007; Accepted: October 26, 2007; Published: December 14, 2007

Copyright: © 2007 Serbus and Sullivan. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was supported by the NIH Ruth L. Kirschstein National Service Award (GM080192-01A1) and by the National Science Foundation (EF-0328363).

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: Khc , Kinesin heavy chain ; mRNP, messenger ribonucleoprotein particle; osk , oskar ; stau , staufen

Introduction

Wolbachia are among the most widespread intracellular bacteria, carried by an estimated 15%–76% of insect species as well as by some crustaceans, mites, and filarial nematodes [1,2]. Wolbachia are closely related to the Rickettsia family, a collection of tick-borne pathogens known for causing typhus and spotted fevers in humans. Wolbachia are also linked to human disease via a symbiotic relationship with pathogenic nematodes [3]. For example, the Wolbachia-bearing nematode Onchocerca volvulus is linked to the condition African river blindness in humans. Of the 18 million people infected by O. volvulus, nearly one million are visually impaired or already blind [4]. Recent work has implicated Wolbachia directly as the cause of ocular inflammation leading to river blindness [5].

The effect of Wolbachia infection on its host is as varied as the hosts are themselves. Wolbachia act as endosymbionts of some host organisms, such as the filarial nematode O. volvulus and the wasp Asobara tabida, which require Wolbachia in order to complete oogenesis properly [3,6]. Wolbachia appear to cause little phenotypic impact in certain hosts, such as in Drosophila melanogaster. In other cases, Wolbachia manipulate the host to their advantage. Wolbachia bias host reproduction to favor infected females by inducing phenotypes such as male-killing, feminization, sperm–egg cytoplasmic incompatibility, and parthenogenesis (virgin birth) [1,2]. This is thought to promote the spread of Wolbachia throughout host populations.

Infectious agents often spread to new hosts by becoming inhaled or ingested by that host. In the case of Wolbachia, however, bacterial transmission occurs within the host maternal germline [1,2]. Though Wolbachia are present in both male and female germlines, the bacteria are removed from sperm cysts at the end of spermatogenesis [7,8], creating a reliance upon maternal transmission. In arthropods, this maternal transmission is accomplished via incorporation of Wolbachia into germline precursor cells, also known as “pole cells” [9–11]. This ensures that infected females resulting from those embryos will carry bacteria in their germlines as well, thus perpetuating the Wolbachia transmission cycle. Wolbachia transmission rates have been reported at over 97% for wild-caught D. melanogaster flies, and at 100% for laboratory-reared D. melanogaster and D. simulans flies [12,13], suggesting that the pole cell–based transmission strategy is highly efficient.

How might Wolbachia ensure their incorporation into host pole cells? Many Wolbachia strains have been reported to concentrate at the posterior of mature oocytes [1,9–11,14–17]. Interestingly, the oocyte posterior pole corresponds to the location where pole cell formation takes place later in embryogenesis. For this reason, the posterior concentration of Wolbachia during oogenesis is thought to promote Wolbachia incorporation into the embryonic germline [9–11]. The cellular and molecular basis underlying this posterior Wolbachia localization in oogenesis is unknown to date, however.

A recent study indicated that Wolbachia can associate with host cell microtubules in D. melanogaster oocytes [18]. These oocytes contain an extensive network of microtubules that serves as a scaffold for cargo transport by motor proteins [19]. Up to stage 6 of oogenesis, microtubule minus ends are generally concentrated at the oocyte posterior with plus ends toward the anterior [20–22]. At stage 7, microtubules reorient such that minus ends are concentrated at the antero-lateral cortex of the oocyte, and plus ends are biased toward the posterior [23–27]. Work from D. melanogaster demonstrated that the wMel Wolbachia strain exhibits a microtubule-dependent concentration at the oocyte anterior from oogenesis stages 3 to 6 [18]. This anterior wMel localization requires the minus end–directed motor cytoplasmic dynein and the associated motor regulatory complex dynactin. However, the plus end–directed motor kinesin-1 is not required for anterior wMel localization [18]. These results suggest that interactions between Wolbachia and specific microtubule motors can direct the subcellular distribution of Wolbachia in oogenesis. This raises the possibility that posterior Wolbachia localization in late-stage oocytes may also rely upon interactions between bacteria, microtubules, and microtubule motor proteins. This also highlights Wolbachia as a means of understanding bacterial manipulation of host microtubules, an interaction that is considerably less well-studied than bacterial exploitation of host actin, such as in engulfment of Salmonella or intracellular propulsion of Rickettsia, Listeria, and Shigella [28,29].

