Avian antimicrobial peptides: in vitro and in ovo characterization and protection from early chick mortality caused by yolk sac infection.

Increasing antibiotic resistance is a matter of grave concern for consumers, public health authorities, farmers, and researchers. Antimicrobial peptides (AMPs) are emerging as novel and effective non-antibiotic tools to combat infectious diseases in poultry. In this study, we evaluated six avian AMPs including 2 truncated cathelicidins, [CATH-1(6-26) and CATH-2(1-15)], and 4 avian ß-defensins (ABD1, 2, 6 and 9) for their bactericidal and immunomodulatory activities. Our findings have shown CATH-1(6-26) and ABD1 being the two most potent avian AMPs effective against Gram-positive and Gram-negative bacteria investigated in these studies. Moreover, CATH-1(6-26) inhibited LPS-induced NO production and exhibited dose-dependent cytotoxicity to HD11 cells. While, ABD1 blocked LPS-induced IL-1ß gene induction and was non-toxic to HD11 cells. Importantly, in ovo administration of these AMPs demonstrated that ABD1 can offer significant protection from early chick mortality (44% less mortality in ABD1 treated group versus the control group) due to the experimental yolk sac infection caused by avian pathogenic Escherichia coli. Our data suggest that in ovo administration of ABD1 has immunomodulatory and anti-infection activity comparable with CpG ODN. Thus, ABD1 can be a significant addition to potential alternatives to antibiotics for the control of bacterial infections in young chicks.

KR-12 coating of polyetheretherketone (PEEK) surface via polydopamine improves osteointegration and antibacterial activity in vivo.

Polyetheretherketone (PEEK) is considered to be a promising bone implant material owing to its biocompatibility and elastic modulus, which is similar to that of natural bone. However, the clinical potential of PEEK is severely limited by its bioinertness, which leads to poor osseointegration, and the lack of antibacterial properties. In this study, the antimicrobial peptide, KR-12, was immobilized on the surface of PEEK implants with the assistance of polydopamine (PDA) to inhibit bacterial infection as well as to promote osteogenesis and osseointegration. Compared to unmodified PEEK, the PEEK with immobilized KR-12 showed significantly improved antibacterial activity against Staphylococcus aureus (ATCC 25923), both in vitro and in vivo. For the in vitro and in vivo evaluation of the osteogenic properties of modified PEEK, rat bone mesenchymal stem cells (rBMSCs) and a rat femoral defect model were used, respectively. The in vitro studies showed that compared to rBMSCs treated with unmodified PEEK, those treated with KR-12-coated PEEK exhibited improved adhesion, proliferation, and osteogenic differentiation. Moreover, micro-computed tomography and histological analysis suggested that the KR-12 coating promoted osteointegration in vivo in rat femurs. Taken together, these results suggest that the KR-12 coating could improve the antibacterial ability of pure or PDA-coated PEEK against Staphylococcus aureus (ATCC 25923), both in vitro and in vivo. Overall, KR-12 combined with the PDA film coating synergistically induced osteogenic effects both in vitro and in vivo. Thus, the surface-modified material, which exhibits both anti-bacterial and osteointegration properties, shows considerable potential for use as an orthopedic implant.

The design of a cell-selective fowlicidin-1-derived peptide with both antimicrobial and anti-inflammatory activities.

Fowlicidin-1 (Fowl-1), a cathelicidin expressed in chicken intestine, is known to have both antimicrobial and anti-inflammatory properties. However, its pharmaceutical development has been ultimately compromised by its high host cytotoxicity. In this study, a series of N- and C-terminal-truncated 19-meric Fowl-1 peptides were synthesized. Among these truncated peptides, Fowl-1 (8-26) exhibited broad-spectrum antimicrobial activity without human erythrocyte cytotoxicity while reducing anti-inflammatory activity. Further, Fowl-1 (8-26)-WRK was designed via Thr5→Trp, Ile7→Arg, and Asn11→Lys substitutions in Fowl-1 (8-26) to exhibit more amphipathicity. The results revealed that it exhibited both antimicrobial and anti-inflammatory properties. This study also demonstrated that the inhibitory activity of Fowl-1 (8-26)-WRK against LPS-induced inflammation was mainly due to the binding of LPS to the peptide. Interestingly, compared with human cathelicidin LL-37 and melittin, Fowl-1 (8-26)-WRK showed more potent activity against drug-resistant bacteria. It was also resistant to physiological salts and human serum and acted synergistically in combination with conventional antibiotics, such as chloramphenicol, ciprofloxacin, and oxacillin, suggesting that combined with conventional antibiotics, it is a promising adjuvant. Furthermore, membrane depolarization, SYTOX Green uptake, and flow cytometry revealed that it kills bacteria by damaging their membrane integrity. Therefore, this study suggests that Fowl-1 (8-26)-WRK has considerable potential for future development as an antimicrobial and anti-inflammatory agent for treating antibiotic-resistant infections.

