Brian F. Tack

Antimicrobial peptides in animals and their role in host defences

Domesticated animals have a large variety of antimicrobial peptides that serve as natural innate barriers limiting microbial infection or, in some instances, act as an integral component in response to inflammation or microbial infection. These peptides differ in size, composition, mechanisms of activity and range of antimicrobial specificities. They are expressed in many tissues, polymorphonuclear leukocytes, macrophages and mucosal epithelial cells. There is a small group of anionic antimicrobial peptides found in ruminants and a much larger group of cationic antimicrobial peptides found in all domesticated animals. The cationic peptides include linear, helical peptides, linear peptides rich in proline and cysteine-stabilized peptides with a beta-sheet and are commonly referred to as cathelicidins and defensins. These peptides are generally broad-spectrum for Gram-positive bacteria, Gram-negative bacteria and fungi (e.g. myeloid antimicrobial peptides, alpha-, beta-defensins, and protegrins) or are specific to one of these groups (e.g. porcine cecropin P1, Bac5, Bac7, PR-39 and prophenin).

Bactericidal Activity of Mammalian Cathelicidin-Derived Peptides

ABSTRACT Endogenous antimicrobial peptides of the cathelicidin family contribute to innate immunity. The emergence of widespread antibiotic resistance in many commonly encountered bacteria requires the search for new bactericidal agents with therapeutic potential. Solid-phase synthesis was employed to prepare linear antimicrobial peptides found in cathelicidins of five mammals: human (FALL39/LL37), rabbit (CAP18), mouse (mCRAMP), rat (rCRAMP), and sheep (SMAP29 and SMAP34). These peptides were tested at ionic strengths of 25 and 175 mM against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. Each peptide manifested activity against P. aeruginosa irrespective of the NaCl concentration. CAP18 and SMAP29 were the most effective peptides of the group against all test organisms under both low- and high-salt conditions. Select peptides of 15 to 21 residues, modeled on CAP18 (37 residues), retained activity against the gram-negative bacteria and methicillin-sensitive S. aureus, although the bactericidal activity was reduced compared to that of the parent peptide. In accordance with the behavior of the parent molecule, the truncated peptides adopted an α-helical structure in the presence of trifluoroethanol or lipopolysaccharide. The relationship between the bactericidal activity and several physiochemical properties of the cathelicidins was examined. The activities of the full-length peptides correlated positively with a predicted gradient of hydrophobicity along the peptide backbone and with net positive charge; they correlated inversely with relative abundance of anionic residues. The salt-resistant, antimicrobial properties of CAP18 and SMAP29 suggest that these peptides or congeneric structures have potential for the treatment of bacterial infections in normal and immunocompromised persons and individuals with cystic fibrosis.

Discovery of new human β-defensins using a genomics-based approach

Abstract Epithelial β-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a

Expression of the Complement Anaphylatoxin C3a and C5a Receptors on Bronchial Epithelial and Smooth Muscle Cells in Models of Sepsis and Asthma

The presence of the complement-derived anaphylatoxin peptides, C3a and C5a, in the lung can induce respiratory distress characterized by contraction of the smooth muscle walls in bronchioles and pulmonary arteries and aggregation of platelets and leukocytes in pulmonary vessels. C3a and C5a mediate these effects by binding to their specific receptors, C3aR and C5aR, respectively. The cells that express these receptors in the lung have not been thoroughly investigated, nor has their expression been examined during inflammation. Accordingly, C3aR and C5aR expression in normal human and murine lung was determined in this study by immunohistochemistry and in situ hybridization. In addition, the expression of these receptors was delineated in mice subjected to LPS- and OVA-induced models of inflammation. Under noninflamed conditions, C3aR and C5aR protein and mRNA were expressed by bronchial epithelial and smooth muscle cells of both human and mouse lung. C3aR expression increased significantly on both bronchial epithelial and smooth muscle cells in mice treated with LPS; however, in the OVA-challenged animals only the bronchial smooth muscle cells showed increased C3aR expression. C5aR expression also increased significantly on bronchial epithelial cells in mice treated with LPS, but was not elevated in either cell type in the OVA-challenged mice. These results demonstrate the expression of C3aR and C5aR by cells endogenous to the lung, and, given the participation of bronchial epithelial and smooth muscle cells in the pathology of diseases such as sepsis and asthma, the data suggest a role for these receptors during lung inflammation.