How else might Wolbachia take advantage of the host cell to promote their posterior localization? It is possible that Wolbachia manipulate oocyte patterning events to their advantage. In Drosophila, the body axes are established via asymmetrical localization of determinant mRNAs in the oocyte [30,31]. For example, the posterior/germline determinant oskar (osk) mRNA concentrates at the oocyte posterior pole. The current model is that from stages 8 to 10A of oogenesis, kinesin-1 transports osk mRNA and associated Staufen (Stau) protein along microtubules toward the posterior cortex, where osk is translated [23–27]. Osk then initates recruitment of numerous mRNAs, proteins, mitochondria, and ribosomes to the oocyte posterior [32]. This multicomponent posterior assembly is referred to as “pole plasm”, and it functions in embryogenesis to specify posterior pole cell fates. Pole plasm is needed for posterior wMel localization in embryos [9]. Perhaps Wolbachia require posteriorly enriched substrates such as osk-induced pole plasm to establish their posterior localization in oogenesis as well.

This study addresses how Wolbachia posterior localization is achieved by examining the roles of microtubules, motor proteins, pole plasm assembly, and Wolbachia. Our findings indicate that during mid- to late oogenesis, kinesin-1 transports wMel Wolbachia toward the posterior cortex where pole plasm components mediate posterior wMel anchorage. The functions of kinesin-1 and pole plasm contribute independently to posterior Wolbachia localization. Furthermore, wMel can direct its localization to the oocyte posterior pole, unlike the homogeneously distributed wRi Wolbachia strain carried by D. simulans. This distinction between posteriorly concentrating and evenly dispersed Wolbachia strains may be due to different abilities of those strains to interact with posterior pole plasm.

Results

Wolbachia Concentrate at the Oocyte Posterior Pole in Mid- to Late Oogenesis

To understand the basis for wMel incorporation into embryonic pole cells, ovaries were stained with propidium iodide. This showed wMel to be anteriorly concentrated in stage 3–6 oocytes (Figure 1A and 1B) and homogeneously distributed in stage 7–9 oocytes (Figure 1E, 1E', 1F, and 1F') [18]. From late stage 9 to stage 12, a subset of wMel bacteria concentrated at the oocyte posterior cortex (Figure 1I, 1I', 1J, and 1J'; Table 1) [10]. wMel posterior localization persisted through early embryogenesis, facilitating wMel incorporation into the pole cells (Figure S1) [9–11]. Thus, concentration of wMel at the posterior of late stage oocytes promotes germline-based transmission of wMel.

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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.

https://doi.org/10.1371/journal.ppat.0030190.g001

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Table 1.

Wolbachia Localization in Late Stage 9 and Stage 10A Oocytes

https://doi.org/10.1371/journal.ppat.0030190.t001

Directed Transport by Kinesin-1 Is Important for Posterior Wolbachia Localization

The redistribution of wMel from the oocyte anterior to posterior suggests that an active localization mechanism is involved. To test a role for microtubule-based transport in posterior wMel localization, oocytes were treated with colcemid and colchicine. Some colcemid-treated oocytes exhibited wMel at both the lateral and posterior cortex (n = 7 of 15 cases; Figure 2A and 2A'), while others displayed a non-cortical, homogeneous distribution of wMel throughout the cytoplasm (n = 8 of 15 cases; Figure 2B and 2B'). Colchicine-treated oocytes displayed similar broad cortical or homogeneous wMel localization (n = 13 of 20 and n = 5 of 20 cases, respectively). This differed from control oocytes that mainly exhibited posterior wMel localization (19 of 22 cases; Figure 2C and 2C'). These data indicate that microtubules are required for focused posterior localization of wMel.

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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.

https://doi.org/10.1371/journal.ppat.0030190.g002

A role for microtubules in wMel localization implies that a posteriorly directed microtubule motor such as kinesin-1 is involved. To determine if kinesin-1 participates in wMel posterior localization, we created germlines mutant for the Kinesin heavy chain (Khc) gene [23,27,33,34]. Khc27 oocytes, null for kinesin function, showed normal anterior wMel localization during early stages (Figure S2). However, stage 10A Khc27 oocytes exhibited abnormal wMel distribution, with wMel absent from the posterior cortex in 83% of oocytes (Figure 2D, 2D', 2F, and 2F'; Table 1). wMel was also strikingly depleted from the posterior half of Khc27 oocytes (Figure 2D and 2F). Thus, kinesin-1 is important to both localize wMel to the posterior cortex and redistribute wMel into the posterior region.

