Broad-spectrum Antimicrobial Peptide Research Articles

Beta‐defensins: what are they REALLY doing in the oral cavity?

Initially identified as broad-spectrum antimicrobial peptides, the members of the β-defensin family have increasingly been observed to exhibit numerous other activities, both in vitro and in vivo, that do not always relate directly to host defense. Much research has been carried out in the oral cavity, where the presence of commensal bacteria further complicates the definition of their role. In addition to direct antimicrobial activity, β-defensins exhibit potent chemotactic activity for a variety of innate immune cells, as well as stimulating other cells to secrete cytokines. They can also inhibit the inflammatory response, however, by the specific binding of microbe-associated molecular patterns. These patterns are also able to induce the expression of β-defensins in gingival epithelial cells, although significant differences are observed between different species of bacteria. Together these results suggest a complex model of a host-defense related function in maintenance of bacterial homeostasis and response to pathogens. This model is complicated, however, by numerous other observations of β-defensin involvement in cell proliferation, wound healing and cancer. Together, the in vitro, in vivo and human studies suggest that these peptides are important in the biology of the oral cavity; exactly how is still subject to speculation.

Regulation of antimicrobial peptide expression in human gingival keratinocytes by interleukin-1α

In the oral cavity, mucosal keratinocytes resist bacterial infection, in part, by producing broad-spectrum antimicrobial peptides (AMPs) including defensin, adrenomedullin and calprotectin. Epidermal keratinocyte expression of many AMPs increases in response to interleukin-1α (IL-1α). IL-1α is produced by epidermal keratinocytes and regulates cell differentiation. To better understand innate immunity in the oral cavity, we sought to determine how IL-1α might regulate expression of AMPs by human gingival keratinocytes (HGKs) using DNA microarray and Western blot analyses. HGKs from three subjects expressed eleven AMPs, including S100A7, S100A8, S100A9, S100A12, secretory leucocyte protease inhibitor, lipocalin 2 (LCN2), cystatin C and β-defensin 2. Of the expressed AMPs, S100A7, S100A12 and LCN2 were up-regulated by IL-1α (inducible AMPs); the other AMPs were considered to be constitutive. Human gingival keratinocytes, therefore, express constitutive and IL-1α-inducible AMPs to provide a rapid and robust innate response to microbial infection.

Structure, Interactions, and Antibacterial Activities of MSI-594 Derived Mutant Peptide MSI-594F5A in Lipopolysaccharide Micelles: Role of the Helical Hairpin Conformation in Outer-Membrane Permeabilization

Lipopolysaccharide (LPS) provides a well-organized permeability barrier at the outer membrane of Gram-negative bacteria. Host defense cationic antimicrobial peptides (AMPs) need to disrupt the outer membrane before gaining access to the inner cytoplasmic membrane or intracellular targets. Several AMPs are largely inactive against Gram-negative pathogens due to the restricted permeation through the LPS layer of the outer membrane. MSI-594 (GIGKFLKKAKKGIGAVLKVLTTG) is a highly active AMP with a broad-spectrum of activities against bacteria, fungi, and virus. In the context of LPS, MSI-594 assumes a hairpin helical structure dictated by packing interactions between two helical segments. Residue Phe5 of MSI-594 has been found to be engaged in important interhelical interactions. In order to understand plausible structural and functional inter-relationship of the helical hairpin structure of MSI-594 with outer membrane permeabilization, a mutant peptide, termed MSI-594F5A, containing a replacement of Phe5 with Ala has been prepared. We have compared antibacterial activities, outer and inner membrane permeabilizations, LPS binding affinity, perturbation of LPS micelles structures by MSI-594 and MSI-594F5A peptides. Our results demonstrated that the MSI-594F5A has lower activities against Gram-negative bacteria, due to limited permeabilization through the LPS layer, however, retains Gram-positive activity, akin to MSI-594. The atomic-resolution structure of MSI-594F5A has been determined in LPS micelles by NMR spectroscopy showing an amphipathic curved helix without any packing interactions. The 3D structures, interactions, and activities of MSI-594 and its mutant MSI-594F5A in LPS provide important mechanistic insights toward the requirements of LPS specific conformations and outer membrane permeabilization by broad-spectrum antimicrobial peptides.

