VOLUME 33, NUMBER 23 THURSDAY, April 4, 2002

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Cellular signaling system
Research focuses on understanding how toxin causes disease

Contributing Editor

Working with Vibrio cholerae, the bacterium that causes the severe diarrheal disease of cholera, UB microbiologists have revealed new information on a cellular signaling system that ultimately will help scientists understand how cholera toxin and virulent proteins of other pathogenic bacteria migrate through their cellular membranes to cause disease.

Using chitinase, a protein known to be secreted by V. cholerae by the same mechanism as cholera toxin, the researchers engineered a series of insertions, deletions and mutations in its amino-acid chain. Using this mutagenic approach, they determined that the extracellular transport signal of chitinase was encoded by amino acids located between positions 75 and 555 on the chain.

Further experiments demonstrated that only a portion of this 480-amino-acid-region was essential for secretion of chitinase.

"In addition to providing new information about the transport of chitinase and cholera toxin, these findings increase our basic understanding of the methods by which proteins in general are transported across membranes, an essential activity for any living cell," said Terry D. Connell, associate professor of microbiology in the School of Medicine and Biomedical Sciences and senior author on the research.

Results of the research appear in the April issue (Vol. 184, No. 8) of the Journal of Bacteriology.

Cholera toxin is known to be secreted from the bacterial cell by a complex secretory machinery. However, rather than concentrating on the secretory mechanism—a focus of several laboratories at other institutions—Connell and colleagues Jason Folster, a doctoral student, and Daniel Metzger, research associate, set out to investigate the structural signals on cholera toxin that initiate its translocation.

The UB scientists, working in the university's Witebsky Center for Microbial Pathogenesis and Immunology, are the only researchers in the U.S. using V. cholerae to study this extracellular transport signal.

"Once the signaling system is understood, there are a variety of methods that can be used to block it, such as providing synthetic peptides to compete with the signal, or other methods that could be devised to disrupt the signal transmission," said Connell. "If you know how the toxins are secreted, you can stop the disease."

Techniques for determining protein function by causing mutations through sequential elimination of amino acids and portions of protein are used widely in microbiology, Connell noted. During initial studies, however, he and colleagues discovered that minor changes in the amino-acid sequence of cholera toxin necessary for identifying the extracellular transport signal often destabilized the protein.

Their focus then shifted to the study of chitinase, another extracellular protein of V. cholerae, which they chose as a model protein for cholera toxin. "We know that chitinase (an enzyme essential in the organism's food chain) is secreted by Vibrio cholerae by the same mechanism that transports cholera toxin, the molecule responsible for eliciting disease," said Connell. "That observation provided strong evidence that cholera toxin and chitinase contained functionally identical extracellular transport signals."

V. cholerae expresses chitinase during its free-living life stage to enable it to degrade chitin, the major component of the shells of crustaceans, which the bacterium uses as a food source. Although chitinase likely does not induce any of the symptoms of cholera, chitin is the major component of the cell wall of many fungi, Connell noted, including those important to disease (athlete's foot and certain opportunistic HIV-related infections, for example) and to agriculture (such as corn fungus), making them scientifically interesting molecules in their own right.

To elucidate the extracellular transport signal of chitinase, Folster induced a series of insertions, deletions and mutations in its amino-acid chain and determined that the extracellular transport signal of the 846-amino-acid chitinase was encoded by amino acids located between amino acid positions 75 and 555.

"The extracellular transport signal of chitinase is located in two non-adjacent sites within this 480-amino-acid-region," Folster said. "In the process of folding during protein maturation, these two regions are brought together to form an active extracellular transport signal. If we can precisely locate that small portion of the protein where the signal is actually formed, we can target it for intervention."

Folster is working with scientists at the Hauptman-Woodward Medical Research Institute to crystallize and resolve the three-dimensional structure of chitinase, which should enable him to identify precisely the structure of the molecule's extracellular transport signal.

The research was supported by grants from the National Institutes of Health and School of Medicine and Biomedical Sciences.