Plicatamide (Phe-Phe-His-Leu-His-Phe-His-dc⌬DOPA), where dc⌬DOPA represents decarboxy-(E)-␣,-dehydro-3,4-dihydroxyphenylalanine, is a potently antimicrobial octapeptide from the blood cells of the solitary tunicate, Styela plicata. Wild type and methicillin-resistant Staphylococcus aureus (MRSA) responded to plicatamide exposure with a massive potassium efflux that began within seconds. Soon thereafter, treated bacteria largely ceased consuming oxygen, and most became nonviable. Native plicatamide also formed cation-selective channels in model lipid bilayers composed of bacterial lipids. Methicillin-resistant S. aureus treated with plicatamide for 5 min contained prominent mesosomes as well as multiple, small dome-shaped surface protrusions that suggested the involvement of osmotic forces in its antimicrobial effects. To ascertain the contribution of the C-terminal dc⌬DOPA residue to antimicrobial activity, we synthesized several analogues of plicatamide that lacked it. One of these peptides, PL-101 (Phe-Phe-His-Leu-His-Phe-HisTyr-amide), closely resembled native plicatamide in its antimicrobial activity and its ability to induce potassium efflux. Plicatamide was potently hemolytic for human red blood cells but did not lyse ovine erythrocytes. The small size, rapid action, and potent anti-staphylococcal activity of plicatamide and PL-101 make them intriguing subjects for future antimicrobial peptide design.Phe-Phe-His-Leu-His-Phe-His-dc⌬DOPA (plicatamide) 1 is a modified octapeptide found in the hemocytes of Styela plicata (1). In the preceding sequence, dc⌬DOPA indicates decarboxy-(E)-␣,-dehydro-3,4-dihydroxyphenylalanine. Although the sequence of plicatamide did not resemble a conventional antimicrobial peptide, we examined its antimicrobial properties because hemocytes are key participants in innate antimicrobial defenses. Despite its small size, plicatamide proved to be a potent, rapidly acting, and broad spectrum antimicrobial. We also prepared the following four synthetic analogues that differed from plicatamide only in their C-terminal residue: tyrosine amide in PL-101; tyrosine acid in PL-102; DOPA (3,4-dihydroxyphenylalanine) acid in PL-103; and DOPA-amide in PL-104. Of these octapeptides, PL-101 most closely simulated the antimicrobial properties of native plicatamide. This report will describe the effects of plicatamide on staphylococci. MATERIALS AND METHODS Peptide PurificationNative plicatamide was purified from freshly harvested hemocytes (blood cells) of S. plicata as described recently (1). We determined their peptide content either by performing quantitative amino acid analysis or by doing analytical reverse phase-HPLC on a C18 column and then computing and comparing the area under the curve (AUC) at 215 nm with the AUC of an appropriate standard previously subjected to quantitative amino acid analysis. Peptide SynthesisThe synthetic peptides used in our initial experiments were customsynthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry at Research Genetics (Huntsville, AL) and puri...
