Every moviegoer knows that the Mafia is a secret terrorist organization, which originated in Italy and spread its criminal shakedown depredations to the U.S. and elsewhere.
In this country, that cosa nostra network seems to be experiencing a decline in its sinister influence, thanks to vigorous law enforcement.
The same can't be said for Mycobacteria, a set of pathogens that deserves to be renamed "Mafiabacteria." Its "don" species is M. tuberculosis (TB), now - with the AIDS epidemic - in recrudescence, following decades of TB control by antibiotics. Another well-known branch of the bacterial mafioso family, M. leprae, wreaks leprosy on human populations, but its prevalence is minimal, at least in industrialized countries.
And then there are M. marinarum and M. ulcerans, both of which cause horrendous, though nonfatal, skin diseases. The former's medical name is swimming-pool granuloma. It attacks mainly ankles, knees and elbows bruised by the rough concrete of infected pools, or by ocean beach surfaces - especially in Hawaii.
The site of that bruise becomes red and inflamed, then breaks into an open ulcer, which spreads down below the epidermal top layer of skin. It may heal in a couple of years, or persist chronically for a decade or more.
From Hawaiian beaches to the banks of the Nile, the scene shifts to M. ulcerans, perpetrator of Buruli ulcer. This affliction owes its name to a district of Uganda where one branch of the Nile flows through a lake. But Buruli ulcer is also a serious disease in coastal Australia. It's thought to follow puncture of the skin by a thorn or insect bite, and tends to strike families, usually children in good health.
Pathogenic microbiologist Pamela Small described the typical stigmata of Buruli ulcer as "open lesions that look very clean. No pus. And that is really a striking feature, where the top skin has just sloughed off, and what you're seeing is underlying tissue. And these sores are painless. They may measure anywhere from the size of a quarter to that of a dinner plate, and can extend to encompass a whole limb. The only treatment for the disease is surgery, excision and skin grafting."
"Right now," Small observed, "the major foci of Buruli ulcer are in West Africa - Ghana, Cote d'Ivoire, Benin and Togo. In fact, within Africa, the epidemiology has shifted from Uganda, and the disease appears to be increasing in severity. It also occurs in South America, and probably in a lot of tropical countries. It's been best studied in Africa and Australia."
Small serves on a task force that the World Health Organization (WHO) has recently set up to track the M. ulcerans disease.
Small is acting head of the Facultative Intracellular Bacteria Unit at the Rocky Mountain Laboratories in Hamilton, Mont. - a field site of the National Institute of Allergy and Infectious Diseases (NIAID). She pointed out that "although it's the title of my unit, this mycobacterium, uniquely, is not intracellular but extracellular. Other mycobacteria get into macrophages and do something that enables to live in them. Mycobacterium ulcerans is probably picked up by a macrophage and kills it."
Seeking A Possibly Nonexistent Toxin
Small came to the Rocky Mountain Laboratories about seven years ago, slated to work on M. tuberculosis. "At that time," she recalled, "there was some interest in the fact that TB might contain a cytotoxic molecule. When you have active pulmonary tuberculosis, there's a lot of necrotic tissue destruction In the lung. I wanted to start on a mycobacterial pathogen that caused disease in healthy - not immunocompromised - humans. I thought maybe I could eventually transfer what I learned from this to TB, and it would be useful."
So Small turned to M. ulcerans, and its suspected cytotoxic molecule. Hunting for a toxin in this organism, she remembered a toxin tip-off - that its skin damage occurred at some distance from the site of bacterial entry.
She spent the first three years at Hamilton trying to isolate this putative toxin from its culture medium, and testing its cytotoxic activity in vitro. "I had made a lot of progress in identifying what the molecule did," she recalled, "but very little in terms of purifying it. We were trying unsuccessfully to clone it."
Enter post-doctoral fellow Kathleen George, who had come out of a TB lab, where she'd learned how to isolate lipids. "Nearly all natural toxins are proteins; and hence clonable," Small observed. "George proved this one to be lipophilic rather than proteinaceous. That was a major breakthrough, and why I made her first author of our paper in the current issue of Science [dated Feb. 5, 1999.]." Its title is "Mycolactone: A polyketide toxin from Mycobacterium ulcerans required for virulence."
Guinea Pigs Served As Guinea Pigs
"We injected it into animals," Small told BioWorld Today, "and we saw that this fatty-type molecule, which we called mycolactone, not only caused toxic effects on cells but actually reproduced most of the pathology of Buruli ulcer on the shaved backs of our guinea pig models, without causing them any pain." But a mutant bacterium, lacking the toxin, did their skin no harm.
The co-authors knew that their toxin consisted of carbon, hydrogen and oxygen. "But we really didn't know what was attached to what," Small recalled. "And the molecule looked so strange that when I showed it the chemists, one of them said, 'Nothing with that structure could possibly cause these effects.' All I knew was that we had a single molecule and it did cause those effects. One clue was that, because there was so much oxygen in it, maybe mycolactone wasn't just a fat. Maybe it was a particular type of fatty molecule called polyketides."
At that point she contacted a specialist on polyketides, Leonard Katz at Abbott Laboratories, in Abbott Park, Ill. On a pro bono basis, he invited a natural-products chemist at Abbott, Geewananda Gunawardana, to analyze Small's molecule.
"So I sent it to him," she recalled. "That same day he replied by e-mail: 'This molecule is definitely a macrolide.' And Gunawardana was able to determine its complete complex structure quite quickly." The chemical formula is C44H70O9.
"Macrolides are a kind of magic molecule, derived from polyketides," Small said. "They include antibiotics, immunosuppressants, antifungal and anti-helmetic agents. Some of these compounds, which are so bioactive, have activities similar to the ones we had found in our mycolactone."
She concluded: "When you have a toxin, there's always a potential for developing an antitoxin as a vaccine. So, I'd like to work with the World Health Organization, and see if somehow we can get funding to explore this area."