The role for kinesin-1 in wMel posterior localization may reflect a direct or indirect Wolbachia localization mechanism. One possibility is that kinesin-1 transports wMel to the posterior as a cargo. However, kinesin-1 also drives bulk cytoplasmic streaming during mid- to late oogenesis [27,35,36]. Perhaps streaming currents sweep wMel passively toward the posterior cortex. To test a requirement for streaming in wMel localization, we examined oocytes carrying the hypomorphic mutations Khc17 and Khc23. These alleles give rise to streaming-capable and streaming-deficient oocytes, respectively [27]. Posterior Wolbachia were exhibited by 70% of Khc17 mutant oocytes and 62% of Khc23 mutant oocytes (Figure 2E; Table 1). The similarity of posterior wMel localization in these Khc mutants suggests streaming is not needed for posterior Wolbachia localization. Rather, as both Khc17 and Khc23 oocytes retain some kinesin-1 function [27,37], these results indicate that wMel is transported toward the posterior as a cargo of kinesin-1.

Pole Plasm Mediates Posterior Concentration of Wolbachia

A dependency of wMel on kinesin-1 for its posterior localization in oogenesis suggests wMel may rely on the kinesin-1 cargoes osk mRNA and Stau as well. Perhaps wMel hitchhikes to the oocyte posterior as a passenger on osk/Stau messenger ribonucleoprotein particles (mRNPs). Alternatively, wMel may require osk-induced pole plasm for efficient anchorage to the oocyte posterior cortex. To test these possibilities, osk and stau were disrupted with maternal-effect mutations. The majority of these mutant oocytes exhibited depletion or absence of wMel from the posterior cortex compared to wild-type (Figure 2G–2I, 2G'–2I'; Table 1), indicating that osk and stau gene products are important for efficient posterior wMel localization. Furthermore, osk and stau mutant oocytes lacking posteriorly concentrated wMel still exhibited a homogeneous bacterial distribution throughout the cytoplasm, differing sharply from the anterior wMel concentrations seen in Khc27 oocytes (compare Figure 2D to 2G). This suggests that kinesin-1 can transport wMel into the posterior half of the oocyte independently of osk/Stau mRNPs. However, kinesin-1 is insufficient to drive robust wMel concentration at the posterior cortex in oocytes with disrupted pole plasm (Figure 2G and 2G'; Table 1). This suggests that pole plasm is important for posterior wMel anchorage.

Kinesin-1 and Pole Plasm Contribute Independently to Posterior Wolbachia Enrichment

To test whether pole plasm is sufficient to drive wMel localization, we examined wMel in oocytes with anteriorly localized pole plasm. To this end, an osk-bicoid 3'UTR transgene was used to target osk mRNA to the oocyte anterior margin [38]. This ectopically localized osk is translated and assembles functional pole plasm at the antero-lateral cortex [38]. wMel co-localized with wild-type Osk protein at the oocyte posterior cortex (Figure 3A–3C, 3A'–3C'). However, wMel did not concentrate at the anterior margin with ectopically localized Osk in osk-bicoid 3'UTR oocytes (Figure 3D–3F, 3D'–3F'), suggesting that pole plasm alone is insufficient to recruit wMel from the cytoplasm. This result, taken together with those above, suggests that individual functions of kinesin-1 and pole plasm are both needed for robust posterior wMel localization in late stages 9 and 10A. This is consistent with a two-step mechanism for wMel localization: kinesin-1-mediated transport of wMel toward the oocyte posterior, followed by pole plasm-mediated anchorage of wMel to the posterior cortex (Figure 4).

