Rainbow trout DUBA inhibits type I interferon signaling by deubiquitinating TRAF3.

Deubiquitinating enzyme A (DUBA), a member of the ovarian tumor (OTU) subfamily of deubiquitinases (DUBs), is recognized for its negative regulatory role in type I interferon (IFN) expression downstream of Toll-like receptor 3 (TLR3). However, its involvement in the TLR3 signaling pathway in fish remains largely unexplored. In this study, we investigated the regulatory role of DUBA (OmDUBA) in the TLR3 response in rainbow trout (Oncorhynchus mykiss). OmDUBA features a conserved OTU domain, and its expression increased in RTH-149 cells following stimulation with the TLR3 agonist poly(I:C). Gain- and loss-of-function experiments demonstrated that OmDUBA attenuated the activation of TANK-binding kinase 1 (TBK1), resulting in a subsequent reduction in type I IFN expression and IFN-stimulated response element (ISRE) activation in poly(I:C)-stimulated cells. OmDUBA interacted with TRAF3, a crucial mediator in TLR3-mediated type I IFN production. Under poly(I:C) stimulation, there was an augmentation in the K63-linked polyubiquitination of TRAF3, a process significantly inhibited upon OmDUBA overexpression. These findings suggest that OmDUBA may function similarly to its mammalian counterparts in downregulating the poly(I:C)-induced type I IFN response in rainbow trout by removing the K63-linked ubiquitin chain on TRAF3. Our study provides novel insights into the role of fish DUBA in antiviral immunity.

Rainbow trout USP4 downregulates LPS-induced inflammation by removing the K63-linked ubiquitin chain on TAK1.

Ubiquitin-specific protease 4 (USP4) is pivotal in negatively regulating the Toll-like receptor (TLR) signaling-mediated innate immune response. Although USP4 has been well studied in mammals, its role in TLR signaling pathways in fish remains largely unknown. In this study, we investigated the role of USP4 (OmUSP4) in regulating TLR response in rainbow trout Oncorhynchus mykiss. OmUSP4 contained the characteristic domains conserved in other USP4s: domain in USP (DUSP), ubiquitin-like (UBL), and the bi-part catalytic domain known as USP. OmUSP4 expression was increased in RTH-149 cells by stimulation with fish-pathogenic bacteria and bacterial ligands. Gain- and loss-of-function experiments revealed that OmUSP4 mitigated the activation of MAPKs and NF-κB, as well as the expression of pro-inflammatory cytokines in LPS-stimulated cells. OmUSP4 interacted with TAK1, a critical mediator in TLR-mediated NF-κB signaling pathways. LPS stimulation increased the K63-linked polyubiquitination of TAK1, which was significantly suppressed when OmUSP4 was compelled to be overexpressed. These results imply that OmUSP4 might function like mammals to downregulate LPS-induced inflammation in rainbow trout by removing the K63-linked ubiquitin chain on TAK1.

Semi-Biosynthetic Production of Surface-Binding Adhesive Antimicrobial Peptides Using Intein-Mediated Protein Ligation.

Microbial infections remain a global health concern, calling for the urgent need to implement effective prevention measures. Antimicrobial peptides (AMPs) have been extensively studied as potential antimicrobial coating agents. However, an efficient and economical method for AMP production is lacking. Here, we synthesized the direct coating adhesive AMP, NKC-DOPA5, composed of NKC, a potent AMP, and repeats of the adhesive amino acid 3,4-dihydroxyphenylalanine (DOPA) via an intein-mediated protein ligation strategy. NKC was expressed as a soluble fusion protein His-NKC-GyrA (HNG) in Escherichia coli, comprising an N-terminal 6× His-tag and a C-terminal Mxe GyrA intein. The HNG protein was efficiently produced in a 500-L fermenter, with a titer of 1.63 g/L. The NKC-thioester was released from the purified HNG fusion protein by thiol attack and subsequently ligated with chemically synthesized Cys-DOPA5. The ligated peptide His-NKC-Cys-DOPA5 was obtained at a yield of 88.7%. The purified His-NKC-Cys-DOPA5 possessed surface-binding and antimicrobial properties identical to those of the peptide obtained via solid-phase peptide synthesis. His-NKC-Cys-DOPA5 can be applied as a practical and functional antimicrobial coating to various materials, such as medical devices and home appliances.

A20 Inhibits LPS-Induced Inflammation by Regulating TRAF6 Polyubiquitination in Rainbow Trout.