An Antibody as Surrogate Receptor Reveals Determinants of Activity of an Innate Immune Peptide Antibiotic

Drug discovery initiatives often depend critically on knowledge of ligand-receptor interactions. However, the identity or structure of the target receptor may not be known in every instance. The concept of receptor surrogate, a molecular environment mimic of natural receptor, may prove beneficial under such circumstances. Here, we demonstrate the potential of monoclonal antibodies (mAbs) to act as surrogate receptors for a class of innate immune peptide antibiotics, a strategy that can help comprehend their action mechanism and identify chemical entities crucial for activity. A panel of antibody surrogates was raised against indolicidin, a tryptophan-rich cationic broad spectrum antimicrobial peptide of innate immune origin. Employing an elegant combination of thermodynamics, crystallography, and molecular modeling, interactions of the peptide with a high affinity anti-indolicidin monoclonal antibody were analyzed and were used to identify a motif that contained almost the entire antibiotic activity of native indolicidin. The analysis clarified the interaction of the peptide with previously proposed targets such as bacterial cell membrane and DNA and could further be correlated with antimicrobial compounds whose actions involve varied other mechanisms. These features suggest a multipronged assault pathway for indolicidin. Remarkably, the anti-indolicidin mAb surrogate was able to isolate additional independent bactericidal sequences from a random peptide library, providing compelling evidence as to the physiological relevance of surrogate receptor concept and suggesting applications in receptor-based pharmacophore research.

Antimicrobial peptides on calcium phosphate-coated titanium for the prevention of implant-associated infections

Prevention of implant-associated infections has been one of the main challenges in orthopaedic surgery. This challenge is further complicated by the concern over the development of antibiotic resistance as a result of using traditional antibiotics for infection prophylaxis. The objective of this study was to develop a technique that enables the loading and local delivery of a unique group of cationic antimicrobial peptides (AMP) through implant surfaces. A thin layer of micro-porous calcium phosphate (CaP) coating was processed by electrolytic deposition onto the surface of titanium as the drug carrier. The broad spectrum AMP Tet213 (KRWWKWWRRC) was selected and loaded onto the CaP coating. SEM, XRD and FTIR analyses confirmed the CaP coating to be micro-porous octacalcium phosphate. By using a luminescence spectrometer technique, it was demonstrated that a 7 μm thick porous CaP coating could load up to 9 μg of AMP/cm2 using a simple soaking technique. The drug-loaded CaP coating (CaP-Tet213) was not cytotoxic for MG-63 osteoblast-like cells. The CaP-Tet213 implants had antimicrobial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria with 106-fold reductions of both bacterial strains within 30 min as assessed by measuring colony-forming units (CFU). Repeated CFU assays on the same CaP-Tet213 specimen demonstrated retention of antimicrobial activity by the CaP-Tet213 surfaces through four test cycles. The susceptibility of bacteria to the CaP-Tet213 surfaces was also evaluated by assessing the inhibition of luminescence of P. aeruginosa containing a luxCDABE cassette at 4 h and 24 h with ∼92% and ∼77% inhibition of luminescence, respectively. It was demonstrated that CaP-Tet213 was a more efficient antimicrobial coating than CaP-MX226, CaP-hLF1-11 or CaP-tobramycin following incubation of CaP implants with equimolar concentrations of Tet213, the commercially developed antimicrobial peptide MX-226, hLF1-11 or tobramycin. A device coated with CaP-Tet213 could be a potential solution for the prevention of the peri-implant infection in orthopaedics.

Survival and adherence of antimicrobial peptide ST4SA, produced by Enterococcus mundtii, at conditions found in the human gastro‐intestinal tract

