An international team of researchers conducted a structural biology study at the U.S. Department of Energy's (DOE) SLAC National Accelerator Laboratory (Menlo Park, CA) to uncover how small insecticidal protein crystals that are naturally produced by bacteria might be tailored to combat dengue fever and the Zika virus. SLAC's x-ray free-electron laser—the Linac Coherent Light Source (LCLS)—offered unprecedented views of the toxin BinAB, which is used as a larvicide in public health efforts against mosquito-borne diseases such as malaria, West Nile virus, and viral encephalitis.
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BinAB is currently ineffective against the Aedes mosquitoes that transmit Zika and dengue fever, and therefore not used to combat these species of mosquitoes at this time. The new information provides clues to how scientists could design a composite toxin that would work against a broader range of mosquito species, including Aedes. Study lead author Jacques-Philippe Colletier, a scientist at the Institut de Biologie Structurale (Grenoble, France), explains that a more detailed look at the protein's structure provides information fundamental to understanding how the crystals kill mosquito larvae—and is a prerequisite for modifying the toxin to adapt it to the research team's needs.
The BinAB crystals 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. But because Aedes larvae can evade one of these steps of intoxication, they are resistant 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.
For public health officials who want to prevent mosquito-borne disease, BinAB could also offer an alternative for controlling certain species of mosquitoes 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. In the LCLS experiments, 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.
|The mosquito larvicide BinAB is composed of two proteins, BinA (yellow) and BinB (blue). Inside bacterial cells, BinAB naturally forms nanocrystals. Using these crystals and the intense x-ray pulses produced by SLAC's Linac Coherent Light Source, the research team was able to shed light on the 3D structure of BinAB and its mode of action. (Image credit: SLAC National Accelerator Laboratory)|
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 mosquitoes. This is work that is ongoing at Institut de Biologie Structurale, the University of California Los Angeles (UCLA), the University of California Riverside, and SLAC. Only coarse details were known about the unique 3D structure and biological behavior of BinAB prior to the experiment at LCLS.
The small size of the crystals made them difficult to study at conventional x-ray sources. So the research team used genetic engineering techniques to increase the size of the crystals, and the bright, fast pulses of light at LCLS allowed the scientists 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 3D map of the electron density of the protein. The technique had so far only been used on test samples where the structure was already known 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," Colletier says. "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 Aedes mosquitoes."
Full details of the work appear in the journal Nature; for more information, please visit http://dx.doi.org/10.1038/nature19825.