Defensins from the tick Ixodes scapularis are effective against phytopathogenic fungi and the human bacterial pathogen Listeria grayi

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Ixodes scapularis is the most common tick species in North America and a vector of important pathogens that cause diseases in humans and animals including Lyme disease, anaplasmosis and babesiosis. Tick defensins have been identified as a new source of antimicrobial agents with putative medical applications due to their wide-ranging antimicrobial activities. Two multigene families of defensins were previously reported in I. scapularis. The objective of the present study was to characterise the potential antimicrobial activity of two defensins from I. scapularis with emphasis on human pathogenic bacterial strains and important phytopathogenic fungi. [Methods]: Scapularisin-3 and Scapularisin-6 mature peptides were chemically synthesised. In vitro antimicrobial assays were performed to test the activity of these two defensins against species of different bacterial genera including Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Listeria spp. as well as Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa along with two plant-pathogenic fungi from the genus Fusarium. In addition, the tissue-specific expression patterns of Scapularisin-3 and Scapularisin-6 in I. scapularis midgut, salivary glands and embryo-derived cell lines were determined using PCR. Finally, tertiary structures of the two defensins were predicted and structural analyses were conducted. [Results]: Scapularisin-6 efficiently killed L. grayi, and both Scapularisin-3 and Scapularisin-6 caused strong inhibition (IC value: ∼1 μM) of the germination of plant-pathogenic fungi Fusarium culmorum and Fusarium graminearum. Scapularisin-6 gene expression was observed in I. scapularis salivary glands and midgut. However, Scapularisin-3 gene expression was only detected in the salivary glands. Transcripts from the two defensins were not found in the I. scapularis tick cell lines ISE6 and ISE18. [Conclusion]: Our results have two main implications. Firstly, the anti-Listeria and antifungal activities of Scapularisin-3 and Scapularisin-6 suggest that these peptides may be useful for (i) treatment of antibiotic-resistant L. grayi in humans and (ii) plant protection. Secondly, the antimicrobial properties of the two defensins described in this study may pave the way for further studies regarding pathogen invasion and innate immunity in I. scapularis.Miray Tonk is a Marie Curie Early Stage Researcher supported by the POSTICK ITN (Post-graduate training network for capacity building to control ticks and tick-borne diseases) within the FP7- PEOPLE – ITN programme (EU Grant No. 238511). This project was partially supported by the Grant Agency of the Czech Republic (GACR P302/11/1901) and with institutional support RVO: 60077344 from Biology Centre, Institute of Parasitology as well as grant ANTIGONE (EU-7FP; 278976). James J. Valdés was sponsored by project CZ.1.07/2.3.00/30.0032, co-financed by the European Social Fund and the state budget of the Czech Republic. Radek Šíma was supported by the Grant 13-12816P (GA CR). Mohammad Rahnamaeian and Andreas Vilcinskas acknowledge the Ministry for Science and Art of the State of Hesse (Germany) for funding the LOEWE Center of Insect Biotechnology and Bioresources. Zdeněk Franta was supported by Alexander von Humboldt Research Fellowship for Postdoctoral Researchers.Peer Reviewe

Proline-Rich Antimicrobial Peptides in Medicinal Maggots of Lucilia sericata Interact With Bacterial DnaK But Do Not Inhibit Protein Synthesis

In the search for new antibiotics to combat multidrug-resistant microbes, insects offer a rich source of novel anti-infectives, including a remarkably diverse array of antimicrobial peptides (AMPs) with broad activity against a wide range of species. Larvae of the common green bottle fly Lucilia sericata are used for maggot debridement therapy, and their effectiveness in part reflects the large panel of AMPs they secrete into the wound. To investigate the activity of these peptides in more detail, we selected two structurally different proline rich peptides (Lser-PRP2 and Lser-PRP3) in addition to the a-helical peptide Lser-stomoxyn. We investigated the mechanism of anti-Escherichia coli action of the PRPs in vitro and found that neither of them interfered with protein synthesis but both were able to bind the bacterial chaperone DnaK and are therefore likely to inhibit protein folding. However, unlike Lser-stomoxyn that permeabilized the bacterial membrane by 1% at the low concentration (0.25 µM) neither of the PRPs alone was able to permeabilize E. coli membrane. In the presence of this Lser-stomoxyn concentration significant increase in anti-E. coli activity of Lser-PRP2 was observed, indicating that this peptide needs specific membrane permeabilizing agents to exert its antibacterial activity. We then examined the AMPs-treated bacterial surface and observed detrimental structural changes in the bacterial cell envelope in response to combined AMPs. The functional analysis of insect AMPs will help select optimal combinations for targeted antimicrobial therap

The ABC transporter DerAB of <i>Lactobacillus casei</i> mediates resistance against insect-derived defensins

