The Drosophila Baramicin polypeptide gene protects against fungal infection

The fruit fly Drosophila melanogaster combats microbial infection by producing a battery of effector peptides that are secreted into the haemolymph. Technical difficulties prevented the investigation of these short effector genes until the recent advent of the CRISPR/CAS era. As a consequence, many putative immune effectors remain to be formally described, and exactly how each of these effectors contribute to survival is not well characterized. Here we describe a novel Drosophila antifungal peptide gene that we name Baramicin A. We show that BaraA encodes a precursor protein cleaved into multiple peptides via furin cleavage sites. BaraA is strongly immune-induced in the fat body downstream of the Toll pathway, but also exhibits expression in other tissues. Importantly, we show that flies lacking BaraA are viable but susceptible to the entomopathogenic fungus Beauveria bassiana. Consistent with BaraA being directly antimicrobial, overexpression of BaraA promotes resistance to fungi and the IM10-like peptides produced by BaraA synergistically inhibit growth of fungi in vitro when combined with a membrane-disrupting antifungal. Surprisingly, BaraA mutant males but not females display an erect wing phenotype upon infection. Here, we characterize a new antifungal immune effector downstream of Toll signalling, and show it is a key contributor to the Drosophila antimicrobial response.     

Insect immunity. Constitutive expression of a cysteine-rich antifungal and a linear antibacterial peptide in a termite insect

Two novel antimicrobial peptides, which we propose to name termicin and spinigerin, have been isolated from the fungus-growing termite Pseudacanthotermes spiniger (heterometabole insect, Isoptera). Termicin is a 36-amino acid residue antifungal peptide, with six cysteines arranged in a disulfide array similar to that of insect defensins. In contrast to most insect defensins, termicin is C-terminally amidated. Spinigerin consists of 25 amino acids and is devoid of cysteines. It is active against bacteria and fungi. Termicin and spinigerin show no obvious sequence similarities with other peptides. Termicin is constitutively present in hemocyte granules and in salivary glands. The presence of termicin and spinigerin in unchallenged termites contrasts with observations in evolutionary recent insects or insects undergoing complete metamorphosis, in which antimicrobial peptides are induced in the fat body and released into the hemolymph after septic injury.     

Molecular characterization of two novel antibacterial peptides inducible upon bacterial challenge in an annelid, the leech Theromyzon tessulatum

Two novel antimicrobial peptides named theromacin and theromyzin were isolated and characterized from the coelomic liquid of the leech Theromyzon tessulatum. Theromacin is a 75-amino acid cationic peptide containing 10 cysteine residues arranged in a disulfide array showing no similarities with other known antimicrobial peptides. Theromyzin is an 86-amino acid linear peptide and constitutes the first anionic antimicrobial peptide observed in invertebrates. Both peptides exhibit activity directed against Gram-positive bacteria. Theromacin and theromyzin cDNAs code precursor molecules containing a putative signal sequence directly followed by the mature peptide. The enhancement of theromacin and theromyzin mRNA levels has been observed after blood meal ingestion and upon bacterial challenge. In situ hybridization revealed that both genes are expressed in large fat cells in contact with coelomic cavities. Gene products were immunodetected in large fat cells, in intestinal epithelia, and at the epidermis level. In addition, a rapid release of the peptides into the coelomic liquid was observed after bacterial challenge. The presence of antimicrobial peptide genes in leeches and their expression in a specific tissue functionally resembling the insect fat body provide evidence for the first time of an antibacterial response in a lophotrochozoan comparable to that of holometabola insects.     

Functional Analysis of PGRP-LA in Drosophila Immunity

PeptidoGlycan Recognition Proteins (PGRPs) are key regulators of the insect innate antibacterial response. Even if they have been intensively studied, some of them have yet unknown functions. Here, we present a functional analysis of PGRP-LA, an as yet uncharacterized Drosophila PGRP. The PGRP-LA gene is located in cluster with PGRP-LC and PGRP-LF, which encode a receptor and a negative regulator of the Imd pathway, respectively. Structure predictions indicate that PGRP-LA would not bind to peptidoglycan, pointing to a regulatory role of this PGRP. PGRP-LA expression was enriched in barrier epithelia, but low in the fat body. Use of a newly generated PGRP-LA deficient mutant indicates that PGRP-LA is not required for the production of antimicrobial peptides by the fat body in response to a systemic infection. Focusing on the respiratory tract, where PGRP-LA is strongly expressed, we conducted a genome-wide microarray analysis of the tracheal immune response of wild-type, Relish, and PGRP-LA mutant larvae. Comparing our data to previous microarray studies, we report that a majority of genes regulated in the trachea upon infection differ from those induced in the gut or the fat body. Importantly, antimicrobial peptide gene expression was reduced in the tracheae of larvae and in the adult gut of PGRP-LA-deficient Drosophila upon oral bacterial infection. Together, our results suggest that PGRP-LA positively regulates the Imd pathway in barrier epithelia.     