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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.

https://doi.org/10.1371/journal.ppat.0030190.g003

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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.

https://doi.org/10.1371/journal.ppat.0030190.g004

Factors Intrinsic to Wolbachia Are Needed for Posterior Wolbachia Localization

The extensive requirement of host components for posterior wMel concentration raises questions about whether wMel contributes to its localization. To investigate this, a trans-infection approach was employed using the host species, D. simulans, that normally carries the wRi Wolbachia strain [39]. In D. simulans oogenesis, wRi exhibited an anterior concentration during stages 3–6 and homogeneous distribution throughout the rest of oogenesis (Figure 1C, 1G, 1G', 1K, and 1K'; Table 1) [18]. Is this lack of posterior concentration due to differences between host oogenesis machinery or between the wRi and wMel strains? To address this, we examined D. simulans oocytes ectopically transformed with wMel [40]. wMel-infected D. simulans oocytes exhibited anterior Wolbachia concentration during early stages, homogeneous distribution in middle stages, and a striking posterior localization in late stages (Figure 1D, 1H, 1H' 1L, and 1L'; Table 1). This demonstrates that host components required for Wolbachia posterior localization are present in both D. melanogaster and D. simulans oocytes. Due to strain-specific differences, however, wMel engages those host components to enhance its posterior concentration in late oogenesis, whereas wRi does not.

Which oocyte components are engaged by wMel but not by wRi? Comparing wMel in osk mutant oocytes to wRi localization in D. simulans reveals a similar homogeneous distribution (Figures 1K and 2G). A speculative interpretation of this similarity is that wMel and wRi are similarly transported into the posterior half of the oocyte by kinesin-1. A further possibility is that wRi is unable to interact with host pole plasm, unlike wMel, which requires pole plasm for efficient posterior localization (Figure 2G and 2G'; Table 1). Perhaps unlike wRi, factors intrinsic to wMel drive interactions with posterior pole plasm that facilitate posterior Wolbachia anchorage (Figure 4).

Discussion

Wolbachia Localization Shares Some Common Features with Other Pathogens

The involvement of kinesin (this study) and dynein [18] in Wolbachia localization during oogenesis is reminiscent of microtubule-based transport employed by a number of human pathogens. Viruses such as herpes simplex virus type 1 rely on dynein and dynactin for their transport to a perinuclear position referred to as their “replication site” [41]. Kinesin transports the viruses back to the cell periphery, enabling their exit from the cell. Bacteria such as Salmonella are transported toward the host cell nucleus in a dynein/dynactin-dependent manner, which then facilitates bacterial replication [41]. Salmonella also actively recruits kinesin-1 to its surrounding membrane [42]. These observations suggest some parallels with wMel, which requires dynein and dynactin for anterior localization during early oogenesis [18] and kinesin-1 for posterior localization in late oogenesis. While the function of Wolbachia anterior localization is unclear, Wolbachia titer increases substantially at that location, suggesting that dynein-driven localization creates a replication site for Wolbachia within the oocyte [18]. Once replicated, kinesin-1-based transport enables Wolbachia to traverse the entire length of the growing oocyte, promoting Wolbachia incorporation into posterior pole cells. Wolbachia may therefore have sophisticated interactions with host motor proteins analogous to those used by other bacteria and viruses. The basis for a switch between dynein- and kinesin-1-dependent Wolbachia localization is currently unknown. In some systems the dynactin complex coordinates alternation of kinesin- and dynein-driven organelle motility [43]. Perhaps a regulatory agent like dynactin directs the changing Wolbachia localization pattern in oogenesis.

Posterior Wolbachia Anchorage May Be a Cooperative Process

Upon reaching the posterior pole, wMel becomes anchored in a pole plasm–mediated manner. How might this occur? The simplest interpretation is that wMel associates directly with pole plasm components. However, a minority of osk null oocytes exhibited weak posterior Wolbachia localization (Table 1), although pole plasm is absent in this mutant background [38]. This suggests that other factors in addition to pole plasm assist posterior Wolbachia anchorage. Perhaps wMel has a dual affinity for pole plasm and an as-yet-unidentified posterior anchor. In such a case, the combined presence of those substrates may be important for robust Wolbachia anchorage to the posterior cortex. Alternatively, pole plasm may indirectly promote Wolbachia localization by stabilizing Wolbachia anchorage sites. A recent report indicated that Osk regulates actin polymerization at the oocyte posterior cortex [44]. It may be that wMel has a high affinity for unknown factors that associate with the posterior actin cortex, creating an indirect dependency of wMel upon posterior Osk.

