Host defence peptides identified in human apolipoprotein B as promising antifungal agents.

Therapeutic options to treat invasive fungal infections are still limited. This makes the development of novel antifungal agents highly desirable. Naturally occurring antifungal peptides represent valid candidates, since they are not harmful for human cells and are endowed with a wide range of activities and their mechanism of action is different from that of conventional antifungal drugs. Here, we characterized for the first time the antifungal properties of novel peptides identified in human apolipoprotein B. ApoB-derived peptides, here named r(P)ApoBLPro, r(P)ApoBLAla and r(P)ApoBSPro, were found to have significant fungicidal activity towards Candida albicans (C. albicans) cells. Peptides were also found to be able to slow down metabolic activity of Aspergillus niger (A. niger) spores. In addition, experiments were carried out to clarify the mechanism of fungicidal activity of ApoB-derived peptides. Peptides immediately interacted with C. albicans cell surfaces, as indicated by fluorescence live cell imaging analyses, and induced severe membrane damage, as indicated by propidium iodide uptake induced upon treatment of C. albicans cells with ApoB-derived peptides. ApoB-derived peptides were also tested on A. niger swollen spores, initial hyphae and branched mycelium. The effects of peptides were found to be more severe on swollen spores and initial hyphae compared to mycelium. Fluorescence live cell imaging analyses confirmed peptide internalization into swollen spores with a consequent accumulation into hyphae. Altogether, these findings open interesting perspectives to the application of ApoB-derived peptides as effective antifungal agents. KEY POINTS: Human cryptides identified in ApoB are effective antifungal agents. ApoB-derived cryptides exert fungicidal effects towards C. albicans cells. ApoB-derived cryptides affect different stages of growth of A. niger. Graphical abstract.

Host defense peptides identified in human apolipoprotein B as novel food biopreservatives and active coating components.

The effectiveness of three novel "host defence peptides" identified in human Apolipoprotein B (ApoB) as novel antimicrobial and antibiofilm agents to be employed in food industry is reported. ApoB-derived peptides have been found to exert significant antimicrobial effects towards Salmonella typhimurium ATCC® 14028 and Salmonella enteritidis 706 RIVM strains. Furthermore, they have been found to retain antimicrobial activity under experimental conditions selected to simulate those occurring during food storage, transportation and heat treatment, and have been found to be endowed with antibiofilm properties. Based on these findings, to evaluate the applicability of ApoB-derived peptides as food biopreservatives, coating solutions composed by chitosan (CH) and an ApoB-derived peptide have been prepared and found to be able to prevent Salmonella cells attachment to different kinds of surfaces employed in food industry. Finally, obtained coating solution has been demonstrated to hinder microbial proliferation in chicken meat samples. Altogether, obtained findings indicate that ApoB-derived peptides are promising candidates as novel biopreservatives for food packaging.

Cryptides Identified in Human Apolipoprotein B as New Weapons to Fight Antibiotic Resistance in Cystic Fibrosis Disease.

Chronic respiratory infections are the main cause of morbidity and mortality in cystic fibrosis (CF) patients, and are characterized by the development of multidrug resistance (MDR) phenotype and biofilm formation, generally recalcitrant to treatment with conventional antibiotics. Hence, novel effective strategies are urgently needed. Antimicrobial peptides represent new promising therapeutic agents. Here, we analyze for the first time the efficacy of three versions of a cryptide identified in human apolipoprotein B (ApoB, residues 887-922) towards bacterial strains clinically isolated from CF patients. Antimicrobial and anti-biofilm properties of ApoB-derived cryptides have been analyzed by broth microdilution assays, crystal violet assays, confocal laser scanning microscopy and scanning electron microscopy. Cell proliferation assays have been performed to test cryptide effects on human host cells. ApoB-derived cryptides have been found to be endowed with significant antimicrobial and anti-biofilm properties towards Pseudomonas and Burkholderia strains clinically isolated from CF patients. Peptides have been also found to be able to act in combination with the antibiotic ciprofloxacin, and they are harmless when tested on human bronchial epithelial mesothelial cells. These findings open interesting perspectives to cryptide applicability in the treatment of chronic lung infections associated with CF disease.

Human apolipoprotein E as a reservoir of cryptic bioactive peptides: The case of ApoE 133-167.

