Biotechnological Method of Preparation and Characterization of Recombinant Antimicrobial Peptide Avicin A from Enterococcus avium.

Avicin A is a bacteriocin from the gram-positive bacterium Enterococcus avium. It exhibits a high microbicidal activity against bacteria of the genus Listeria, a causative agent of the severe human infection listeriosis. We developed a biotechnological method for obtaining avicin A and characterized its structure and biological activity. We also proposed a possible mechanism of the antimicrobial action of avicin A.

Minibactenecins ChBac7.Nα and ChBac7. Nß - Antimicrobial Peptides from Leukocytes of the Goat Capra hircus.

Antimicrobial peptides (AMPs) of neutrophils play an important role in the animal and human host defenses. We have isolated two AMPs (average molecular masses of 2895.5 and 2739.3 Da), with potent antimicrobial activity from neutrophils of the domestic goat (Capra hircus). A structural analysis of the obtained peptides revealed that they encompass N-terminal fragments (1-21 and 1-22) of the proline-rich peptide bactenecin 7.5. The primary structure of caprine bactenecin 7.5 had been previously deduced from the nucleotide sequence, but the corresponding protein had not been isolated from leukocytes until now. The obtained caprine AMPs were designated as mini-batenecins (mini-ChBac7.5Nα and mini-ChBac7.5Nß), analogously to the reported C-terminal fragment of the ovine bactenecin 7.5 named Bac7.5mini [Anderson, Yu, 2003]. Caprine mini-ChBac7.5Nα and mini-ChBac7.5Nß exhibit significant antimicrobial activity against Gram-negative bacteria, including drug-resistant strains of Pseudomonas aeruginosa, Klebsiella spp., Acinetobacter baumannii at a range of concentrations of 0.5-4 µM, as well as against some species of Gram-positive bacteria (Listeria monocytogenes EGD, Micrococcus luteus). The peptides demonstrate lipopolysaccharide-binding activity. Similarly to most proline-rich AMPs, caprine peptides inactivate bacteria without appreciable damage of their membranes. Mini-ChBac7.5Nα and mini-ChBac7.5Nß have no hemolytic effect on human red blood cells and are nontoxic to various cultured human cells. Therefore, they might be considered as promising templates for the development of novel antibiotic pharmaceuticals. Isolation of highly active fragments of the antimicrobial peptide from goat neutrophils supports the hypothesis that fragmentation of cathelicidin-related AMPs is an important process that results in the generation of potent effector molecules, which are in some cases more active than full-size AMPs. These truncated AMPs may play a crucial role in host defense reactions.

[STRESS-PROTECTIVE EFFECT OF HUMAN LACTOFERRIN].

Lactoferrin - multifunctional glycoprotein of the transferrin family with a molecular mass of about 80 kDa. We studied the effect of human lactoferrin on stress-induced changes in the level of corticosterone and adrenocorticotropic hormone (ACTH) and redistribution of leukocytes in the blood of rats. The used model of stress - swimming in cold water (1-4 °C) for 2 minutes. Corticosterone and ACTH levels in the plasma were determined by enzyme immunoassay. It has been established that preventive intraperitoneal administration of lactoferrin has reduced stress-induced increase in the concentration of corticosterone in 30 minutes after the stress and has normalized stress-inducing changes in the number of neutrophils in the blood after 30 minutes and 3 hours after exposure of stress, but has not affected the level of ACTH. The results of this study suggest that lactoferrin can act as an adaptogen during experimental stress.

[Interaction of Arenicin-1 with C1q Protein].

The interaction between arenicin-1, that is an antimicrobial peptide from polychaeta Arenicola marina, and human complement system protein C1q was studied using enzyme-linked receptor sorbent assay and ELISA. We revealed that arenicin-1 and C1q form complex that is stable in high ionic strength condition 0.5 M NaCl. The ability of C1q to interact with arenicin-1 is comparable with the binding activity of C1q towards another antimicrobial peptide, porcine cathelicidin protegrin-1, which has a similar spatial arrangement with arenicin-1. Namely, both arenicin-1 and protegrin-1 form cystine-stabilized antiparallel ß-hairpin structure.

Acipensins - Novel Antimicrobial Peptides from Leukocytes of the Russian Sturgeon Acipenser gueldenstaedtii.