Characterization of bactericidal efficiency, cell selectivity, and mechanism of short interspecific hybrid peptides.

Facing rising global antibiotics resistance, physical membrane-damaging antimicrobial peptides (AMPs) represent promising antimicrobial agents. Various strategies to design effective hybrid peptides offer many advantages in overcoming the adverse effects of natural AMPs. In this study, hybrid peptides from different species were investigated, and three hybrid antimicrobial peptides, LI, LN, and LC, were designed by combining the typical fragment of human cathelicidin-derived LL37 with either indolicidin, pig nematode cecropin P1 (CP-1) or rat neutrophil peptide-1 (NP-1). In an aqueous solution, all hybrid peptides had an unordered conformation. In simulated membrane conditions, the hybrid peptide LI displayed more ß-turn and ß-hairpin structures, whereas LN and LC folded into α-helix structures. The three interspecific hybrid peptides LI, LN, and LC exhibited different levels of antimicrobial activity against Gram-positive and Gram-negative bacteria. LI demonstrated the highest antimicrobial activity and cell selectivity. The results of the swimming motility indicated that LI repressed bacterial motility in a concentration-dependent method. Endotoxin binding assay demonstrated that hybrid peptide LI conserved the binding ability to LPS (polyanionic lipopolysaccharides) of its parental peptides. Fluorescence assays, flow cytometry, and SEM further revealed that hybrid peptide LI acted through different bacteriostatic mechanisms than LL37 and indolicidin and that LI killed bacterial cells via membrane damage. In summary, this study demonstrated that hybrid peptide LI produced by interspecific hybrid synthesis possessed strong cell selectivity and is a promising therapeutic candidate for drug-resistant bacteria infection.

As-CATH4 and 5, two vertebrate-derived natural host defense peptides, enhance the immuno-resistance efficiency against bacterial infections in Chinese mitten crab, Eriocheir sinensis.

Host defense peptides (HDPs), a class of conserved components of animal innate immune system, possess direct antimicrobial activities against invading pathogens and broadly participate in boosting and modulating host immune responses. Cathelicidins is an important family of HDPs that has been identified exclusively in vertebrates. Considering the relatively conserved innate immune system between invertebrates and vertebrates, it is speculated that HDPs from vertebrates may also possess modulating functions on invertebrate innate immune system. In the present study, two novel cathelicidins (As-CATH4 and 5), which had been identified from the Chinese alligator in our previous study, were employed to investigate their functions as novel peptide immunostimulants in Chinese mitten crab. As-CATH4 and 5 exhibited potent, broad-spectrum, and rapid antimicrobial activities against all the tested aquatic pathogenic bacteria. Unlike traditional antibiotics, they target on bacterial cell membrane, induce membrane permeabilization and cell disruption, and ultimately result in cell death. The antimicrobial effect is far more rapid than traditional antibiotics. Therefore they are unlikely to induce bacteria resistance. After the crabs were administered with As-CATH4 and 5, the activities of lysozyme, acid phosphatase and alkaline phosphatase were significantly enhanced, which indicated that the immune system of crabs could be activated by As-CATH4 and 5. In bacteria challenge test, As-CATH4 and 5 could significantly decrease the bacterial numbers in crabs, and increase the survival rates of crabs in both pre-stimulation and co-stimulation groups. All of the results above indicated the great potential of As-CATH4 and 5 as novel peptide immunostimulants in the crab aquaculture.

Microwave-Assisted Synthesis of Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are emerging as one of the unsurpassed therapeutic tools to treat various devastating diseases that are affecting millions of lives. Conventional synthesis of peptides requires longer times, and hence automated microwave technology could be regarded as an alternative implement which offers advantages like less reaction times and higher yields. In this sense, we herein describe a methodology to prepare AMPs through solid-phase peptide synthesis under microwave conditions. We have used LL37 as an example to discuss the synthetic protocol including the difficulties involved in the preparation of so-called long and difficult peptides and also remedial procedures to overcome these obstacles.

Immobilization and orientation-dependent activity of a naturally occurring antimicrobial peptide.