The ubiquitin-editing enzyme A20 is known to inhibit the NF-κB transcription factor in the Toll-like receptor (TLR) pathways, thereby negatively regulating inflammation. However, its role in the TLR signaling pathway in fish is still largely unknown. Here, we identified a gene encoding A20 (OmA20) in rainbow trout, Oncorhynchus mykiss, and investigated its role in TLR response regulation. The deduced amino acid sequence of OmA20 contained a conserved N-terminal ovarian tumor (OTU) domain and seven C-terminal zinc-finger (ZnF) domains. Lipopolysaccharide (LPS) stimulation increased OmA20 expression in RTH-149 cells. In LPS-stimulated RTH-149 cells, gain- and loss-of-function experiments revealed that OmA20 inhibited MAPK and NF-κB activation, as well as the expression of pro-inflammatory cytokines. OmA20 interacted with TRAF6, a key molecule involved in the activation of TLR-mediated NF-κB signaling pathways. LPS treatment increased the K63-linked polyubiquitination of TRAF6 in RTH-149 cells, which was suppressed when OmA20 was forced expression. Furthermore, mutations in the OTU domain significantly decreased deubiquitination of the K63-linked ubiquitin chain on TRAF6, indicating that deubiquitinase activity is dependent on the OTU domain. These findings suggest that OmA20, like those of mammals, reduces LPS-induced inflammation in rainbow trout, most likely by regulating K63-linked ubiquitination of TRAF6.

Adhesive Antimicrobial Peptides Containing 3,4-Dihydroxy-L-Phenylalanine Residues for Direct One-Step Surface Coating.

Bacterial colonization and transmission via surfaces increase the risk of infection. In this study, we design and employ novel adhesive antimicrobial peptides to prevent bacterial contamination of surfaces. Repeats of 3,4-dihydroxy-L-phenylalanine (DOPA) were added to the C-terminus of NKC, a potent synthetic antimicrobial peptide, and the adhesiveness and antibacterial properties of the resulting peptides are evaluated. The peptide is successfully immobilized on polystyrene, titanium, and polydimethylsiloxane surfaces within 10 min in a one-step coating process with no prior surface functionalization. The antibacterial effectiveness of the NKC-DOPA5-coated polystyrene, titanium, and polydimethylsiloxane surfaces is confirmed by complete inhibition of the growth of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus within 2 h. The stability of the peptide coated on the substrate surface is maintained for 84 days, as confirmed by its bactericidal activity. Additionally, the NKC-DOPA5-coated polystyrene, titanium, and polydimethylsiloxane surfaces show no cytotoxicity toward the human keratinocyte cell line HaCaT. The antimicrobial properties of the peptide-coated surfaces are confirmed in a subcutaneous implantation animal model. The adhesive antimicrobial peptide developed in this study exhibits potential as an antimicrobial surface-coating agent for efficiently killing a broad spectrum of bacteria on contact.

Molecular cloning and functional analysis of deubiquitinase CYLD in rainbow trout, Oncorhynchus mykiss.

Deubiquitinase cylindromatosis (CYLD) inhibits MAPK and NF-κB activation pathways by deubiquitinating upstream regulatory factors. Although CYLD has been identified and actively studied in mammals, nothing is known about its putative function in fish. In this study, we identified the gene encoding CYLD (OmCYLD) from rainbow trout, Oncorhynchus mykiss, and examined its role during pathogenic infections. The deduced amino acid sequence of OmCYLD contains conserved CAP-Gly and USP domains. In RTH-149 cells, the expression of OmCYLD was increased by stimulation with Edwardsiella tarda and Streptococcus iniae. Gain-of-function and loss-of-function experiments showed that OmCYLD down-regulates the activation of MAPK and NF-κB and the expression of pro-inflammatory cytokines in E. tarda-stimulated RTH-149 cells. These findings suggest that OmCYLD might function like those of mammals to negatively regulate bacteria-triggered signaling pathway in fish.

Molecular cloning and functional characterization of TRAF6 and TAK1 in rainbow trout, Oncorhynchus mykiss.

