"Armed and dangerous. Shoot to kill!"
That alert — familiar to moviegoers and cops — warns of a convict who breaks jail by stealing a guard's weapon and gunning him down before escaping. It has striking parallels to the tuberculosis (TB) pathogen, Mycobacterium tuberculosis. This microbial murderer slips out of the macrophage cell, where it's been holed up for years, and goes on a lethal rampage of infecting healthy cells in the body.
The antibiotic guards that have held the TB bug in check for decades are now succumbing to the bacterium's multidrug resistance genes. So tuberculosis is re-emerging worldwide as a major infectious-disease threat. (See BioWorld Today, June 8, 1998, p. 1.)
Relief may be on the way from an unsuspected source. Not just one more antibiotic compound, but the immune system's own antibacterial protein, granulysin by name.
Stanford immunology researcher Alan Krensky identified the protein a decade ago in two of the body's pathogen-wasting cells — cytotoxic T lymphocytes (CTLs) and natural killer cells (NKs). "But it was only a couple of years ago," Krensky told BioWorld Today, "that we learned it was a killer molecule, which we named granulysin."
Just how granulysin does its pathogen-cleansing work is still on Krensky's drawing board at Stanford University, and on the drawing board of his close collaborator, dermatologist and microbiologist Robert Modlin, at the University of California/Los Angeles (UCLA).
"We've just started on those killing-mechanism studies," Krensky said, "and we already know three different ways granulysin does it. One is the fatty acid ceramide, a known inducer of cell killing. The second activates the caspase enzymes, which in turn activate apoptosis — programmed cell death.
"Thirdly, granulysin directly damages lipid membranes, not by poking holes in them but deforming their surface. All three modes," he observed, "are relevant to microbes, such as M. tuberculosis, and some to different pathogenic systems, such as tumors and fungi."
UCLA's Modlin is senior author, and Krensky a co-author, of a paper in today's Science, dated Oct. 2, 1998. It bears the title: "An antimicrobial activity of cytolytic T cells mediated by granulysin."
In five in vitro experiments, the co-authors cultured M. tuberculosis strains, together with escalating dosages of recombinant granulysin. It killed up to 90 percent of the bacteria within 72 hours. However, the same titers of granulysin bounced harmlessly off the TB bacilli, infecting their favorite hide-out cells, phagocytic macrophages.
Assuming that the reason for this failure was inability of the antibacterial protein to penetrate the intracellular compartment, the co-authors summoned another protein, perforin, to the scene. This molecule, which also inhabits the T-cell granules that harbor granulysin, pokes holes in bacterial membranes. By combining the two proteins in a single killer package, the team succeeded in breaking and entering the bacterium's safe house, and significantly reducing its viability.
TB Only One Of Many Target Germs
Numerous other microbes besides the one inflicting tuberculosis hole up in intracellular compartments: Listeria monocytogenes causes life-threatening meningitis, endocarditis and blood-poisoning; and Trypanosoma cruzi wreaks Chagas' disease in South America. Both escape from the phagocytic vacuoles into the cytoplasm of infected hosts. Staphylococcus aureus (toxic shock), Salmonella typhimurium and Escherichia coli (both food poisoning) are other armed and dangerous escapee pathogens.
In these and other bacteria, the co-authors report that recombinant granulysin "caused a three-orders-of-magnitude reduction in the microbes' population. It also killed pathogenic fungi and parasites."
What about viruses, especially HIV, which infects T cells, the very home of granulysin?
"We're very interested in granulysin's effect on HIV," observed Krensky. "There are preliminary results that it has some activity on HIV isolates, but it's really much too early to say anything definitive. However, we're certainly looking into that. We're also interested in its possibile usefulness in treating other diseases, such as cancer."
A single CTL or NK cell contains thousands of granulysin-harboring granules. They resemble the vesicles that ship messenger chemicals across nerve synapses.
"Granulysin is the only antimicrobial protein from T cells that's known," Modlin told BioWorld Today. "In a single granule, besides the granulysins, are two other active molecules tightly packed together. They contain perforin, the pore-forming molecule, and certain proteins called granzymes. After the perforins form a pore in the target cell, the granzymes enter, start cleaving other proteins, and then initiate that cell's programmed death by apoptosis.
"We have filed a patent application on granulysin," Modlin said, "and one of the possibilities is that it would be developed as an antimicrobial agent. The other application: that it would serve as an important marker of protective immunity — a vaccine — in response to a pathogen."
In Offing: Start A Company Or Find A Licensee
"We don't know that [it would], yet; but it's the direction we want to go to determine if granulysin is required for protection, and we want to see if it can be developed as an antimicrobial therapeutic."
As a next step, Modlin continued, "We need to identify the mouse gene, so we can do the in vivo stuff. It's important for us to make a gene knockout mouse, but also we would like to attempt to use it as a murine model for treating infection." Human granulysin resides on chromosome 2.
"There are two things we want to do in vivo," Modlin summed up. "First, knock out the granulysin gene in mice and see if they're more susceptible to infection. Second, infect mice, and then figure out a way to target granulysin to the site of infection and see if that resolves it.
"Nothing is planned yet," he added. "We're contemplating either starting a company or hoping to interest an existing company in forming a partnership so we'd be able to do those in vivo experiments. And we're hoping that our paper being in Science will receive some attention."
Meanwhile, one month ago, Stanford's Office of Technology Licensing mailed out a message to 50 companies active in the field of infectious diseases. It summarized the granulysin data now reported in Science, and offered licenses to the pending patents. The licensing liaison officer may be reached at (650) 725-9407, or firstname.lastname@example.org. *