A novel endogenous antimicrobial peptide targeting Candida albicans from Huangshui: Isolation and characterization

Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

Hyphal Invasion of Candida albicans Inhibits the Expression of Human β-Defensins in Experimental Oral Candidiasis

Antimicrobial activity and mechanism of PDC213, an endogenous peptide from human milk

Antibiotics are often used to treat oral infections. Unfortunately, excessive antibiotic use can adversely alter oral microbiomes and promote the development of antibiotic-resistant microorganisms, which can be difficult to treat. An alternate approach could be to induce the local transcription and expression of endogenous oral antimicrobial peptides (AMPs). To assess the feasibility and benefits of this approach, we conducted literature searches to identify (i) the AMPs expressed in the oral cavity; (ii) the methods used to induce endogenous AMP expression; and (iii) the roles that expressed AMPs may have in regulating oral inflammation, immunity, healing, and pain. Search results identified human neutrophil peptides (HNP), human beta defensins (HBD), and cathelicidin AMP (CAMP) gene product LL-37 as prominent AMPs expressed by oral cells and tissues. HNP, HBD, and LL-37 expression can be induced by micronutrients (trace elements, elements, and vitamins), nutrients, macronutrients (mono-, di-, and polysaccharides, amino acids, pyropeptides, proteins, and fatty acids), proinflammatory agonists, thyroid hormones, and exposure to ultraviolet (UV) irradiation, red light, or near infrared radiation (NIR). Localized AMP expression can help reduce infection, inflammation, and pain and help oral tissues heal. The use of a specific inducer depends upon the overall objective. Inducing the expression of AMPs through beneficial foods would be suitable for long-term health protection. Additionally, the specialized metabolites or concentrated extracts that are utilized as dosage forms would maintain the oral and intestinal microbiome composition and control oral and intestinal infections. Inducing AMP expression using irradiation methodologies would be applicable to a specific oral treatment area in addition to controlling local infections while regulating inflammatory and healing processes.

Tuberculosis (TB) continues to be a devastating infectious disease and remerges as a global health emergency due to an alarming rise of antimicrobial resistance to its treatment. Despite of the serious effort that has been applied to develop effective antitubercular chemotherapies, the potential of antimicrobial peptides (AMPs) remains underexploited. A large amount of literature is now accessible on the AMP mechanisms of action against a diversity of pathogens; nevertheless, research on their activity on mycobacteria is still scarce. In particular, there is an urgent need to integrate all available interdisciplinary strategies to eradicate extensively drug-resistant Mycobacterium tuberculosis strains. In this context, we should not underestimate our endogenous antimicrobial proteins and peptides as ancient players of the human host defense system. We are confident that novel antibiotics based on human AMPs displaying a rapid and multifaceted mechanism, with reduced toxicity, should significantly contribute to reverse the tide of antimycobacterial drug resistance. In this review, we have provided an up to date perspective of the current research on AMPs to be applied in the fight against TB. A better understanding on the mechanisms of action of human endogenous peptides should ensure the basis for the best guided design of novel antitubercular chemotherapeutics.

BACKGROUND: Resistance of microorganisms caused dangerous to human health infections to traditional antibiotics is a serious problem for healthcare. In this regard, the development of new effective antimicrobial drugs and therapeutic approaches is an urgent task. Antimicrobial peptides (AMPs) are considered a promising alternative to traditional antibiotic in the fight against resistant microorganisms.
 AIM: The aim of this work is to study the effect of new synthesized AMPs of the medicinal leech Hirudo medicinalis (including under conditions of development of oxidative/halogenative stress) on the functional activity of neutrophils granular proteins the main effector cells of the immune system.
 MATERIALS AND METHODS: Myeloperoxidase peroxidase activity was assessed by the rate of o-dianisidine oxidation. Neutrophil elastase activity was determined by the fluorescence method using a specific substrate MeOSuc-AAPV-AMC. Lactoferrin iron-binding activity was assessed spectrophotometrically by the change in absorption of protein solution after addition of Fe3+ salt. Lysozyme activity was determined by the rate of M. lysodeikticus bacterial cells lysis.
 RESULTS: Native AMPs 536_1 and 19347_2 inhibited and 12530 increased myeloperoxidase peroxidase activity, this tendency persisted after these AMPs modification by hypochlorous acid (HOCl). In contrast to the native AMP halogenated AMP 3967_1 acquired the ability to enhance myeloperoxidase enzymatic activity. In the presence of AMP 3967_1 neutrophil elastase amidolytic activity increased insignificantly, while AMP 19347_2 inhibited neutrophil elastase activity. After HOCl modification these AMPs retained their ability to regulate neutrophil elastase activity. Synergistic effects (~20%) against gram-positive bacteria M. lysodeikticus were revealed for combination of lysozyme with AMPs 12530 and 3967_1. Inhibition lysozyme antimicrobial activity was observed in the presence of AMPs 19347_2 and 536_1, however the severity of this effect decreased after AMPs modification by HOCl. After HOCl modification AMP 3967_1 increased, while AMP 12530 on the contrary acquired the ability to inhibit lysozyme mucolytic activity.
 CONCLUSIONS: The use of drugs based on studied AMPs of medicinal leech will have a beneficial effect on the bodys fight against infectious agents due to the antimicrobial action of AMPs themselves. But in addition studied AMPs are capable to modulate the biological activity of own endogenous antimicrobial proteins and peptides: to enhance it, if it is necessary to eliminate pathogen and to inhibit if it necessary to protect against damage to the bodys own tissues.