TRAF6 and TAK1 are known to play important roles in vertebrate innate immunity as molecular bridge, linking upstream toll-like receptors (TLRs) with the downstream MAPK and NF-κB signalling pathways. However, their roles in TLR signalling pathway have yet to be fully described in fish. Here we identified genes encoding TRAF6 (OmTRAF6) and TAK1 (OmTAK1) from rainbow trout, Oncorhynchus mykiss, and examined their roles during pathogenic infections. The deduced amino acid sequences of OmTRAF6 and OmTAK1 contained the characteristic domains conserved in the TRAF and TAK1 families, respectively (OmTRAF6: RING, two TRAF-type zinc fingers, CCR and MATH domains; OmTAK1: STKc and CCR domains). In RTH-149 cells, the expression of OmTRAF6 and OmTAK1 was increased by stimulation with Edwardsiella tarda and LPS. Silencing of OmTRAF6 and OmTAK1 in RTH-149 cells negatively regulated the LPS-induced phosphorylation of p38 MAPK and JNK. TAK1 inhibitor (5z)-7-Oxozeaenol significantly decreased the LPS-induced activation of NF-κB in RTH-149 cells. In addition, silencing of OmTRAF6 and OmTAK1 significantly decreased the expression of MAPKs and NF-κB downstream target genes induced by LPS in RTH-149 cells. These findings suggest that OmTRAF6 and OmTAK1 might function like those of mammals to regulate bacteria-triggered signalling pathway in fish.

Anti-Photoaging Effect of Jeju Putgyul (Unripe Citrus) Extracts on Human Dermal Fibroblasts and Ultraviolet B-induced Hairless Mouse Skin.

Ultraviolet (UV) radiation stimulates the expression of matrix metalloproteinases (MMPs) and inflammatory cytokines. These signaling pathways participate in the degradation of the extracellular matrix and induce inflammatory responses that lead to photoaging. This study evaluated the antioxidant activity and the effect on MMPs and procollagen of putgyul extract in vitro. The anti-photoaging activity of putgyul extracts was estimated in vivo using hairless mice (HR-1). The putgyul extracts reduced MMP-1 production and increased the content of procollagen type I carboxy-terminal peptide in human dermal fibroblasts. Ultravilot-B (UVB)-induced expression of inflammatory cytokines and MMPs was detected in mice, and putgyul extracts suppressed the expression. These results suggest that putgyul extract inhibits photoaging by inhibiting the expression of MMPs that degrade collagen and inhibiting cytokines that induce inflammatory responses. The mouse model also demonstrated that oral administration of putgyul extracts decreased wrinkle depth, epidermal thickness, collagen degradation, and trans-epidermal water loss, and increased ß-glucosidase activity on UVB exposed skin. Putgyul extract protects against UVB-induced damage of skin and could be valuable in the prevention of photoaging.

Molecular cloning and functional analysis of nucleotide-binding oligomerization domain-containing protein 1 in rainbow trout, Oncorhynchus mykiss.

NOD1 has important roles in innate immunity as sensor of microbial components derived from bacterial peptidoglycan. In this study, we identified genes encoding components of the NOD1 signaling pathway, including NOD1 (OmNOD1) and RIP2 (OmRIP2) from rainbow trout, Oncorhynchus mykiss, and investigated whether OmNOD1 has immunomodulating activity in a rainbow trout hepatoma cell line RTH-149 treated with NOD1-specific ligand (iE-DAP). The deduced amino acid sequence of OmNOD1 contained conserved CARD, NOD and LRR domains. Loss-of-function and gain-of-function experiments indicated that OmNOD1 is involved in the expression of pro-inflammatory cytokines. Silencing of OmNOD1 in RTH-149 cells treated with iE-DAP decreased the expression of IL-1ß, IL-6, IL-8 and TNF-α. Conversely, overexpression of OmNOD1 resulted in up-regulation of IL-1ß, IL-6, IL-8 and TNF-α expression. In addition, RIP2 inhibitor (gefitinib) significantly decreased the expression of these pro-inflammatory cytokines induced by iE-DAP in RTH-149 cells. These findings highlight the important role of NOD1 signaling pathway in fish in eliciting innate immune response.

Acute and subchronic (28 days) oral toxicity studies of Codonopsis lanceolata extract in Sprague-Dawley rats.

Codonopsis lanceolata is a perennial plant that has been used as a food and in traditional medicine for the treatment of cough, bronchitis, and inflammation in East Asia including Korea, Japan, and China. However, information regarding its toxicity is limited. Therefore, we performed a safety evaluation of aqueous C. lanceolata root extract (CLE) in Sprague-Dawley rats. Assessment of acute toxicity revealed that CLE did not influence mortality, clinical appearance, body weight gain, or necropsy findings at a dose of 5000 mg/kg body weight. In the subchronic oral toxicity, data revealed that several significant alteration in food consumption, water consumption, protein excretion, WBCs levels, TGs, BUN levels, and the absolute and relative weights in the liver, spleen and lungs. However, these changes were transient and were not considered treatment related because they showed no apparent dose dependent. These results suggest that CLE (1250, 2500, and 5000 mg/kg body weight/day) administered orally does not cause acute or subchronic toxicity to male or female rats. The 50% lethal dose (LD50) of CLE was determined to be greater than 5000 mg/kg.

