Mass Spectral Verification of CAMP Harvesting by Hydrogel Particles.

<p>Mass spectra of particle eluents following harvesting from of American alligator plasma spiked with 3 known CAMPs (Buforin, SMAP-29 and Indolicidin). The CAMP peaks in the spectra are identified and labeled with their corresponding identity.</p

CAMP prediction results.

<p>Using 3 different web-based CAMP prediction applications (<i>CAMP database, AntiBP2 and APD2</i>) each peptide was scored and given a prediction of whether it would have antimicrobial activity (AMP) or not (Non-AMP).</p><p>* <i>AntiBP2</i> requires the sequence length of ≥ 15 amino acids for a prediction score.</p><p><i>CAMP</i> database: <a href="http://www.camp.bicnirrh.res.in/" target="_blank">http://www.camp.bicnirrh.res.in/</a>.</p><p><i>AntiBP2:</i><a href="http://www.imtech.res.in/raghava/antibp2/" target="_blank">http://www.imtech.res.in/raghava/antibp2/</a>.</p><p><i>APD2:</i><a href="http://aps.unmc.edu/AP/prediction/prediction_main.php" target="_blank">http://aps.unmc.edu/AP/prediction/prediction_main.php</a>.</p><p>CAMP prediction results.</p

Sequences and Physico-chemical Properties of Novel Alligator CAMPs.

<p>The physico-chemical properties for eight novel alligator CAMPs identified via the process. The peptide name is determined based on the parent protein with the amino acid sequence numbers in the subscript.</p><p>* These peptides were <i>de novo</i> identified.</p><p>Sequences and Physico-chemical Properties of Novel Alligator CAMPs.</p

Bioprospecting the American Alligator (<i>Alligator mississippiensis</i>) Host Defense Peptidome

<div><p>Cationic antimicrobial peptides and their therapeutic potential have garnered growing interest because of the proliferation of bacterial resistance. However, the discovery of new antimicrobial peptides from animals has proven challenging due to the limitations associated with conventional biochemical purification and difficulties in predicting active peptides from genomic sequences, if known. As an example, no antimicrobial peptides have been identified from the American alligator, <i>Alligator mississippiensis</i>, although their serum is antimicrobial. We have developed a novel approach for the discovery of new antimicrobial peptides from these animals, one that capitalizes on their fundamental and conserved physico-chemical properties. This sample-agnostic process employs custom-made functionalized hydrogel microparticles to harvest cationic peptides from biological samples, followed by <i>de novo</i> sequencing of captured peptides, eliminating the need to isolate individual peptides. After evaluation of the peptide sequences using a combination of rational and web-based bioinformatic analyses, forty-five potential antimicrobial peptides were identified, and eight of these peptides were selected to be chemically synthesized and evaluated. The successful identification of multiple novel peptides, exhibiting antibacterial properties, from <i>Alligator mississippiensis</i> plasma demonstrates the potential of this innovative discovery process in identifying potential new host defense peptides.</p></div

Antibacterial Performance Data for Alligator CAMPs.

<p>Antibacterial activities against <i>E. coli, B. cereus, P. aeruginosa</i> and <i>S. aureus</i> are expressed in terms of EC₅₀ (µM) values with corresponding 95% confidence interval (CI) range. The human CAMP LL-37 is used as a standard for assessing antibacterial performance [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117394#pone.0117394.ref030" target="_blank">30</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117394#pone.0117394.ref031" target="_blank">31</a>]. <b>NA</b> = no significant activity at the highest peptide concentration tested against the listed bacteria.</p><p>Antibacterial Performance Data for Alligator CAMPs.</p

Potencies of LL-37 and Five Novel Alligator CAMPs.

<p>Comparison of the antibacterial effectiveness of each peptide against <i>E. coli, B. cereus, P. aeruginosa</i> and <i>S. aureus</i>, expressed in terms of EC₅₀ (µM) values. Bacterial survival results generated for each CAMP are fit to a variable-slope sigmoidal regression model to reveal bacterial survival curves to ascertain EC₅₀ values.</p

Mass Spectral Verification of CAMP Harvesting by Hydrogel Particles.

<p>Mass spectra of particle eluents following harvesting from of American alligator plasma spiked with 3 known CAMPs (Buforin, SMAP-29 and Indolicidin). The CAMP peaks in the spectra are identified and labeled with their corresponding identity.</p

Bioprospecting Approach to CAMP discovery.

<p>(A) Hydrogel microparticles are introduced into the plasma sample. (B) The particles capture small cationic peptides which are present in the sample, while excluding high molecular weight proteins. (C) The particles are then recovered, (D) captured low molecular weight peptides are eluted from the particles and (E) analyzed by high-resolution MS/MS.</p

Discovery of Novel Antimicrobial Peptides from <i>Varanus komodoensis</i> (Komodo Dragon) by Large-Scale Analyses and De-Novo-Assisted Sequencing Using Electron-Transfer Dissociation Mass Spectrometry

Komodo dragons are the largest living lizards and are the apex predators in their environs. They endure numerous strains of pathogenic bacteria in their saliva and recover from wounds inflicted by other dragons, reflecting the inherent robustness of their innate immune defense. We have employed a custom bioprospecting approach combining partial de novo peptide sequencing with transcriptome assembly to identify cationic antimicrobial peptides from Komodo dragon plasma. Through these analyses, we identified 48 novel potential cationic antimicrobial peptides. All but one of the identified peptides were derived from histone proteins. The antimicrobial effectiveness of eight of these peptides was evaluated against <i>Pseudomonas aeruginosa</i> (ATCC 9027) and <i>Staphylococcus aureus</i> (ATCC 25923), with seven peptides exhibiting antimicrobial activity against both microbes and one only showing significant potency against <i>P. aeruginosa</i>. This study demonstrates the power and promise of our bioprospecting approach to cationic antimicrobial peptide discovery, and it reveals the presence of a plethora of novel histone-derived antimicrobial peptides in the plasma of the Komodo dragon. These findings may have broader implications regarding the role that intact histones and histone-derived peptides play in defending the host from infection. Data are available via ProteomeXChange with identifier PXD005043