The probiotic strain Enterococcus mundtii ST4SA, isolated from soy beans, produces a broad-spectrum antimicrobial peptide. The aim of this study was to determine if peptide ST4SA could withstand conditions simulating those found in the gastro-intestinal tract (GIT). Antimicrobial activity of peptide ST4SA has been monitored by growth inhibition of Enterococcus faecium on plates and leakage of β-galactosidase from damaged cells. The ability of peptide ST4SA to adhere to target cells, which is the first step in cell destruction, has been determined by calculating the percentage of active peptide that remained in the cell-free supernatant. Seventy-five percent of peptide ST4SA adhered to E. faecium HKLHS at 37 °C, 88% adhered to the cells at pH 8.0 and 10.0, and 75% adhered to the cells at pH 4.0 and 6.0 at 37 °C. Complete adherence of peptide ST4SA to E. faecium HKLHS was recorded in the presence of 3.0%, 5.0% and 10.0% (v/v) pancreatic juice, 0.3%, 0.5% and 1.0% (v/v) oxbile and 1% (w/v) NaCl, MgCl(2) and KCl. Peptide ST4SA survived conditions associated with the GIT and may be used to prevent or retard the growth of intestinal microbiota.

Isolation and characterization of the tissue and development-specific potato snakin-1 promoter inducible by temperature and wounding

Snakin-1 (StSN1) is a broad-spectrum antimicrobial peptide isolated from Solanum tuberosum . Homologous proteins have been identified in a wide range of species but there is no apparent consensus in the roles they play. A 1394 bp fragment of the 5'upstream region of StSN1 gene, designated PStSN1, was isolated from the potato genome and sequenced. Bioinformatics analyses revealed a total of 55 potential regulatory motifs related to tissue-specificity, stress, defence and hormone responsiveness, among others. PStSN1 spatial and temporal activity was studied in transgenic Arabidopsis plants expressing a reporter gene under this promoter control (PStSN1::GUS). Histochemical staining revealed PStSN1::GUS expression in the root vasculature, cotyledons, young leaves and floral organs. Moreover, GUS staining was detected in young developmental stages gradually decreasing as the plant aged. Stress treatments on transgenic plants showed that PStSN1 activity was induced by high/low temperature and wounding. The characterization of PStSN1 in a model plant establishes a framework for the understanding of its possible biological functions and provides a potential tool for plant modification through genetic engineering.

Paneth's disease

In about 70% of patients Crohn's disease (CD) affects the small intestine. This disease location is stable over time and associated with a genetic background different from isolated colonic disease. A characteristic feature of small intestinal host defense is the presence of Paneth cells at the bottom of the crypts of Lieberkühn. These cells produce different broad spectrum antimicrobial peptides (AMPs) most abundantly the α-defensins HD-5 and -6 (DEFA5 und DEFA6). In small intestinal Crohn's disease both these PC products are specifically reduced. As a functional consequence, ileal extracts from Crohn's disease patients are compromised in clearing bacteria and enteroadherent E. coli colonize the mucosa. Mechanisms for defective antimicrobial Paneth cell function are complex and include an association with a NOD2 loss of function mutation, a disturbance of the Wnt pathway transcription factor TCF7L2 (also known as TCF4), the autophagy factor ATG16L1, the endosomal stress protein XBP1, the toll-like receptor TLR9, the calcium mediated potassium chanel KCNN4 as well as mutations or inactivation of HD5. Thus we conclude that small intestinal Crohn's disease is most likely a complex disease of the Paneth cell: Paneth's disease.

The POU transcription factor Drifter/Ventral veinless regulates expression of Drosophila immune defense genes.

Innate immunity operates as a first line of defense in multicellular organisms against infections caused by different classes of microorganisms. Antimicrobial peptides (AMPs) are synthesized constitutively in barrier epithelia to protect against microbial attack and are also upregulated in response to infection. Here, we implicate Drifter/Ventral veinless (Dfr/Vvl), a class III POU domain transcription factor, in tissue-specific regulation of the innate immune defense of Drosophila. We show that Dfr/Vvl is highly expressed in a range of immunocompetent tissues, including the male ejaculatory duct, where its presence overlaps with and drives the expression of cecropin, a potent broad-spectrum AMP. Dfr/Vvl overexpression activates transcription of several AMP genes in uninfected flies in a Toll pathway- and Imd pathway-independent manner. Dfr/Vvl activates a CecA1 reporter gene both in vitro and in vivo by binding to an upstream enhancer specific for the male ejaculatory duct. Further, Dfr/Vvl and the homeodomain protein Caudal (Cad) activate transcription synergistically via this enhancer. We propose that the POU protein Dfr/Vvl acts together with other regulators in a combinatorial manner to control constitutive AMP gene expression in a gene-, tissue-, and sex-specific manner, thus promoting a first-line defense against infection in tissues that are readily exposed to pathogens.