Endogenous peptides are valuable targets in the analysis of biological processes. The tear film contains proteins and peptides released by the tear duct mucosal cells, including antimicrobial peptides involved in the protection against exogenous pathogens; however, the peptide content of the tear liquid remains poorly characterized. We analyzed naturally occurring peptides isolated from human basal tears. Mass spectrometry analysis of endogenous peptides presents a number of drawbacks, including size heterogeneity and nonpredictable fragmentation patterns, among others. Therefore, CID, ETD, and HCD methods were used for the characterization of the tear peptide content. The contribution of DMSO as an additive of the chromatographic solvents was also evaluated. We identified 157, 131, and 122 peptides using CID-, ETD-, and HCD-based methods, respectively. Altogether, 234 different peptides were identified, leading to the generation of the biggest data set of endogenous tear peptides to date. The antimicrobial activity prediction analysis performed in silico revealed different putative antimicrobial peptides. Two of the extracellular glycoprotein lacritin peptides were de novo synthesized, and their antimicrobial activity was confirmed in vitro. Our findings demonstrate the benefits of using different fragmentation methods for the analysis of endogenous peptides and provide a useful approach for the discovery of peptides with antimicrobial activity.

New endogenous antimicrobial peptides (AMPs) derived from chromogranin A (CgA) are secreted by nervous, endocrine and immune cells during stress. They display antimicrobial activities by lytic effects at micromolar range using a pore-forming mechanism against Gram-positive bacteria, filamentous fungi and yeasts. These AMPs can also penetrate quickly into neutrophils (without lytic effects), where, similarly to "cell penetrating peptides", they interact with cytoplasmic calmodulin, and induce calcium influx via Store Operated Channels therefore triggering neutrophils activation. Staphylococcus aureus and Salmonella enteritis are bacteria responsible for severe infections. We investigated here the effects of S. aureus and S. enteritis bacterial proteases on CgA-derived peptides and evaluated their antimicrobial activities. We showed that the Glu-C protease produced by S. aureus V8 induces the loss of the AMPs antibacterial activities and produces new antifungal peptides. In addition, four antimicrobial CGA-derived peptides (chromofungin, procatestatin, human/bovine catestatin) are degraded when treated with bacterial supernatants from S. aureus and S. enteritis, whereas, cateslytin, the short active form of catestatin, resists to this degradation. Finally, we demonstrate that several antimicrobial CgA-derived peptides are able to act synergistically with antibiotics against bacteria and fungi indicating their roles in innate defense.

Surgical site infections represent a significant clinical problem. Herein, we report a nanofiber dressing for topical codelivery of immunomodulating compounds including 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and VID400, a CYP24A1 inhibitor in a sustained manner, for inducing the expression of the endogenous cathelicidin antimicrobial peptide (CAMP) gene encoding the hCAP18 protein, which is processed into the LL-37 peptide. Nanofiber wound dressings with coencapsulation of 1,25(OH)2D3 and VID400 were generated by electrospinning. Both 1,25(OH)2D3 and VID400 were coencapsulated into nanofibers with loading efficiencies higher than 90% and exhibited a prolonged release from nanofiber membranes longer than 28 days. Incubation with 1,25(OH)2D3/VID400-coencapsulated poly(ϵ-caprolactone) nanofiber membranes greatly induced the hCAP18/LL-37 gene expression in monocytes, neutrophils, and keratinocytes in vitro. Moreover, the administration of 1,25(OH)2D3/VID400-coencapsulated nanofiber membranes dramatically promoted the hCAP18/LL-37 expression in dermal wounds created in both human CAMP transgenic mice and human skin tissues. The 1,25(OH)2D3- and VID400-coencapsulated nanofiber dressings enhanced innate immunity via the more effective induction of antimicrobial peptide than the free drug alone or 1,25(OH)2D3-loaded nanofibers. Together, 1,25(OH)2D3/VID400-embedded nanofiber dressings presented in this study show potential in preventing surgical site infections.