Fat body and hemocyte contribution to the antimicrobial peptide synthesis in <Emphasis Type="Italic">Calliphora vicina</Emphasis> R.-D. (Diptera: Calliphoridae) larvae

Antimicrobial peptides accumulated in the hemolymph in response to infection are a key element of insect innate immunity. The involvement of the fat body and hemocytes in the antimicrobial peptide synthesis is widely acknowledged, although release of the peptides present in the hemolymph from the immune cells was not directly verified so far. Here, we studied the presence of antimicrobial peptides in the culture medium of fat body cells and hemocytes isolated from the blue blowfly Calliphora vicina using complex of liquid chromatography, mass spectrometry, and antimicrobial activity assays. Both fat body and hemocytes are shown to synthesize and release to culture medium defensin, cecropin, diptericins, and proline-rich peptides. The spectra of peptide antibiotics released by the fat body and hemocytes partially overlap. Thus, the results suggest that insect fat body and blood cells are capable of releasing mature antimicrobial peptides to the hemolymph. It is notable that the data obtained demonstrate dramatic difference in the functioning of insect antimicrobial peptides and their mammalian counterparts localized into blood cells’ phagosomes where they exert their antibacterial activity.     

Hepcidin, a urinary antimicrobial peptide synthesized in the liver

Cysteine-rich antimicrobial peptides are abundant in animal and plant tissues involved in host defense. In insects, most are synthesized in the fat body, an organ analogous to the liver of vertebrates. From human urine, we characterized a cysteine-rich peptide with three forms differing by amino-terminal truncation, and we named it hepcidin (Hepc) because of its origin in the liver and its antimicrobial properties. Two predominant forms, Hepc20 and Hepc25, contained 20 and 25 amino acid residues with all 8 cysteines connected by intramolecular disulfide bonds. Reverse translation and search of the data bases found homologous liver cDNAs in species from fish to human and a corresponding human genomic sequence on human chromosome 19. The full cDNA by 5' rapid amplification of cDNA ends was 0.4 kilobase pair, in agreement with hepcidin mRNA size on Northern blots. The liver was the predominant site of mRNA expression. The encoded prepropeptide contains 84 amino acids, but only the 20-25-amino acid processed forms were found in urine. Hepcidins exhibited antifungal activity against Candida albicans, Aspergillus fumigatus, and Aspergillus niger and antibacterial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and group B Streptococcus. Hepcidin may be a vertebrate counterpart of cysteine-rich antimicrobial peptides produced in the fat body of insects.     

Antifungal activity of Latarcin 1 derived cell-penetrating peptides against Fusarium solani

Cell-penetrating peptides and antimicrobial peptides share physicochemical characteristics and mechanisms of interaction with biological membranes, hence, termed as membrane active peptides. The present study aims at evaluating AMP activity of CPPs. LDP-NLS and LDP are Latarcin 1 derived cell-penetrating peptides and in the current study we have evaluated antifungal and cell-penetrating properties of these CPPs in Fusarium solani. We observed that LDP-NLS and LDP exhibited excellent antifungal activity against the fungus. Cellular uptake experiments with LDP-NLS and LDP showed that LDP-NLS acted as a CPP but LDP uptake into fungal spores and hyphae was negligible. CPP and AMP activity of mutated version of LDP-NLS was also evaluated and it was observed that both the activities of the peptide were compromised, signifying the importance of arginines and lysines present in LDP-NLS for initial interaction of membrane active peptides with biological membranes. Dextrans and Propidium Iodide uptake studies revealed that the mode of entry of LDP-NLS into fungal hyphae is through pore formation. Also, both LDP-NLS and LDP showed no cytotoxicity when infiltered into leaf tissues. Overall, our results suggest that LDP-NLS and LDP are selectively cytotoxic to F. solani and can be a potent peptide based antifungal agents.     

Apoptosis-inducing antifungal peptides and proteins

Despite the availability of various classes of antimycotics, the treatment of patients with systemic fungal infections is challenging. Therefore the development of new antifungals is urgently required. Promising new antifungal candidates are antimicrobial peptides. In the present review, we provide an overview of antifungal peptides isolated from plants, insects, amphibians and mammals that induce apoptosis. Their antifungal spectrum, mode of action and toxicity are discussed in more detail.     