Phylloseptin-1 (PSN-1) from Phyllomedusa sauvagei skin secretion: A novel broad-spectrum antimicrobial peptide with antibiofilm activity

Amphibian skin secretions have proven to be rich sources of antimicrobial peptides that are proposed to be fundamental components of the innate immune system. As amphibian skin is a multi-functional organ playing, among other things, a crucial role in respiration, it has been deemed that a core biological role for such peptides is control of microbial flora on this surface. To date, however, antimicrobial efficacy has been universally determined by means of establishing minimum inhibitory concentrations (MICs) using planktonic organisms rather than those within a biofilm such as would occur on this exposed surface. Here we describe the identification and structural characterisation of a novel 19 amino acid residue antimicrobial peptide of the phylloseptin family, named PSN-1, from the skin secretion of the waxy monkey frog, Phyllomedusa sauvagei. PSN-1 displayed broad-spectrum activity against a range of planktonic organisms with a high potency (MIC 5 μM) against Staphylococcus aureus. In a specific bioassay with the same organism grown as a biofilm, the minimal biofilm eradication concentration (MBEC) was found to be of the same high potency (5 μM). The present data would suggest that evaluation of actions and potency of amphibian skin secretion antimicrobial peptides might best be achieved by evaluating MBEC rather than MIC using planktonic organisms and that data arising from such studies may have more biological relevance in reflecting the purpose for which they have evolved through natural selection.

High‐throughput discovery of broad‐spectrum peptide antibiotics

Membrane-permeabilizing peptide antibiotics are an underutilized weapon in the battle against drug-resistant microorganisms. This is true, in part, because of the bottleneck caused by the lack of explicit design principles and the paucity of simple high-throughput methods for selection. In this work, we characterize the requirements for broad-spectrum antimicrobial activity by membrane permeabilization and find that different microbial membranes have very different susceptibilities to permeabilization by individual antimicrobial peptides. Broad-spectrum activity requires only that an AMP have at least a small amount of membrane-permeabilizing activity against multiple classes of microbes, a feature that we show to be rare in a peptide library containing many members with species-specific activity. We compare biological and vesicle-based high-throughput strategies for selecting such broad-spectrum AMPs from combinatorial peptide libraries and demonstrate that a simple in vitro, lipid vesicle-based high-throughput screen is the most effective strategy for rapid discovery of novel, broad-spectrum antimicrobial peptides.

Fluorescence and UV Resonance Raman Study of Peptide−Vesicle Interactions of Human Cathelicidin LL-37 and Its F6W and F17W Mutants

LL-37 is a broad-spectrum human antimicrobial peptide in the cathelicidin family. Potency assays in the form of minimal inhibitory concentration and vesicle leakage indicate that the single-tryptophan mutants, F6W and F17W, are as effective at killing bacteria and disrupting membranes as the native, tryptophan-free LL-37 peptide. Steady-state fluorescence and UV resonance Raman spectroscopy of F6W and F17W reveal molecular details of these tryptophan residues. The local environment polarity, hydrogen bond strength of the indole N-H moiety, and rotational freedom decrease for both F6W and F17W in the presence of carbonate ions relative to in pure distilled water; these results are consistent with burial of the hydrophobic region of alpha-helical LL-37 in oligomeric cores induced in the presence of carbonate ions. Differences in the spectroscopic properties of the carbonate-induced alpha-helical forms of F6W and F17W reflect the presence of a local lysine residue near F6W that makes the microenvironment of F6W more polar than that of F17W. In the presence of lipid vesicles, the mutants undergo additional loss of environment polarity, hydrogen bond strength, and rotational freedom. Quenching experiments utilizing brominated lipids reveal that the tryptophan residues in both mutants are essentially equidistant from the bilayer center and that bromines closer to the bilayer center, in the 9,10 positions, quench fluorescence more efficiently than those closer to the headgroups (6,7 positions). These results support carpeting or toroidal pore mechanisms of membrane disruption by LL-37 and demonstrate that the combination of tryptophan mutants and sensitive spectroscopic tools may provide important molecular clues about antimicrobial action.

