Cholera Protein Structure--a Target For Vaccines & Antibiotics--described By TSRI Scientists

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, Ph.D., "is a disease that could use better vaccines."

Tainer, who is an investigator in TSRI's Department of Molecular Biology and a member of The Skaggs Institute for Chemical Biology at TSRI, determined the atomic structure of the pilus filaments with TSRI Senior Research Associate Lisa Craig, Ph.D., 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.

"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.

The Structure and How It Was Solved

The pili are used by several types of 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.

However, solving the structure of these proteins has not been 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. 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. The structures gave 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.

The research article, "Type IV Pilin Structure and Assembly: X-ray and EM Analyses of Vibrio cholerae Toxin Coregulated Pilus and Pseudomonas aeruginosa PAK Pilin" is authored by Lisa Craig, Ronald Taylor, Michael Pique, Brian Adair, Andrew Arvai, Mona Singh, Jane Lloyd, David Shin, Elizabeth Getzoff, Mark Yeager, Katrina Forest, and John Tainer and appears in the May 23, 2003 issue of the journal Molecular Cell.

The research was funded by the National Institutes of Health, The Skaggs Institute for Research, and the Canadian Institutes of Health Research.

More on Cholera: A Deadly Disease of Developing Nations

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. In the developing world and in areas with poor sewage treatment, cholera is still a major public health problem, and these areas frequently support outbreaks of disease. Cholera can be a deadly problem for children in third world countries. Epidemic outbreaks in the past have involved changes to the pilin protein, now structurally characterized by Scripps researchers.

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. According to the Centers for Disease Control and Prevention, the only cholera vaccine licensed in the United States has been discontinued because it offers only brief and incomplete immunity. For more information, please see: http://www.cdc.gov/ncidod/dbmd/diseaseinfo/cholera_g.htm.