Antimicrobial peptides (AMPs) play an important role in the innate defense mechanisms in humans and animals. We have isolated and studied a set of antimicrobial peptides from leukocytes of the Russian sturgeon Acipenser gueldenstaedtii belonging to a subclass of chondrosteans, an ancient group of bony fish. Structural analysis of the isolated peptides, designated as acipensins (Ac), revealed in leukocytes of the Russian sturgeon six novel peptides with molecular masses of 5336.2 Da, 3803.0 Da, 5173.0 Da, 4777.5 Da, 5449.4 Da, and 2740.2 Da, designated as Ac1-Ac6, respectively. Complete primary structures of all the isolated peptides were determined, and the biological activities of three major components - Ac1, Ac2, and Ac6 - were examined. The peptides Ac1, Ac2, Ac3, Ac4, and Ac5 were found to be the N-terminal acetylated fragments 1-0, 1-5, 1-9, 1-4, and 1-1 of the histone H2A, respectively, while Ac6 was shown to be the 62-5 fragment of the histone H2A. The peptides Ac1 and Ac2 displayed potent antimicrobial activity towards Gram-negative and Gram-positive bacteria (Escherichia coli ML35p, Listeria monocytogenes EGD, MRSA ATCC 33591) and the fungus Candida albicans 820, while Ac6 proved effective only against Gram-negative bacteria. The efficacy of Ac 1 and Ac2 towards the fungus and MRSA was reduced upon an increase in the ionic strength of the solution. Ac1, Ac2, and Ac6, at concentrations close to their minimum inhibitory concentrations, enhanced the permeability of the E.coli ML35p outer membrane to the chromogenic marker, but they did not affect appreciably the permeability of the bacterial inner membrane in comparison with a potent pore-forming peptide, protegrin 1. Ac1, Ac2, and Ac6 revealed no hemolytic activity against human erythrocytes at concentrations of 1 to 40 µM and had no cytotoxic effect (1 to 20 µM) on K-562 and U-937 cells in vitro. Our findings suggest that histone-derived peptides serve as important anti-infective host defense molecules.

Lactoferrin from canine neutrophils: isolation and physicochemical and antimicrobial properties.

Lactoferrin has been isolated from canine leukocytes for the first time. Lactoferrin was identified by N-terminal amino acid sequence and by capability to capture ferric cations resulting in a complex with absorbance maximum at 460-470 nm. It is demonstrated that canine lactoferrin resembles the human homolog in some physicochemical properties, i.e. molecular weight, carbohydrate presence, and conditions of protein-iron complex dissociation. Bactericidal activity of dog lactoferrin was demonstrated on the gram-negative bacterium Escherichia coli and gram-positive bacterium Listeria monocytogenes. Bactericidal activity of canine lactoferrin is similar to that of human lactoferrin.

Effects of defensin and lactoferrin on functional activity of endothelial cells in vitro.

We studied the effects of antibacterial peptides and proteins (defensins and lactoferrins) on functional activity of endothelial cells in vitro: proliferative activity and adhesion of human endothelial ECV-304 cells to the matrix were evaluated, alpha-Defensin (NP-2) from rabbit neutrophils, total alpha-defensin (HNP 1-3) from human neutrophils, and lactoferrins from porcine neutrophils and human milk were studied. Defensins stimulated and lactoferrin in doses of 1-10 microg/ml inhibited proliferation and adhesion of endothelial cell. The stimulatory effect of defensins on proliferation and adhesion was reproduced in fibroblast culture. Lactoferrins did not modify proliferation of fibroblasts, but suppressed their adhesion. These data suggest that antibiotic proteins and peptides are prospective objects for the creation of drugs regulating angiogenesis.

alpha-Defensins from blood leukocytes of the monkey Papio hamadryas.

Three antimicrobial peptides named PHD1-3 (Papio hamadryas defensin) have been isolated from hamadryas baboon blood leukocytes using preparative electrophoresis and reverse-phase HPLC. The primary structures of these peptides have been determined by automated Edman degradation and mass-spectrometry. The results suggest that the peptides belong to the alpha-defensin family. Structural homology analysis reveals that among alpha-defensins from other animal species, PHD3 is the most closely related to RMAD5 (rhesus macaque alpha-defensin) (90% homology) from rhesus macaque leukocytes and also highly similar to human alpha-defensin HD5 (60% homology), which is produced by intestinal Paneth cells. The homology of PHD3 with human neutrophil alpha-defensin HNP1 (human natural peptide) was 30%. The primary structures of PHD1 and PHD2 are most similar to RED1 (rhesus enteral defensin), one of six enteral alpha-defensins of rhesus monkeys. PHD1-3 have been shown to be active against the Gram-positive bacteria Listeria monocytogenes and Staphylococcus aureus, the Gram-negative bacterium Escherichia coli, and the fungus Candida albicans, similarly to the human HNP1 defensin.