Innate antimicrobial host defense in small intestinal Crohn's disease

Paneth cells (PCs) are specialized epithelial cells predominantly found in the small intestinal crypts of Lieberkuehn. They produce different broad spectrum antimicrobial peptides most abundantly the α-defensins HD-5 and -6 (DEFA5 und DEFA6). Both these PC products show a specific reduction in small intestinal Crohn′s disease (CD) – a form of inflammatory bowel disease (IBD). Their decrease is independent of current inflammation and an association with a NOD2 frameshift mutation has been demonstrated. More recently, another independent and even more frequent mechanism has been found which is linked to diminished levels of the Wnt pathway transcription factor TCF7L2 (also known as TCF4). Besides regulating the expression of HD-5 and HD-6 as TCF4 target genes, the Wnt pathway also orchestrates Paneth cell differentiation and maturation and controls stem cell maintenance in the small intestine. Besides NOD2 (which is predominantly expressed in PC) and ATG16L1 (inter alia important in the exocytosis of PC products), TCF4 is the third gene which is associated with small intestinal CD and Paneth cell antimicrobial function. Thus, Paneth cells seem to be key player emphazising a paramount importance of antimicrobial host defense in small intestinal CD pathogenesis.

Shosaikoto increases calprotectin expression in human oral epithelial cells

Oral epithelial cells help to prevent against bacterial infection in the oral cavity by producing antimicrobial peptides (AMPs). A broad-spectrum AMP, calprotectin (a complex of S100A8 and S100A9 proteins), is expressed by oral epithelial cells and is up-regulated by interleukin-1alpha (IL-1alpha). Shosaikoto (SST) is a traditional Japanese herbal medicine that has immunomodulatory effects and is reported to enhance the levels of IL-1alpha in epithelial cells. The purpose of this study was to investigate the effect of SST on the expression of calprotectin and other AMPs through the regulation of IL-1alpha in oral epithelial cells. Human oral epithelial cells (TR146) were cultured with SST (at concentrations ranging from 10 to 250 microg/mL) in the presence or absence of anti-IL-1alpha or IL-1 receptor antagonist. The expression of S100A8- and S100A9-specific mRNAs was examined by northern blotting. Calprotectin expression and IL-1alpha secretion were investigated by immunofluorescent staining or ELISA. The expression of other AMPs and IL-1alpha was analyzed by RT-PCR and by quantitative real-time PCR. Shosaikoto (25 microg/mL) significantly increased the expression of S100A8- and S100A9-specific mRNAs and calprotectin protein. Shosaikoto increased S100A7 expression, but had no effect on the expression of other AMPs. The expression of IL-1alpha-specific mRNA and its protein were slightly increased by SST. A neutralizing antibody against IL-1alpha or IL-1 receptor antagonist inhibited SST up-regulated S100A8/S100A9 mRNA expression. These results suggest that SST increases the expression of calprotectin and S100A7 in oral epithelial cells. In response to SST, up-regulation of calprotectin may be partially induced via IL-1alpha.

Broad-Spectrum Antimicrobial Peptides by Rational Combinatorial Design and High-Throughput Screening: The Importance of Interfacial Activity

We recently described 10 peptides selected from a 16,384-member combinatorial library based on their ability to permeabilize synthetic lipid vesicles in vitro. These peptides did not share a common sequence motif, length, or net charge; nonetheless, they shared a mechanism of action that is similar to the natural membrane permeabilizing antimicrobial peptides (AMP). To characterize the selected peptides and to compare the activity of AMPs in vivo and in vitro, we report on the biological activity of the same selected peptides in bacteria, fungi, and mammalian cells. Each of the peptides has sterilizing activity against all classes of microbes tested, at 2-8 microM peptide, with only slight hemolytic or cytotoxicity against mammalian cells. Similar to many natural AMPs, bacteria are killed within a few minutes of peptide addition, and the lethal step in vivo is membrane permeabilization. Single D-amino acid substitutions eliminated or diminished the secondary structure of the peptides, and yet, they retained activity against some microbes. Thus, secondary structure and biological activity are not coupled, consistent with the hypothesis that AMPs do not form pores of well-defined structure in membranes but rather destabilize membranes by partitioning into membrane interfaces and disturbing the organization of the lipids, a property that we have called "interfacial activity". The observation that broad-spectrum activity, but not all antimicrobial activity, is lost by small changes to the peptides suggests that the in vitro screen is specifically selecting for the rare peptides that have broad-spectrum activity. We put forth the hypothesis that methods focusing on screening peptide libraries in vitro for members with the appropriate interfacial activity can enable the design, selection, and discovery of novel, potent, and broad-spectrum membrane-active antibiotics.