Orientation and Dynamics of an Antimicrobial Peptide in the Lipid Bilayer by Solid-State NMR Spectroscopy

The orientation and dynamics of an 18-residue antimicrobial peptide, ovispirin, has been investigated using solid-state NMR spectroscopy. Ovispirin is a cathelicidin-like model peptide (NH(2)-KNLRRIIRKIIHIIKKYG-COOH) with potent, broad-spectrum bactericidal activity. (15)N NMR spectra of oriented ovispirin reconstituted into synthetic phospholipids show that the helical peptide is predominantly oriented in the plane of the lipid bilayer, except for a small portion of the helix, possibly at the C-terminus, which deviates from the surface orientation. This suggests differential insertion of the peptide backbone into the lipid bilayer. (15)N spectra of both oriented and unoriented peptides show a reduced (15)N chemical shift anisotropy at room temperature compared with that of rigid proteins, indicating that the peptide undergoes uniaxial rotational diffusion around the bilayer normal with correlation times shorter than 10(-4) s. This motion is frozen below the gel-to-liquid crystalline transition temperature of the lipids. Ovispirin interacts strongly with the lipid bilayer, as manifested by the significantly reduced (2)H quadrupolar splittings of perdeuterated palmitoyloleoylphosphatidylcholine acyl chains upon peptide binding. Therefore, ovispirin is a curved helix residing in the membrane-water interface that executes rapid uniaxial rotation. These structural and dynamic features are important for understanding the antimicrobial function of this peptide.

Killing of African Trypanosomes by Antimicrobial Peptides

Antimicrobial peptides are components of the innate immune systems of a wide variety of eukaryotic organisms and are being developed as antibiotics in the fight against bacterial and fungal infections. We explored the potential activities of antimicrobial peptides against the African trypanosome Trypanosoma brucei, a vector-borne protozoan parasite that is responsible for significant morbidity and mortality in both humans and animals. Three classes of mammalian antimicrobial peptides were tested: alpha-defensins, beta-defensins, and cathelicidins. Although members of all 3 classes of antimicrobial peptides showed activity, those derived from the cathelicidin class were most effective, killing both insect and bloodstream forms of the parasite. The mechanism of action of the cathelicidins against T. brucei involves disruption of surface membrane integrity. Administration of cathelicidin antimicrobial peptides to mice with late-stage T. brucei infection acutely decreased parasitemia and prolonged survival. These results highlight the potential use of antimicrobial peptides for the treatment of African trypanosomiasis.

Susceptibilities of Oral Bacteria and Yeast to Mammalian Cathelicidins

ABSTRACT The effects of cathelicidins against oral bacteria and clinically important oral yeasts are not known. We tested the susceptibilities of Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum,Porphyromonas gingivalis, Streptococcus sanguis, Candida krusei, Candida tropicalis and Candida albicans to the following cathelicidins: FALL39, SMAP29, and CAP18. SMAP29 and CAP18 were antimicrobial, whereas FALL39 did not exhibit antimicrobial activity. Future studies are needed to determine the potential use of these antimicrobial peptides in prevention and treatment of oral infections.

Host defense functions of proteolytically processed and parent (unprocessed) cathelicidins of rabbit granulocytes.