De novo generation of short antimicrobial peptides with enhanced stability and cell specificity.

OBJECTIVES: Though antimicrobial peptides (AMPs) show great potential as novel antibiotics, therapeutic applications are hindered by their low stability, toxicity and high manufacturing cost. Various chemical modification strategies are employed to overcome these problems. However, chemical modifications often significantly increase the manufacturing cost of AMPs with only limited pharmacokinetic advantages. Therefore, we developed AMPs with enhanced stability and cell specificity that can be economically produced. METHODS: Peptides were designed by systematic amino acid arrangement without the incorporation of both non-natural amino acids and peptidomimetics. Antimicrobial activities were measured against Gram-positive bacteria, Gram-negative bacteria and fungi by MIC evaluation under both standard and physiologically relevant conditions. Cytotoxicity towards human cells was evaluated to verify selective antimicrobial activity. The antibacterial mechanism of the peptides was elucidated by ß-galactosidase assay and scanning electron microscopy. RESULTS: Among the designed peptides, GNU6 and GNU7 showed potent antimicrobial activity against bacteria and fungi and maintained their activity in the presence of 150 mM NaCl and 10% serum. These peptides were not digested by exposure to trypsin, chymotrypsin and aureolysin for up to 12 h and showed potent antimicrobial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. Moreover, they did not affect the viability of erythrocytes, keratinocytes and fibroblasts up to 128 mg/L. A membrane permeabilization assay and scanning electron microscopy analysis showed that GNU6 and GNU7 compromised membrane integrity and function in microorganisms. CONCLUSIONS: This study suggests that GNU6 and GNU7 might overcome serious problems that currently prevent the clinical use of AMPs and be developed as novel antimicrobial agents.

Efficacy of the designer antimicrobial peptide SHAP1 in wound healing and wound infection.

Infected wounds cause delay in wound closure and impose significantly negative effects on patient care and recovery. Antimicrobial peptides (AMPs) with antimicrobial and wound closure activities, along with little opportunity for the development of resistance, represent one of the promising agents for new therapeutic approaches in the infected wound treatment. However, therapeutic applications of these AMPs are limited by their toxicity and low stability in vivo. Previously, we reported that the 19-amino-acid designer peptide SHAP1 possessed salt-resistant antimicrobial activities. Here, we analyzed the wound closure activities of SHAP1 both in vitro and in vivo. SHAP1 did not affect the viability of human erythrocytes and keratinocytes up to 200 µM, and was not digested by exposure to proteases in the wound fluid, such as human neutrophil elastase and Staphylococcus aureus V8 proteinase for up to 12 h. SHAP1 elicited stronger wound closure activity than human cathelicidin AMP LL-37 in vitro by inducing HaCaT cell migration, which was shown to progress via transactivation of the epidermal growth factor receptor. In vivo analysis revealed that SHAP1 treatment accelerated closure and healing of full-thickness excisional wounds in mice. Moreover, SHAP1 effectively countered S. aureus infection and enhanced wound healing in S. aureus-infected murine wounds. Overall, these results suggest that SHAP1 might be developed as a novel topical agent for the infected wound treatment.

Identification of marker compounds in fermented Benincasa hispida and validation of the method for its analysis.

Fermentation is a process that improves health functionality by inducing the production and increase of bioactive compounds. In this study, to standardize the fermentation process for Benincasa hispida, marker compounds that are increased or produced during fermentation were identified based on UPLC-QTOF-MS/MS. Analysis method verification and content analysis were conducted using HPLC-PDA. The marker compounds produced or increased in content were identified as 2-furoic acid, 2,3-dihydroxybenzoic acid, and rubinaphthin A by comparing their retention times, UV and MS spectra, and molecular formulas with those reported in previous studies. In addition, the increase in the content of the marker compounds by fermentation was confirmed, and the analytical method was validated by measuring its specificity, linearity, limit of detection and quantitation, precision, and accuracy. These results suggest that the developed fermentation process, marker compound identification, and verified analysis method can be applied to develop potential functional food ingredients from fermented B. hispida.

Rainbow trout peptidoglycan recognition protein has an anti-inflammatory function in liver cells.