A naturally occurring antimicrobial peptide, SMAP-29, was synthesized with an n-terminal or c-terminal cysteine, termed c_SMAP and SMAP_c, respectively, for site-directed immobilization to superparamagnetic beads. Immobilized SMAP orientation-dependent activity was probed against multiple bacteria of clinical interest including Acinetobacter baumannii, Pseudomonas aeruginosa, Bacillus anthracis sterne and Staphylococcus aureus. A kinetic microplate assay was employed to reveal both concentration and time-dependent activity for elucidation of minimum bactericidal concentration (MBC) and sub-lethal effects. Immobilized SMAP activity was equivalent or reduced compared with soluble SMAP_c and c_SMAP regardless of immobilization orientation, with only one exception. A comparison of immobilized SMAP_c and c_SMAP activity revealed a bacteria-specific potency dependent on immobilization orientation, which was contrary to that seen in solution, wherein SMAP_c was more potent against all bacteria than c_SMAP. Sub-MBC kinetic studies displayed the influence of peptide exposure to the cells with multiple bacteria exhibiting increased susceptibility and efficacy at lower concentrations upon extended exposure (i.e. MBC enhancement). For instances in which complete killing was not achieved, two predominant effects were evident: retardation of growth rate and an increased lag phase. Both effects, seen independently and concomitantly, indicate some degree of induced cellular damage that can serve as a predictor toward eventual cell death. SMAP_c immobilized on glass through standard silanization chemistry was also investigated to ascertain the influence of substrate on activity against select bacteria.

Structure-activity relationships of a snake cathelicidin-related peptide, BF-15.

Cathelicidin-BF15 (BF-15) is a 15-mer peptide derived from Cathelicidin-BF (BF-30), which is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity. Since BF-15 retains most part of the antimicrobial activity of BF-30 but has significantly reduced haemolytic activity and a much shorter sequence length (and less cost), it is a particularly attractive template around which to design novel antimicrobial peptides. However, the structure-activity relationship of it is still unknown. We designed and synthesized a series of C-terminal amidated analogs of BF-15 based on its amphipathic α-helix structure. And we characterized their antimicrobial potency and haemolytic activity. We identified the amidated BF-15 (analog B1) with potent antimicrobial activity against several antibiotic-resistant bacteria (MICs between 1 and 64 µg/mL, 2-16-folds higher than BF-30) and much lower haemolytic activity. The subsequent circular dichroism study results showed a typical α-helix pattern of analog B1 and the content of the α-helix structure of it increased significantly comparing with BF-30, which indicates the peptide sequence of BF-15 may provide a major contribution to the α-helix content of the whole BF-30 sequence. The peptide induced chaotic membrane morphology and cell debris as determined by electron microscopy. This suggests that the antimicrobial activity of B1 is based on cytoplasmic membrane permeability. Taken together, our results suggested that peptide B1 should be considered as an excellent candidate for developing therapeutic drugs.

Tammar wallaby mammary cathelicidins are differentially expressed during lactation and exhibit antimicrobial and cell proliferative activity.

Cathelicidins secreted in milk may be central to autocrine feedback in the mammary gland for optimal development in addition to conferring innate immunity to both the mammary gland and the neonate. This study exploits the unique reproductive strategy of the tammar wallaby (Macropus eugenii) model to analyse differential splicing of cathelicidin genes and to evaluate the bactericidal activity and effect of the protein on mammary epithelial cell proliferation. Two linear peptides, Con73 and Con218, derived from the heterogeneous carboxyl end of cathelicidin transcripts, MaeuCath1 and MaeuCath7 respectively, were evaluated for antimicrobial activity. Both Con73 and Con218 significantly inhibited the growth of Staphylococcus aureus, Pseudomonas aureginosa, Enterococcus faecalis and Salmonella enterica. In addition both MaeuCath1 and MaeuCath7 stimulated proliferation of primary tammar wallaby mammary epithelial cells (WallMEC). Lactation-phase specific alternate spliced transcripts were determined for MaeuCath1 showing utilisation of both antimicrobial and proliferative functions are required by the mammary gland and the suckled young. The study has shown for the first time that temporal regulation of milk cathelicidins may be crucial in antimicrobial protection of the mammary gland and suckled young and mammary cell proliferation.

Anti-tuberculosis activity of α-helical antimicrobial peptides: de novo designed L- and D-enantiomers versus L- and D-LL-37.

With the emergence of multi-drug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (Mtb), a new class of antimycobacterial agents with very different modes of action compared to classical antibiotics, are urgently needed. In this study, a series of 26-residue, amphipathic, α-helical antimicrobial peptides consisting of all D-amino acid residues and synthetic human L-LL37 (L-enantiomer) and D-LL37 (D-enantiomer) were investigated against M. tuberculosis susceptible strain (H37Rv) and a clinical multi-drug resistant strain (Vertulo). Minimal inhibitory concentrations (MICs) were determined through a peptide killing assay. D5, the most active analog against M. tuberculosis had a MIC value of 11.2 µM (35.2 µg/ml) against H37Rv strain and 15.6 µM (49 µg/ml) against the MDR strain. Peptide D1 had similar activity as D5 against the MDR strain (57 µg/mL), a 9-fold improvement in hemolytic activity and a 7.4-fold better therapeutic index compared to D5. Surprisingly, LL37 enantiomers showed little to no activity compared to the de-novo designed α-helical antimicrobial peptides.

