Identification of defensin-encoding genes of Picea glauca: characterization of PgD5, a conserved spruce defensin with strong antifungal activity

Background: Plant defensins represent a major innate immune protein superfamily that displays strong inhibitory effects on filamentous fungi. The total number of plant defensins in a conifer species is unknown since there are no sequenced conifer genomes published, however the genomes of several angiosperm species provide an insight on the diversity of plant defensins. Here we report the identification of five new defensin-encoding genes from the Picea glauca genome and the characterization of two of their gene products, named PgD5 and endopiceasin. Results: Screening of a P. glauca EST database with sequences of known plant defensins identified four genes with homology to the known P. glauca defensin PgD1, which were designated PgD2-5. Whereas in the mature PgD2-4 only 7-9 amino acids differed from PgD1, PgD5 had only 64% sequence identity. PgD5 was amplified from P. glauca genomic DNA by PCR. It codes for a precursor of 77-amino acid that is fully conserved within the Picea genus and has similarity to plant defensins. Recombinant PgD5, produced in Escherichia coli, had a molecular mass of 5.721 kDa, as determined by mass spectrometry. The PgD5 peptide exhibited strong antifungal activity against several phytopathogens without any effect on the morphology of the treated fungal hyphae, but strongly inhibited hyphal elongation. A SYTOX uptake assay suggested that the inhibitory activity of PgD5 could be associated with altering the permeability of the fungal membranes. Another completely unrelated defensin gene was identified in the EST library and named endopiceasin. Its gene codes for a 6-cysteine peptide that shares high similarity with the fungal defensin plectasin. Conclusions: Screening of a P. glauca EST database resulted in the identification of five new defensin-encoding genes. PgD5 codes for a plant defensin that displays non-morphogenic antifungal activity against the phytopathogens tested, probably by altering membrane permeability. PgD5 has potential for application in the plant biotechnology sector. Endopiceasin appears to derive from an endo- or epiphytic fungal strain rather than from the plant itself. Keywords: Spruce defensins, Endophyte, Antifungal activity, Membrane permeabilizatio

Antimicrobial peptides effectively kill a broad spectrum of Listeria monocytogenes and Staphylococcus aureus strains independently of origin, sub-type, or virulence factor expression

<p>Abstract</p> <p>Background</p> <p>Host defense peptides (HDPs), or antimicrobial peptides (AMPs), are important components of the innate immune system that bacterial pathogens must overcome to establish an infection and HDPs have been suggested as novel antimicrobial therapeutics in treatment of infectious diseases. Hence it is important to determine the natural variation in susceptibility to HDPs to ensure a successful use in clinical treatment regimes.</p> <p>Results</p> <p>Strains of two human bacterial pathogens, <it>Listeria monocytogenes </it>and <it>Staphylococcus aureus</it>, were selected to cover a wide range of origin, sub-type, and phenotypic behavior. Strains within each species were equally sensitive to HDPs and oxidative stress representing important components of the innate immune defense system. Four non-human peptides (protamine, plectasin, novicidin, and novispirin G10) were similar in activity profile (MIC value spectrum) to the human β-defensin 3 (HBD-3). All strains were inhibited by concentrations of hydrogen peroxide between 0.1% – 1.0%. Sub-selections of both species differed in expression of several virulence-related factors and in their ability to survive in human whole blood and kill the nematode virulence model <it>Caenorhabditis elegans</it>. For <it>L. monocytogenes</it>, proliferation in whole blood was paralleled by high invasion in Caco-2 cells and fast killing of <it>C. elegans</it>, however, no such pattern in phenotypic behavior was observed for <it>S. aureus </it>and none of the phenotypic differences were correlated to sensitivity to HDPs.</p> <p>Conclusion</p> <p>Strains of <it>L. monocytogenes </it>and <it>S. aureus </it>were within each species equally sensitive to a range of HDPs despite variations in subtype, origin, and phenotypic behavior. Our results suggest that therapeutic use of HDPs will not be hampered by occurrence of naturally tolerant strains of the two species investigated in the present study.</p

The heme sensing response regulator HssR in Staphylococcus aureus but not the homologous RR23 in Listeria monocytogenes modulates susceptibility to the antimicrobial peptide plectasin

<p>Abstract</p> <p>Background</p> <p>Host defence peptides (HDPs), also known as antimicrobial peptides (AMPs), have emerged as potential new therapeutics and their antimicrobial spectrum covers a wide range of target organisms. However, the mode of action and the genetics behind the bacterial response to HDPs is incompletely understood and such knowledge is required to evaluate their potential as antimicrobial therapeutics. Plectasin is a recently discovered HDP active against Gram-positive bacteria with the human pathogen, <it>Staphylococcus aureus </it>(<it>S. aureus</it>) being highly susceptible and the food borne pathogen, <it>Listeria monocytogenes </it>(<it>L. monocytogenes</it>) being less sensitive. In the present study we aimed to use transposon mutagenesis to determine the genetic basis for <it>S. aureus </it>and <it>L. monocytogenes </it>susceptibility to plectasin.</p> <p>Results</p> <p>In order to identify genes that provide susceptibility to plectasin we constructed bacterial transposon mutant libraries of <it>S. aureus </it>NCTC8325-4 and <it>L. monocytogenes </it>4446 and screened for increased resistance to the peptide. No resistant mutants arose when <it>L. monocytogenes </it>was screened on plates containing 5 and 10 fold Minimal Inhibitory Concentration (MIC) of plectasin. However, in <it>S. aureus</it>, four mutants with insertion in the heme response regulator (<it>hssR</it>) were 2-4 fold more resistant to plectasin as compared to the wild type. The <it>hssR </it>mutation also enhanced resistance to the plectasin-like defensin eurocin, but not to other classes of HDPs or to other stressors tested. Addition of plectasin did not influence the expression of <it>hssR </it>or <it>hrtA</it>, a gene regulated by HssR. The genome of <it>L. monocytogenes </it>LO28 encodes a putative HssR homologue, RR23 (in <it>L. monocytogenes </it>EGD-e lmo2583) with 48% identity to the <it>S. aureus </it>HssR, but a mutation in the <it>rr23 </it>gene did not change the susceptibility of <it>L. monocytogenes </it>to plectasin.</p> <p>Conclusions</p> <p><it>S. aureus </it>HssR, but not the homologue RR23 from <it>L. monocytogenes</it>, provides susceptibility to the defensins plectasin and eurocin. Our data suggest that a functional difference between response regulators HssR and RR23 is responsible for the difference in plectasin susceptibility observed between <it>S. aureus </it>and <it>L. monocytogenes</it>.</p