A tick on a blade of grass, waiting for passing prey. [Matt Pinski]

Blood-sucking ticks spread a wider range of microbes to humans and other vertebrates than do any other known insect, causing a multitude of diseases in people and livestock. But a question that has puzzled scientists is how do the ticks protect themselves from potential pathogens they encounter on and in their hosts? A research team led by Seemay Chou, PhD, assistant professor of biochemistry and biophysics at the University of California, San Francisco (UCSF) and Chan Zuckerberg Biohub Investigator, has now discovered that an antibacterial enzyme, Dae2, contained in the ticks, protects them from bacteria found on human skin, but still lets them carry Borrelia burdorferi, the bacterium that causes Lyme disease in humans and animals. The scientists say the ticks acquired the gene for this antibacterial enzyme some 40 million years ago, from an unknown species of bacteria, through horizontal gene transfer.

“Bacteria exchange DNA with each other all the time, but what’s remarkable is that 40 million years ago a gene in bacteria jumped across kingdoms all the way into ticks,” said Chou, who is senior author of the team’s published paper in Cell. “The ticks effectively stole a page out of the bacteria’s playbook, repurposing their arsenal to use against them … Solving this puzzle is an important step toward the longer-term goal of preventing the spread of debilitating diseases like Lyme.” The team described its results in a report titled, “Ticks Resist Skin Commensals with Immune Factor of Bacterial Origin.”

Ticks are notorious for transmitting pathogens, including the Lyme disease bacterium. But ticks themselves live dangerous lives, spending most of their time looking for suitable hosts across very different habitats and seasons. Once they encounter a reptile, bird, or a mammal, such as a human, they become intimately connected with that host, and with all of the host’s bacteria and viruses, potentially for days on end. “Hard ticks, such as the black-legged tick vector of Lyme disease, Ixodes scapularis, have especially prolonged continuous blood meals that can last over a week, during which ticks must embed within the skin of their hosts and maintain an intimate, perilous attachment,” the authors wrote. “Field microbiome studies of hard ticks suggest that they encounter a diverse assemblage of environmental and host-associated bacteria, including species commonly found on the skin of mammalian hosts.”

This diagram shows the interaction between ticks, Dae2, skin bacteria, and Lyme bacteria. [Hayes et al./Cell]
Given this exposure, how does the tick’s immune system keep the organism itself safe from infection by pathogens? The latest work by Chou’s team provides an answer to this puzzle. The research, Chou said, reveals that ticks are exquisitely constructed blood-sucking machines, with immune systems specially tailored for this unique lifestyle. Their defense strategies are carried out both inside and outside their bodies, she said, killing even our resident microbes as they feed.

Five years ago, Chou and colleagues had identified a gene, dae2, in tick DNA, which produces a microbe-killing protein. The tick gene originally evolved in bacteria, where the protein it encoded worked as an offensive agent against other bacteria. Several hundred million years ago, at around the time that the ancestors of some of today’s ticks began feeding on blood, those ticks “stole” the gene, making it a part of their own genomes. “We previously found that I. scapularis and other ticks acquired a potent antibacterial enzyme approximately 40 mya by horizontal gene transfer of an interbacterial competition toxin gene from bacteria,” the investigators wrote in their Cell paper.

For their newly reported studies the team expanded on that work to demonstrate that without the protection offered by the dae2 gene, ticks are vulnerable to infection with Staphylococcus, one of the most common types of commensal bacteria that essentially cover our skin surface, but generally don’t cause us any harm.

“This is the first time anybody’s identified a natural pathogen of ticks, and established a mechanism for it,” said Chou, whose work is supported by a Sanghvi-Agarwal Innovator Award. “Ticks pass more microbes to humans, livestock, and other animals than any other known arthropod, but now their own vulnerabilities are on the table.”

Chou says dae2 represents a rare example of horizontal gene transfer from a bacterium to an animal, and the fact that this transfer occurred as blood-feeding evolved might not be a coincidence. “I’ve always wondered why blood-feeding is even a thing,” said Chou. Not only does blood take a lot of energy to process into useful food, but biting on and attaching to much larger animals “seems inherently like a really risky sport.” With a strong, dae2-enhanced immune system, she said, tick species could have flourished, expanding to fill their bloody ecological niche.