One apparent conflict with these selective anchorage hypotheses is the finding that some colcemid- and colchicine-treated oocytes exhibit Wolbachia in association with the lateral cortex of the oocyte (Figure 2A). One interpretation of this result is that Wolbachia may have a general affinity for cortical actin independent of pole plasm. In such a scenario, one would predict that kinesin must normally drive wMel away from the lateral cortex and restrict it to the oocyte posterior where wMel is permitted to bind actin. This type of model has previously been proposed in the context of osk mRNA localization to the posterior pole [24,27]. If this prediction is accurate for wMel also, then oocytes lacking kinesin function should exhibit wMel localization to the antero-lateral cortex. However, wMel did not concentrate on the cortex of Khc null oocytes (Figure 2D). This suggests wMel does not have a general affinity for the actin cortex analagous to osk mRNA. An alternative interpretation of cortical wMel localization in colchicine- and colcemid-treated oocytes is that the drug treatments permitted microtubule remnants to remain along the cortex of some oocytes [21]. Those microtubule remnants could serve as a substrate for short-range wMel transport by kinesin-1, giving rise to a cortical wMel localization pattern. This possibility is consistent with the other findings of this study that favor kinesin-based wMel transport to the oocyte posterior, followed by selective wMel anchorage at the posterior pole.

Wolbachia Localization Is Distinct from Other Factors in the Oocyte

The study presented here is one of the few to examine host–pathogen interactions in a developmental context. What emerges from this analysis is that the Wolbachia localization pattern is unique and does not follow specific morphogens or organelles during oogenesis. The Wolbachia localization pattern is distinct from mitochondria, which are concentrated on the posterior side of the oocyte nucleus during early stages, homogeneously distributed during mid-oogenesis, and posteriorly concentrated in stages 9 and 10 [45]. The anterior localization of Wolbachia precedes that of the determinant bicoid mRNA, which concentrates anteriorly from stages 6 to 14 of oogenesis [46]. Wolbachia posterior localization also appears later than osk mRNA, which concentrates posteriorly from stages 3 to 6, anteriorly in stage 8, and posteriorly again from stages 8 to 10 of oogenesis [47,48]. Furthermore, our study indicates that Wolbachia do not localize to the posterior cortex in association with osk/Stau mRNPs. Taken together, these observations suggest that the demands of replication and localization are unique to Wolbachia and may preclude these bacteria from hitchhiking on morphogens or organelles.

Posterior Localization as an Adaptive Strategy for Wolbachia

The posterior localization strategy described in our report is exhibited by Wolbachia strains carried within multiple Drosophila and Hymenopteran species [1,9–11,14–17]. This recurrent localization pattern may reflect bacterial adaptations to the host environmental conditions. D. simulans allows wRi to persist at a high titer during embryogenesis, which is sufficient to promote wRi incorporation into posterior pole cells [10]. This environment may provide little incentive for wRi to evolve or retain a posterior localization strategy. The wMel strain, by contrast, is maintained at lower concentrations in D. melanogaster embryos [10]. This may pressure wMel to evolve and/or retain mechanisms that drive its posterior localization in oogenesis, thus enhancing its incorporation into embryonic pole cells. Taking advantage of kinesin-1 and pole plasm assembly at the oocyte posterior, as demonstrated by this study, provides an excellent means by which Wolbachia can accomplish this goal.

Materials and Methods

Fly strains.

wMel Wolbachia were crossed into wild-type D. melanogaster flies carrying the markers and balancers w; Sp/Cyo, Sb/Tm6Hu. This infected stock was used to cross wMel into all the D. melanogaster mutants used for this study, ensuring that all carried wMel strains of a comparable genetic background.

Immunolabeling.

Ovaries were dissected and fixed using standard methods [23], then stained and imaged as previously [18]. Rabbit anti-Osk antibodies were used at 1:3000 [49]. Embryos were dechorionated with 50% bleach, fixed 20 min in a 1:1 mixture of 3.7% formaldehyde and heptane, and devitellinized by vigorous agitation in methanol. Embryos were stained with rabbit anti-Vasa at 1:2000 [50] and mouse anti-Hsp60 (Sigma) at 1:100 [18] in PBS/0.1% Triton, followed by 1:500 dilutions of Alexa-488- and Alexa-594-conjugated secondary antibodies (Molecular Probes).

Microtubule inhibitor treatment.

Flies were starved 18 h, then fed 24–48 h with yeast paste containing 50 μM colcemid, 50 μM colchicine in DMSO, or comparable dilutions of DMSO alone. Mispositioning of the oocyte nucleus served as an internal control to verify that microtubule disruption had occurred [21,51].