Bioactive peptides derived from the receptor-binding region of human apolipoprotein E have previously been reported. All these peptides, encompassing fragments of this region or designed on the basis of short repeated cationic sequences identified in the same region, show toxic activities against a broad spectrum of bacteria and interesting immunomodulatory effects. However, the ability of these molecules to exert antibiofilm properties has not been described so far. In the present work, we report the characterization of a novel peptide, corresponding to residues 133 to 167 of human apolipoprotein E, here named ApoE (133-167). This peptide, besides presenting interesting properties comparable with those reported for other ApoE-derived peptides, such as a direct killing activity against a broad spectrum of bacteria or the ability to downregulate lipopolysaccharide-induced cytokine release, is also endowed with significant antibiofilm properties. Indeed, the peptide is able to strongly affect the formation of the extracellular matrix and also the viability of encapsulated bacteria. Noteworthy, ApoE (133-167) is not toxic toward human and murine cell lines and is able to assume ordered conformations in the presence of membrane mimicking agents. Taken together, collected evidences about biological and structural properties of ApoE (133-167) open new perspectives in the design of therapeutic agents based on human-derived bioactive peptides.

Insights into the anticancer properties of the first antimicrobial peptide from Archaea.

BACKGROUND: The peptide VLL-28, identified in the sequence of an archaeal protein, the transcription factor Stf76 from Sulfolobus islandicus, was previously identified and characterized as an antimicrobial peptide, possessing a broad-spectrum antibacterial activity. METHODS: Through a combined approach of NMR and Circular Dichroism spectroscopy, Dynamic Light Scattering, confocal microscopy and cell viability assays, the interaction of VLL-28 with the membranes of both parental and malignant cell lines has been characterized and peptide mechanism of action has been studied. RESULTS: It is here demonstrated that VLL-28 selectively exerts cytotoxic activity against murine and human tumor cells. By means of structural methodologies, VLL-28 interaction with the membranes has been proven and the binding residues have been identified. Confocal microscopy data show that VLL-28 is internalized only into tumor cells. Finally, it is shown that cell death is mainly caused by a time-dependent activation of apoptotic pathways. CONCLUSIONS: VLL-28, deriving from the archaeal kingdom, is here found to be endowed with selective cytotoxic activity towards both murine and human cancer cells and consequently can be classified as an ACP. GENERAL SIGNIFICANCE: VLL-28 represents the first ACP identified in an archaeal microorganism, exerting a trans-kingdom activity.

Leguminous Seedborne Pathogens: Seed Health and Sustainable Crop Management.

Pulses have gained popularity over the past few decades due to their use as a source of protein in food and their favorable impact on soil fertility. Despite being essential to modern agriculture, these species face a number of challenges, such as agronomic crop management and threats from plant seed pathogens. This review's goal is to gather information on the distribution, symptomatology, biology, and host range of seedborne pathogens. Important diagnostic techniques are also discussed as a part of a successful process of seed health certification. Additionally, strategies for sustainable control are provided. Altogether, the data collected are suggested as basic criteria to set up a conscious laboratory approach.

Surveillance of Landraces' Seed Health in South Italy and New Evidence on Crop Diseases.

During the last three years, more than 300 landraces belonging to different plant species have been the main focus of an Italian valorization research project (AgroBiodiversità Campana, ABC) aiming at analyzing, recovering, preserving, and collecting local biodiversity. In this context, phytosanitary investigation plays a key role in identifying potential threats to the preservation of healthy seeds in gene banks and the successful cultivation of landraces. The surveillance carried out in this study, in addition to highlighting the expected presence of common species-specific pathogens such as Ascochyta pisi in peas, Ascochyta fabae in broad beans, and Macrophomina phaseolina, Xanthomonas axonopodis pv. phaseoli, and Xanthomonas fuscans subsp. fuscans in beans, pointed to the presence of novel microorganisms never detected before in the seeds of some hosts (Apiospora arundinis in common beans or Sclerotinia sclerotiorum and Stemphylium vesicarium in broad beans). These novel seedborne pathogens were fully characterized by (i) studying their morphology, (ii) identifying them by molecular methods, and (iii) studying their impact on adult crop plants. For the first time, this study provides key information about three novel seedborne pathogens that can be used to correctly diagnose their presence in seed lots, helping prevent the outbreaks of new diseases in the field.

Identification and Characterization of New Seedborne Pathogens in Phaseolus vulgaris Landraces of Southern Italy.

The diagnostic survey of seedborne fungal pathogens is fundamental for symptomless material stored in gene banks to avoid the diffusion of pathogens by germplasm distribution and propagation. In this work, seeds of Southern Italian landraces of the common bean (Phaseolus vulgaris L.) belonging to the gene bank at CREA (Italy) were inspected to assess their phytosanitary status. The phytopathological analysis revealed the presence of the most common pathogens associated with common bean seeds such as Fusarium spp., Macrophomina phaseolina, Rhizoctonia solani, Colletotrichum lindemuthianum and Diaporthe/Phomopsis complex. However, new fungi able to completely inhibit seed germination were also observed. The most aggressive were isolated, and the morpho-pathological characterization, DNA sequencing and phylogenetic analysis allowed us to define the strains as Botryosphaeria dothidea CREA OF 360.4 and Diplodia mutila CREA OF 420.36. These two plant pathogens are generally associated with grapevines and other fruit trees. Pathogenicity tests were carried out along with a transmissibility test in which the transmission of the pathogens to the seedlings was proven. Host range experiments revealed the ability of these pathogens to infect crops such as pepper and melon. To our knowledge, this is the first time that B. dothidea and D. mutila were detected on the common bean.

