Cathelicidin-like Peptide Research Articles

Aquiluscidin, a Cathelicidin from Crotalus aquilus, and the Vcn-23 Derivative Peptide, Have Anti-Microbial Activity against Gram-Negative and Gram-Positive Bacteria.

Anti-microbial peptides play a vital role in the defense mechanisms of various organisms performing functions that range from the elimination of microorganisms, through diverse mechanisms, to the modulation of the immune response, providing protection to the host. Among these peptides, cathelicidins, a well-studied family of anti-microbial peptides, are found in various animal species, including reptiles. Due to the rise in anti-microbial resistance, these compounds have been suggested as potential candidates for developing new drugs. In this study, we identified and characterized a cathelicidin-like peptide called Aquiluscidin (Aq-CATH) from transcripts obtained from the skin and oral mucosa of the Querétaro's dark rattlesnake, Crotalus aquilus. The cDNA was cloned, sequenced, and yielded a 566-base-pair sequence. Using bioinformatics, we predicted that the peptide precursor contains a signal peptide, a 101-amino-acid conserved cathelin domain, an anionic region, and a 34-amino-acid mature peptide in the C-terminal region. Aq-CATH and a derived 23-amino-acid peptide (Vcn-23) were synthesized, and their anti-microbial activity was evaluated against various species of bacteria in in vitro assays. The minimal inhibitory concentrations against bacteria ranged from 2 to 8 μg/mL for both peptides. Furthermore, at concentrations of up to 50 μM, they exhibited no significant hemolytic activity (<2.3% and <1.2% for Aquiluscidin and Vcn-23, respectively) against rat erythrocytes and displayed no significant cytotoxic activity at low concentrations (>65% cell viability at 25 µM). Finally, this study represents the first identification of an antimicrobial peptide in Crotalus aquilus, which belongs to the cathelicidin family and exhibits the characteristic features of these peptides. Both Aq-CATH and its derived molecule, Vcn-23, displayed remarkable inhibitory activity against all tested bacteria, highlighting their potential as promising candidates for further antimicrobial research.

In vitro efficacy of different PEGylation designs on cathelicidin-like peptide with high antibacterial and antifungal activity

Recent reports on antibiotic resistance have highlighted the need to reduce the impact of this global health issue through urgent prevention and control. The World Health Organization currently considers antibiotic resistance as one of the most dangerous threats to global health. Therefore, Antimicrobial peptides (AMPs) are promising for the development of novel antibiotic molecules due to their high antimicrobial effects, non-inducing antimicrobial resistance (AMR) properties, and broad spectrum. Hence, in this study, we developed novel antimicrobial peptide/polymer conjugates to reduce the adverse effects of TN6 (RLLRLLLRLLR) peptide. We demonstrate how our constructs function in vitro in terms of antimicrobial activity, hemolytic activity, cytotoxicity, and protease resistance. Our findings show that our molecules are effective against different types of microorganisms such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant S. aureus, vancomycin-resistant Enteroccus faecium, and Candida albicans, which are known to be pathogenic and antibiotic-resistant. Our constructs generally showed low cytotoxicity relative to the peptide in HaCaT and 3T3 cells. Especially these structures are very successful in terms of hemotoxicity. In the bacteremia model with S. aureus, the naked peptide (TN6) was hemotoxic even at 1 µg/mL, while the hemotoxicity of the conjugates was considerably lower than the peptide. Remarkably in this model, the hemolytic activity of PepC-PEG-pepC conjugate decreased 15-fold from 2.36 to 31.12 µg/mL compared to the bacteria-free 60-min treatment. This is proof that in the case of bacteremia and sepsis, the conjugates specifically direct to bacterial cell membranes rather than red blood cells. In addition, the PepC-PEG-pepC conjugate is resistant to plasma proteases. Moreover, morphological and intracellular damage of the peptide/conjugates to Escherichia coli are demonstrated in SEM and TEM images. These results suggest our molecules can be considered potential next-generation broad-spectrum antibiotic molecule/drug candidates that might be used in clinical cases such as bacteremia and sepsis.