A possible molecular mechanism for the interaction of defensin with the sensory neuron membrane.

A local membrane potential clamping method was used to study the effects of defensin NP-1 on the membranes of rat spinal ganglion neurons. NP-1 led to decreases in the effective charge for the activation gating system. This process depended on the NP-1 concentration. Use of the Hill equation showed that Kd was 2.10(-12) M and the Hill coefficient was 0.9. The structure of the defensin molecule was optimized using quantum chemical calculations based on a molecular mechanics method. The results obtained from these calculations suggested that a single hydroxyl group directed towards the outer part of thedefensin molecule and forming the carboxyl group of amino acid Glu14 could form a hydrogen bond with the active center of the membrane receptor. This explains the experimentally observed 1:1 stoichiometry of the ligand-receptor binding interaction between the defensin and the sensory neuron membrane.

Isolation and some physical and chemical properties of elastase and cathepsin G from dog neutrophils.

A new method for isolation of leukocyte serine proteinases has been developed. Elastase (EC 3.4.21.37) and cathepsin G (EC 3.4.21.20) have been isolated from dog neutrophils and purified to homogeneous state. The results of inhibitor analysis indicate that the enzymes belong to the group of serine proteinases. Some physical and chemical characteristics of the purified enzymes have been determined. The molecular weights of the enzymes are 24.5-26 kD for the elastase and 23.5-25.5 kD for the cathepsin G. The cathepsin G is a glycoprotein, while the elastase molecule lacks carbohydrate components. The cathepsin G exhibits a broad pH optimum of catalytic activity in the range of 7.0-9.0; the pH optimum for the elastase is 8.0-8.5. The Michaelis constant of the elastase for N-t-Boc-L-alanine p-nitrophenyl ester is 0.10 mM; the Michaelis constant of the cathepsin G for N-benzoyl-L-tyrosine ethyl ester is 0.42 mM.

Circular minidefensins and posttranslational generation of molecular diversity.

We purified two new minidefensins (RTD-2 and RTD-3) from the bone marrow of rhesus monkeys. Both were circular octadecapeptides that contained three intramolecular disulfide bonds and were homologous to RTD-1, a circular (theta) defensin previously described by Tang et al. (Science, 286, 498-502, 1999). However, whereas the 18 residues of RTD-1 represent spliced nonapeptide fragments derived from two different demidefensin precursors, RTD-2 and -3 comprise tandem nonapeptide repeats derived from only one of the RTD-1 precursors. Thus, circular minidefensins are products of a novel posttranslational system that generates effector molecule diversity without commensurate genome expansion. A system wherein two demidefensin genes can produce three circular minidefensins might allow n such genes to produce (n/2)(n+1) peptides.

Purification and properties of proline-rich antimicrobial peptides from sheep and goat leukocytes.

We purified three proline-rich antimicrobial peptides from elastase-treated extracts of sheep and goat leukocytes and subjected two of them, OaBac5alpha and ChBac5, to detailed analysis. OaBac5alpha and ChBac5 were homologous to each other and to bovine Bac5. Both exhibited potent, broad-spectrum antimicrobial activity under low-concentration salt conditions. While the peptides remained active against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Listeria monocytogenes in 100 mM NaCl, they lost activity against Staphylococcus aureus and Candida albicans under these conditions. ChBac5 was shown to bind lipopolysaccharide, a property that could enhance its ability to kill gram-negative bacteria. Proline-rich Bac5 peptides are highly conserved in ruminants and may contribute significantly to their innate host defense mechanisms.

Isolation and characterization of myeloperoxidase from leukocytes of rat peritoneal fluid.

Myeloperoxidase (MPO) was isolated from rat peritoneal leukocytes with a yield of 51% and A430/A280 = 0.75 - 0.80, and its physicochemical properties were studied. The molecular weight of the MPO is about 150 kD. The MPO was assayed for amino acid content. We used substrate mixture containing phenol, 4-aminoantipyrine, and H2O2 to detect 10(-10) M of the enzyme. The MPO was localized in rat blood neutrophils using polyclonal anti-MPO antibodies and secondary fluorescein isothiocyanate-labeled antibodies. Immunofluorimetric assay (IFMA) was developed for quantitative measurement of the MPO. The MPO and leukocytes can iodinate BSA using NaI or thyroxine as the source of iodine.

Interleukin-1 and defensins in thermoregulation, stress, and immunity.