Occurrence of Macrophomina phaseolina on Chickpea in Italy: Pathogen Identification and Characterization.

Climate change has led to the spread of plant pathogens in novel environments, causing dramatic crop losses and economic damage. Botryosphaeriaceae represents a massive fungal family, containing a huge number of plant pathogens, which are able to infect several hosts. Among them, Macrophomina phaseolina is a necrotrophic fungus, responsible for several plant diseases, including the soft stem rot of common bean, crown rot on strawberry and charcoal rot of several legumes. Here, Macrophomina, causing crown charcoal rot in chickpeas, was isolated from symptomatic plants in Cicerale (SA), Campania, South Italy. Morphological and molecular characterization was carried out and pathogenicity tests were performed. Phylogenetic analyses were performed comparing Macrophomina strains coming from different geographic areas and hosts. The experiments confirmed the pathogenicity of the isolate CREA OF 189.2 on chickpea, while host range highlighted the polyphagous nature of this strain; thus, symptoms were reported on lentils, common bean and cantaloupe. The multidisciplinary approach allows us to increase the knowledge about this emerging pathogen. To the best of our knowledge, this is the first report on Macrophomina phaseolina from chickpeas in Italy.

Synthetic Antibiotic Derived from Sequences Encrypted in a Protein from Human Plasma.

Encrypted peptides have been recently found in the human proteome and represent a potential class of antibiotics. Here we report three peptides derived from the human apolipoprotein B (residues 887-922) that exhibited potent antimicrobial activity against drug-resistant Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococci both in vitro and in an animal model. The peptides had excellent cytotoxicity profiles, targeted bacteria by depolarizing and permeabilizing their cytoplasmic membrane, inhibited biofilms, and displayed anti-inflammatory properties. Importantly, the peptides, when used in combination, potentiated the activity of conventional antibiotics against bacteria and did not select for bacterial resistance. To ensure translatability of these molecules, a protease resistant retro-inverso variant of the lead encrypted peptide was synthesized and demonstrated anti-infective activity in a preclinical mouse model. Our results provide a link between human plasma and innate immunity and point to the blood as a source of much-needed antimicrobials.

Fighting multidrug resistance with a fruit extract: anti-cancer and anti-biofilm activities of Acca sellowiana.

In this study, the efficacy of Acca sellowiana fruit acetonic extract on human MDR cancer cells was tested for the first time, and it was demonstrated that the fruit extract is effective on both sensitive and resistant tumor cells. The effects of A. sellowiana extract on bacterial biofilm were also examined for the first time. By crystal violet assays and confocal microscopy analyses, it was demonstrated that the plant extract is able to strongly inhibit biofilm formation of both sensitive and resistant bacterial strains. Furthermore, antimicrobial activity assays and TEM analyses clearly demonstrated the effectiveness of plant extract on planktonic bacterial cells in both sensitive and resistant strains. Altogether, these findings intriguingly expand the panel of activities of A. sellowiana fruit extract with respect to previous reports, and open interesting perspectives to its therapeutic applications.

Transglutaminase-mediated crosslinking of a host defence peptide derived from human apolipoprotein B and its effect on the peptide antimicrobial activity.

Background Microbial transglutaminase (mTG) has been successfully used to produce site-specific protein conjugates derivatized at the level of Gln and/or Lys residues for different biotechnological applications. Here, a recombinant peptide identified in human apolipoprotein B sequence, named r(P)ApoBL and endowed with antimicrobial activity, was studied as a possible acyl acceptor substrate of mTG with at least one of the six Lys residues present in its sequence. Methods The enzymatic crosslinking reaction was performed in vitro using N,N-dimethylcasein, substance P and bitter vetch (Vicia ervilia) seed proteins, well known acyl donor substrates in mTG-catalyzed reactions. Mass spectrometry analyses were performed for identifying the Lys residue(s) involved in the crosslinking reaction. Finally, bitter vetch protein-based antimicrobial films grafted with r(P)ApoBL were prepared and, their biological activity evaluated. Results r(P)ApoBL was able to be enzymatically modified by mTG. In particular, it was demonstrated the highly selective crosslinking of the peptide under study by mTG at level of Lys-18. Interestingly, the biological activity of the peptide when grafted into protein-based films was found to be lost following mTG-catalyzed crosslinking. Conclusions r(P)ApoBL was shown to be an effective acyl acceptor substrate of mTG. The involvement of Lys-18 in the enzymatic reaction was demonstrated. In addition, films grafted with r(P)ApoBL in the presence of mTG lost antimicrobial property. General significance A possible role of mTG as biotechnological tool to modulate the r(P)ApoBL antimicrobial activity was hypothesized, and a potential use in food packaging of protein-based films grafted with r(P)ApoBL was suggested.