Antimicrobial peptides in the oral environment: expression and function in health and disease

The oral cavity is a unique environment in which antimicrobial peptides play a key role in maintaining health and may have future therapeutic applications. Present evidence suggests that alpha-defensins, beta-defensins, LL-37, histatin, and other antimicrobial peptides and proteins have distinct but overlapping roles in maintaining oral health and preventing bacterial, fungal, and viral adherence and infection. The expression of the inducible hBD-2 in normal oral epithelium, in contrast to other epithelia, and the apparent differential signaling in response to commensal and pathogenic organisms, provides new insights into innate immunity in this body site. Commensal bacteria are excellent inducers of hBD-2 in oral epithelial cells, suggesting that the commensal bacterial community acts in a manner to benefit the overall innate immune readiness of oral epithelia. This may have major significance for understanding host defense in the complex oral environment.     

The imd gene is required for local Cecropin expression in Drosophila barrier epithelia

Surfaces of higher eukaryotes are normally covered with microorganisms but are usually not infected by them. Innate immunity and the expression of gene-encoded antimicrobial peptides play important roles in the first line of defence in higher animals. The immune response in Drosophila promotes systemic expression of antimicrobial peptides in response to microbial infection. We now demonstrate that the epidermal cells underlying the cuticle of larvae respond to infected wounds by local expression of the genes for the antimicrobial peptide cecropin A. Thus, the Drosophila epidermis plays an active role in the innate defence against microorganisms. The immune deficiency (imd) gene was found to be a crucial component of the signal-induced epidermal expression in both embryos and larvae. In contrast, melanization, which is part of the wound healing process, is not dependent on the imd gene, indicating that the signalling pathways promoting melanization and antimicrobial peptide gene expression can be uncoupled.     

Differential regulation of beta-defensin expression in human skin by microbial stimuli

In response to infection, epithelia mount an innate immune response that includes the production of antimicrobial peptides. However, the pathways that connect infection and inflammation with the induction of antimicrobial peptides in epithelia are not understood. We analyzed the molecular links between infection and the expression of three antimicrobial peptides of the beta-defensin family, human beta-defensin (hBD)-1, hBD-2, and hBD-3 in the human epidermis. After exposure to microbe-derived molecules, both monocytes and lymphocytes stimulated the epidermal expression of hBD-1, hBD-2, and hBD-3. The induced expression of hBD-3 was mediated by transactivation of the epidermal growth factor receptor. The mechanisms of induction of hBD-1 and hBD-3 were distinct from each other and from the IL-1-dependent induction of hBD-2 expression. Thus during inflammation, epidermal expression of beta-defensins is mediated by at least three different mechanisms.     

Synthesis of new antifungal peptides selective against Cryptococcus neoformans

Many drugs are available for the treatment of systemic or superficial mycoses, but only a limited number of them are effective antifungal drugs, devoid of toxic and undesirable side effects. Furthermore, resistance development and fungistatic rather than fungicidal activities represent limitations of current antifungal therapy. Therefore there remains an urgent need for a new generation of antifungal agents. According to a polypharmacological approach, the present work concerns the synthesis and antifungal activity of a set of peptides designed to simultaneously target the fungal cell surface and lanosterol demethylase, a key enzyme involved in ergosterol synthesis. Our peptides include amino acid sequences characteristic of membrane-active antimicrobial peptides (AMP), and due to the presence of His residues, they carry the imidazole ring characteristic of azole compounds. The peptides synthesized by us, were tested against different yeast species, and displayed general antifungal activity, with a therapeutically promising antifungal specificity against Cryptococcus neoformans.     

Immune response of Drosophila melanogaster to infection with the flagellate parasite Crithidia spp

Insects are able to recognize invading microorganisms and to mount an immune response to bacterial and fungal infections. Recently, the fruitfly Drosophila melanogaster has emerged as a promising invertebrate model to investigate innate immunity because of its well-characterized genetics. Insects are also vectors of numerous parasites which can trigger an immune response. We have investigated the interaction of Drosophila melanogaster with the flagellate protozoan Crithidia spp. We show that a per os parasitic infection triggers the synthesis of several antimicrobial peptides. By reverse phase HPLC and mass spectrometry, peptides were shown to be present in the hemolymph and not in the gut tissue, suggesting the presence of immune messengers between the site of the infection, namely the gut, and the fat body, the main site of synthesis for antimicrobial peptides. Interestingly, we have identified one molecule which is specifically induced in the hemolymph after infection with Crithidia, but not with bacteria, suggesting that Drosophila can discriminate between pathogens. When flagellates were injected into the hemolymph, a low synthesis of antimicrobial peptides was observed together with phagocytosis of parasites by circulating hemocytes. The data presented here suggest that Drosophila-Crithidia spp. represents an interesting model to study host defense against protozoan parasites.     

