Growing up in east Texas, Mark Merchant spent much of his youth hunting and fishing in marshes that alligators called home.
Now, he is looking to the reptiles as a potential source for therapeutics against a host of microbial infections for which modern medicine seems to have no answer.
Using proteomics technology, Merchant, an assistant professor at McNeese State University, and colleagues at Louisiana State University are studying whether proteins in alligator serum can be turned into treatments for such pathogens as methicillin-resistant Staphylococcus aureus and Candida albicans, and infections associated with diabetic ulcers and severe burns.
Preliminary studies have also shown that alligator proteins have some efficacy against a strain of HIV.
While any therapy based on alligator blood protein is still many years away, the work of Merchant and his co-researchers has drawn interest from pharmaceutical companies, Merchant told ProteoMonitor.
Growing up on the Gulf Coast and now living along the Louisiana bayou, Merchant said he has been around alligators all his life. In the wild, alligators with missing limbs are a common sight, he said. Territorial and aggressive by nature, the reptiles get into nasty smackdowns during which legs, arms, and tails get bitten and torn off. But in spite of their injuries and microbe-rich marshland habitat, they rarely develop serious infections.
Merchant wanted to know why: Did alligator blood kill bacteria?
“The answer was, yes, and it kills all kinds of bacteria,” Merchant said. “And it kills them very rapidly, it kills them without prior exposure, it kills them in a concentration-dependent manner … and it kills them in a temperature-dependent manner.”
At last week’s American Chemical Society’s spring meeting in New Orleans, Merchant’s collaborator Lancia Darville, a graduate student in chemistry at LSU, said that while humans exhibit some of these traits — their bodies can fight off some pathogens without first having been previously exposed to them — alligators are more highly evolved in that respect.
“For example, as humans, usually we have to be exposed to, let’s say, the flu virus in order for our bodies to learn how to fight that flu virus off,” Darville said at a press conference at the meeting. “Whereas the gators, they tend to be exposed to something and their bodies just know exactly what to produce to fight that off.”
It turns out alligator blood contains serum complement, a group of proteins that “acts coordinately in a cascade-like fashion to kill microbes in a non-specific manner,” Merchant said.
Alligator blood “kills all kinds of bacteria, and it kills them very rapidly, it kills them without prior exposure, it kills them in a concentration-dependent manner … and it kills them in a temperature-dependent manner.”
While this complement works fine for the alligator, its proteins are highly unstable and would have “zero shelf life” if used to develop therapeutics for humans, Merchant said. Further, if the serum complement were to enter the human body, it “would elicit a tremendous immune response from the host,” which could turn lethal, Merchant said.
So he and his colleagues turned their attention to alligator leukocytes and the cationic peptides in them. While other research had identified antimicrobial peptides from other organisms, it wasn’t clear that alligator serum contained such peptides.
But in a study published in 2006, Merchant and others reported that alligator leukocyte extracts showed “substantial antimycotic activities” against six of eight Candida yeast species, as well as antimicrobial efficacy against 10 of 12 bacterial species, and “moderate activity” against HIV-1 and herpes simplex-1.
“These activities are most likely due to the presence of cationic antimicrobial peptides,” Merchant and his co-researchers concluded in the study.
Working on that hypothesis, Merchant and his current collaborators are now trying to isolate the peptides. They were reluctant to speak in detail about their unpublished findings, but Merchant said that preliminary analysis indicates four proteins that have activity. “What we need to do is isolate these guys and start to determine which ones are responsible for which activity,” he said.
There Will Be Gator Blood
McNeese State has an alligator farm, but for this project, Merchant wanted blood samples only from alligators living in the wild. So at night, he goes out in a boat into the marshes, armed with a spotlight and a cable snare. After he’s caught one of the animals, he pulls them into the boat, sticks a needle into a spinal vein, and draws blood.
Merchant does some initial separation of the serum, but the bulk of the proteomics work is handled by Darville, who separates the proteins in leukocytes and serum by 1D- and 2D-PAGE. She then tryptically digests them, and sequences the peptides on an Applied Biosystems QSTAR XL LC-MS/MS system.
Darville also has been using cationic exchange separation, which allows for the separation of “just those cationic peptides [that] we believe are present in the alligator’s serum,” she said at the ACS meeting. “Once we’re able to separate those, ultimately, we can determine the sequence as well as the structures of these peptides, which would allow us to develop these as potential drugs.”
However, Darville warned that cationic peptides are not easily adaptable for clinical use. While they may be effective as antimicrobials, they can be toxic to humans. The first cationic peptides were discovered about 20 years ago, and though pharmaceutical firms have been researching their clinical utility, only a few have made it into clinical trials, and a very small number of treatments using them have made it to market.
Darville’s work now involves sequencing of the peptides, with a longer-term goal of determining the structure of the proteins, so that they can be mimicked as potential drugs. Kermit Murray, a professor of mass spectrometry at LSU who has been advising Darville on the project, said at the meeting last week that deciphering the peptides’ structure is “tough going,” because the reptile genome hasn’t been extensively studied.
“So when we do the bioinformatics end of the study, there’s not quite as many proteins in the database for the reptiles, alligators specifically, compared to the other organisms,” he said. “The closest well-studied organism would be the African clawed frog. So it’s a little bit of an initial feeling-about-in-the-dark.”
Darville told ProteoMonitor that she is using additional separation techniques to purify the peptides so that the sequences can be determined with greater precision.
“We may not necessarily be able to get a very specific protein hit in the database,” she said.
The researchers said that it could take a decade before a product stemming from the research hits the market, if one ever does. In addition to therapeutics targeted to humans, Merchant said that undisclosed veterinary pharma firms have also expressed interest.
“If they can help humans, why can’t it help dogs, or horses, small animal, and large animal, both as therapeutic agents for difficult bacterial agents?” he asked.