Cost-effective production of recombinant peptides in Escherichia coli.

Among bioactive peptides, cationic antimicrobial peptides (AMPs), also referred to as host defence peptides (HDPs), are valuable tools to treat infections, being able to kill a wide variety of microbes directly and/or modulate host immunity. HDPs have great therapeutic potential against antibiotic-resistant bacteria, viruses and even parasites. However, high manufacturing costs have greatly limited their development as drugs, thus highlighting the need to develop novel and competitive production strategies. Here, a cost-effective procedure was established to produce the high amounts of peptides required for basic and clinical research. Firstly, a novel culture medium was designed, which was found to support significantly higher cell densities and recombinant expression levels of peptides under test compared to conventional media. The procedure has been also efficiently scaled up by using a 5 L fermenter, while the costs have been lowered significantly by developing a successful auto-induction strategy, which has been found to support higher yields of target constructs and cell biomass compared to conventional strategies based on expression induction by IPTG. Interestingly, it was estimated that by increasing production scale from 100 to 1000 mg/batch, unit costs decreased strongly from 253 to 42 €/mg. These costs appear highly competitive when compared to chemical synthesis strategies. Altogether, the data indicate that the strategy represents an important starting point for the future development of large-scale manufacture of HDPs.

Effects of human antimicrobial cryptides identified in apolipoprotein B depend on specific features of bacterial strains.

Cationic Host Defense Peptides (HDPs) are endowed with a broad variety of activities, including direct antimicrobial properties and modulatory roles in the innate immune response. Even if it has been widely demonstrated that bacterial membrane represents the main target of peptide antimicrobial activity, the molecular mechanisms underlying membrane perturbation by HDPs have not been fully clarified yet. Recently, two cryptic HDPs have been identified in human apolipoprotein B and found to be endowed with a broad-spectrum antimicrobial activity, and with anti-biofilm, wound healing and immunomodulatory properties. Moreover, ApoB derived HDPs are able to synergistically act in combination with conventional antibiotics, while being not toxic for eukaryotic cells. Here, by using a multidisciplinary approach, including time killing curves, Zeta potential measurements, membrane permeabilization assays, electron microscopy analyses, and isothermal titration calorimetry studies, the antimicrobial effects of ApoB cryptides have been analysed on bacterial strains either susceptible or resistant to peptide toxicity. Intriguingly, it emerged that even if electrostatic interactions between negatively charged bacterial membranes and positively charged HDPs play a key role in mediating peptide toxicity, they are strongly influenced by the composition of negatively charged bacterial surfaces and by defined extracellular microenvironments.

Novel human bioactive peptides identified in Apolipoprotein B: Evaluation of their therapeutic potential.

Host defence peptides (HDPs) are short, cationic amphipathic peptides that play a key role in the response to infection and inflammation in all complex life forms. It is increasingly emerging that HDPs generally have a modest direct activity against a broad range of microorganisms, and that their anti-infective properties are mainly due to their ability to modulate the immune response. Here, we report the recombinant production and characterization of two novel HDPs identified in human Apolipoprotein B (residues 887-922) by using a bioinformatics method recently developed by our group. We focused our attention on two variants of the identified HDP, here named r(P)ApoBL and r(P)ApoBS, 38- and 26-residue long, respectively. Both HDPs were found to be endowed with a broad-spectrum antimicrobial activity while they show neither toxic nor haemolytic effects towards eukaryotic cells. Interestingly, both HDPs were found to display a significant anti-biofilm activity, and to act in synergy with either commonly used antibiotics or EDTA. The latter was selected for its ability to affect bacterial outer membrane permeability, and to sensitize bacteria to several antibiotics. Circular dichroism analyses showed that SDS, TFE, and LPS significantly alter r(P)ApoBL conformation, whereas slighter or no significant effects were detected in the case of r(P)ApoBS peptide. Interestingly, both ApoB derived peptides were found to elicit anti-inflammatory effects, being able to mitigate the production of pro-inflammatory interleukin-6 and nitric oxide in LPS induced murine macrophages. It should also be emphasized that r(P)ApoBL peptide was found to play a role in human keratinocytes wound closure in vitro. Altogether, these findings open interesting perspectives on the therapeutic use of the herein identified HDPs.