Antimicrobial peptides: agents of border protection for companion animals.

Over the past 20 years, there have been significant inroads into understanding the roles of antimicrobial peptides in homeostatic functions and their involvement in disease pathogenesis. In addition to direct antimicrobial activity, these peptides participate in many cellular functions, including chemotaxis, wound healing and even determination of canine coat colour. Various biological and genetic approaches have helped to elucidate the role of antimicrobial peptides with respect to innate immunity and host defense. Associations of antimicrobial peptides with various skin diseases, including psoriasis, rosacea and atopic dermatitis, have been documented in humans. In the longer term, therapeutic modulation of antimicrobial peptide expression may provide effective new treatments for disease. This review highlights current knowledge about antimicrobial peptides of the skin and circulating leukocytes, with particular focus on relevance to physiology and disease in companion animals

Probing protein sequences as sources for encrypted antimicrobial peptides

Starting from the premise that a wealth of potentially biologically active peptides may lurk within proteins, we describe here a methodology to identify putative antimicrobial peptides encrypted in protein sequences. Candidate peptides were identified using a new screening procedure based on physicochemical criteria to reveal matching peptides within protein databases. Fifteen such peptides, along with a range of natural antimicrobial peptides, were examined using DSC and CD to characterize their interaction with phospholipid membranes. Principal component analysis of DSC data shows that the investigated peptides group according to their effects on the main phase transition of phospholipid vesicles, and that these effects correlate both to antimicrobial activity and to the changes in peptide secondary structure. Consequently, we have been able to identify novel antimicrobial peptides from larger proteins not hitherto associated with such activity, mimicking endogenous and/or exogenous microorganism enzymatic processing of parent proteins to smaller bioactive molecules. A biotechnological application for this methodology is explored. Soybean (Glycine max) plants, transformed to include a putative antimicrobial protein fragment encoded in its own genome were tested for tolerance against Phakopsora pachyrhizi, the causative agent of the Asian soybean rust. This procedure may represent an inventive alternative to the transgenic technology, since the genetic material to be used belongs to the host organism and not to exogenous sources

Synthesis and cellular penetration properties of new phosphonium based cationic amphiphilic peptides

A new category of phosphonium based cationic amphiphilic peptides has been developed and evaluated as potential antimicrobial peptides and cell penetrating peptides. The required building blocks were conveniently accessible from cysteine and could be applied in a solid phase peptide synthesis protocol for incorporation into peptide sequences. Evaluation of the antimicrobial properties and cellular toxicity of these phosphonium based peptides showed that these “soft” cationic side-chain containing peptides have poor antimicrobial properties and most of them were virtually non toxic (on HEK cells tested at 256 and 512 μM) and non-haemolytic (on horse erythrocytes tested at 512 μM), hinting at an interesting potential application as cell penetrating peptides. This possibility was evaluated using fluorescent peptide derivatives and showed that these phosphonium based peptide derivatives were capable of entering HEK cells and depending on the sequence confined to specific cellular areas

Effects of Cations and PH on Antimicrobial Activity of Thanatin and s-Thanatin against _Escherichia coli_ ATCC25922 and _B. subtilis_ ATCC 21332

Thanatin and s-thanatin were insect antimicrobial peptides which have shown potent antimicrobial activities on a variety of microbes. In order to investigate the effect of cations and pH on the activity of these peptides against Gram-negative bacteria and Gram-positive bacteria, the antimicrobial activities of both peptides were studied in increasing concentrations of monovalent cations (K^+^ and Na^+^), divalent cations (Ca^2+^ and Mg^2+^) and H^+^. The NCCLS broth microdilution method showed that both peptides were sensitive to the presence of cations. The divalent cations showed more antagonized effect on the activity against Gram-negative bacteria than the monovalent cations, since the two peptides lost the ability to inhibit bacterial growth at a very low concentration. In addition, the activities of both peptides tested were not significantly affected by pH. Comparing to studies of other antibacterial peptide activities, our data support a hypothesis that positive ions affect the sensitivity to cation peptides

Isolation and characterisation of host defence peptides ostricacins from ostrich heterophils : a thesis presented in partial fulfilment of the requirements for the degree of Masterate of Technology in Biotechnology at Massey University, Palmerston North, New Zealand

Articles in Appendix 3 removed due to copyright. Articles available in print copy held in the library.Host defence peptides are ubiquitous components of innate immunity within all living organisms. These peptides are small, positively charged and amphiphilic molecules. The biological roles of these peptides are direct antimicrobial activity against pathogens and to induce the innate and adaptive immune response within the host. The research presented in this thesis was focused on isolating host defence peptides from ostrich blood and characterising their antimicrobial properties. Four ostrich (β-defensins, named ostricacins-1-4 (Osp-1-4), were successfully purified from ostrich blood. These peptides contained 36-42 amino acid residues, with the main residues including: arginines, lysines, glycines and cysteines. The molecular weight of these four ostricacins ranged between 4-5 kDa. They displayed antimicrobial activity against Gram-negative bacteria and Gram-positive bacteria with minimum inhibitory concentration (MIC), ranging between 1-12 μg/ml. In addition, Osp-2 displayed antimicrobial activity against yeast, with MIC of 6.2 μg/ml. Osp-1 and Osp-2 were further characterised with the investigation of the effects of cationic ions and temperature changes on their antimicrobial activity against Gram-negative bacteria and Gram-positive bacteria. The antimicrobial activity of both peptides significantly declined with the presence of cationic ions. Both peptides were relatively stable when heated to temperatures between 30-70°C. Finally, an investigation of the mode of action of Osp-1 and Osp-2 against Gram-negative bacteria was carried out. Both peptides were compared with a sheep cathelicidin, SMAP-29, and a human α-defensin, HNP-1. SMAP-29 showed the strongest affinity to LPS and it was the most potent peptide to cause disruption of the outer and cytoplasmic membrane. The two ostricacins showed stronger affinity than HNP-1 and they also indicated partial permeabilisation of the outer membrane and a slight depolarisation of the cytoplasmic membrane. HNP-1 did not indicate disruption of the outer membranes or depolarisation of the cytoplasmic membrane. Further investigation indicated that the partial disruption allowed the ostricacins to pass through the membranes and interact with the intracellular components. However, these peptides could not inhibit the bacterial colony forming potential, and therefore, they were considered bacteriostatic. It is recommended that further research be carried out to investigate the feasibility of ostricacins in adding value to existing topical products