Our previous studies suggested that IL-1 and defensins, which play a critical role in mechanisms of host resistance, participated in the realization of stress reaction. The present report describes functional relations between IL-1 and defensins during stress reactions and the influence of defensins administration on thermoregulation and the IL-1 level in blood. It is shown that stress-induced shifts in the humoral immune response are accompanied by diverse changes in the IL-1 alpha level in blood and LAF production by peritoneal macrophages, which are possibly associated with the intensity and strength of applied stressors. Functional interrelations between actions of defensins and IL-1 during stress reaction and in the conditions of an LPS-induced rise in body temperature was demonstrated. These results suggest that antibacterial peptides defensins may be involved in thermoregulation in case of increased body temperature probably influencing the blood level of IL-1.

Prophenin-1, an exceptionally proline-rich antimicrobial peptide from porcine leukocytes.

We purified and characterized an unusual antimicrobial peptide, prophenin-1 (PF-1), from porcine leukocytes. The peptide had a mass of 8,683 and contained 79 residues, including 42 (53.2%) prolines and 15 (19.0%) phenylalanines. Its N-terminal 60 residues consisted of three perfect and three nearly perfect repeats of a decamer, FPPPNFPGPR. Prophenin-1 was encoded on a cathelin-containing precursor and showed substantially more activity against E. coli, a Gram-negative bacterium, than against Listeria monocytogenes, a Gram-positive organism, in vitro.

Gallinacins: cysteine-rich antimicrobial peptides of chicken leukocytes.

We purified three homologous antimicrobial peptides ('gallinacins') from chicken leukocytes, examined their antimicrobial activity in vitro, and established their primary sequences by a combination of gas phase microsequencing and on-line LC-ESI-MS analysis of endo- and exoprotease peptide digests. The peptides contained 36-39 amino acid residues, were relatively cationic due to their numerous lysine and arginine residues, and each contained 3 intramolecular cystine disulfide bonds. Gallinacins showed primary sequence homology to the recently delineated beta-defensin family, heretofore found only in the respiratory epithelial cells and neutrophils of cattle, suggesting that beta-defensins originated at least 250 million years ago, before avian and mammalian lineages diverged. The 9 invariant residues (6 cysteines, 2 glycines and 1 proline) common to avian gallinacins and bovine beta-defensins are likely to constitute the essential primary structural motif of this ancient family of host-defense peptides.

Primary structure of three cationic peptides from porcine neutrophils. Sequence determination by the combined usage of electrospray ionization mass spectrometry and Edman degradation.

The primary structure of three major cationic peptides from porcine neutrophils has been determined. The sequencing was made by the combined use of electrospray ionization mass spectrometry and Edman degradation. The determined sequences unambiguously show that these peptides can not be considered as defensins.

Protegrins: leukocyte antimicrobial peptides that combine features of corticostatic defensins and tachyplesins.

Porcine leukocytes contained three homologous peptides, PG-1, 2 and 3, that manifested potent microbicidal activity against Escherichia coli, Listeria monocytogenes and Candida albicans in vitro. The peptides ('protegrins') were composed of 16 (PG-2) or 18 amino acid residues, and, like tachyplesins (broad-spectrum antibiotic peptides of horseshoe crab hemocytes), they contained two intramolecular cystine disulfide bonds. Considerably smaller than defensins, protegrins nevertheless showed substantial homology to them, especially to the 'corticostatic' rabbit defensin, NP-3a. The relatively simple structure of protegrins should provide useful prototypes for constructing congeners with selectively enhanced host defense activities.

Haematological reactions of rabbits infected intravenously with Listeria strains of different virulence.

Listeria strains of different virulence were injected intravenously into rabbits of both sexes (2-4 kg). The infectious dose was 10(8) cells/kg. Blood samples were taken from the ear wein one day and immediately before the infection, then 3 h, 1, 2, 3, 6 and 10 days after it. Total white blood cell counts and their changes were determined and the number of lysosomal granules in neutrophils were counted. A marked monocytic reaction was observed after the injection of virulent Listeria monocytogenes 18 and 87/5467, partly virulent Listeria ivanovii, non-pathogenic Listeria seeligeri 87/5626 and 87/5575, and Listeria murrayi G44 strains. A late slow growth was provoked by Listeria innocua C644 and a very slight reaction by Listeria welshimeri 1830 strains. There was no change after injection of L. innocua strain 10. The number of lysosomal granules decreased significantly and remained at a low level for 6 days after the infection of L. monocytogenes strain 18 and for 3 days after the injection of L. innocua strain 10.