The so-called Bt protein (Bt), produced by the bacterium Bacillus thuringiensis, is toxic to insects and widely used as an alternative to chemical pesticides in organic farming and in other crops.  Because the mechanism the toxin uses to enter insect cells is not fully understood, strategies to prevent insects from becoming resistant to it are difficult to develop.

University of California San Diego researcher Raffi Aroian and colleagues have discovered the first step the toxin takes to enter the insect target cells.  The results of the work will be published in the Feb. 11 issue of the journal Science.

Rita Teutonico, program director in the eukaryotic genetics program at the National Science Foundation, which supports the project, said: “Dr. Aroian is uncovering the way pests become resistant to Bt proteins.  Understanding how resistance evolves could alleviate concern about this natural pesticide losing its effectiveness.”

The work also confirms that the Bt protein is not toxic to vertebrates, including animals and humans, since they lack the sugar molecules to which the toxin binds.

As a bonus, the Bt protein holds promise as a pesticide against roundworms, because the worm’s sugar molecules are very similar to those the toxin binds to in insects.

Related news release from UC San Diego:

Discovery may help extend life of natural pesticide

San Diego, California
February 10, 2005

By Sherry Seethaler

A team led by biologists at the University of California, San Diego has discovered a molecule in roundworms that makes them susceptible to Bacillus thuringiensis toxin, or Bt toxin—a pesticide produced by bacteria and widely used by organic farmers and in genetically engineered crops to ward off insect pests.

Their findings should facilitate the design and use of Bt toxins to prevent insects, which the researchers believe also possess the molecule, from developing resistance to Bt, extending the life of this natural pesticide.

The study, published February 11 in the journal Science, details the structure of a molecule to which Bt attaches, or “binds,” in the lining of the intestines of insects and roundworms. The molecule is a glycolipid—a lipid attached to a tree-like arrangement of sugars. Because changes in the sugars impact Bt’s ability to bind, the researchers believe that their discovery will make it possible to develop better pesticides and lead to new treatments for parasitic infections that affect close to two billion people worldwide.

“Our previous findings with the roundworm C. elegans strongly suggested that specific sugar structures are likely critical for Bt toxin susceptibility,” said Joel Griffitts, the first author on the paper and a former graduate student with UCSD biology professor Raffi Aroian. “This latest paper demonstrates what these sugars actually do. They provide a receptor for the toxin that allows the toxin to recognize its “victim”—a roundworm or an insect. This paper also brings us from the conceptual realm to the chemical nature of these sugar structures—how their atoms are arranged, and how the toxin binds to them.”

“Bt toxin, which is produced by a soil bacterium, is toxic to insects and roundworms, but not to vertebrates, which accounts for its popularity as a pesticide,” explained Aroian, who led the team. “But the development of insect resistance to Bt is a major threat to its long term use. Our findings make it possible to understand resistance at the molecular level and should improve resistance management.”

In collaboration with Paul Cremer and Tinglu Yang, coauthors on the paper and chemists at Texas A&M University, Griffitts and Aroian found that Bt toxin directly binds glycolipids. However, in each of the four Bt resistant mutants tested—bre-2, bre-3, bre-4 and bre-5—the researchers found that there was either zero or dramatically reduced binding of glycolipids to Bt toxin. They concluded that the defective sugar structure of the glycolipid receptor in each of the mutants prevents Bt from binding.

Other members of the research team, coauthors Stuart Haslam and Anne Dell, biologists at Imperial College London; Barbara Mulloy, a biochemist at the Laboratory for Molecular Structure, National Institute for Biological Standards and Control in Hertfordshire, England; and Howard Morris, a biochemist at the M-SCAN Mass Spectrometry Research and Training Centre in Berkshire England, determined the chemical structure of the normal glycolipid receptor that binds Bt toxin.

Elements of this structure are found in both insects and nematodes, but are not found in vertebrates at all, which may be one reason these proteins are safe to vertebrates. This work furthermore opens up the possibility of using Bt toxins against roundworms that parasitize humans.

“These parasites infect nearly one-third of the human population and pose a significant health problem in developing countries,” said Aroian. “Perhaps one-day vertebrate-safe Bt toxins could be used as human therapies against these parasites.”

Griffitts and Aroian credit the flexibility of the roundworm C. elegans as an experimental system, particularly the ease of manipulating it genetically, in making it possible to find and characterize the structure of the long sought-after Bt receptor. However, their results apply to insects as well. Michael Adang and Stephan Garczynski, coauthors and entomologists at the University of Georgia, showed that the glycolipid receptor is present in the tobacco hornworm, an insect pest that is susceptible to Bt toxins used commercially in plants.

“It will now be possible to monitor insect populations near fields where Bt is used and catch insect resistance in its early stages by looking for changes in glycolipids,” said Aroian. “If changes are detected, switching to another pesticide, perhaps even another variety of Bt that works through a different mechanism, could prevent the resistance genes from becoming widespread.”

According to the researchers, prior work indicates that there are other receptors that also contribute to Bt resistance. Combining pesticides that work through different receptors or designing pesticides that can work through more than one receptor type could thwart the development of resistance.

“This paper presents an intriguing question,” said Griffitts. “In light of findings by insect biologists that certain proteins function as important Bt toxin receptors in some cases, how might glycolipid and protein receptors cooperate to engage this intoxication program? If the field can figure this out, it might allow for the engineering of toxins that can utilize either type of receptor alternatively, such that host resistance would require the mutation of both receptor types. This means that resistance would be exponentially less probable.”

The study was funded by the National Science Foundation, the Burroughs-Wellcome Foundation and the Beckman Foundation.

