Cellular signaling system
Research focuses on understanding how toxin causes disease
By LOIS
BAKER
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
mechanisma focus of several laboratories at other institutionsConnell
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.