Spring 2007, Issue 7

Parasitic Worms and Botulism

Charles ShoemakerCharles B. Shoemaker, PhD, came to the Cummings School of Veterinary Medicine in 2003 to set up the Molecular Helminthology Laboratory with colleague Patrick Skelly, PhD. Shoemaker is intrigued by the remarkable lives of helminths, parasitic worms that infect millions of people worldwide. Some of these parasites can live for decades in a human host without being recognized by the immune system. Uncovering the mechanisms behind this cloaking ability has the potential not only to aid in the creation of vaccines against parasitic infections but also to advance our understanding of the human immune system. 

Shoemaker also studies neurotoxins produced by the bacterium Clostridium botulinum. Botulinum toxins cause paralysis, asphyxia, and death. Because this bacterium survives only in anaerobic environments, the toxin is usually contracted through consumption of contaminated food (canned foods in particular). Although intoxication can be mitigated or prevented by antitoxin administration if the patient receives treatment shortly after exposure, no therapy exists for reversing botulism poisoning. A patient’s only chance of survival is to be put on an artificial lung until the toxin dissipates, which can take weeks to months. Shoemaker leads several projects in the Botulism Therapeutics Program of the Microbiology and Botulism Research Unit, which is directed by Saul Tzipori. “Our primary focus is to try to develop therapies for treating people who have been exposed and have begun to show symptoms,” says Shoemaker.

After receiving his PhD in biochemistry from the University of Iowa in 1979, Shoemaker played a principal role in elaborating the mechanism of retroviral DNA integration while working in David Baltimore’s laboratory at MIT. Shoemaker was one of the original scientists at Genetics Institute, and he joined the Department of Tropical Public Health at Harvard University in 1987. From 1995 through 2003 Shoemaker split his time between Harvard and AgResearch Limited in New Zealand.

 “We quickly take on new technologies and apply them in novel ways to problems that are relevant to human diseases,” Shoemaker comments. One approach that he believes has great potential for use in many fields is phage display, a biotechnology in which a bacteriophage (a virus that infects bacteria) is genetically modified to display proteins of interest on its surface. The pool of modified phage (known as a “library”) is then incubated with a target, such as a protein for which a binding agent is sought. After everything that doesn’t bind has been rinsed away, an enriched pool of “binding” phage is recovered. Further enrichments yield cleaner pools of phage (sublibraries) that display the binding agent of interest, which can then be genetically sequenced.

Molecular Helminthology

The Molecular Helminthology Lab focuses on interactions between helminth parasites and their mammalian hosts. “The remarkable thing about helminths is how long individual worms can live within a host,” says Shoemaker. “In my mind the most remarkable one is the schistosome, a blood fluke flatworm. They have been known to live for many decades inside a human.” The World Health Organization estimates that schistosomes currently infect several hundred million people, and hundreds of thousands die of schistosomiasis each year. Worms living in the vascular system as male–female pairs can survive for many years during which the female produces hundreds of eggs per day. The primary damage caused by schistosome infection arises from the host's immune response to parasite eggs within its tissue.

“These worms live for a long period of time sitting in what could be viewed as the most immunologically dangerous area, the bloodstream, with all the effectors, the antibodies, the T cells, everything migrating around them, and yet they don’t seem to be noticed by the host. They’re cloaked in some way,” Shoemaker comments. “A big part of our work has been to try to understand how they can remain hidden and to find out what they do present to the host on their extensive exposed surface.” Shoemaker’s research group is using phage display technology to investigate the schistosome’s host–parasite interface.  “We’re using the worms themselves to find things that bind to worms,” says Shoemaker. “Then we use the single-chain antibodies that recognize the living worm and find out what the parasite does expose to the host.”

Botulism Therapeutics

The primary goal of the Botulism Therapeutics Program is to develop and evaluate antidotes for each of the various botulinum neurotoxins. “The toxin itself consists of two parts—a heavy chain and a light chain,” explains Shoemaker. “The heavy chain is the part of the protein that gets the toxin into the body and then into the neurons. Once it gets into the neurons, then the light chain, which is a protease—a protein-degrading enzyme—disassociates from the heavy chain and begins degrading a protein that is essential for neurotransmission. It doesn’t kill the neurons; it just sits in there, and as the neuron tries to make more of this key protein, the protease degrades it. The neurons can’t recover until the protease disappears, which can take months.”

One of Shoemaker’s projects uses phage display technology to identify antibody fragments that are especially potent at neutralizing the toxin. Researchers immunized sheep against the toxin, isolated the sheep’s antibody-producing cells, amplified the coding DNA for the antigen-combining site of the antibody, displayed it on phage, and incubated the phage with live toxin. Toxin-bound proteins are now being screened to identify antibody fragments that are potent at neutralizing the toxin. The hope is that these antitoxin fragments will become components of new therapeutic biomolecules.

Ira Herman of the Sackler School of Graduate Biomedical Sciences is collaborating with Shoemaker on the proteomic analysis of botulinum intoxication for the Botulism Therapeutics Program. Also contributing to the work are Jong-Beak Park, Michael Berne, and Jon DeGnore. “We’re looking at proteomic correlations with the pathology and stability of the toxin,” says Shoemaker. “If we can identify normal cellular pathways that contribute to the disease, we can hopefully develop new therapeutic approaches that would either reduce the pathology or accelerate the turnover of the toxin.” Shoemaker hopes to expand collaborations with Sackler School researchers, especially on a new project that will use RNA interference techniques to specifically inhibit expression of individual genes within neurons, followed by a check for recovery from the toxin. Shoemaker would welcome opportunities to collaborate with researchers with expertise in cell biology and confocal microscopy.

For more information, please see http://www.tufts.edu/vet/facpages/shoemaker_c.html.


 

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