Getting a good look at these long thin strands is not easy because their shape is not uniform. But University of Virginia researcher Edward Egelman has developed a way to view the filaments in three dimensions. Egelman’s new imaging approach will help researchers develop improved drug therapies and vaccines to prevent the outbreak of highly infectious diseases such as gonorrhea and traveler’s diarrhea.
Pili Structure Revealed
One of the major questions regarding pili on gonorrhea bacteria relates to the bacteria’s ability to continually change the pili’s amino acids to avoid the body’s immune response. By changing the amino acid sequence, the bacteria are never eliminated completely from the body and their continual presence can result in repeated infections that can lead to infertility, arthritis, and meningitis.
To determine how bacteria alter their pili, Egelman and a group of researchers from Simon Fraser University, the Burnham Institute, and The Scripps Research Institute took two-dimensional images of the pili with an electron microscope. They then loaded data from these images into Egelman’s new image analysis program—the iterative helical real space reconstruction (IHRSR) method.
The computer program solves a series of mathematical equations based on an initial guess at what the pili’s structure might be. The guess is “based on prior knowledge of the number of subunits per length in the strand,” says Egelman, a professor in the University of Virginia’s Biochemistry and Molecular Genetics Department. As the computer solves the equations, it is actually testing different ribbon-like symmetries to see which fits best with the original data from the electron micrographs. This procedure results in the three-dimensional structure of the filament under study.
Based on the three-dimensional reconstructions of gonorrhea pili, Egelman and his colleagues have developed a detailed molecular structure for pili and from this structure offer a basis for developing vaccines against pili. In addition, grooves found on the pili’s surface could also provide targets for specially designed drugs that could block pathogens.
The research group chose the IHRSR method because it does a better job than other imaging approaches of reconstructing highly variable helical polymers (a class of large complexes to which pili belong). “Previous methods for image reconstruction require highly ordered filaments, but most filaments [such as pili] are not ordered,” explains Egelman. The program also permits studying variations within filaments rather than studying only the average structure of all filaments, as was the case with prior programs.
Generation of the three-dimensional reconstructions requires fast computers. “The approach we have taken to helical reconstruction would not have been possible in 1968 [when the first three-dimensional reconstruction was generated by electron microscopy], or even in 1988. In fact, there are things that we can do today that could not have easily been done just 5 years ago,” Egelman says. The reconstruction programs run on computers built by members of Egelman’s laboratory.
Tracking Down Traveler’s Diarrhea
ETEC attach to the intestinal lining using pili. Once attached, the bacteria multiply and produce toxins that stimulate an outpouring of intestinal fluids, which cause diarrhea and can result in dehydration. Bullitt’s structure work has helped Stephen Savarino at the Naval Medical Research Center develop a vaccine that is expected to block both bacterial attachment and toxin activity. Savarino’s goal is to interrupt the infection at its earliest stages to reduce the number and severity of ETEC diarrhea cases.
Before Egelman’s IHRSR approach, Bullitt used reconstruction methods that required her to know the exact helical symmetry of the filament she was studying. “If it’s a perfect helix, you can tell the symmetry, but this is not usually the case,” she explains. In reality, Bullitt had a difficult time getting clear two-dimensional images of the filaments because they were so small. “For 2 years I tried to determine the helical symmetry but was never confident it was right,” she says.
At an NIH study section meeting, Bullitt overheard Egelman describing his new approach to a colleague and asked if she could try it. Egelman agreed, and in collaboration with Bullitt, he expanded the program so it could be applied to a wider variety of samples.
“This method helps researchers more efficiently determine the structure of filaments and more efficiently create a body of knowledge large enough to target disease-causing bacteria,” says Bullitt. She adds that Egelman’s approach does not require researchers to possess in-depth knowledge of helical structures. “You don’t have to be an expert, which means you are more quickly at the stage of what the answer means rather than struggling to get the answer,” she says.
Egelman will continue to refine the IHRSR technique by working on a number of systems related to disease-causing bacteria as well as those related to movement and muscle action. He hopes to further disseminate the program, which is now used by nearly two dozen laboratories worldwide, by making it more user-friendly. Additional improvements will include better image resolution and a new program to align images and refine the three-dimensional structures. “This will be the main way of looking at helical structures in 10 years,” says Bullitt.
This work is supported in part by the National Institute of Biomedical Imaging and Bioengineering, the National Institute of General Medical Sciences, and the National Institute of Allergy and Infectious Diseases.
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