Structure-function relationship of king cobra cathelicidin.

King cobra cathelicidin (OH-CATH) is composed of 34 amino acid residues having strong antibacterial and very weak hemolytic activities as reported by us recently. OH-CATH can be served as a valuable template to develop novel therapeutic drugs. In this study, OH-CATH and six of its analogs were synthesized to explore their structure-function relationships based on their bactericidal and hemolytic activities. Experimental results of OH-CATH(3-34) and OH-CATH(5-34) indicated that the N-terminal 4 amino acid residues of OH-CATH played an important role on its hemolytic activity but had weak effects on its bactericidal activity. Among OH-CATH and its analogs, OH-CATH(5-34) had the lowest hemolytic activity while maintained strong antimicrobial activity. To evaluate its potential usage, the biological activities of OH-CATH(5-34) were compared with those of pexiganan. The bactericidal activity of OH-CATH(5-34) against 5 different species (11 laboratory strains) was 2-4 times stronger than that of pexiganan (4-16 microg/ml vs 8-32 microg/ml). Hemolytic activity of OH-CATH(5-34) against human erythrocytes was 0.69% while that of pexiganan was 16.5% at the dosage of 200 microg/ml. OH-CATH(5-34) showed very weak cytotoxic activities against primary rabbit ventricular endothelial cells and four human cancer cell lines whereas pexiganan showed strong cytotoxic activity against these five cell lines (IC(50)=20-90 microg/ml). The intravenous LD(50) value of OH-CATH(5-34) on mice was 7-fold higher than that of pexiganan (175 mg/kg vs 25mg/kg). Taken together, our results suggested that OH-CATH(5-34) should be considered as an excellent candidate for developing therapeutic drugs.

Global reemergence of tuberculosis: are host defense peptides an option to ameliorate disease burden?

Tuberculosis is the most relevant infectious disease worldwide according to the estimates of the World Health Organization, and despite being a curable disease, it requires a 6-9-month therapy with multiple antibiotics. Intermittent drug therapy due to noncompliance or poor delivery of therapy promotes the emergence of bacterial strains showing resistance to multiple drugs and the rise of extremely drug-resistant strains. Moreover, increased antibiotic resistance has been observed for several microorganisms, including extremely drug-resistant tuberculosis, vancomycin-resistant Enterococcus faecalis, or methicillin-resistant Staphylococcus aureus. In vitro, cathelicidin induction results in enhanced mycobacterial clearance, and synthetic human neutrophil peptides had a rather modest bactericidal effect in Mycobacterium tuberculosis-infected mice. In vivo therapeutic efficacy of improved molecules that show enhanced bactericidal action in vitro remains to be tested.

Activity of antimicrobial peptides in the presence of polysaccharides produced by pulmonary pathogens.

Antimicrobial peptides (AMPs) are secreted in the airway and contribute to initial defence against inhaled pathogens. Infections of the respiratory tract are a major cause of morbidity and mortality in preterm newborns and in patients with cystic fibrosis (CF). In this latter group, the state of chronic lung infection is due to the ability of bacteria to grow as mucoid biofilm, a condition characterised by overproduction and release of polysaccharides (PSs). In this study, we investigate the effect of PSs produced by lung pathogens such as Pseudomonas aeruginosa, Klebsiella pneumoniae and members of the Burkholderia cepacia complex on the antibacterial activity of structurally different peptides. The AMPs tested in this study include the cathelicidin LL-37 and the beta-defensin hBD-3 from humans, both released at the alveolar level, as well as peptides from other mammals, i.e. SMAP-29, PG-1 and Bac7(1-35). Susceptibility assays, time killing and membrane permeabilization kinetics experiments were carried out to establish whether PSs produced by lung pathogens may be involved in the poor defence reaction of infected lungs and thus explain infection persistence. All the PSs investigated inhibited, albeit to a different extent, the antibacterial activity of the peptides tested, suggesting that their presence in the lungs of patients with CF may contribute to the decreased defence response of this district upon infection by PS-producing microorganisms. The results also show that inhibition of the antibacterial activity is not simply due to ionic interaction between the negatively charged PSs and the cationic AMPs, but it also involves other structural features of both interactors.