Background and Objective Both chronic and aggressive periodontal disease are associated with vitamin D deficiency. The active form of vitamin D, 1,25(OH)2D3, induces the expression of the antimicrobial peptide LL‐37 and innate immune mediators in cultured human gingival epithelial cells (GECs). The aim of this study was to further delineate the mechanism by which vitamin D enhances the innate defense against the development of periodontal disease (PD). Materials and Methods Wild‐type C57Bl/6 mice were made deficient in vitamin D by dietary restriction. Cultured primary and immortalized GEC were stimulated with 1,25(OH)2D3, followed by infection with Porphyromonas gingivalis, and viable intracellular bacteria were quantified. Conversion of vitamin D3 to 25(OH)D3 and 1,25(OH)2D3 was quantified by ELISA. Effect of vitamin D on basal IL‐1α expression in mice was determined by topical administration to the gingiva of wild‐type mice, followed by qRT‐PCR. Results Dietary restriction of vitamin D led to alveolar bone loss and increased inflammation in the gingiva in the mouse model. In primary human GEC and established human cell lines, treatment of GEC with 1,25(OH)2D3 inhibited the intracellular growth of P. gingivalis. Cultured GEC expressed two 25‐hydroxylases (CYP27A1 and CYP2R1), as well as 1‐α hydroxylase, enabling conversion of vitamin D to both 25(OH)D3 and 1,25(OH)2D3. Topical application of both vitamin D3 and 1,25(OH)2D3 to the gingiva of mice led to rapid inhibition of IL‐1α expression, a prominent pro‐inflammatory cytokine associated with inflammation, which also exhibited more than a 2‐fold decrease from basal levels in OKF6/TERT1 cells upon 1,25(OH)2D3 treatment, as determined by RNA‐seq. Conclusion Vitamin D deficiency in mice contributes to PD, recapitulating the association seen in humans, and provides a unique model to study the development of PD. Vitamin D increases the activity of GEC against the invasion of periodontal pathogens and inhibits the inflammatory response, both in vitro and in vivo. GEC can convert inactive vitamin D to the active form in situ, supporting the hypothesis that vitamin D can be applied directly to the gingiva to prevent or treat periodontal disease.
Because tunicates rely on innate immunity, their hemocytes are important contributors to host defense. Styela clava, a solitary ascidian, have eight hemocyte subtypes. Extracts of their total hemocyte population contained multiple small (2-4 kDa) antimicrobial peptides. When purified, these fell into two distinct families that were named styelins and clavanins.Styelins A-E are phenylalanine-rich, 32 residue peptides with activity against marine bacteria and human pathogens. They show considerable sequence homology to pleurocidins, antimicrobial peptides of the flounder, Pseudopleuronectes americanus. Styelin D, one of the five styelins identified by peptide isolation and cDNA cloning, was remarkable in containing 12 post-translationally modified residues, including a 6-bromotryptophan, two monohydroxylysines, four 3,4-dihydroxyphenylalanines (DOPA), four dihydroxylysines and one dihydroxyarginine. These modifications enhanced Styelin D's bactericidal ability at acidic pH and high salinity. A novel histochemical stain for DOPA suggested that Styelin D was restricted to granulocytes.Clavanins A-E are histidine-rich, 23 residue peptides that are C-terminally amidated and most effective at acidic pH. Clavaspirin is a newly described family member that also has potent cytotoxic properties. By immunocytochemistry, clavanins were identified in the granules of five eosinophilic granulocyte subtypes and in macrophage cytoplasm.Transmission and scanning electron micrographs of methicillin-resistant Staphylococcus aureus (MRSA) and E. coli that had been treated with Styelin D and clavaspirin suggested that both peptides induced osmotic disregulation. Treated bacteria manifested cytoplasmic swelling and extrusion of cytoplasmic contents through their peptidoglycan cell wall. The diverse array of antimicrobial peptides in S. clava hemocytes constitutes an effective host defense mechanism.
Lethal systemic fungal infections of Candida species are increasingly common, especially in immune compromised patients. By in vitro screening of small molecule mimics of naturally occurring host defense peptides (HDP), we have identified several active antifungal molecules, which also exhibited potent activity in two mouse models of oral candidiasis. Here we show that one such compound, C4, exhibits a mechanism of action that is similar to the parent HDP upon which it was designed. Specifically, its initial interaction with the anionic microbial membrane is electrostatic, as its fungicidal activity is inhibited by cations. We observed rapid membrane permeabilization to propidium iodide and ATP efflux in response to C4. Unlike the antifungal peptide histatin 5, it did not require energy-dependent transport across the membrane. Rapid membrane disruption was observed by both fluorescence and electron microscopy. The compound was highly active in vitro against numerous fluconazole-resistant clinical isolates of C. albicans and non-albicans species, and it exhibited potent, dose-dependent activity in a mouse model of invasive candidiasis, reducing kidney burden by three logs after 24 hours, and preventing mortality for up to 17 days. Together the results support the development of this class of antifungal drug to treat invasive candidiasis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.