Potent and Broad-Spectrum Antimicrobial Activity of CXCL14 Suggests an Immediate Role in Skin Infections

The skin is constantly exposed to commensal microflora and pathogenic microbes. The stratum corneum of the outermost skin layer employs distinct tools such as harsh growth conditions and numerous antimicrobial peptides (AMPs) to discriminate between beneficial cutaneous microflora and harmful bacteria. How the skin deals with microbes that have gained access to the live part of the skin as a result of microinjuries is ill defined. In this study, we report that the chemokine CXCL14 is a broad-spectrum AMP with killing activity for cutaneous gram-positive bacteria and Candida albicans as well as the gram-negative enterobacterium Escherichia coli. Based on two separate bacteria-killing assays, CXCL14 compares favorably with other tested AMPs, including human beta-defensin and the chemokine CCL20. Increased salt concentrations and skin-typical pH conditions did not abrogate its AMP function. This novel AMP is highly abundant in the epidermis and dermis of healthy human skin but is down-modulated under conditions of inflammation and disease. We propose that CXCL14 fights bacteria at the earliest stage of infection, well before the establishment of inflammation, and thus fulfills a unique role in antimicrobial immunity.

Inhibition of Bacillus cereus and Bacillus weihenstephanensis in raw vegetables by application of washing solutions containing enterocin AS-48 alone and in combination with other antimicrobials

Enterocin AS-48 is a broad-spectrum cyclic antimicrobial peptide produced by Enterococcus faecalis. In the present study, the bacteriocin was tested alone and in combination with other antimicrobials for decontamination of Bacillus inoculated on alfalfa, soybean sprouts and green asparagus. Washing with enterocin AS-48 solutions reduced viable cell counts of Bacillus cereus and Bacillus weihenstephanensis by 1.0–1.5 and by 1.5–2.38 log units right after application of treatment, respectively. In both cases, the bacteriocin was effective in reducing the remaining viable population below detection levels during further storage of the samples at 6 °C, but failed to prevent regrowth in samples stored at 15 or 22 °C. Application of washing treatments containing enterocin AS-48 in combination with several other antimicrobials and sanitizers (cinnamic and hydrocinnamic acids, carvacrol, polyphosphoric acid, peracetic acid, hexadecylpyridinium chloride and sodium hypochlorite) greatly enhanced the bactericidal effects. The combinations of AS-48 and sodium hypochlorite, peracetic acid or hexadecylpyridinium chloride provided the best results. After application of the combined treatments on alfalfa sprouts contaminated with B. cereus or with B. weihenstephanensis, viable bacilli were not detected or remained at very low concentrations in the treated samples during a 1-week storage period at 15 °C. Inhibition of B. cereus by in situ produced bacteriocin was tested by cocultivation with the AS-48 producer strain E. faecalis A-48-32 inoculated on soybean sprouts. Strain A-48-32 was able to grow and produce bacteriocin on sprouts both at 15 and 22 °C. At 15 °C, growth of B. cereus was completely inhibited in the cocultures, while a much more limited effect was observed at 22 °C. The results obtained for washing treatments are very encouraging for the application of enterocin AS-48 in the decontamination of sprouts. Application of washing treatments containing AS-48 alone can serve to reduce viable cell counts of bacilli in samples stored under refrigeration, while application of combined treatments should be recommended to avoid proliferation of the surviving bacilli under temperature-abuse conditions.