Marine invertebrates lack an acquired, memory-type immunity based on T-lymphocyte subsets and clonally derived immunoglobulins (72). This differs from the vertebrate immune system, which is characterized by somatic gene rearrangement, clonal selection, and expansion and a discriminative ability that includes lymphocytes, among other factors, which impart specificity and memory (71). Marine invertebrates rely solely on innate immune mechanisms that include both humoral and cellular responses. Humoral immunity in marine invertebrates is characterized by antimicrobial agents present in the blood cells and plasma (92), along with reactions such as hemolymph coagulation or melanization (79, 85). Cellular immunity in marine invertebrates is based on cell defense reactions, including encapsulation, nodule formation, and phagocytosis (92). The cellular component of marine invertebrate immunity is mediated by hemocytes, motile cells that phagocytize microbes and secrete soluble antimicrobial and cytotoxic substances into the hemolymph (53). This differs from insects, especially Drosophila melanogaster, which rely largely on the challenge-induced synthesis of antimicrobial peptides by the fat body (30, 88) and use exclusion, via a tough exoskeleton, as their major antimicrobial defense. The circulating hemolymph in marine invertebrates contains biologically active substances such as complement, lectins, clotting factors, and antimicrobial peptides (57). All of these factors contribute to a self-defense system in marine invertebrates against invading microorganisms, which can number up to 106 bacteria/ml and 109 virus/ml of seawater (2). The survival of marine invertebrates in this environment suggests that their innate immune system is effective and robust (52). Antimicrobial peptides are a major component of the innate immune defense system in marine invertebrates. They are defined as molecules less than 10 kDa in mass which show antimicrobial properties (12) and provide an immediate and rapid response to invading microorganisms (8). The major classes of antimicrobial peptides include (i) α-helices, (ii) β-sheet and small proteins, (iii) peptides with thio-ether rings, (iv) peptides with an overrepresentation of one or two amino acids, (v) lipopeptides, and (vi) macrocyclic cystine knot peptides (24). There is evidence that antimicrobial peptides are widespread in invertebrates (15), especially in tissues such as the gut and respiratory organs in marine invertebrates, where exposure to pathogenic microorganisms is likely. In spite of variations in structure and size, the majority of antimicrobial peptides are amphiphilic, displaying both hydrophilic and hydrophobic surfaces. These peptides generally act by forming pores in microbial membranes or otherwise disrupting membrane integrity (82), which is facilitated by their amphiphilic structure. This mode of action is unlikely to lead to the development of resistance (9, 58), although it must be noted that this presumption is debatable (10). Recently, cationic antimicrobial peptides have been reported to be involved in many aspects of innate host defenses, associated with processes such as acute inflammation (25). The value of antimicrobial peptides in innate immunity lies in their ability to function without either high specificity or memory, and their small size makes them easy to synthesize (72). In addition, many antibacterial peptides show remarkable specificity for prokaryotes with low toxicity for eukaryotic cells (97). This is a characteristic that has favored their investigation and exploitation as potential new antibiotics (97). The recent appearance of a growing number of bacteria resistant to conventional antibiotics has become a serious medical problem. To overcome this resistance, the development of antibiotics with novel mechanisms of action is a pressing issue (48). Endogenous antimicrobial peptides are exciting candidates as new antibacterial agents due to their broad antimicrobial spectra, highly selective toxicities, and the difficulty for bacteria to develop resistance to these peptides (11, 26, 47). The ocean covers 71% of the surface of the earth and contains approximately half of the total global biodiversity, with estimates ranging between 3 and 500 × 106 different species (28). Marine macrofauna alone comprise 0.5 to 10 × 106 species (23). Therefore, the marine environment, especially marine invertebrates that rely solely on innate immune mechanisms for host defense, is a spectacular resource for the development of new antimicrobial compounds. This minireview will encompass what is known about gene-encoded antimicrobial peptides from marine invertebrates, covering the phyla Arthropoda, Chordata, and Mollusca (Table ​(Table11). TABLE 1. Antimicrobial peptides from marine invertebrates

