Discovery of Novel Antimicrobial Peptides from Varanus komodoensis (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 Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 25923), with seven peptides exhibiting antimicrobial activity against both microbes and one only showing significant potency against P. aeruginosa. 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.

Helical cationic antimicrobial peptide length and its impact on membrane disruption.

Cationic antimicrobial peptides (CAMPs) are important elements of innate immunity in higher organisms, representing an ancient defense mechanism against pathogenic bacteria. These peptides exhibit broad-spectrum antimicrobial activities, utilizing mechanisms that involve targeting bacterial membranes. Recently, a 34-residue CAMP (NA-CATH) was identified in cDNA from the venom gland of the Chinese cobra (Naja atra). A semi-conserved 11-residue pattern observed in the NA-CATH sequence provided the basis for generating an 11-residue truncated peptide, ATRA-1A, and its corresponding D-peptide isomer. While the antimicrobial and biophysical properties of the ATRA-1A stereoisomers have been investigated, their modes of action remain unclear. More broadly, mechanistic differences that can arise when investigating minimal antimicrobial units within larger naturally occurring CAMPs have not been rigorously explored. Therefore, the studies reported here are focused on this question and the interactions of full-length NA-CATH and the truncated ATRA-1A isomers with bacterial membranes. The results of these studies indicate that in engineering the ATRA-1A isomers, the associated change in peptide length and charge dramatically impacts not only their antimicrobial effectiveness, but also the mechanism of action they employ relative to that of the full-length parent peptide NA-CATH. These insights are relevant to future efforts to develop shorter versions of larger naturally occurring CAMPs for potential therapeutic applications.

Peptides from American alligator plasma are antimicrobial against multi-drug resistant bacterial pathogens including Acinetobacter baumannii.

BACKGROUND: Our group has developed a new process for isolating and identifying novel cationic antimicrobial peptides from small amounts of biological samples. Previously, we identified several active antimicrobial peptides from 100 µl of plasma from Alligator mississippiensis. These peptides were found to have in vitro antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In this work, we further characterize three of the novel peptides discovered using this process: Apo5, Apo6, and A1P. RESULTS: We examined the activity of these peptides against multi-drug resistant strains and clinical isolates of common human pathogens. We investigated their structural characteristics using circular dichroism and tested for membrane disruption and DNA binding. These peptides were found to have strong in vitro activity against multi-drug resistant and clinically isolated strains of S. aureus, Escherichia coli, P. aeruginosa, and Acinetobacter baumannii. Apo5 and Apo6, peptides derived from alligator apolipoprotein C-1, depolarized the bacterial membrane. A1P, a peptide from the serpin proteinase inhibitor, did not permeabilize membranes. Performing circular dichroism analysis, Apo5 and Apo6 were found to be predominantly helical in SDS and TFE buffer, while A1P has significantly different structures in phosphate buffer, SDS, and TFE. None of these peptides were found to be hemolytic to sheep red blood cells or significantly cytotoxic up to 100 µg/ml after 24 h exposure. CONCLUSIONS: Overall, we suggest that Apo5 and Apo6 have a different mode of action than A1P, and that all three peptides make promising candidates for the treatment of drug-resistant bacteria, such as A. baumannii.

Cranberry proanthocyanidins have anti-biofilm properties against Pseudomonas aeruginosa.

BACKGROUND: Bacteria within a biofilm are phenotypically more resistant to antibiotics, desiccation, and the host immune system, making it an important virulence factor for many microbes. Cranberry juice has long been used to prevent infections of the urinary tract, which are often related to biofilm formation. Recent studies have found that the A-type proanthocyanidins from cranberries have anti-biofilm properties against Escherichia coli. METHODS: Using crystal violet biofilm staining, resazurin metabolism assays, and confocal imaging, we examined the ability of A-type proanthocyanidins (PACs) to disrupt the biofilm formation of Pseudomonas aeruginosa. We used mass spectrometry to analyze the proteomic effects of PAC treatment. We also performed synergy assays and in vitro and in vivo infections to determine whether PACs, alone and in combination with gentamicin, could contribute to the killing of P. aeruginosa and the survival of cell lines and G. mellonella. RESULTS: Cranberry PACs reduced P. aeruginosa swarming motility. Cranberry PACs significantly disrupted the biofilm formation of P. aeruginosa. Proteomics analysis revealed significantly different proteins expressed following PAC treatment. In addition, we found that PACs potentiated the antibiotic activity of gentamicin in an in vivo model of infection using G. mellonella. CONCLUSIONS: Results suggest that A-type proanthocyanidins may be a useful therapeutic against the biofilm-mediated infections caused by P. aeruginosa and should be further tested.