Microscopy and image analysis.

Images were acquired on a Leica DM IRB confocal microscope using a 63× oil objective and zoom factor of 1.5. Each oocyte was imaged as a z-series stack of 7–14 images spaced at 1.5-μm intervals. Optical sections deeper than 4.5 μm into the oocyte were examined for the presence of posterior Wolbachia. Oocytes were categorized in Table 1 as showing strong posterior localization if they exhibited striking Wolbachia staining, which consisted of either an intense linear array of Wolbachia puncta or a crescent-shaped area saturated with Wolbachia staining along the posterior cortex for four out of five consecutive z-sections. Oocytes were designated as showing weak posterior localization if they exhibited a.) at least one z-section with striking posterior localization, or b.) at least two z-sections with a higher Wolbachia density along the posterior cortex than in the cytoplasm of the cell. Oocytes were categorized as showing no posterior localization if they did not meet the above conditions. Wolbachia density was not analyzed in this study because oocytes carrying high bacterial loads exhibited saturation of Wolbachia labeling at the posterior pole that disrupted bacterial quantitation.

Supporting Information

Figure S1. wMel and Pole Plasm Localization in Early Embryos

Embryos are shown (A–C) prior to meiosis, (D–F) in cycle 11, (G–I) in cycle 14, and (J–L) during gastrulation. Posterior is facing down. Panel columns, left to right: (A, D, G, J) anti-Hsp60 staining indicating wMel [18,52], (B, E, H, K) anti-Vasa labeling pole plasm and pole cells [50], and (C, F, I, L) merged images showing anti-Hsp60 (yellow) and anti-Vasa (cyan). (A–C) At the beginning of embryogenesis, wMel is enriched at the posterior relative to the rest of the cortex (B, C). (D–F) wMel is incorporated into pole cells and (G–I) persists in pole cells as they multiply. (J–L) wMel is strongly concentrated in pole cells as they migrate into the embryo. Scale bar = 50 μm.

https://doi.org/10.1371/journal.ppat.0030190.sg001

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Figure S2. Khc27 Oocyte Infected with wMel

Propidium iodide labeling of stage 5–6 oocytes shows anterior wMel localization. Scale bar = 25 μm.

https://doi.org/10.1371/journal.ppat.0030190.sg002

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Accession Numbers

The NCBI Entrez (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene) accession numbers for the genes and gene products discussed in this paper are Hsp60 (P10809), Kinesin heavy chain (P17210), Oskar (P25158), Staufen (P25159), and Vasa (P09052).

Acknowledgments

We thank Kostas Bourtzis, Byeong-Jik Cha, Anne Ephrussi, Paul Lasko, Herve Mercot, and Bill Saxton for reagents. Thanks also to Elise and Patrick Ferree, Jian Cao, Catharina Lindley, Anne Royou, Bill Saxton, and Susan Strome for their assistance.

Author Contributions

LRS and WS conceived and designed the experiments. LRS performed the experiments. LRS and WS analyzed the data. LRS and WS contributed reagents, materials, and analysis tools. LRS wrote the paper. LRS and WS edited the paper.