Ever since the discovery of endogenous host defense antimicrobial peptides it has been discussed how these evolutionary conserved molecules avoid to induce resistance and to remain effective. Human ß-defensin 1 (hBD1) is an ubiquitously expressed endogenous antimicrobial peptide that exhibits qualitatively distinct activities between its oxidized and reduced forms. Here, we explore these antimicrobial mechanisms. Surprisingly, using electron microscopy we detected a so far unknown net-like structure surrounding bacteria, which were treated with the reduced but not the oxidized form of hBD1. A transmigration assay demonstrated that hBD1-derived nets capture bacteria and inhibit bacterial transmigration independent of bacterial killing. The presence of nets could completely prevent migration of hBD1 resistant pathogens and are stable in the presence of human duodenal secretion with a high amount of proteases. In contrast to HD6, cysteins are necessary for net formation. This redox-dependent function serves as an additional mechanism of action for hBD1 and differs from net formation by other defensins such as Paneth cell-derived human α-defensin 6 (HD6). While hBD1red and hBD1ox have distinct antimicrobial profiles and functions, only the reduced form provides additional host protection by entrapping bacteria in extracellular net structures preventing bacterial invasion. Better understanding of the modes of action of endogenous host peptides will help to find new antimicrobial strategies.

In the past decade, there has been fast-growing interest among researchers to discover bioactive peptides from edible insects and to evaluate their potential applications in the management of human, livestock, and plant health. This review summarizes current knowledge of insect-derived peptides and their potential role in tackling human health issues and solving agriculture problems by protecting crops and livestock against their pathogens. Numerous bioactive peptides have been identified from edible insect species, including peptides that were enzymatically liberated from insect proteins and endogenous peptides that occur naturally in insects. The peptides exhibited diverse bioactivities, encompassing antioxidant, anti-angiotensin-converting enzyme, anti-dipeptidyl peptidase-IV, anti-glucosidase, anti-lipase, anti-lipoxygenase, anti-cyclooxygenase, anti-obesity, and hepatoprotective activities. Such findings point to their potential contribution to solving human health problems related to inflammation, free radical damage, diabetes, hypertension, and liver damage, among others. Although most of the experiments were performed in vitro, evidence for the in vivo efficacy of some peptides is emerging. Evidence of the protective effects of insect-derived endogenous antimicrobial peptides in combating farm animal and plant pathogens is available. The ability of insect-derived endogenous neuropeptides to protect plants against herbivorous insects has been demonstrated as well. Nevertheless, the potency of peptides identified from insect protein hydrolysates in modulating livestock and plant health remains a knowledge gap to be filled.

A large number of bioactive peptides derived from breast milk have been identified to be multifunctional having anti-inflammatory, immunoregulatory and antimicrobial activities. Here, we report that an endogenous peptide located at β-casein 211-225 amino acid from human breast milk (hereafter called CAMP211-225) presents specific antimicrobial activity against pathogenic E. coli and Y. enterocolitica. CAMP211-225 is a novel peptide that occurs at higher levels in preterm milk than in term milk. The minimal inhibitory concentrations (MIC) of CAMP211-225 against E. coli and Y. enterocolitica are 3.125 μg ml-1 and 6.25 μg ml-1, respectively, and the antimicrobial activity of CAMP211-225 was also confirmed by a disk diffusion assay. Further studies using fluorescence staining, scanning electron microscopy and a DNA-binding assay revealed that CAMP211-225 kills bacteria through a membrane-disrupting mechanism, but not by binding to intracellular nucleic acids. Neonatal necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease in neonatal intensive care units. In our study, CAMP211-225 administration effectively reduced ileal mucosa damage in an experimental NEC mice model. These results suggest that the antimicrobial peptide CAMP211-225 may have potential value in the prevention and treatment of neonatal infections.

Human antimicrobial peptides in ocular surface defense.

The development of antimicrobial resistance by pathogenic bacteria has fuelled the search for alternatives to conventional antibiotics. Endogenous antimicrobial peptides have the potential to be used as new antimicrobial substances because they have low minimum inhibitory concentration in vitro, have broad-spectrum activity, neutralise lipopolysaccharides, promote wound healing and have synergistic effects with conventional antibiotics. Farm animals, in particular the blood that is a by-product of the meat and poultry industries, are an abundant, and currently underutilised, source of such antimicrobial peptides. These antimicrobial peptides could be isolated and developed into high-value products such as biopreservatives, topical neutraceutical products and pharmaceuticals. There have been some clinical trials of antimicrobial peptides as pharmaceutical products, but up to now, the trials have shown disappointing results. Further research and development is still needed before such peptides can be commercialised and full advantage taken of this waste product of the meat and poultry industries.