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Peptide Prospecting of Komodo Dragon Plasma IDs Possible Antimicrobial Agents


NEW YORK (GenomeWeb) – Researchers from George Mason University have identified 48 antimicrobial peptides in the plasma of Komodo dragons.

Detailed in a study published last month in the Journal of Proteome Research, the peptides were discovered using a mass spec-based "bioprospecting" approach developed by the GMU scientists, and could prove useful in fighting infectious diseases.

In a test of eight of the identified peptides, the researchers determined that seven of them showed antimicrobial activity against both Pseudomonas aeruginosa and Staphylococcus aureus, with one effective against only P. aeruginosa.

Barney Bishop, associate professor of protein biochemistry at GMU and first author on the paper, noted as well that many of the antimicrobial peptides he and his colleagues have discovered in this and other work appear to be effective in antibiotic-resistance bacterial strains.

As the authors wrote, cationic antimicrobial peptides (CAMPs) are key components of the immune systems of higher animals and, as such, represent a potential source of new antimicrobial agents. However, isolating and identifying these molecules is challenging due to their low abundance, the often small size of available samples, and the need to analyze them in their native, intact form without trypsin digestion.

To tackle these challenges, Bishop and his colleague Monique van Hoek, an associate professor at GMU's National Center for Biodefense and Infectious Diseases and senior author on the JPR study, developed a middle-down proteomics approach that uses hydrogel nanotechnology based on materials developed at GMU — and currently being commercialized by GMU spin-out Ceres Nanoscience — to capture CAMP-like peptides in their native form followed by de novo mass spec sequencing using electron-transfer dissociation. They then use web-based prediction tools and sequence analyses to identify likely CAMPs based on attributes like size, charge, and similarity to other potential CAMPs. Once they have identified likely CAMPs, they then synthesize them for further testing.

The researchers first put forth this "bioprospecting" workflow in a 2015 PLOS One paper in which they identified 45 potentially antimicrobial peptides from American alligator plasma, which is known to have antimicrobial properties. Various aspects of the Komodo dragon lifestyle suggested that it, too, could be a rich source of CAMPs.

"They live in an environment that's challenging from a bacterial standpoint," he said. "They eat carrion. Their oral cavities have a bunch of bacteria, over 57 strains, at least in one animal tested, the majority of which can be pathogenic. Yet, despite bleeding gums and wounds from fights with each other, they appear to be doing just fine."

Using hydrogel microparticles seeded with baits with negatively charged acidic groups to attract the cationic peptides most likely to be CAMPs, the researchers collected and sequenced 193 peptides in unstimulated plasma and 259 in plasma treated with lipopolysaccharide, which stimulates immune responses.

They then used their method to analyze these molecules, identifying a total of 48 candidates likely to have antimicrobial activity.

Interestingly, Van Hoek said, 47 of these 48 peptides were histone-derived.

"That was surprising to us," she said, noting that other histone-derived peptides have been identified previously, including from rainbow trout skin secretions and the stomach lining of the toad Bufo gargarizans, but that these peptides appeared to have little in common structurally with those identified in the JPR study.

"Our scripts are designed to take the sequences that we identified from the mass-spec data and [characterize] their physical and chemical properties," Bishop said. "And these scripts also take the same sequences and query them with prediction algorithms that are based on databases of [antimicrobial] peptides. The script then compiles the data into a unified spreadsheet that compares what the prediction algorithms [say] are probably anti-microbial, versus [those identified by] physical-chemical properties."

"The algorithms have their shortcomings," he added, noting that he and his colleagues are looking for hits based not on just one database or algorithm, but multiple.

Once the researchers identify the peptides they are interested in exploring further, they generate them synthetically. This ensures that they can have a sufficient supply of them for testing, a key consideration given that they are typically present in low abundance and that, in the case of endangered species like the Komodo dragon, samples are limited.

More generally, the low samples requirement is a key advantage of the GMU approach, Bishop suggested.

Some previously developed proteomic "bioprospecting" approaches have tried to compensate for the challenges of identifying and the testing low abundance CAMP candidates by starting with very large sample sizes, but this can limit the organisms available to such an analysis.

"We're using around 100 microliters of plasma," Bishop said. "So we're not drawing a lot of blood from the animal."

Van Hoek also noted that this approach opens up their technique to very small organisms, "where it might be possible to get a blood sample but not of a very large volume."

Bishop said it was too early to say how the molecules they have discovered will compare to conventional antibiotics, adding that, ultimately, a treatment would likely consist of agents inspired by these peptides, or combinations of multiple peptides, or peptides and existing drugs.

He noted that one significant finding was that many of the peptides they identified in their reptile work have shown activity against antibiotic-resistant bacterial strains.

Specifically, Van Hoek said that alligator peptides had shown activity against bacteria including multi-drug resistant Acinetobacter, Pseudomonas, and methicillin-resistant Staphylococcus aureus (MRSA).

She said the researchers are now preparing to test the peptides they have identified in models, including animal models, to show they have activity against infections and generate evidence supporting clinical testing of the agents.