ABSTRACT Members of the cathelicidin family are present in all mammals studied. Generally, these proteins contain a conserved N-terminal domain and a structurally and functionally divergent C-terminal region that expresses antibacterial or other activities when proteolytically released. Rabbit granulocytes produce CAP18, a cathelicidin that conforms to this structural and functional organization, and also 15-kDa protein isoforms (p15s) that share several key structural features with other cathelicidins but apparently do not undergo processing with release of an active peptide. To further define the importance of proteolysis in the antibacterial activities of these proteins, we have purified from granulocytes proCAP18, its C-terminal peptide (CAP18p), and two p15 isoforms to apparent homogeneity. Of these four polypeptides, only CAP18p was independently cytotoxic to encapsulated Escherichia coli (90% inhibitory concentration, ∼600 nM) but it was ∼50-fold less potent on a molar basis than the bactericidal/permeability-increasing protein (BPI). However, all four cathelicidin species, notably including proCAP18, exhibited antibacterial synergy with BPI, and the p15s also displayed synergy with CAP18p in the absence of BPI. Subnanomolar concentrations of proCAP18 blocked lipopolysaccharide-induced chemiluminescence of human leukocytes, showing a molar potency more than 100-fold greater than that of CAP18p (∼20 nM) or BPI (∼50 nM). Thus, while independent bactericidal activity of cathelicidins requires processing, other host-defense functions do not and are more potently expressed by the unprocessed protein than by the C-terminal peptide.

Developmental expression and distribution of sheep β-defensin-2

The aim of this study was to define the ontogeny of sheep β-defensin-2 (SBD-2) mRNA and peptide in selected tissues of fetal, neonatal and adult sheep by real-time PCR and immunohistochemistry, respectively. Fetal and neonatal lambs had significantly greater SBD-2 tissue distribution than adult sheep. For all ages, the intestines had consistent SBD-2 mRNA expression while extra intestinal expression was sporadic and weak. In adult sheep, SBD-2 mRNA levels decreased from the jejunum caudally to the rectum and a pooled sample from all age groups showed a similar tendency. SBD-2 immunoreactive cells were predominantly in the crypts and base of villi in the small intestine and in a modest number of glands in the large intestine. Interestingly, ileal follicle-associated epithelium lacked detectable SBD-2 immunoreactivity. SBD-2 mRNA and peptide expression are greatest in the intestinal tract and tissue distribution progressively decreases with maturity.

SMAP29 congeners demonstrate activity against oral bacteria and reduced toxicity against oral keratinocytes

INTRODUCTIONnCathelicidins are antimicrobial peptides found in epithelial and mucosal tissues as well as the secondary granules of neutrophils. SMAP29, a sheep cathelicidin, has differential antimicrobial properties against various pathogens, including periodontal organisms. The purpose of this study was to evaluate the antimicrobial properties and cytotoxicity of SMAP29, SMAP28, and three congeners (SMAP18A, SMAP18D, and SMAP14A).nnnMETHODSnThe peptides at concentrations ranging from 0.25 to 250 microg/ml were tested for their activity against multiple strains of Streptococcus mutans, Streptococcus sanguis, Actinomyces israelii, Actinomyces naeslundii, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Peptostreptococcus micros, and Porphyromonas gingivalis using a radial diffusion assay. Cytotoxicity of keratinocytes was evaluated by measuring lactate dehydrogenase release after incubation with the individual peptides.nnnRESULTSnSMAP28, thought to be the biologically active peptide, was the most potent antimicrobial (range of minimum inhibitory concentrations 0.06-7.03 microg/ml, P < 0.05); however, the activity of SMAP28 and SMAP29 was strongly associated (r = 0.933). The congeners also demonstrated antimicrobial activity against the bacteria tested (range of minimum inhibitory concnetrations 0.21-79 microg/ml). Overall, F. nucleatum was the most susceptible organism, while P. gingivalis was the least susceptible. Keratinocyte cytotoxicity was dependent on peptide length and dose. SMAP28 was the most cytotoxic, while SMAP14A was the least cytotoxic.nnnCONCLUSIONnThe antimicrobial activities against oral microorganisms and the minimal toxicity seen in this study suggest that the congeners of SMAP29 may serve as an alternative to traditional antibiotics in the prevention and treatment of periodontal and other oral diseases.