Bce-like systems mediate resistance against antimicrobial peptides in Firmicutes bacteria. Lactobacillus casei BL23 encodes an “orphan” ABC transporter that, based on homology to BceAB-like systems, was proposed to contribute to antimicrobial peptide resistance. A mutant lacking the permease subunit was tested for sensitivity against a collection of peptides derived from bacteria, fungi, insects, and humans. Our results show that the transporter specifically conferred resistance against insect-derived cysteine-stabilized αβ defensins, and it was therefore renamed DerAB for defensin resistance ABC transporter. Surprisingly, cells lacking DerAB showed a marked increase in resistance against the lantibiotic nisin. This could be explained by significantly increased expression of the antimicrobial peptide resistance determinants regulated by the Bce-like systems PsdRSAB (formerly module 09) and ApsRSAB (formerly module 12). Bacterial two-hybrid studies in Escherichia coli showed that DerB could interact with proteins of the sensory complex in the Psd resistance system. We therefore propose that interaction of DerAB with this complex in the cell creates signaling interference and reduces the cell’s potential to mount an effective nisin resistance response. In the absence of DerB, this negative interference is relieved, leading to the observed hyperactivation of the Psd module and thus increased resistance to nisin. Our results unravel the function of a previously uncharacterized Bce-like orphan resistance transporter with pleiotropic biological effects on the cell.This work was financially supported by DFG grant MA2837/3-2 (to T.M.) and by funds from the former Spanish Ministry of Science and Innovation and FEDER (grant AGL2010-15679) and the Generalitat Valenciana (grant ACOMP2012/137) (to M.Z.). A.R.-G. thanks the Federation of European Microbiological Societies for research grant FEMS-RG-2014-0067. Q.Z. is financially supported by a stipend from the China Scholarship Council (CSC).Peer reviewe

Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens

The potential of metchnikowin, a 26-amino acid residue proline-rich antimicrobial peptide synthesized in the fat body of Drosophila melanogaster was explored to engineer disease resistance in barley against devastating fungal plant pathogens. The synthetic peptide caused strong in vitro growth inhibition (IC50 value ∼1 μM) of the pathogenic fungus Fusarium graminearum. Transgenic barley expressing the metchnikowin gene in its 52-amino acid pre-pro-peptide form under the control of the inducible mannopine synthase (mas) gene promoter from the Ti plasmid of Agrobacterium tumefaciens displayed enhanced resistance to powdery mildew as well as Fusarium head blight and root rot. In response to these pathogens, metchnikowin accumulated in plant apoplastic space, specifying that the insect signal peptide is functional in monocotyledons. In vitro and in vivo tests revealed that the peptide is markedly effective against fungal pathogens of the phylum Ascomycota but, clearly, less active against Basidiomycota fungi. Importantly, germination of the mutualistic basidiomycete mycorrhizal fungus Piriformospora indica was affected only at concentrations beyond 50 μM. These results suggest that antifungal peptides from insects are a valuable source for crop plant improvements and their differential activities toward different phyla of fungi denote a capacity for insect peptides to be used as selective measures on specific plant diseases

Bioactivity of Natural and Engineered Antimicrobial Peptides from Venom of the Scorpions Urodacus yaschenkoi and U. manicatus

The spread of multidrug-resistant human pathogens has drawn attention towards antimicrobial peptides (AMPs), which are major players in the innate immune systems of many organisms, including vertebrates, invertebrates, plants and microbes. Scorpion venom is an abundant source of novel and potent AMPs. Here, we investigated natural and engineered AMPs from the scorpions Urodacus yaschenkoi and U. manicatus to determine their antimicrobial spectra as well as their hemolytic/cytotoxic activity. None of the AMPs were active against fungi, but many of them were active at low concentrations (0.25–30 µM) against seven different bacteria. Hemolytic and cytotoxic activities were determined using pig erythrocytes and baby hamster kidney cells, respectively. The amino acid substitutions in the engineered AMPs did not inhibit cytotoxicity, but reduced hemolysis and therefore increased the therapeutic indices. The phylogenetic analysis of scorpion AMPs revealed they are closely related and the GXK motif is highly conserved. The engineered scorpion AMPs offer a promising alternative for the treatment of multidrug-resistant bacterial infections and could be modified further to reduce their hemolytic/cytotoxic activity

Proline-Rich Antimicrobial Peptides in Medicinal Maggots of Lucilia sericata Interact With Bacterial DnaK But Do Not Inhibit Protein Synthesis

In the search for new antibiotics to combat multidrug-resistant microbes, insects offer a rich source of novel anti-infectives, including a remarkably diverse array of antimicrobial peptides (AMPs) with broad activity against a wide range of species. Larvae of the common green bottle fly Lucilia sericata are used for maggot debridement therapy, and their effectiveness in part reflects the large panel of AMPs they secrete into the wound. To investigate the activity of these peptides in more detail, we selected two structurally different proline rich peptides (Lser-PRP2 and Lser-PRP3) in addition to the a-helical peptide Lser-stomoxyn. We investigated the mechanism of anti-Escherichia coli action of the PRPs in vitro and found that neither of them interfered with protein synthesis but both were able to bind the bacterial chaperone DnaK and are therefore likely to inhibit protein folding. However, unlike Lser-stomoxyn that permeabilized the bacterial membrane by 1% at the low concentration (0.25 µM) neither of the PRPs alone was able to permeabilize E. coli membrane. In the presence of this Lser-stomoxyn concentration significant increase in anti-E. coli activity of Lser-PRP2 was observed, indicating that this peptide needs specific membrane permeabilizing agents to exert its antibacterial activity. We then examined the AMPs-treated bacterial surface and observed detrimental structural changes in the bacterial cell envelope in response to combined AMPs. The functional analysis of insect AMPs will help select optimal combinations for targeted antimicrobial therap