Physicochemical-guided design of cathelicidin-derived peptides generates membrane active variants with therapeutic potential

The spread of multi-drug resistance and the slow pace at which antibiotics come onto the market are undermining our ability to treat human infections, leading to high mortality rates. Aiming to overcome this global crisis, antimicrobial peptides are considered promising alternatives to counter bacterial infections with multi-drug resistant bacteria. The cathelicidins comprise a well-studied class of AMPs whose members have been used as model molecules for sequence modifications, aiming at enhanced biological activities and stability, along with reduced toxic effects on mammalian cells. Here, we describe the antimicrobial activities, modes of action and structural characterization of two novel cathelicidin-like peptides, named BotrAMP14 and CrotAMP14, which were re-designed from snake batroxicidin and crotalicidin, respectively. BotrAMP14 and CrotAMP14 showed broad-spectrum antibacterial activity against susceptible microorganisms and clinical isolates with minimal inhibitory concentrations ranging from 2–35.1 μM. Moreover, both peptides had low cytotoxicity against Caco-2 cells in vitro. In addition, in vivo toxicity against Galleria mellonella moth larvae revealed that both peptides led to>76% larval survival after 144 h. Microscopy studies suggest that BotrAMP14 and CrotAMP14 destabilize E. coli membranes. Furthermore, circular dichroism and molecular dynamics simulations indicate that, in a membrane-like environment, both peptides adopt α-helical structures that interact with bilayer phospholipids through hydrogen bonds and electrostatic interaction. Thus, we concluded that BotrAMP14 and CrotAMP14 are helical membrane active peptides, with similar antibacterial properties but lower cytotoxicity than the larger parent peptides batroxicidin and crotalicidin, having advantages for drug development strategies.

Structural determinants conferring unusual long life in human serum to rattlesnake-derived antimicrobial peptide Ctn[15-34

Ctn[15-34], a downsized version of the snake venom cathelicidin-like peptide crotalicidin (Ctn), shows an unusually high lifespan (t1/2 , approximately 12 h) in human serum, which significantly adds to its promise as an antimicrobial and antitumor agent. Herein we investigate the role of Ctn[15-34] structure on serum survival. Using a set of analogs, we show that C-terminal amidation, as well as the specific layout of the Ctn[15-34] sequence-a helical N-terminal domain followed by a hydrophobic domain-is crucial for slow degradation, and any change in their arrangement results in significantly lower t1/2 . Aside from the privileged primary structure, features such as self-aggregation can be ruled out as causes for the long serum life. Instead, studies in other protease-rich fluids suggest a key role for certain human serum components. Finally, we demonstrate that Ctn[15-34] is able to induce bacterial death even after 12-hour pre-incubation in serum, in agreement with the proteolytic data. Altogether, the results shed light on the uncommon stability of Ctn[15-34] in human serum and confirm its potential as an anti-infective lead.

The first identified cathelicidin from tree frogs possesses anti-inflammatory and partial LPS neutralization activities

As of February 2017, approximately 7639 amphibian species have been described in the AmphibiaWeb database. However, only 20 cathelicidin-like antimicrobial peptides have been identified to date from 10 amphibian species. Half of these peptides were identified from genome sequences and have not yet been functionally characterized. In this study, a novel cathelicidin-like peptide designated cathelicidin-PP was purified from the skin of tree frog Polypedates puerensis. Cathelicidin-PP is a 32 residue peptide of sequence ASENGKCNLLCLVKKKLRAVGNVIKTVVGKIA. Circular dichroism spectroscopy indicated that cathelicidin-PP mainly adopts a β-sheet structure in membrane-mimetic solutions. Cathelicidin-PP exhibits potent antimicrobial activity against bacteria and fungi, especially Gram-negative bacteria. Meanwhile, it shows low cytotoxicity toward mammalian cells. Scanning electron microscopy analysis indicated that cathelicidin-PP kills bacteria through the disruption of the bacterial cell membrane integrity. Furthermore, cathelicidin-PP exerts significant anti-inflammatory functions by inhibiting the lipopolysaccharide (LPS)-mediated generation of nitric oxide and pro-inflammatory cytokines, tumor necrosis factor-α, interleukin-1β, and interleukin-6. The MAPKs (ERK, JNK, and p38) and NF-κB signaling pathways are involved in the anti-inflammatory effect. Cathelicidin-PP caused partial neutralization of LPS in a dose-dependent manner. Quantitative PCR indicated that infection of tree frogs with bacteria causes increased expression of cathelicidin-PP in immune-related tissues. Taken together, cathelicidin-PP is the first identified cathelicidin-like peptide from tree frogs. Our findings demonstrate that in addition to direct bactericidal capacity, cathelicidin-PP also possesses immunomodulatory properties, including partial neutralization of LPS, and inhibiting the production of inflammatory cytokines.