Cathelicidin antimicrobial peptide from Alligator mississippiensis has antibacterial activity against multi-drug resistant Acinetobacter baumanii and Klebsiella pneumoniae.

Alligator mississippiensis (American alligator), a member of order Crocodilia, lives in bacteria-laden environments but is not often known to succumb to bacterial infections. Their serum has been shown to have antibacterial activity beyond that of human serum, and it is believed that this activity is partially due to cationic antimicrobial peptides (CAMPs). CAMPs are produced by many organisms as part of the innate immune system. CAMPs are attractive possible therapies against multi-drug resistant bacteria, such as those found in biofilm-infected war wounds, because they seldom cause genetic resistance in bacteria and are effective against antibiotic resistant bacteria. In this work, we identified, synthesized, and characterized a cathelicidin and two shorter fragments from the American alligator. We discovered the cathelicidin using Basic Local Alignment Search Tool (BLAST) alignment and by comparing A. mississippiensis expressed sequence tags (ESTs) with propeptide cathelicidins of other reptiles. We analyzed the structure using bioinformatics tools and circular dichroism and predicted that the full-length cathelicidin peptide has a mixed structure, with an N-terminal α-helix and a center Pro hinge. In minimal inhibitory concentration (MIC) assays, it was determined that the cathelicidin and the two shorter fragments have strong activity against multiple Gram-negative bacteria, including clinical isolates of multi-drug resistant (MDR) Acinetobacter baumannii and carbapenem-resistant Klebsiella pneumoniae. Using the ethidium bromide uptake assay, it was found that these peptides permeabilize the bacterial membrane and are less sensitive to salt inhibition than many other known CAMPs. The alligator cathelicidin peptides were not hemolytic against sheep red blood cells at 300 µg/ml and were not significantly cytotoxic against A549 human lung epithelial cells after 24 h exposure in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. These alligator cathelicidin peptides have activity similar to other CAMPs from reptiles such as NA-CATH. It is possible that the alligator cathelicidins play an important role in the innate immune response of A. mississippiensis, similar to LL-37 in humans. In addition, due to their activities against MDR bacteria and lack of cytotoxicity, the AM-CATH peptides could be an attractive platform for further development as a potential therapeutic.

Snake Cathelicidin NA-CATH and Smaller Helical Antimicrobial Peptides Are Effective against Burkholderia thailandensis.

Burkholderia thailandensis is a Gram-negative soil bacterium used as a model organism for B. pseudomallei, the causative agent of melioidosis and an organism classified category B priority pathogen and a Tier 1 select agent for its potential use as a biological weapon. Burkholderia species are reportedly "highly resistant" to antimicrobial agents, including cyclic peptide antibiotics, due to multiple resistance systems, a hypothesis we decided to test using antimicrobial (host defense) peptides. In this study, a number of cationic antimicrobial peptides (CAMPs) were tested in vitro against B. thailandensis for both antimicrobial activity and inhibition of biofilm formation. Here, we report that the Chinese cobra (Naja atra) cathelicidin NA-CATH was significantly antimicrobial against B. thailandensis. Additional cathelicidins, including the human cathelicidin LL-37, a sheep cathelicidin SMAP-29, and some smaller ATRA peptide derivatives of NA-CATH were also effective. The D-enantiomer of one small peptide (ATRA-1A) was found to be antimicrobial as well, with EC50 in the range of the L-enantiomer. Our results also demonstrate that human alpha-defensins (HNP-1 & -2) and a short beta-defensin-derived peptide (Peptide 4 of hBD-3) were not bactericidal against B. thailandensis. We also found that the cathelicidin peptides, including LL-37, NA-CATH, and SMAP-29, possessed significant ability to prevent biofilm formation of B. thailandensis. Additionally, we show that LL-37 and its D-enantiomer D-LL-37 can disperse pre-formed biofilms. These results demonstrate that although B. thailandensis is highly resistant to many antibiotics, cyclic peptide antibiotics such as polymyxin B, and defensing peptides, some antimicrobial peptides including the elapid snake cathelicidin NA-CATH exert significant antimicrobial and antibiofilm activity towards B. thailandensis.

Bioprospecting the American alligator (Alligator mississippiensis) host defense peptidome.

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, Alligator mississippiensis, 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 de novo 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 Alligator mississippiensis plasma demonstrates the potential of this innovative discovery process in identifying potential new host defense peptides.