Peptidoglycan recognition proteins (PGRPs) are innate immune molecules that are structurally conserved through evolution in both invertebrate and vertebrate animals. PGRPs exert diverse host-defense functions both through direct antibacterial activity and through indirect effects, including the induction of antimicrobial peptides and the modulation of inflammation and immune responses. In this study, we identified the gene encoding a long form of PGRP (OmPGRP-L1) from the rainbow trout, Oncorhynchus mykiss, and investigated whether it has immunomodulating activity in a rainbow trout hepatoma cell line RTH-149 challenged with fish pathogenic bacteria. OmPGRP-L1 contains the conserved PGRP domain and the four Zn(2+)-binding amino acid residues required for amidase activity. In RTH-149 cells, OmPGRP-L1 expression was increased by bacterial stimulation. Loss-of-function and gain-of-function experiments indicated that OmPGRP-L1 is involved in the expression of pro-inflammatory cytokines. Silencing of OmPGRP-L1 in RTH-149 cells challenged with Edwardsiella tarda dramatically increased the expression of IL-1ß and TNF-α. In contrast, overexpression of OmPGRP-L1 or its amidase-inactive mutant OmPGRP-L1(C472S) resulted in down-regulation of IL-1ß and TNF-α expression. When overexpressed in RTH-149 cells, OmPGRP-L1 inhibited NF-κB activity with or without bacterial stimulation. Collectively, these findings suggest that OmPGRP-L1 has an anti-inflammatory function, independent of its amidase activity, possibly via NF-κB inhibition in liver cells.

Antioxidant activity and anti-adipogenic effects of wild herbs mainly cultivated in Korea.

Wild herbs, which are edible plants that grow in mountainous areas, have diverse biological effects such as anti-obesity and anti-cancer activities. The aim of this study was to evaluate the total phenolic and flavonoid contents as well as the antioxidant activity of methanol extracts of Aster scaber, Ligularia fischeri, Kalopanax pictus, Codonopsis lanceolata, and Cirsium setidens and to assess their effects on lipid accumulation and reactive oxygen species (ROS) production during adipogenesis of 3T3-L1 cells. The results revealed that among the five studied wild herb extracts, Ligularia fischeri showed the highest total phenolic contents (215.8 ± 14.2 mg GAE/g) and Aster scaber showed the highest total flavonoid content (103.9 ± 3.4 mg RE/g). Furthermore, Aster scaber and Ligularia fischeri extracts showed higher antioxidant activity than the other wild herbs. Regarding anti-adipogenic activity, the Cirsium setidens extract significantly inhibited lipid accumulation (~80%) and ROS production (~50%) during adipogenesis of 3T3-L1 cells compared with control cells. These results suggest that wild herbs could be used for the development of functional foods as well as health promoting and pharmaceutical agents.

Novel Genetically Engineered Probiotics for Targeted Elimination of Pseudomonas aeruginosa in Intestinal Colonization.

The intestinal carriage rates of Pseudomonas aeruginosa are notably elevated in immunosuppressed individuals and hospitalized patients, increasing the risk of infection and antibiotic-associated diarrhea. A potential solution to this issue lies in autonomous antibacterial therapy, remaining inactive until a pathogen is detected, and releasing antibacterial compounds on demand to eliminate the pathogen. This study focuses on the development of genetically engineered probiotics capable of detecting and eradicating P. aeruginosa by producing and secreting PA2-GNU7, a P. aeruginosa-selective antimicrobial peptide (AMP), triggered by the presence of P. aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone (3OC12HSL). To achieve this goal, plasmid-based systems were constructed to produce AMPs in response to 3OC12HSL and secrete them into the extracellular medium using either the microcin V secretion system or YebF as a carrier protein. Following the transfer of these plasmid-based systems to Escherichia coli Nissle 1917 (EcN), we successfully demonstrated the ability of the engineered EcN to express and secrete PA2-GNU7, leading to the inhibition of P. aeruginosa growth in vitro. In addition, in a mouse model of intestinal P. aeruginosa colonization, the administration of engineered EcN resulted in reduced levels of P. aeruginosa in both the feces and the colon. These findings suggest that engineered EcN holds promise as a potential option for combating intestinal P. aeruginosa colonization, thus mitigating the risk of future endogenous infections in vulnerable patients.

A Novel Peptide as a Specific and Selective Probe for Klebsiella pneumoniae Detection.

