Abstract
Wolbachia pipientis is an incredibly widespread bacterial symbiont of insects, present in an estimated 25-52% of species worldwide. Wolbachia is faithfully maternally transmitted both in a laboratory setting and in the wild. In an established infection, Wolbachia is primarily intracellular, residing within host-derived vacuoles that are associated with the endoplasmic reticulum. However, Wolbachia also frequently transfer between host species, requiring an extracellular stage to their life cycle. Indeed, Wolbachia has been moved between insect species for the precise goal of controlling populations. The use of Wolbachia in this application requires we better understand how it initiates and establishes new infections. Here we designed a novel method for live-tracking Wolbachia during infection using a combination of stains and microscopy. We show that live Wolbachia are taken up by host cells at a much faster rate than dead Wolbachia cells, indicating that Wolbachia play a role in their own uptake and that Wolbachia colonization is not just a passive process. We also show that the host actin cytoskeleton must be intact for this to occur, and that drugs that disrupt the actin cytoskeleton effectively abrogate Wolbachia uptake. The development of this live infection assay will assist in future efforts to characterize Wolbachia factors used during host infection.
Introduction
Wolbachia are alpha-proteobacteria, part of the anciently intracellular Anaplasmataceae, and related to the important human pathogens Anaplasma, Rickettsia and Ehrlichia (1). However, Wolbachia do not infect mammals, but instead are well known for their reproductive manipulations of insect populations, inducing phenotypes such as male-killing, feminization, or sperm-egg incompatibility (2–7). Wolbachia is an incredibly successful infection; the symbiont is both vertically and horizontally transmitted and manipulates host reproduction to persist in populations (2, 5, 8–10). In the last decade, Wolbachia have been shown to provide a benefit to insects, where the infection can inhibit RNA virus replication within the host, a phenomenon known as pathogen blocking (11). Because insects are vectors for disease, and Wolbachia alter the ability of these vectors to harbor important human pathogens, Wolbachia are being used to control the spread of diseases such as dengue (12, 13). For Wolbachia to establish itself in an insect population it must invade host cells, persist during infection, and be transmitted to the next generation. Therefore, a prerequisite for the use of Wolbachia in vector control in insects is the establishment of infection.
Although Wolbachia cannot be cultivated ex vivo, they can survive for weeks in cell culture media when removed from host cells (2). Additionally, Wolbachia can be found in host hemolymph, suggesting that during natural infection of whole organisms, they can be found outside of host cells (2). However, Wolbachia entry into the host cell is still relatively uncharacterized. What little we do know about the infection process comes from elegant trans-well assays where Wolbachia passively exiting host cells, then passing through a 10 mm barrier, were able to colonize uninfected Drosophila cells (14). The infection timeline calculated based on this approach between 3 hours and 6 days, a relatively long time course for most of the Anaplasmataceae (15). In that same work, the authors investigated the importance of clathrin-mediated endocytosis and showed that this host process alone cannot account for all Wolbachia entry. In this study, we therefore had two goals: to determine Wolbachia’s contribution to host uptake and to identify a more precise timeline for direct infection of host cells.
Using both infected and uninfected Drosophila melanogaster JW18 cells, we designed a protocol that allows for live visualization of Wolbachia establishment. We first asked if live Wolbachia are taken up by host cells more rapidly than Wolbachia that had been killed. We then established a timeline and quantified the relative numbers of live vs. killed Wolbachia entering host cells. Our results suggest that Wolbachia indeed do contribute to their own uptake, with live Wolbachia colonizing cells more quickly and at higher infection rates than their dead counterparts. We then treated uninfected JW18 cells with cytochalasin-D, which inhibits the polymerization of filamentous actin, and challenged them with live Wolbachia. We also show that pre-treatment of host cells with the actin inhibiting drug cytochalasin-D inhibits the uptake of live Wolbachia dramatically, identifying the host actin cytoskeleton as important for the internalization process. We therefore conclude that Wolbachia are not only passively taken up by Drosophila but also actively facilitate uptake.