When she first began working with dae2 in the deer tick Ixodes scapularis, Chou thought ticks’ acquisition of the gene must have something to do with protection against tick-dwelling bacteria like B. burgdorferi. Carrying out multiple experiments, Chou and colleagues tried to find a mechanism for the gene to inhibit this bacterium. “It makes no sense for ticks to have acquired this immune effector to kill off the bacteria that it’s most notoriously known to stably associate with,” she said. With this notion in mind, the researchers began the much more complicated process of looking for bacteria that ticks are not known to live with peaceably. When co-first authors Beth M. Hayes, PhD, and Atanas D. Radkov, PhD, proposed exploring the idea that dae2 might protect against Staphylococcus bacteria, “I actually pooh-poohed the idea—I bet a beer against it,” said Chou.

Seemay Chou, PhD, examining ticks in a test tube. [Lauren J Young, NPR Science Friday]
But when the scientists introduced the Dae2 protein to cultured staphylococcal bacteria, they were surprised by the result. “The tube went from murky to clear in like, a second,” Chou said. “It was astounding how fast it worked … I was kind of glad to have lost this bet. After two years of trying to figure out what was going on, it all started falling together.”

As detailed in the newly released Cell paper, the researchers then carried out a broad series of experiments, first comparing dae2 genes in a range of tick species with the bacterial tae genes from which they were originally derived. “In this study, we consider the possibility that the Dae2 immune factor protects against the natural pathogens of ticks themselves by examining the specificity and biological function of this toxin in the tick disease vector I. scapularis,” they wrote. With these comparisons and high-resolution protein structures in hand, they used computer models of these proteins to compare their shape and orientation when they came in contact with molecules found in bacterial cell walls.

They then tested the proteins directly against actual molecules extracted from bacterial cell walls. They found that while the Dae2 protein could quickly degrade this material, taken from common skin bacteria, Tae2 could not. Dae2 also killed a wide range of bacteria, notably three very common species that are symbiotic partners of human skin. “… our analyses led to two major conclusions: expanded Dae2 specificity allows the eukaryotic enzymes to target a broader and distinct set of microbes compared to prokaryotic Tae2 enzymes, and Dae2 kills common mammalian skin commensals with incredible potency,” they wrote.

The researchers in addition checked whether Dae2 carried by the ticks could reach our skin. They found Dae2 protein in tick salivary glands and saliva, and observed that, from there, the protein was transferred to the ticks’ blood meal hosts. In a separate set of in vivo experiments, they investigated the effects of blocking dae. They used RNAi interference to knock down Dae2 in a group of ticks before allowing them to feed on mice. They also blocked pre-existing dae protein in tick midguts or saliva by immunizing mouse hosts against the protein, before allowing the ticks to feed. The results of these in vivo tests confirmed higher levels of Staphylococcus bacteria in the ticks that had reduced or absent dae2, compared with the control ticks that had a functional protein. Ticks without Dae protection also stayed smaller and gained less weight than ticks with Dae2, “suggesting increased host bacteria interfered with feeding,” the team noted.

“Together, our findings highlight the inseparability of animals from their microbiota: animals confront not only each other but also their associated microbes,” the scientists concluded, “For blood-feeding parasites, such as ticks, successful navigation of such complex, interkingdom clashes is critical for survival … Thus, this work identifies a major microbial threat to ticks and identifies an immune mechanism ticks have acquired to cope with it.”

“This is a new way of thinking about how ticks interact with microbes,” said Chou. Microbes borne by ticks cause disease in humans and animals worldwide, but that’s only half the story, she said. “Their commensal is our pathogen, and our commensal is their pathogen.” The net result is a well-honed biochemical system that benefits ticks and B. burgdorferi alike. The findings support a growing idea among biologists that the key to controlling tick-borne diseases may be controlling the ticks themselves, not just treating the diseases. This is what the research team is exploring next.

“There are all these old studies that show that tick saliva directly enables disease transmission, so now we’re really interested in digging into some of the mechanics underlying that,” commented Chou. “We’ve also started to look more closely at the bite site itself. That’s basically ground zero for all these different interactions.”

So while ticks are currently winning the evolutionary war against bacteria and humans, there remains a whole battleground yet to be explored. “The beautiful thing about vector biology is that it’s like a huge puzzle that we’re slowly piecing together to understand how it fits into one big picture,” Chou said.

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