Klebsiella pneumoniae is infamous for generating hospital-acquired infections, many of which are difficult to treat due to the bacterium's multidrug resistance. A sensitive and robust detection method of K. pneumoniae can help prevent a disease outbreak. Herein, we used K. pneumoniae cells as bait to screen a commercially available phage-displayed random peptide library for peptides that could be used to detect K. pneumoniae. The biopanning-derived peptide TSATKFMMNLSP, named KP peptide, displayed a high selectivity for the K. pneumoniae with low cross-reactivity to related Gram-negative bacteria. The specific interaction between KP peptide and K. pneumoniae lipopolysaccharide resulted in the peptide's selectivity against K. pneumoniae. Quantitative analysis of this interaction by enzyme-linked immunosorbent assay revealed that the KP peptide possessed higher specificity and sensitivity toward K. pneumoniae than commercially available anti-Klebsiella spp. antibodies and could detect K. pneumoniae at a detection limit of 104 CFU/mL. These results suggest that KP peptide can be a promising alternative to antibodies in developing a biosensor system for K. pneumoniae detection.

Buforins: histone H2A-derived antimicrobial peptides from toad stomach.

Antimicrobial peptides (AMPs) constitute an important component of the innate immune system in a variety of organisms. Buforin I is a 39-amino acid AMP that was first isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans. Buforin II is a 21-amino acid peptide that is derived from buforin I and displays an even more potent antimicrobial activity than its parent AMP. Both peptides share complete sequence identity with the N-terminal region of histone H2A that interacts directly with nucleic acids. Buforin I is generated from histone H2A by pepsin-directed proteolysis in the cytoplasm of gastric gland cells. After secretion into the gastric lumen, buforin I remains adhered to the mucous biofilm that lines the stomach, thus providing a protective antimicrobial coat. Buforins, which house a helix-hinge-helix domain, kill a microorganism by entering the cell without membrane permeabilization and thus binding to nucleic acids. The proline hinge is crucial for the cell penetrating activity of buforins. Buforins also are known to possess anti-endotoxin and anticancer activities, thus making these peptides attractive reagents for pharmaceutical applications. This review describes the role of buforins in innate host defense; future research paradigms; and use of these agents as human therapeutics.

Molecular cloning and characterization of peptidoglycan recognition proteins from the rockfish, Sebastes schlegeli.

Peptidoglycan recognition proteins (PGRPs) are innate immune molecules that are structurally conserved through evolution in both invertebrate and vertebrate animals. Here we report the identification and characterization of two long forms of PGRP (SsPGRP-L1 and SsPGRP-L2) from the rockfish, Sebastes schlegeli. The deduced amino acid sequences of SsPGRP-L1 and SsPGRP-L2, 466 and 482 residues respectively, contain the conserved PGRP domain and the four Zn(2+)-binding amino acid residues required for amidase activity. In addition to peptidoglycan-lytic amidase activity, recombinant SsPGRPs have broad-spectrum antimicrobial activity like zebrafish PGRPs. RT-PCR analysis of total RNA shows that the expression patterns of SsPGRP-L1 and SsPGRP-L2 genes are different, though they are widely expressed in the tissues that come in contact with bacteria. Overall, these data suggest that rockfish PGRPs are involved in the innate host defense of S. schlegeli against bacterial infections.

Development of a novel hybrid antimicrobial peptide for targeted killing of Pseudomonas aeruginosa.

The emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa, coupled with shrinking antibiotic pipelines, has increased the demand for new antimicrobials with novel mechanisms of action. As the indiscriminate nature of broad-spectrum antimicrobial toxicity may have negative clinical consequences and increase the incidence of resistance, we have developed a P. aeruginosa-selective antimicrobial peptide capable of preferentially killing P. aeruginosa relative to benign microorganisms. A targeting peptide (PA2) that binds specifically to OprF porin on P. aeruginosa was identified by phage display peptide library screening, and a hybrid peptide was constructed by addition of the targeting peptide to GNU7, a potent antimicrobial peptide. The resulting hybrid peptide PA2-GNU7 exhibited potent antimicrobial activity against P. aeruginosa without causing host toxicity. Confocal laser scanning microscopy analysis and time-kill experiments demonstrated that PA2-GNU7 exhibited a high degree of specificity for P. aeruginosa, and rapidly and selectively killed P. aeruginosa cells in mixed cultures. In addition, in vivo treatment efficacy of PA2-GNU7 was significantly greater than that of conventional antibiotics in a mouse model of MDR P. aeruginosa infection. Taken together, the data suggest that PA2-GNU7 may be a promising template for further development as a novel anti-MDR P. aeruginosa therapeutic agent.