Inner Workings: A big role for a microbial parasite

Cell biologist William Sullivan first turned his attention to a microbial parasite called Wolbachia as an excuse to take his students on a field trip. After reading a paper that suggested Wolbachia was making its way up the California coast via infected Drosophila, Sullivan, who is at the University of California, Santa Cruz, thought the class could explore whether the parasite had reached Santa Cruz County. “We would go to local wineries, collect flies, and bring them back to characterize them,” he recalls.

The petal-like structures are heartworm oocytes (red), each with a large nucleus (green). Wolbachia parasites (green flecks) have started to invade. In the center is actin (blue), a protein that forms cell filaments. Image courtesy of Frédéric Landmann and William Sullivan.

Sullivan learned a lot about the area’s vineyards and even more about Wolbachia, an intracellular parasite that relies on host cells for survival. His work led to a 2002 paper in Science (1) detailing how the parasite manipulates male chromosomes to gain a selective advantage, and gave rise to Sullivan’s abiding fascination with the biology of this highly successful endosymbiotic interloper.

An estimated two-thirds of all insects harbor Wolbachia inside their cells. For some hosts, the microbial pest is a plus: mosquitos that carry it are rendered resistant to RNA viruses, such as dengue and Zika. For other hosts, Wolbachia is a necessity: the threadlike “filarial” worms that cause African River blindness and elephantiasis can’t survive without it.

In the last decade, Sullivan and his colleagues have used their expertise in advanced molecular imaging to shed light on how Wolbachia engineers this interdependence by subverting its host’s cellular machinery. Their observations could do more than reveal the organism’s idiosyncrasies: they could help guide the development of therapeutics that will more effectively stop the parasite in its path.

Parasitic Chicanery

Sullivan has always had an eye for the unusual. In wasps, bees, and ants, unfertilized eggs develop into males. As part of one of his favorite projects, Sullivan examined wasp eggs microscopically and discovered that the female nucleus spawns thousands of little satellite nuclei that then race to assume the role of centrosomes: a pair of structures, traditionally supplied by the sperm, which anchors the cell-division machinery. “I remember seeing this and thinking, this is like cell biology from Mars,” says Sullivan.

Like the unfertilized wasp eggs, Wolbachia also dabbles in developmental devilry. But instead of making males, the microbial parasite gets rid of them. Although males can carry Wolbachia, only females can transmit the bacterium to the next generation. “So Wolbachia do all these amazing things to favor females,” says Sullivan.

The parasites can kill males outright or convert them into females. But the trick that captured Sullivan’s attention is a form of selective sterility called “cytoplasmic incompatibility.” For insects that carry Wolbachia, if an infected male mates with an uninfected female, the resulting embryos will die. However, if both the male and the female are infected, the embryos are fine. So for females, Sullivan says, “you’re better off infected, because all of your eggs will hatch no matter who you mate with.” This mechanism promotes Wolbachia’s rapid spread throughout the population.

To explore the molecular basis of this Machiavellian maneuver, Sullivan used fluorescent markers to differentially “paint” the maternal and paternal chromosomes in a fertilized fly egg. And he discovered that dad’s genetic material gets tangled up and never makes it past the first round of cell division. For fly embryos, that molecular misstep is fatal.

Sullivan’s insights were a fast track to publication (1). “I sent it in, it got reviewed and it came back ‘accepted.’ It was like, ‘whoa,’” he says. “When I do something on the cell cycle, I work for 10 years and it goes through five rounds of revision.” He suspects the rarity of studies on this quirky creature's cell biology boosted interest.

Since then, Sullivan has moved on to explore how being infected enables a female to “rescue” the compromised male chromosomes, allowing for normal embryonic development. Based on his observations, it appears that Wolbachia’s meddling delays replication of the dad’s DNA. Inside an uninfected egg, these dawdling paternal chromosomes remain a step behind their maternal counterparts. Unprepared for mitosis as a result, they get abandoned in a snarled jumble. But when the female also carries Wolbachia, Sullivan suspects the assembly of the structure that pulls the chromosomes apart is itself waylaid, giving the male chromosomes time to catch up.

“Wolbachia are master manipulators,” says biologist Jack Werren of the University of Rochester. “They have perfected the ability to control many aspects of cell biology. If people like Bill can dig in there and figure out how they do it, that could provide new tools for controlling insects and understanding cell biology.”

Learning more about Wolbachia’s artifice could also expose the parasite’s own Achilles heel. Sullivan and his team have shown that Wolbachia move around host cells by latching onto microtubules: protein filaments that form a sort of intracellular commuter rail system (2). “You can watch them run back and forth on microtubules,” he says. “Like little microtubule mites.”

In the early embryo, Wolbachia ride these microtubule rails south, to the region that will give rise to the reproductive cells of the developing organism. Thus, eliminating their microtubule handhold could prevent transmission to the next generation.

In this Drosophila oocyte, parasitic Wolbachia (red dots) are making their way to the posterior of the egg, the region that will develop into the germ line. Image courtesy of Laura Serbus and William Sullivan.

Evolving New Therapies

Reigning in this bacterial parasite becomes particularly important for the treatment of diseases caused by filarial worms, from African river blindness to heartworm in dogs. These worms have evolved a dependence on their microbial copilots. “So if you kill the Wolbachia, you kill the worm,” says Barton Slatko of New England Biolabs. Current antifilarial drugs do not affect adult worms, which live in the body for 12–15 years. For the first time, with the help of Wolbachia, there could be a treatment that wipes out adult worms in one fell swoop, suggests Slatko.

In Sullivan’s experience, when filarial worms are stripped of their Wolbachia, “apoptosis goes through the roof.” In addition to triggering this rampant cell suicide, removing Wolbachia derails the formation of the head-to-tail body axis in the worm embryo. “As far as I know, no other endosymbiont is involved in embryogenesis,” says Sullivan.

Sullivan speculates that over time, Wolbachia has “taken over essential processes that the worm was perfectly capable of doing,” a situation he likens to the mafia commandeering a popular nightclub. “It’s like, ‘you didn’t ask for us, but we’re here and we’ve made ourselves indispensable,’” he says. “I don’t have a term for it, but I’m calling it ‘evil symbiosis.’”

Sullivan is now searching for compounds to combat Wolbachia’s evil ways. Using flies he and his students collected on their field trips to Big Sur and the Channel Islands, Sullivan established Wolbachia-infested cell lines he can use to screen for chemicals that exterminate the parasite without damaging its host (3). “There’s really a big push to make drugs for these diseases,” says Sullivan, who has received support from the Anti-Wolbachia (A•WOL) Consortium, a program funded by the Bill & Melinda Gates Foundation.

At the same time, others are considering using Wolbachia to prevent the spread of dengue and perhaps even Zika, because infection with the endosymbiont renders mosquitos resistant to RNA viruses. Wolbachia-infected Aedes aegypti have already been released in countries, including Australia, Brazil, Colombia, and Vietnam. Because Zika is transmitted by the same mosquito as dengue, the propagation of Wolbachia-infected Aedes aegypti could also, in principle, curb the spread of this troubling virus.

Sullivan sees enormous potential in vector control endeavors (4), and a promising area for fledgling biologists. He has started teaching a course on neglected diseases, an experience he himself finds educational. “Every week the students give presentations and I say, ‘That’s really interesting, can you give me that paper?’” Perhaps one such paper will be the spark for Sullivan’s next intellectual odyssey.