References

  1. 1. Tram U, Ferree PM, Sullivan W (2003) Identification of Wolbachia--host interacting factors through cytological analysis. Microbes Infect 5: 999–1011.
  2. 2. Stouthamer R, Breeuwer JA, Hurst GD (1999) Wolbachia pipientis: microbial manipulator of arthropod reproduction. Annu Rev Microbiol 53: 71–102.
  3. 3. Hise AG, Gillette-Ferguson I, Pearlman E (2004) The role of endosymbiotic Wolbachia bacteria in filarial disease. Cell Microbiol 6: 97–104.
  4. 4. 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.
  5. 5. Saint Andre A, Blackwell NM, Hall LR, Hoerauf A, Brattig NW, et al. (2002) The role of endosymbiotic Wolbachia bacteria in the pathogenesis of river blindness. Science 295: 1892–1895.
  6. 6. Pannebakker BA, Loppin B, Elemans CP, Humblot L, Vavre F (2007) Parasitic inhibition of cell death facilitates symbiosis. Proc Natl Acad Sci U S A 104: 213–215.
  7. 7. Bressac C, Rousset F (1993) The reproductive incompatibility system in Drosophila simulans: DAPI-staining analysis of the Wolbachia symbionts in sperm cysts. J Invertebr Pathol 61: 226–230.
  8. 8. Clark ME, Veneti Z, Bourtzis K, Karr TL (2002) The distribution and proliferation of the intracellular bacteria Wolbachia during spermatogenesis in Drosophila. Mech Dev 111: 3–15.
  9. 9. Hadfield SJ, Axton JM (1999) Germ cells colonized by endosymbiotic bacteria. Nature 402: 482.
  10. 10. Veneti Z, Clark ME, Karr TL, Savakis C, Bourtzis K (2004) Heads or tails: host-parasite interactions in the Drosophila-Wolbachia system. Appl Environ Microbiol 70: 5366–5372.
  11. 11. Kose H, Karr TL (1995) Organization of Wolbachia pipientis in the Drosophila fertilized egg and embryo revealed by an anti-Wolbachia monoclonal antibody. Mech Dev 51: 275–288.
  12. 12. McGraw EA, O'Neill SL (2004) Wolbachia pipientis: intracellular infection and pathogenesis in Drosophila. Curr Opin Microbiol 7: 67–70.
  13. 13. Hoffmann AA, Hercus M, Dagher H (1998) Population dynamics of the Wolbachia infection causing cytoplasmic incompatibility in Drosophila melanogaster. Genetics 148: 221–231.
  14. 14. Dedeine F, Vavre F, Fleury F, Loppin B, Hochberg ME, et al. (2001) Removing symbiotic Wolbachia bacteria specifically inhibits oogenesis in a parasitic wasp. Proc Natl Acad Sci U S A 98: 6247–6252.
  15. 15. Stouthamer R, Breeuwert JA, Luck RF, Werren JH (1993) Molecular identification of microorganisms associated with parthenogenesis. Nature 361: 66–68.
  16. 16. Breeuwer JA, Werren JH (1990) Microorganisms associated with chromosome destruction and reproductive isolation between two insect species. Nature 346: 558–560.
  17. 17. Zchori-Fein E, Roush RT, Rosen D (1998) Distribution of parthenogenesis-inducing symbionts in ovaries and eggs of Aphytis (Hymentoptera: Aphelinidae). Curr Microbiol 36: 1–8.
  18. 18. Ferree PM, Frydman HM, Li JM, Cao J, Wieschaus E, et al. (2005) Wolbachia utilizes host microtubules and Dynein for anterior localization in the Drosophila oocyte. PLoS Pathog 1: e14.
  19. 19. Steinhauer J, Kalderon D (2006) Microtubule polarity and axis formation in the Drosophila oocyte. Dev Dyn 235: 1455–1468.
  20. 20. Megraw TL, Kaufman TC (2000) The centrosome in Drosophila oocyte development. Curr Top Dev Biol 49: 385–407.
  21. 21. Theurkauf WE, Smiley S, Wong ML, Alberts BM (1992) Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport. Development 115: 923–936.
  22. 22. Mahowald AP, Strassheim JM (1970) Intercellular migration of centrioles in the germarium of Drosophila melanogaster. An electron microscopic study. J Cell Biol 45: 306–320.
  23. 23. Brendza RP, Serbus LR, Duffy JB, Saxton WM (2000) A function for kinesin I in the posterior transport of oskar mRNA and Staufen protein. Science 289: 2120–2122.
  24. 24. Cha BJ, Serbus LR, Koppetsch BS, Theurkauf WE (2002) Kinesin I-dependent cortical exclusion restricts pole plasm to the oocyte posterior. Nat Cell Biol 4: 592–598.
  25. 25. Clark I, Giniger E, Ruohola-Baker H, Jan LY, Jan YN (1994) Transient posterior localization of a kinesin fusion protein reflects anteroposterior polarity of the Drosophila oocyte. Curr Biol 4: 289–300.