Materials and Methods
Insect Cell Culture
Drosophila melanogaster JW18 cells were tetracycline treated to remove Wolbachia infection. Infection status was confirmed by PCR after four passages in the presence of antibiotic. Cells with and without Wolbachia were maintained in the dark, in T25 unvented cell culture flasks at room temperature. Schneider’s Insect Medium was used and supplemented with 10% heat-treated Fetal Bovine Serum and 1% α/α (penicillin/streptomycin) solution.
Wolbachia Isolation and Overlay for Rate of Uptake Screen
To visualize the rate of uptake of Wolbachia, ~2000 uninfected JW18 host cells were seeded into a CellVis 96 well glass bottom plate with fresh Schneider’s media and incubated in the dark at room temperature overnight to allow them to settle. The following day, a confluent T25 flask of JW18 Wolbachia+ cells were first counted, then transferred into a 15ml conical tube containing 500μl sterile glass beads and SYTOX Orange (ThermoFisher) dye (250nM). To release Wolbachia from JW18 host cells, the conical tubes containing glass beads were then vortexed for 4 pulses of 5 seconds each. Lysate was divided into 500μl aliquots, and each placed onto an Ultrafree MC-SV Durapore PVDF 5.0μm centrifugal filter column (Millipore). Columns were spun at 20,000g at 10°C for 10 minutes to purify Wolbachia from host cells (2). Resulting pellets were then transferred into a new 1.5ml centrifuge tube containing fresh Schneider’s media and resuspended. Extractions from the same T25 were divided such that half would be used immediately (live) and the other frozen at −20°C for at least 3 days (killed) to serve as a control for the infection. To prevent bleaching, 50μl of ProLong Live (ThermoFisher) solution was added to each tube (250μL volume total) prior to imaging then incubated away from light for 15min, as per manufacturer’s instructions. JW18 cells are not all infected at the same density (16, 17), and because live Wolbachia cannot be quantified by qPCR, we controlled for the multiplicity of infection by counting the number of infected JW18 host cells that we lysed before isolating Wolbachia, and by using the matched extracted Wolbachia sample as a dead control for each experiment. The Wolbachia were then added to each seeded well at an MOI of 20:1 host cell equivalents. That is, for each seeded JW18 cell, we overlayed the Wolbachia contents from 20 infected JW18 cells. The plate was then gently spun down for 5min at 500rpm to ensure that Wolbachia and uninfected JW18 host cells were proximal. For cytochalasin D treatments, the same infection protocol was followed but preceding overlay, host cells were treated with 15 μg/mL cytochalasin-D in Schneider’s media for 30 minutes.
Live/Dead Staining of Isolated Wolbachia
Wolbachia isolated from infected JW18 cells were stained with a 1:1 mixture of SYTO9 (ThermoFisher) and Propidium Iodide (ThermoFisher) either immediately following isolation (live) or after storage at −20°C for at least 3 days prior to imaging (dead). Stained cells were incubated away from light for 20min prior to imaging using the same magnification, intensities, and exposure times.
Microscopy and Analysis of Images
All images were obtained using a Nikon Ti2 Eclipse inverted microscope at 100x magnification with a Nikon DS-Qi2 camera attachment. Intensities and exposure times are as follows: green/FITC for 200ms with SOLA pad at 4%, red/Texas Red for 400ms with SOLA pad at 8%. All Z stacks were taken with a step size of 0.5μm.
Nikon NiE Elements software was used to first deconvolute all Z stacks (Automatic 3D Deconvolution) and a NiE Elements GA3 program was used to collect internalized Wolbachia data using the threshold parameters specified in Supplemental Table 1 for each image.
Counts and volume data from Nikon NiE Elements were tested for normalcy and overdispersion in R and a zero-inflated GLM with a Poisson distribution (zeroinfl in the PSCL library) was used to test for significant differences in the number and volume of total Wolbachia inside of host cells over time in each condition. A logistic regression (glm with family = binomial) was used to predict infection status of cells based on Wolbachia volume and number.