Related news release from UC San Diego:

San Diego, California
August 2, 2004

UC San Diego biologists identify genetic mechanisms conferring resistance to Bt toxins

Biologists at the University of California, San Diego have discovered the genetic and molecular means by which roundworms, and probably insects, can develop resistance to the most widely used biologically produced insecticide—crystalline toxins from the bacterium Bacillus thuringiensis, or Bt.

Such Bt toxins, which are safe to humans and other vertebrates and far more environmentally friendly than pesticides, have been sprayed on crops by organic farmers for decades. They have also played an important role in Africa in controlling insects that carry disease and are now being used in genetically modified corn, cotton and other crops to control caterpillars and beetles. But as the use of Bt toxins expands worldwide, scientists fear their long-term effectiveness will be threatened by the development of Bt-resistant strains.

The achievement by the UCSD biologists, reported in the August 3 issue of Science, provides important molecular and genetic information that will help scientists develop strategies to delay or circumvent the evolution of Bt-resistant strains of roundworms and insects.

“There are insects in the wild now that contain gene variants that allow them to be resistant to Bt toxins, but fortunately they are small in number,” says Raffi V. Aroian, an assistant professor of biology at UCSD who headed the study. “However, as more crops with Bt genes are planted, it’s only a matter of time before populations of Bt-resistant insects grow numerous enough to become economically troublesome to farmers hoping to control these insects.”

In their study, the researchers examined mutant genes they discovered in the roundworm C. elegans that confer resistance to a particular Bt toxin known as Cry5B. Joel S. Griffitts, a graduate student at UCSD and the lead author of the study, cloned one of these five mutant genes, which the scientists named bre for Bt resistance, then compared differences in the proteins produced by the mutant gene and the corresponding normal gene. That comparison allowed the UCSD researchers, which included postdoctoral fellow Johanna L. Whitacre and technician Daniel E. Stevens, to conclude that the roundworm’s Bt toxin resistance resulted from the loss of a galactosyltransferase, an enzyme that adds carbohydrates to proteins and lipids.

Their discovery prompted the scientists to hypothesize that crystalline Bt toxins—which act by attacking and dissolving the intestines of their hosts—normally recognize the outer surface of intestinal cells by means of carbohydrates or sugars.  When the galactyosyltransferase gene is missing, these sugars are not made and the toxin fails to recognize its host.

Whether this enzyme is essential for many other Bt toxins remains to be determined. But the UCSD scientists discovered that their mutant roundworms were also resistant to a Bt toxin that is lethal to beetles, suggesting that the development of resistance by the loss of carbohydrate-modifying enzyme is relevant to insects as well. Furthermore the three dimensional structure of diverse insecticidal Bt toxins contains a fold that is predicted to bind precisely the sugar modification made by the galactosyltransferase, raising the possibility that this mechanism of resistance could be widespread.

The discovery that the loss of a general modifier like a galactosyltransferase can allow an organism to develop resistance to Bt toxin is not good news.

“For people using Bt toxins to control insects, this is a particularly threatening scenario,” says Aroian. “The reason is that with one swoop, you can knock out the binding of multiple toxins to multiple receptors. But now that we know this mechanism of resistance, we can devise strategies to cope with this.”

One possible strategy, Aroian says, is for scientists to modify the toxins such that they can bind to the inner lining of the insects’ or roundworms’ guts independent of this carbohydrate modification.

In their study, the UCSD scientists showed visually, using toxins labeled with fluorescent dyes fed to normal and resistant forms of C.  elegans, that the Bt toxin is taken up into the gut cells of a normal roundworm but not a resistant roundworm. If the toxin is not recognized, as is the case in resistant animals, it simply passes through the lumen of the gut and is defecated without entering the gut cells.  

“This provides strong evidence for our model, which essentially is that if you don’t have this carbohydrate enzyme, you don’t make a carbohydrate that the toxin needs to recognize the surface of the gut,” says Aroian. “We also provide, using ‘mosaic analysis,’ definitive molecular evidence that Bt toxins target the gut. Scientists have long known that these toxins targeted the gut. But this, at a molecular level, conclusively proves it.”

The UCSD team’s discovery also sheds light on the puzzling and sometimes contradictory findings of previous attempts to pinpoint a mechanism for the development of Bt resistance in insects.
 
“For a couple of decades now,” says Griffitts, the senior author of the paper, “researchers have been grinding up insect guts and finding components of those extracts that stick to Bt toxins. And over the last decade, they’ve found multiple proteins, some of which appear unrelated, that bind to Bt toxins. This study may explain those seemingly contradictory results. These proteins, which may look very different structurally, may have the same binding motif because of carbohydrate modification.”

The discovery of this motif, or mechanism of action, in C. elegans demonstrates the many advantages of this roundworm to researchers. “The kind of analysis that can be done in C. elegans can’t be done as easily in insects,” says Aroian. “We have a complete genetic and physical map of C. elegans, we can breed them in the laboratory easily, they grow fast, having only a three-and-a-half day life cycle, they’re transparent, so we can easily see their internal structures and they eat bacteria, so we can express the Bt toxin right in their food source.”

The discovery of the resistance mechanism in C. elegans will not only help farmers control insects. It will also help scientists employ Bt toxins in the growing problem of roundworm, or nematode, infestations in plants, animals and humans.

“Even if Bt toxins weren’t used to fight insects, nematodes are a huge problem,” says Aroian. “At last estimate, which was 13 years ago, they caused $80-billion worth of crop damage per year. And the damages will become worse, because the main chemical now used to control them in agriculture, methyl bromide, has been banned by the Montreal protocol.  They are also a human health problem—a quarter of the world’s population are infected with animal parasitic nematodes.”

The UCSD study was supported by the National Science Foundation, the Burroughs Wellcome Fund and the Beckman Foundation.