  26. 26. Pokrywka NJ, Stephenson EC (1995) Microtubules are a general component of mRNA localization systems in Drosophila oocytes. Dev Biol 167: 363–370.
  27. 27. Serbus LR, Cha BJ, Theurkauf WE, Saxton WM (2005) Dynein and the actin cytoskeleton control kinesin-driven cytoplasmic streaming in Drosophila oocytes. Development 132: 3743–3752.
  28. 28. Ly KT, Casanova JE (2007) Mechanisms of Salmonella entry into host cells. Cell Microbiol 9: 2103–2111.
  29. 29. Suzuki T, Sasakawa C (2001) Molecular basis of the intracellular spreading of Shigella. Infect Immun 69: 5959–5966.
  30. 30. Riechmann V, Ephrussi A (2001) Axis formation during Drosophila oogenesis. Curr Opin Genet Dev 11: 374–383.
  31. 31. van Eeden F, St Johnston D (1999) The polarisation of the anterior-posterior and dorsal-ventral axes during Drosophila oogenesis. Curr Opin Genet Dev 9: 396–404.
  32. 32. Mahowald AP (2001) Assembly of the Drosophila germ plasm. Int Rev Cytol 203: 187–213.
  33. 33. Chou TB, Noll E, Perrimon N (1993) Autosomal P[ovoD1] dominant female-sterile insertions in Drosophila and their use in generating germ-line chimeras. Development 119: 1359–1369.
  34. 34. Golic KG, Lindquist S (1989) The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59: 499–509.
  35. 35. Palacios IM, St Johnston D (2002) Kinesin light chain-independent function of the Kinesin heavy chain in cytoplasmic streaming and posterior localisation in the Drosophila oocyte. Development 129: 5473–5485.
  36. 36. Theurkauf WE (1994) Premature microtubule-dependent cytoplasmic streaming in cappuccino and spire mutant oocytes. Science 265: 2093–2096.
  37. 37. Brendza KM, Rose DJ, Gilbert SP, Saxton WM (1999) Lethal kinesin mutations reveal amino acids important for ATPase activation and structural coupling. J Biol Chem 274: 31506–31514.
  38. 38. Ephrussi A, Lehmann R (1992) Induction of germ cell formation by oskar. Nature 358: 387–392.
  39. 39. Iturbe-Ormaetxe I, Riegler M, O'Neill SL (2005) New names for old strains? Wolbachia wSim is actually wRi. Genome Biol 6: 401. author reply 401.
  40. 40. Poinsot D, Bourtzis K, Markakis G, Savakis C, Mercot H (1998) Wolbachia transfer from Drosophila melanogaster into D. simulans: host effect and cytoplasmic incompatibility relationships. Genetics 150: 227–237.
  41. 41. Henry T, Gorvel JP, Meresse S (2006) Molecular motors hijacking by intracellular pathogens. Cell Microbiol 8: 23–32.
  42. 42. Henry T, Couillault C, Rockenfeller P, Boucrot E, Dumont A, et al. (2006) The Salmonella effector protein PipB2 is a linker for kinesin-1. Proc Natl Acad Sci U S A 103: 13497–13502.
  43. 43. Welte MA (2004) Bidirectional transport along microtubules. Curr Biol 14: R525–R537.
  44. 44. Vanzo N, Oprins A, Xanthakis D, Ephrussi A, Rabouille C (2007) Stimulation of endocytosis and actin dynamics by Oskar polarizes the Drosophila oocyte. Dev Cell 12: 543–555.
  45. 45. Cox RT, Spradling AC (2003) A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis. Development 130: 1579–1590.
  46. 46. St Johnston D, Driever W, Berleth T, Richstein S, Nusslein-Volhard C (1989) Multiple steps in the localization of bicoid RNA to the anterior pole of the Drosophila oocyte. Development 107(Suppl): 13–19.
  47. 47. Ephrussi A, Dickinson LK, Lehmann R (1991) Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell 66: 37–50.
  48. 48. Clegg NJ, Frost DM, Larkin MK, Subrahmanyan L, Bryant Z, et al. (1997) maelstrom is required for an early step in the establishment of Drosophila oocyte polarity: posterior localization of grk mRNA. Development 124: 4661–4671.
  49. 49. Markussen FH, Michon AM, Breitwieser W, Ephrussi A (1995) Translational control of oskar generates short OSK, the isoform that induces pole plasma assembly. Development 121: 3723–3732.
  50. 50. Lasko PF, Ashburner M (1990) Posterior localization of vasa protein correlates with, but is not sufficient for, pole cell development. Genes Dev 4: 905–921.
  51. 51. Koch EA, Spitzer RH (1983) Multiple effects of colchicine on oogenesis in Drosophila: induced sterility and switch of potential oocyte to nurse-cell developmental pathway. Cell Tissue Res 228: 21–32.
  52. 52. Taylor MJ, Hoerauf A (1999) Wolbachia bacteria of filarial nematodes. Parasitol Today 15: 437–442.