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RESEARCH BRIEFS
Study Finds Polyvalent Inhibitor Of Anthrax ToxinIf there is any good news about anthrax, it is that Bacillus anthracis can be killed with antibiotics. However, as can be seen in the recent Florida outbreak, the tragic news is that once the mild, flulike, symptoms become apparent, it is too late for medical intervention.
Disrupting anthrax toxin assembly on the cell membrane. The membrane-bound proteolytic fragment of the protective antigen, PA63, resides as a heptamer complex on the cell surface. Under normal conditions, the catalytic toxin subunits, edema factor (EF) and lethal factor (LF), bind to the PA63 multimer. In the presence of the long, Velcro-like polyvalent decoy, binding of EF and LF is blocked. Illustration adapted from original by R. John Collier Now Harvard scientists have found a chink in the anthrax armor. In an article appearing in the October Nature Biotechnology, researchers in the lab of R. John Collier, the Maude and Lillian Presley professor of microbiology and molecular genetics, report a strategy to neutralize anthrax toxin in the body (see also Anthrax, page 1). And the technique they used may have an even broader applicability--for example, in cancer therapy. The anthrax bacterium secretes three toxin components into the host bloodstream. One of them, protective antigen, assembles into a heptamer pre-pore at the cell surface. Then two enzymatic moieties, edema factor and lethal factor, bind to the pre-pore and are internalized. Once in the endosome, the protective antigen multimer transforms into a pore, through which edema factor and lethal factor reach the cytoplasm. Collier and postdoc Michael Mourez reasoned that if they could flood the protective antigen binding site with a nontoxic decoy polypeptide, they could block edema factor-lethal factor binding and thwart the entire process. To isolate such a peptide the researchers employed a phage display library, constructed to express random dodecapeptides, and exploited a quirk of the toxin's biochemistry: once on the cell surface, protective antigen is cleaved, exposing the edema factor- lethal factor binding site. The researchers used a differential screening procedure, selecting all the phage particles that bound to PA63, the proteolytic fragment, then subtracted from that set those that bound protective antigen. The phages remaining expressed peptides specific for the edema factor-lethal factor site. The researchers synthesized one peptide, but its binding to protective antigen was weak, making it impractical for use as a therapeutic agent. To increase binding avidity, they coupled multiple copies of the peptide onto a polyacrylamide backbone. This polyvalent synthetic molecule is much like a long, thin strip of Velcro and, like Velcro, the collective action of the many weak hooks, or peptide molecules, produced a tight grip: binding between peptide and protective antigen increased 7,500-fold. For this work Collier teamed up with Harvard chemist George Whitesides, who devised a way to graft small molecules onto polyacrylamide carriers. Administered in vivo, the inhibitor blocked toxin action in an animal model for intoxication. Normally, rats die within hours after injection with a mixture of toxin subunits. But when challenged by toxin that was followed minutes later with an injection of the polyvalent ligand (itself completely innocuous), the rats were protected. "It's a nice proof of principle," said Collier, noting that this is the first application of the polyvalency technology that has worked in vivo. It could be used to increase the therapeutic potency of antitumor peptides or growth receptor agonists, for example. The Collier lab members are old hands at thwarting anthrax. In work published in the April 27 issue of Science, they produced a mutant form of protective antigen. When injected into a rat along with wild-type toxin, the toxin complex formed but was unable to make the critical transformation into the channel that allows entry of the lethal and edema factors. Like the polyvalent decoy, the protective antigen mutant protected rats against intoxication. In addition, because the mutant elicits a protective immune response, it is double acting. Both discoveries harbor potential to block toxin in infected individuals and might provide a therapeutic strategy when antibiotic treatment is no longer an option. "If the protective antigen mutant works in an infection model at a very late stage, it could go straight into production," Collier said. "We know already that the molecule is safe, and with mutations it has got to be safer." Asked if the polyvalent ligand can be tested in humans, Collier said, "There will be a lot of hoops to jump through first, but we'll push forward."
Worm Model Identified For Gram-positive Virulence FactorsMammalian model systems for Enterococcus and other Gram-positive human pathogens are cumbersome, costly, and not always amenable to genetic manipulation. The worm C. elegans offers a practical alternative. In a study published in the Sept. 11 Proceedings of the National Academy of Sciences, Frederick Ausubel, HMS professor of genetics at Massachusetts General Hospital, and coworkers showed that the nematode provides a model host system for several Gram-positive bacteria, opening the door to further investigations into host- pathogen interaction. The study focused on the enterococcal strain E. faecalis. Under the microscope, the guts of nematodes fed this pathogen were grossly distended and filled with bacteria. Unlike related, noninfectious strains that colonize the gut, E. faecalis did not clear the stomach once it was removed from the culture medium, indicating persistent infection. It is generally accepted that virulence factors from Gram-negative bacteria strains, such as Pseudomonas, cause disease in hosts as evolutionarily distinct as mouse, nematode, plants, and insects. Extending these results to Gram-positive bacteria, the investigators demonstrated that two E. faecalis proteins that contribute to virulence in mammalian models are also lethal to C. elegans. Mutations in their genes allowed prolonged survival in both the mouse and nematode. One is the E. faecalis gene product Cyl, which lyses eukaryotic cells. When either the Cyl gene or a second enterococcus gene, gelE, were disrupted, the researchers observed a decreased virulence in the mouse model. Conversely, the researchers also showed that C. elegans could be used to identify hitherto unknown Gram-positive virulence factors. By screening an E. faecalis Tn917 transposon library, they isolated 20 bacterial mutants that were less virulent when tested in the nematode system. One of these, with a mutation in a gene homologous to sucrose-6-phosphate hydrolase (ScrB), also prolonged survival in a mouse model. Ausubel and collaborators found that the virulence of some species depends upon the growth medium, leading them to predict that "the list of pathogens able to use C. elegans as a model host will expand as different conditions are tested."
Racial Disparity in Flu Shots QuantifiedElderly African Americans are less likely to get a flu shot than their white counterparts, says a study in the Sept. 26 Journal of the American Medical Association. HSPH and HMS researchers report that influenza vaccination rates among African Americans were 46 percent versus 68 percent among whites. This gap was observed regardless of health care insurance, be it managed care or traditional fee-for-service. Eric Schneider, HMS instructor in medicine; Arnold Epstein, the John H. Foster professor of health policy and management and chair of that department at HSPH; Paul Cleary, HMS professor of medical sociology in Health Care Policy; and Alan Zaslavsky, HMS associate professor of statistics in Health Care Policy, evaluated data from the 1996 Medicare Current Beneficiary Survey, selecting 13,674 respondents 65 years old or older who identified their race as African American or white. The researchers showed that while African Americans were less likely to solicit medical attention, differences in attitude toward medical care could not account for the observed disparity in immunization patterns. Managed care plans offer flu shots free of charge. And while they have been somewhat more effective in getting people out to be vaccinated (71 percent versus 65 percent for fee-for-service enrollees), Schneider, lead author of the study, said, "Health plans are not having an impact on racial disparities or the 'flu shot gap.'" --Briefs by Anne Mahon |
