Critical Step Traced in Anthrax Infection

Pore Protein Plays Active Role In Toxin’s Entry into Target Cells

Scientists at HMS and Albert Einstein College of Medicine have revealed details of a key step in the entry of anthrax toxin into human cells. The work, which grew out of an ongoing effort to produce a better anthrax therapeutic, shows that the protective antigen component of the bacterial toxin plays an active role in transferring the other two toxin components through the cell membrane. The findings, which may provide insight into the broader question of how proteins cross cell membranes, appear in the July 29 issue of Science.

Photo by Graham Ramsay A recent study led by R. John Collier shows that protective antigen, one component of the three-part anthrax toxin, plays an active role in translocating the other two through the target cell membrane.

Tag-team Toxin
An anthrax bacterium secretes three nontoxic proteins that assemble into a toxic complex on the surface of the host cell to set off a chain of events leading to cell toxicity and death. Protective antigen (PA) is one of these proteins, and after binding to the cell, seven copies of it assemble into a specific complex that is capable of forming a pore in a cellular membrane. The pore permits the other two proteins, lethal factor (LF) and edema factor (EF), to enter the cell interior, where the factors interfere with metabolic processes, leading to the infected individual’s death.

Details surrounding this process are continuing to be uncovered in the lab of HMS researcher R. John Collier, the senior author on the current study and the Maude and Lillian Presley professor of microbiology and molecular genetics. “Until now, we have not known whether the PA pore serves simply as a passive conduit, or alternatively, plays an active role in shepherding the unfolded LF and EF molecules through,” Collier said. The findings now show it is the latter—the pore takes an active role in protein translocation.

The Amino Acid Clamp
In their investigation, the scientists used cysteine-scanning mutagenesis to identify the hydrophobic, or “greasy,” amino acid phenylalanine in protective antigen’s pore-forming domain. Seven of these amino acids project into the lumen of the pore and form a collection of greasy residues, nicknamed by the scientists “the phi-clamp.” Because the water-filled lumen of the membrane pore is smaller than the folded lethal factor and edema factor, these proteins must first unfold before being actively translocated through the heptameric channel. The clamp appears to work as a chaperone, interacting with the hydrophobic sequences on the two factors as they unfold during translocation. The researchers demonstrated that the phi-clamp was critical to infection by mutating the region and thereby blocking translocation of the toxin proteins.

These experimental results extend and explain a 1999 discovery by the Collier lab identifying a set of mutations in protective antigen that prevent translocation, some of which represented a new type of antitoxin that may be useful in anthrax treatment.

“I believe discovery of the phi-clamp will prove to be one of the high points along the path to understanding how translocation occurs in this system.”

In the recent work, Collier and his colleagues found that the phi-clamp composes the main conductance-blocking site for model cations and hydrophobic drugs, and it is one of their targets for further investigation. “I believe discovery of the phi-clamp will prove to be one of the high points along the path to understanding how translocation occurs in this system,” Collier said.

One of the greatest strengths of the experiment, according to Collier, was the integrative use of technologies applied to the testing procedures. Both cellular systems and model electrophysiological membrane systems were used to test the potency of the anthrax toxin. “We tried to bridge reductionist science with the in vivo situation. We have to do both to make correlations,” he said.

The researchers, who were funded by the National Institutes of Health and the National Science Foundation, will continue to study protein unfolding in the translocation of anthrax infection, which may prove to be relevant in other biological systems. “This is only a partial picture,” Collier said. “There are still major outstanding questions about the overall process that need to be addressed.”

—Rachel Patzer

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