Results
We were able to successfully isolate live Wolbachia from JW18 cells and using a freeze method we were able to kill the population of bacteria for our controls. The efficacy of this method was visualized using live/dead staining (Fig. 1). No propidium iodide staining was observed for live, freshly-isolated Wolbachia but all cells subjected to freeze-treatment internalized the dye, suggesting the cell membranes were now permeable to propidium iodide staining. Each of the samples were used in live microscopy during infection of Drosophila melanogaster cells. Briefly, Wolbachia (either live or dead) were stained using SYTOX dye and overlayed onto Wolbachia- free JW18 cells. The process was visualized using ProLong Live reagent and fluorescent light microscopy and pictures were taken of live cells every two hours for 4 hours. Live Wolbachia could readily be observed entering host cells by 2 hours post infection, whereas we rarely observed internalized killed Wolbachia (Fig. 2). This result confirmed that we could visualize the infection process and that we could quantify Wolbachia’s contribution using a killed control.
We next proceeded to perform 3 paired experiments, using live and killed counterparts, and counted a total of 454 cells during three four-hour infection time courses (Table 1). Experiments were paired such that the same Wolbachia isolated in one experiment were freeze-killed and used as a control for that same experiment. No effect of experiment on the number and volume of Wolbachia found within host cells over time was observed (ANOVA; df = 2, F = 1.368, p = 0.256 and df =2, F = 0.311; p = 0.733). However, out of an abundance of caution, and to ensure we could directly compare MOIs, we analyzed the data in aggregate as well as in pairwise fashion, comparing each heat-killed control to each live sample. The two measures we took were of Wolbachia counts within cells (individual bacteria-shaped objects) and Wolbachia volumes (the calculated volumes, based on pixels, for each of those objects). Both measures were significantly correlated with each other (Fig. 3A; Pearson correlation R = 0.92; p < 2.2e-16). We therefore present data from the Wolbachia counts in the main text (please see Supplementary Figure 1 for volume-based data).
Live Wolbachia are internalized more quickly and at higher numbers than their dead counterparts
Over the course of the infection, we saw an increase in the total number and volume of Wolbachia internalized per cell (with median peaks at around 13 and3 in the live vs. dead Wolbachia overlays, respectively) (Table 1). In aggregate, across all the sampled cells, the number of total Wolbachia internalized over the time course of infection was greatest for the treatment with live Wolbachia (Fig. 3B; χ2= 5.6407, df = 1, p = 0.0175). Indeed, a logistic regression significantly predicts infection status based on both time (estimate = 0.507, stderr = 0.070, z = 7.256, p = 3.89e-13) and whether the assay involved live Wolbachia (estimate = 0.723, stderr = 0.211, z = 3.429, p = 0.0006). For two of the three experiments, the largest number and volume of internalized Wolbachia was observed in the live overlay after 2 hours (Fig. 3C; 23.1 and 12.4 Wolbachia, on average, observed within a host cell). The numbers stabilized at an average of 8.3 Wolbachia per host cell at 4 hrs (Table 1).
Because our data are necessarily zero-inflated (due to the large number of uninfected cells in the dead overlay and at early timepoints), we used a zero-inflated GLM to compare number of Wolbachia internalized across time for each condition. We observed a significant effect of both time (estimate = −0.079, stderr = 0.014, z = −5.707, p = 1.15e-08) and whether the bacteria were live or dead (estimate = −0.474, stderr = 0.192, z =−2.464, p = 0.0137) on number of Wolbachia internalized, corroborating our observations.
Cytochalasin treatment reduces Wolbachia uptake
Previously, Wolbachia has been shown to use the host cytoskeleton for localization within oocytes and for maternal transmission (5, 10). Therefore, we sought to confirm the fraction of uptake of Wolbachia we observed that was based on the host cell cytoskeleton. We treated uninfected JW18-tet cells with cytochalasin prior to infection with Wolbachia and, as expected, observed a dramatic decrease in internalization (Fig. 4A). The median counts observed were generally 1 or zero for each time point (mean of Wolbachia within host cells per time; 0hrs μ = 2.33; 2hrs μ = 0.167; 4 hrs μ = 1.62). Indeed, when compared to overlays using dead Wolbachia, the cytochalasin D treated samples are statistically indistinguishable (estimate = − 2.055, stderr = 1712.3, z =−0.001, p = 0.950) (Fig. 4B).
