Whooping cough is on the rise this year in the U.S., infecting bothyoung and old. To blunt this attack by the Bordatella pertussisbacterium, researchers are moving up their heaviest guns, notablymass spectrometry.Though the disease is potentially life-threatening, the number ofdeaths in this country has dwindled to near-vanishing, thanks tohealth care support for the pathogen's sufferers.Meanwhile, in less-developed parts of the world, pertussis kills anestimated 340,000 victims a year, most of them children. "It's acombination of malnutrition," says bacterial toxinologist ErikHewlett, "with respiratory death from oxygen starvation due tocoughing, secondary infections such as pneumonia, or brain damageassociated with the disease process." He explains that themalnutrition and dehydration occur "because these young childrencough and vomit; they can't keep food down."Hewlett, associate dean for research at the University of VirginiaSchool of Medicine, Charlottesville, is senior author of a paper onthe subject in the Oct. 20 issue of Science. Its title: "Internal LysinePalmitoylation in Adenylate Cyclase Toxin From Bordatellapertussis."Like many other infectious bacteria, Hewlett told BioWorld Today,B. pertussis operates by secreting a toxin that lacerates cells liningthe respiratory tract. (See BioWorld Today, Jan. 5, 1994, p. 1.) Butthis virulence factor is helpless without a second protein to activatethe toxin, so it can penetrate the membrane of its target cells.Acylation Turns Toxin ToxicThis activator is encoded by an additional gene, Hewlett explained,and what it apparently does is hitch a fatty acid onto the toxinprotein, after it's expressed. That post-translational process is calledacylation. For B. pertussis, he said, "no one had been able toidentify what the actual modification was that occurred in real life_ in vivo in the organism."His paper in Science reports that the add-on lipid is palmitic acid. Itpinpoints the fatty acid's attachment site on the 1,706-amino-acidjumbo protein to a lysine at position 983. "So far as we can find,"Hewlett said, "there are no other documented examples of apalmitic acid attached to an internal lysine like this."He added, "The work we reported in Science illustrates wonderfullythe power of mass spectrometry. It made possible comparing aninactive and an active form of this toxin from B. pertussis. What ourspectrometrist, Don Hunt, found _ using literally picomolequantities of the protein _ was an additional mass of 238 massunits. He sequenced this, also on the mass spec," Hewlett continued,"and it turned out to be a palmitoyl group on that lysine 983. Thatis, the activated adenylate cyclase toxin."Hunt, a co-author of the Science paper, described his versatilespectrometric analyses and sequencing technology earlier thismonth to the Cancer Vaccine 1994 symposium in New York. (SeeBioWorld Today, Oct. 5, p. 1.)A group in France," Hewlett went on, "found that adenylate cyclasetoxin is a protective antigen. So it's reasonable to consider it forinclusion in acellular pertussis vaccines." These, he explained, aresubunit vaccines now being field-tested in various countries. Theyincorporate one or more immunogens derived from B. pertussis, butso far not adenylate cyclase."One of the things we're trying to do now," Hewlett said, "is to usea peptide with that fatty-acid acyl group on it, to immunize mice,instead of using the whole activated toxin molecule. Perhaps thatwill be effective; we don't have any data on it yet."He noted that, "This family of bacterial toxins that require auxiliaryactivators includes E. coli hemolysin, on which we're also workingat the moment. In that bacterium's invasive form, this toxincontributes to frequently fatal septicemia. Other bacteria secretingsuch virulence factors," he added, "bring pneumonia in cattle andmastitis in ewes, for example."Back on the pertussis agenda, Hewlett continued, "we'd like toknow better how this little fatty acid, attached to this one aminoacid, enables this large toxin to insert into the membrane of thetarget cell and intoxicate it. That activity is a very hot area ineukaryotic intercellular trafficking of protein," he said, "and amajor thrust of what we're doing. We don't understand yet how itgets inside a cell, which is obviously a critical step in the infectionprocess."To elucidate the specificity of palmitic acid, "one of the things we'dlike to do in future is substitute other lipid groups _ for instance,myristyl, farnesyl, geranyl _ on one or another lysine, and see ifthey work just as well."`Acellular' Vaccines: Fewer Sore ArmsField tests of "acellular" pertussis vaccines are under way inSweden, Germany, Italy and various West African sites, Hewlettsaid. "They are comparing whole-cell vaccine, such as is used in theU.S., with the subunit versions, to see whether they can do as wellor better. One advantage: They are less reactogenic, because theydon't have so much endotoxin in them, so will cause fewer sorearms."The cafeteria of five acellular ingredients, being tried in variouscombinations, includes pertussis toxin, filamentous hemagglutinin,pertactin (another bacterial surface component), and two surfacefimbri."The Swedish field trials, sponsored by the U.S. National Institutesof Health and Centers for Disease Control and Prevention," Hewlettsaid, "will be over next year, and we'll have their results. The ideais that there will be continuing evolution; perhaps adenylate cyclasetoxin will be added in future to these combined vaccines." n

-- David N. Leff Science Editor

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