WS07.5 Novel antimicrobial peptides to fight infections in patients with cystic fibrosis

Objectives: In view of the current limitations to treat and reduce pulmonary infections and chronic inflammation in Cystic Fibrosis (CF) patients effectively, we explored the potential of a novel therapeutic approach based upon antimicrobial peptides (AMPs). Novel cathelicidin-like peptides have been designed and were tested for their antimicrobial and cytotoxic properties in vitro and ex vivo. Methods: Several well-established microbiological assays (colony counts, bioscreen kinetics) were applied using different media to test the killing efficacy of the different peptide designs against several isolates of CF-relevant bacterial and fungal species. Different mammalian cell culture systems (bronchial cell lines, primary human nasal cells) were used to screen for toxicity using the WST-1 conversion assay (cell viability). Results: Newly developed AMPs were found to display extraordinary strong antimicrobial activities against several clinical isolates of multi-resistant Pseudomonas aeruginosa and Staphylococcus aureus and were shown to be effective against pathogens present in CF-patient-derived pulmonary lavage and sputum. In addition, these peptides possessed strong antifungal properties against Candida albicans and Aspergillus fumigatus, two important fungal pathogens associated with CF. In vitro studies revealed that these AMPs are active under physiological conditions (in the presence of NaCl, CaCl2) and at the lower pH that typically exists in theCFlung. Furthermore, these peptides exhibitedlowcytotoxicity towards several mammalian cell types (e.g. primary human nasal epithelial cells). Conclusion: Novel cathelicidin-inspired peptides show potential as a new anti-infectious therapy to effectively reduce a broad-range of microbial infections in the upper and lower airways of patients suffering from CF. This novel therapeutic approach may help to limit pulmonary inflammation and tissue damage, and will improve clinical outcome in CF-patients.

The immune modulatory peptide FhHDM-1 secreted by the helminth Fasciola hepatica prevents NLRP3 inflammasome activation by inhibiting endolysosomal acidification in macrophages.

The NLRP3 inflammasome is a multimeric protein complex that controls the production of IL-1β, a cytokine that influences the development of both innate and adaptive immune responses. Helminth parasites secrete molecules that interact with innate immune cells, modulating their activity to ultimately determine the phenotype of differentiated T cells, thus creating an immune environment that is conducive to sustaining chronic infection. We show that one of these molecules, FhHDM-1, a cathelicidin-like peptide secreted by the helminth parasite, Fasciola hepatica, inhibits the activation of the NLRP3 inflammasome resulting in reduced secretion of IL-1β by macrophages. FhHDM-1 had no effect on the synthesis of pro-IL-1β. Rather, the inhibitory effect was associated with the capacity of the peptide to prevent acidification of the endolysosome. The activation of cathepsin B protease by lysosomal destabilization was prevented in FhHDM-1-treated macrophages. By contrast, peptide derivatives of FhHDM-1 that did not alter the lysosomal pH did not inhibit secretion of IL-1β. We propose a novel immune modulatory strategy used by F. hepatica, whereby secretion of the FhHDM-1 peptide impairs the activation of NLRP3 by lysosomal cathepsin B protease, which prevents the downstream production of IL-1β and the development of protective T helper 1 type immune responses that are detrimental to parasite survival.-Alvarado, R., To, J., Lund, M. E., Pinar, A., Mansell, A., Robinson, M. W., O'Brien, B. A., Dalton, J. P., Donnelly, S. The immune modulatory peptide FhHDM-1 secreted by the helminth Fasciola hepatica prevents NLRP3 inflammasome activation by inhibiting endolysosomal acidification in macrophages.