Discussion
All intracellular bacteria must manipulate the host to enter, persist, replicate, and be transmitted. For those that move between hosts, they must be able to survive extracellularly and reinfect host cells. Orientia tsutsugamushi, which causes scrub typhus, utilizes clathrin-mediated endocytosis to enter a host cell, escapes the endosome and utilizes dynein and microtubules to transport itself to the nucleus for replication, then exits the host cell enveloped in host membrane to continue its life cycle (18). For the endosymbiont Wolbachia, little is known about its ex vivo lifestyle, although more than a decade ago, Wolbachia pipientis strain wAlbB was observed to survive outside of host cells for one month and be infectious at least 7 days after extraction from the host (19). Additionally, Wolbachia can be purified from Drosophila host hemolymph and used to inject fruit fly abdomens, after which they establish the canonical infection of the ovaries, suggesting that the microbe has a free-living form conducive to horizontal transfer (20). Understanding how Wolbachia moves between hosts and establishes infection is a primary research goal for the use of Wolbachia in vector control.
Here we used cultured Drosophila cells to identify a timeline of Wolbachia entry into host cells, and to characterize Wolbachia’s role in its own uptake. We show that a population of live Wolbachia can directly infect established cultured JW18 host cells in as few as 2 hours (Fig. 2, Fig. 3). We show that this population of live Wolbachia enters host cells much more rapidly, and in greater numbers compared to killed controls (Fig. 2, Fig. 3, Table 1). This strongly implies that Wolbachia play a role in their own uptake; if this was purely a passive process, the live and dead populations would enter host cells at comparable rates and in similar numbers. This work confirms a previous study that has shown that heat-killed Wolbachia directly placed into uninfected cell culture are not detectable by either fluorescence in-situ hybridization (FISH) or PCR after incubation for 5 days, while live Wolbachia are easily detectable by both methods after this period of time (19). Our results are the first to show a direct comparison between the rates of initial colonization of live and dead Wolbachia by direct infection in cell culture in less than 24 hours.
Previous work has established that Wolbachia interacts with the host actin cytoskeleton, both requiring it for efficient host colonization and transmission and using a secreted effector that bundles host actin to facilitate transmission (10, 21). Work from the Sullivan lab clearly established that Wolbachia utilize dynamin and clathrin-mediated endocytosis to enter host cells, but inhibition of these pathways did not prevent entry completely (22). This indicates that Wolbachia may utilize redundant routes of entry. To build on and expand the known Wolbachia routes of entry, we next endeavored to show that the host cell actin cytoskeleton is needed for this initial entry process to occur. We treated uninfected JW18 cells with the drug cytochalasin-D, then exposed these treated cells to live Wolbachia as before. Importantly, live Wolbachia were unable to enter the treated cells (Fig. 4), and indeed we observed numbers even more reduced compared to the dead Wolbachia controls. This result suggests that the host actin cytoskeleton must be functional for the initial colonization step to occur.
When Wolbachia finds itself outside of host cells, it may alter its protein expression, likely to allow it to survive extracellularly. Understanding the changes in Wolbachia gene expression during ex vivo growth and during host infection will help identify Wolbachia loci that are important for host internalization. Many bacterial pathogens, for example Coxiella, use acidification of the vacuole to detect host uptake, and it may be that Wolbachia similarly responds to the acidification of its host-derived vacuole after uptake.
In conclusion, this work shows that Wolbachia promotes its own uptake by host cells, which helps elucidate how this bacterial symbiont has become such a globally successful infection. We also show that the host actin cytoskeleton is necessary for Wolbachia uptake, which further expands knowledge necessary to use the microbe in vector control efforts.
Supplementary Figure 1. Live Wolbachia are internalized more quickly when volume is used to quantifyWolbachia load. Numbers calculated based on live imaging of SYTOX stained Wolbachia over a four-hour time-course. Across three independent experiments, we consistently observed higher Wolbachia volumes per cell when Wolbachia was live relative to heat-killed (dead) matched samples.
Acknowledgements
We thank Dr. Bill Sullivan for the JW18 cell lines used in this work and Newton laboratory members for their feedback on early drafts of this manuscript. This work was supported by NIH grant R01 AI144430 to ILGN.