Scientists Describe Cholera Protein Structure—a Target for Vaccines and Antibiotics

By Jason Socrates Bardi

A group of researchers from The Scripps Research Institute (TSRI) has solved structures of a bacterial protein called pilin, which is required for infection by pathogens that cause human diseases like meningitis, gonorrhea, diarrheal diseases, pneumonia, and cholera.

In the latest issue of the journal Molecular Cell, the TSRI group reports two key structures of these pilins and discoveries about their assembly into fibrous "pili." Because a whole class of bacterial pathogens require the assembly of pilin into the hair-like pilus filaments on their surface in order for them to move around, attach to, and infect host cells, the authors believe that this research provides essential knowledge to help scientists develop novel antibiotics and vaccines against these deadly and emerging bacterial diseases.

This work directly focuses on two such pathogens—Pseudomonas aeruginosa, which causes severe lung infections in cystic fibrosis patients, AIDS patients, and other immunocompromised individuals, and Vibrio cholerae, which causes cholera, a potentially fatal diarrheal disease that primarily afflicts people in developing countries.

"Cholera," says TSRI Professor John Tainer, "is a disease that could use better vaccines."

In the developing world and in areas with poor sewage treatment, cholera is still a major public health problem, and can be a deadly for children in third world countries. Although cholera was once common in this country, modern water treatment has virtually eliminated the disease domestically, though it is still a concern for U.S. world travelers.

Cholera is caused by an acute intestinal infection with the bacterium Vibrio cholerae. This usually occurs after someone has eaten food or drank water contaminated with the pathogen.

Cholera infections are sometimes mild, but result in watery diarrhea, vomiting, and severe fluid loss about five percent of the time. These cases are life-threatening and deadly where treatment through simple rehydration with a sugar and salt mixture is not available. There is currently no effective vaccine available for this disease.

The Structure and How It Was Solved

Pili are key structures of the bacterium Vibrio cholerae and several other types of bacteria. They enable the bacteria to crawl around and stick to the intestine, lung, and other mucosal surfaces, and to pick up foreign genes and DNA, bringing them aboard to potentially increase the bacteria's pathogenicity.

In cholera, these pili are essential for the infection because they allow the bacteria to clump together and form a colony that protects them from the human immune response. This makes pili a good target for vaccine design, since blocking them should block the bacterium's ability to cause infection.

Tainer, who is an investigator in TSRI's Department of Molecular Biology and The Skaggs Institute for Chemical Biology, worked on solving the atomic structure of the pilus filaments with Senior Research Associate Lisa Craig, and three other key researchers—TSRI Professor Mark Yeager, computational expert and director of graphics development at TSRI Michael Pique, and Dartmouth Medical School Professor Ronald Taylor.

However, solving the structure of these proteins was not easy because of their size and shape. The pili themselves are assembled from thousands of copies of a single pilin subunit protein stacked together to resemble a microscopic thread—they are several hundred times longer than they are wide.

These structures are too large and flexible to be solved with the traditional techniques of structural biology used to study small proteins.

Yet members of the research team were aware that solving the structures was an important goal. "If we can understand their atomic structure, we can go after developing vaccines that are highly specific," says Craig, who is first author on the paper.

So in the current study, the TSRI team was creative and combined more than one approach.

The group first solved the structure of the individual pilin proteins from the V. cholerae bacterium using x-ray crystallography—a technique where scientists first make crystals of molecules like proteins or DNA and then expose them to x-rays. The pattern of diffracted x-rays can then be collected and analyzed to determine the structure of the molecules in the crystal. Although a fragment of the V. cholerae pilin protein was missing in their structure, they were able to infer this structure by solving a full length structure of a pilin subunit from P. aeruginosa, which is important in infections of children with cystic fibrosis.

Craig, Yeager, and Tainer then used a technique called electron microscopy to understand how the pilin proteins were organized in the pilus filaments. Electron microscopy uses a beam of electrons to magnify protein assemblies and other tiny structures up to one hundred thousand times onto a digital camera or a photographic plate.

The integration of x-ray crystallography and electron microscopy allowed Craig, Pique, and Tainer to build a model of the pili otherwise impossible at that level of molecular detail.

And the structures give new insights into how the pili assemble and how they contribute to the pathogenesis of the bacteria—as well as providing a unique molecular map of these proteins that should aid in the design of new vaccines and therapeutics.





Professor John Tainer led a group of researchers that solved structures in bacteria that may become targets for drug development. Photo by Biomedical Graphics.




Research Associate Lisa Craig is first author on the recent paper the group published in the journal Molecular Cell. Photo by Jason S. Bardi.





The structure and assembly of Vibrio cholerae toxin coregulated pilus (Panel a) was based on the crystal structures of V. cholerae pilin (red, Panels b and c) and full length PAK pilin (blue, Panel c), as well as crystal packing interactions and cryo-electron microscopy of toxin coregulated pilus filaments. Image courtesy of Lisa Craig.