Sequential gene activation by ecdysteroids in polytene chromosomes ofDrosophila melanogaster

Summary Changes in polytene chromosome 3 L puffing patterns in the fat body ofDrosophila melanogaster larvae and prepupae are compared to those in the salivary gland. While some general features are common to the two tissues, there are differences which reflect their different developmental roles. In vitro experiments with fat body chromosomes show that they have a distinct response to ecdysteroids which is different from that of salivary gland chromosomes, and which does not,in this culture system, reproduce the changes observed in normal development. In short term culture experiments, the fat body chromosomes appear more sensitive to ecdysteroids than the salivary gland chromosomes and, although 20-OH ecdysone is more active than ecdysone in these assays, the possibility is not excluded that ecdysone has a role in normal development as it appears to alter gene activity at physiological levels in these cells.     

Innate immunity: involvement of new neuropeptides

Secretory granules of chromaffin cells from the adrenal medulla store catecholamines and a variety of peptides that are secreted in the extracellular medium during exocytosis. Among these fragments, several natural peptides displaying antimicrobial activities at the micromolar range have been isolated and characterized. We have shown that these peptides, derived from the natural processing of chromogranins (CGs), proenkephalin-A (PEA) and free ubiquitin (Ub), are released into the circulation and display antibacterial and antifungal activities. In this review we focus on three naturally secreted antimicrobial peptides corresponding to CGA1–76 (vasostatin-I), the bisphosphorylated form of PEA209–237 (enkelytin) and Ub. In addition, the antimicrobial properties of the synthetic active domains of vasostatin-I (CGA47–66 or chromofungin) and Ub (Ub65–76 or ubifungin) are reported.     

Structure-activity relationship study: short antimicrobial peptides.

Many short antimicrobial peptides (< 18mer) have been identified for the development of therapeutic agents. However, Structure-activity relationship (SAR) studies about short antimicrobial peptides have not been extensively performed. To investigate the relationship between activity and structural parameters such as an alpha-helical structure, a net positive charge and a hydrophobicity, we synthesized and characterized diastereomers, scramble peptides and substituted peptides of the short antimicrobial peptide identified by combinatorial libraries. Circular dichroism (CD) spectra and in vitro activity indicated that an alpha-helical structure correlated with the antimicrobial activity and a beta-sheet structure also satisfied a structural requirement for antimicrobial activity. Most peptides consisting of L-amino acids lost antifungal activity in the presence of heat-inactivated serum, while active diastereomers and a scramble peptide with the beta-sheet structure retained antifungal activity in the same condition.     

Lead optimization of antifungal peptides with 3D NMR structures analysis

Antimicrobial peptides are key components of the innate immune response in most multicellular organisms. These molecules are considered as one of the most innovative class of anti-infective agents that have been discovered over the last two decades, and therefore, as a source of inspiration for novel drug design. Insect cystine-rich antimicrobial peptides with the CS alpha beta scaffold (an alpha-helix linked to a beta-sheet by two disulfide bridges) represent particularly attractive templates for the development of systemic agents owing to their remarkable resistance to protease degradation. We have selected heliomicin, a broad spectrum antifungal CS alpha beta peptide from Lepidoptera as the starting point of a lead optimization program based on phylogenic exploration and fine tuned mutagenesis. We report here the characterization, biological activity, and 3D structure of heliomicin improved analogs, namely the peptides ARD1, ETD-135, and ETD-151. The ARD1 peptide was initially purified from the immune hemolymph of the caterpillars of Archeoprepona demophoon. Although it differs from heliomicin by only two residues, it was found to be more active against the human pathogens Aspergillus fumigatus and Candida albicans. The peptides ETD-135 and ETD-151 were engineered by site-directed mutagenesis of ARD1 in either cationic or hydrophobic regions. ETD-135 and ETD-151 demonstrated an improved antifungal activity over the native peptides, heliomicin and ARD1. A comparative analysis of the 3D structure of the four molecules highlighted the direct impact of the modification of the amphipathic properties on the molecule potency. In addition, it allowed to characterize an optimal organization of cationic and hydrophobic regions to achieve best antifungal activity.