Engineering a Protein to Combat Dengue Fever, Zika

Researchers make breakthrough understanding the structure of a protein that has proven effective in controlling many species of mosquitoes

Life cycle of the bacterium that kills mosquito larvae. Image credit: UCLA, UC Riverside, and the Institute of Structural Biology, Grenoble, France.

RIVERSIDE, Calif. ( — A team of scientists, including several from the University of California, Riverside, has uncovered how small insecticidal protein crystals that are naturally produced by bacteria might be tailored to combat mosquito-borne illnesses such as dengue fever and Zika.

The protein crystals are currently used as a larvicide in public health efforts against mosquito-borne diseases such as malaria, filariasis, West Nile virus and viral encephalities. However, they are ineffective against the Aedes mosquitos that transmit Zika virus and dengue fever.

The new information, outlined in a paper published Sept. 28  in Nature, provides clues to how scientists could design modified proteins that would work against a broader range of mosquito species.

“Understanding the structure of this protein has proved very elusive,” said Brian Federici, a distinguished professor of the graduate division in the Department of Entomology at UC Riverside. “Others have tried over the last 20 years without success. Now, we can design experiments that will allow us to target virtually all mosquitoes.”

The protein crystals, known as BinAB, are produced by Lysinibacillus sphaericus bacteria, which release the crystals along with spores at the end of their life cycle. Mosquito larvae eat the crystals along with the spores, and then die.

BinAB is inactive in the crystalline state and does not work on contact. For the crystals to dissolve, they must be exposed to alkaline conditions, such as those in a mosquito larva’s gut. The binary protein is then activated, recognized by a specific receptor at the surface of cells and internalized.

Brian Federici. Photo credit: I. Pittalwala, UC Riverside.

Brian Federici. Photo credit: I. Pittalwala, UC Riverside.

Because many species of Aedes larvae can evade one of these steps of intoxication, they are not susceptible to BinAB. These larvae do not express the correct receptors at the surface of their intestinal cells. Many other insect species, small crustaceans and humans also lack these receptors, as well as alkaline digestive systems, making this binary protein safe for non-target insects and humans.

“Part of the appeal is that the larvicide’s safe because it’s so specific, but that’s also part of its limitation,” said Michael Sawaya, a scientist at the UCLA-DOE Molecular Biology Institute and co-author on the paper.

For public health officials who want to prevent mosquito-borne disease, BinAB could also offer an alternative for controlling certain species of mosquitos that have begun to show resistance to other forms of chemical control.

The research team already knew the larvicide is composed of a pair of proteins, BinA and BinB, that pair together in crystals and are later activated by larval digestive enzymes.

To further investigate, they conducted experiments on an X-ray free-electron laser – the Linac Coherent Light Source (LCLS) – at the U.S. Department of Energy SLAC National Accelerator Laboratory in Menlo Park, California.

They learned the molecular basis for how the two proteins paired with each other – each performing an important, unique function. Previous research had determined that BinA is the toxic part of the complex, while BinB is responsible for binding the toxin to the mosquito’s intestine. BinB ushers BinA into the cells; once inside, BinA kills the cell.

The scientists also identified four “hot spots” on the proteins that are activated by the alkaline conditions in the larval gut. Altogether, they trigger a change from a nontoxic form of the protein to a version that is lethal to mosquito larvae.

Using the information gathered during the crystallography study, the research team has already begun to engineer a form of the BinAB proteins that will work against more species of mosquitos. This is work that is ongoing at Institut de Biologie Structurale in Grenoble, France, UCLA, UC Riverside and SLAC.

Only preliminary details were known about the unique three-dimensional structure and biological behavior of BinAB prior to the experiment at LCLS.

“We chose to look at the BinAB larvicide because it is so widely used, yet the structural details were a mystery,” Federici said.

The small size of the crystals made it difficult to study them at conventional X-ray sources. However, using genetic engineering techniques, Federici’s team was able to increase the size of the crystals eight-fold. This enabled the scientists to use the bright, fast pulses of light at LCLS to collect detailed structural data from the tiny crystals before X-rays damaged their samples.

The researchers used a crystallography technique called de novo phasing. This involves tagging the crystals with heavy metal markers, collecting tens of thousands of X-ray diffraction patterns, and combining the information collected to obtain a three-dimensional map of the electron density of the protein.

“This is the first time we’ve used de novo phasing on a crystal of great interest at an X-ray free-electron laser,” said Sebastien Boutet, a Stanford SLAC scientist and an author of the paper.

The technique had so far only been used on test samples where the structure was already known, in order to prove that it would work.

“The most immediate need is to now expand the spectrum of action of the BinAB toxin to counter the progression of Zika, in particular,” said Jacques-Philippe Colletier, a scientist at the Institut de Biologie Structurale and lead author on the paper. “BinAB is already effective against Culex [carrier of West Nile encephalitis] and Anopheles [carrier of malaria] mosquitoes. With results of the study, we now feel more confident that we can design the protein to target all Aedes mosquitoes.”

Three of the co-authors of the paper have ties to Federici’s lab: Robert Hice, Hyun-Woo Park and Dennis Bideshi.

Additional contributors to the research include scientists from the Howard Hughes Medical Institutes at UCLA, Lawrence Berkeley National Laboratory, and Stanford University.

The Collaborative Innovation Award program of Howard Hughes Medical Institute (HCIA-HHMI), W.M Keck Foundation, National Institutes of Health, National Science Foundation, France Alzheimer Foundation, Agence Nationale de la Recherche, and DOE’s Office of Science and Office of Basic Energy Sciences supported the research.


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