Anti-lipopolysaccharide factor in Litopenaeus vannamei ( LvALF): A broad spectrum antimicrobial peptide essential for shrimp immunity against bacterial and fungal infection

Antimicrobial peptides are an essential component of the innate immune system of most organisms. Expressed sequence tag analysis from various shrimp ( Litopenaeus vannamei) tissues revealed transcripts corresponding to two distinct sequences ( LvALF1 and LvALF2) with strong sequence similarity to anti-lipopolysaccharide factor (ALF), an antimicrobial peptide originally isolated from the horseshoe crab Limulus polyphemus. Full-length clones contained a 528 bp transcript with a predicted open reading frame coding for 120 amino acids in LvALF1, and a 623 bp transcript with a predicted open reading frame coding for 93 amino acids in LvALF2. A reverse genetic approach was implemented to study the in vivo role of LvALF1 in protecting shrimp from bacterial, fungal and viral infections. Injection of double-stranded RNA (dsRNA) corresponding to the LvALF1 message resulted in a significant reduction of LvALF1 mRNA transcript abundance as determined by qPCR. Following knockdown, shrimp were challenged with low pathogenic doses of Vibrio penaeicida, Fusarium oxysporum or white spot syndrome virus (WSSV) and the resulting mortality curves were compared with controls. A significant increase of mortality in the LvALF1 knockdown shrimp was observed in the V. penaeicida and F. oxysporum infections when compared to controls, showing that this gene has a role in protecting shrimp from both bacterial and fungal infections. In contrast, LvALF1 dsRNA activated the sequence-independent innate anti-viral immune response giving increased protection from WSSV infection.

Detection of the pediocin gene pedA in strains from human faeces by real-time PCR and characterization of Pediococcus acidilacticiUVA1

BackgroundBacteriocin-producing lactic acid bacteria are commonly used as natural protective cultures. Among them, strains of the genus Pediococcus are particularly interesting for their ability to produce pediocin, a broad spectrum antimicrobial peptide with a strong antagonistic activity against the food-borne pathogen Listeria monocytogenes. Furthermore, there is increasing interest in isolating new bacteriocin-producing strains of human intestinal origin that could be developed for probiotic effects and inhibition of pathogenic bacteria in the gut. In this work, we typed a new strain, co-isolated from baby faeces together with a Bifidobacterium thermophilum strain, and characterized its proteinaceous compound with strong antilisterial activity.ResultsThe newly isolated strain UVA1 was identified as a Pediococcus acidilactici by carbohydrate fermentation profile, growth at 50°C and 16S rDNA sequencing. The partially purified bacteriocin was heat resistant up to 100°C, active over a wide range of pH (2 to 9) and susceptible to proteolytic enzymes. The molecular weight, estimated by SDS-PAGE, was similar to that of pediocin AcH/PA-1 (4.5 kDa). P. acidilactici UVA1 harboured a 9.5-kb plasmid that could be cured easily, which resulted in the loss of the antimicrobial activity. Southern hybridization using the DIG-labelled pedA-probe established that the bacteriocin gene was plasmid-borne as for all pediocin described so far. Nucleotide sequence of the whole operon (3.5 kb) showed almost 100 % similarity to the pediocin AcH/PA-1 operon. The mRNA transcript for pedA could be detected in P. acidilactici UVA1 but not in the cured derivative, confirming the expression of the pedA-gene in UVA1. Using a new real-time PCR assay, eleven out of seventeen human faecal samples tested were found to contain pedA-DNA.ConclusionWe identified and characterised the first pediocin produced by a human intestinal Pediococcus acidilactici isolate and successfully developed a new real-time PCR assay to show the large distribution of pedA-containing strains in baby faecal samples.

Peptide Inhibitors of Streptococcus mutans in the Control of Dental Caries

Peptides have been investigated as potential inhibitors of Streptococcus mutans, the main cause of dental caries, and have demonstrated considerable promise. In a human trial, topical application to tooth surfaces of a synthetic peptide inhibitor (p1025) of S. mutans adhesion prevented recolonisation with the oral pathogen following treatment with chlorhexidine gluconate (a broad spectrum antiseptic compound). An important feature of this treatment is that the duration of protection extends well beyond the period in which p1025 is applied. The specific targeting of S. mutans which allows other members of the oral flora associated with health to recolonise the oral cavity and competitively exclude S. mutans may explain the extended protection. Further in vitro studies have identified several other peptides which may have potential as inhibitors of S. mutans. Of particular interest are studies that demonstrate that competence stimulating peptides of S. mutans act as inhibitors of S. mutans growth and that peptides derived from the competence stimulating peptides can be used as a means of specifically targeting broad spectrum antimicrobial peptides.