The cathelicidin-like peptide derived from panda genome is a potential antimicrobial peptide

A novel cathelicidin-like antimicrobial peptide was identified by mining genome of panda. This peptide (cathelicidin-AM) was synthesized. It showed potential antimicrobial activities against wide spectrum of microorganisms including Gram-negative and -positive bacteria, and fungi. It had similar antimicrobial abilities against both standard and clinically isolated drug-resistant strains. Cathelicidin-AM could rapidly exert its antibacterial activities. It just took less than 1 h to kill all Staphylococcus sciuri at the concentration of 2, 4 or 10 times of minimal inhibitory concentration (MIC) while clindamycin took 6 h. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis indicated that cathelicidin-AM killed bacteria by directly affecting bacterial cell wall and membrane. Phylogenetic analysis revealed that the panda cathelicidin had the nearest evolution relationship with dog cathelicidin. The current work provides a novel cathelicidin-like peptide with strong antimicrobial abilities.

Identification, characterization and expression of cathelicidin in the pouch young of tammar wallaby (Macropus eugenii)

Antimicrobial peptides, such as cathelicidin, are an evolutionarily old defense system. However they have more complex actions than just simply their antimicrobial effects, including immunoregulation and interaction with the adaptive immune system. In this study we have characterized several novel cathelicidin-like peptides from the tammar wallaby (Macropus eugenii). The tammar cathelicidin-like (MaeuCath) mRNA were isolated based on the conservation of the cathelin-like amino terminus. Mature MaeuCath peptides were positively charged with hydrophobic carboxyl tails, features that are fundamental for antimicrobial function. MaeuCath1 was induced in tammar leukocytes in response to pathogen-associated molecular patterns from both gram positive and negative bacteria. In addition, we also examined the expression of MaeuCath1 in the primary and secondary lymphoid organs of the tammar neonate throughout early pouch life. The results from this study demonstrate the importance that MaeuCath1 may play in innate defense of the marsupial young, especially in the mucosal organs. Such expression of antimicrobial peptides may form part of the immune strategies of marsupials for neonatal survival during their post-partum development.

Expression of SMAP-29 cathelicidin-like peptide in bacterial cells by intein-mediated system

In this work, the intein fusion approach was used for expression and purification of cathelicidin-like peptide SMAP-29 from Escherichia coli cultures. To overcome the high toxicity of the antimicrobial peptide against host cells, both C- and N-terminal fusions with Sce VMA intein were evaluated. The fusion of SMAP-29 with the N-terminus of intein had a dramatic lethal effect. In contrast, chimeric constructs harboring SMAP-29 linked to the C-terminus of intein displayed no significant inhibition of bacterial growth. Expression of intein-SMAP fusion protein was then induced in ER2566 E. coli strain by IPTG addition and different experimental conditions were tested in order to optimize the recovery of the soluble protein complex. Peptide purification was carried out by affinity chromatography: the chitin binding domain linked to intein was used to immobilize the chimeric protein on a chitin column and intein-mediated splicing of target peptide was obtained by thiol addition. Microbroth dilution assay showed that recombinant SMAP-29 displayed a high, dose-dependent bactericidal activity. These data demonstrate that the fusion of SMAP-29 with C-intein was able to inactivate the antimicrobial properties of the cathelicidin peptide allowing the expression of fusion protein in the host cell. The intein-mediated purification supplied an effective way to recover the fusion partner in its proper biologically active form.

Possible antimicrobial compounds from the pouch of the koala, Phascolarctos cinereus

The antimicrobial activity of secretions of the pouch of the koala, Phascolarctos cinereus has been documented in thepresence and absence of young. These secretions have been analysed by polyacrylamide gel electrophoresis, high performanceliquid chromatography and mass spectrometry (MS) including time of flight (TOF), MS-MS and Edman sequencing. Gel electrophoresis of reduced samples revealed the presence of a four polypeptides in active and inactive secretions. The sequence data obtained from all four do not appear to be either defensin or cathelicidin-like peptides and show little homology with knownantimicrobial protein/peptide sequence available on public accessdatabases. It is proposed that marsupials regulate microbialpopulations in the pouch via cleavage of large, inactive molecules to produce small active peptides.