Robert I. Lehrer

Interaction of human defensins with Escherichia coli. Mechanism of bactericidal activity.

Defensins are small, cysteine-rich antimicrobial peptides that are abundant in human, rabbit, and guinea pig neutrophils (PMN). Three defensins (human neutrophil peptide defensin [HNP]-1, HNP-2, and HNP-3) constitute between 30 and 50% of the total protein in azurophil granules of human PMN. We examined the mechanism of HNP-mediated bactericidal activity against Escherichia coli ML-35 (i-, y-, z+) and its pBR322-transformed derivative, E. coli ML-35p. Under conditions that supported bactericidal activity, HNP-1 sequentially permeabilized the outer membrane (OM) and inner membrane (IM) of E. coli. Coincident with these events, bacterial synthesis of DNA, RNA, and protein ceased and the colony count fell. Although these events were closely coupled under standard assay conditions, OM permeabilization was partially dissociated from IM permeabilization when experiments were performed with E. coli that had been plasmolyzed by mannitol. Under such conditions, the rate and extent of bacterial death more closely paralled loss of IM integrity than OM permeabilization. Electron microscopy of E. coli that had been killed by defensins revealed the presence of striking electron-dense deposits in the periplasmic space and affixed to the OM. Overall, these studies show that HNP-mediated bactericidal activity against E. coli ML-35 is associated with sequential permeabilization of the OM and IM, and that inner membrane permeabilization appears to be the lethal event.

Modulation of the in vitro candidacidal activity of human neutrophil defensins by target cell metabolism and divalent cations.

We tested the in vitro susceptibility of Candida albicans to three defensins from human neutrophilic granulocytes (HNP-1, 2, and 3), a homologous defensin from rabbit leukocytes (NP-1), and four unrelated cationic peptides. Although the primary amino acid sequences of HNP-1, 2, and 3 are identical except for a single amino-terminal amino acid alteration, HNP-1 and HNP-2 killed C. albicans but HNP-3 did not. C. albicans blastoconidia were protected from HNP-1 when incubations were performed in the absence of oxygen or in the presence of inhibitors that blocked both of its mitochondrial respiratory pathways. Neither anaerobiosis nor mitochondrial inhibitors substantially protected C. albicans exposed to NP-1, poly-L-arginine, poly-L-lysine, or mellitin. Human neutrophilic granulocyte defensin-mediated candidacidal activity was inhibited by both Mg2+ and Ca2+, and was unaffected by Fe2+. In contrast, Fe2+ inhibited the candidacidal activity of NP-1 and all of the model cationic peptides, whereas Mg2+ inhibited none of them. These data demonstrate that susceptibility of C. albicans to human defensins depends both on the ionic environment and on the metabolic state of the target cell. The latter finding suggests that leukocyte-mediated microbicidal mechanisms may manifest oxygen dependence for reasons unrelated to the production of reactive oxygen intermediates by the leukocyte.

Defensins : a family of antimicrobial and cytotoxic peptides

Defensins are antimicrobial and cytotoxic peptides that contain 29-35 amino acid residues, including 6 invariant cysteines that form 3 intramolecular disulfide bonds. They constitute more than 5% of the total cellular protein of human and rabbit neutrophils (PMN), and are also produced by rabbit lung macrophages and by murine and human small intestinal Paneth cells. Defensins exerted antimicrobial effects in vitro against many Gram-positive and Gram-negative bacteria, fungi, mycobacteria and some enveloped viruses, and were cytotoxic to a wide range of normal and malignant targets, including cells resistant to TNF-alpha and NK-cytolytic factor. Human and rabbit defensins formed voltage-sensitive channels in a variety of planar lipid bilayers when a negative voltage of approximately 70-90 mV was applied to the contralateral side. These channels showed modest anion selectivity and their formation was strongly influenced by defensin concentration. Although most other channel-forming peptides have prominent alpha-helical domains, the structure of defensin molecules is primarily composed of antiparallel beta-sheets. Studies with various prokaryotic and eukaryotic cells provided convincing evidence that defensins killed these targets by forming voltage-regulated channels in the susceptible cells membrane. The broad spectrum of defensin-susceptible targets and the abundance of defensins in specialized host defense cells of the blood, lungs and intestinal tract suggest that defensins could play a significant role in innate immunity to infection and neoplasia.

Concurrent assessment of inner and outer membrane permeabilization and bacteriolysis in E. coli by multiple-wavelength spectrophotometry

We developed a dual wavelength spectrophotometric assay that permitted beta-lactamase and beta-galactosidase activities to be measured concurrently in a single sample. We also constructed a target cell, E. coli ML-35p, that was substantially cryptic for its periplasmic beta-lactamase and cytoplasmic beta-galactosidase unless outer membrane (beta-lactamase) or inner membrane (beta-galactosidase) permeabilization occurred. By applying the spectrophotometric assay to whole target cells, we could ascertain the kinetics of inner and outer membrane permeabilization by biological agents, including serum, polymyxin B and mellitin. By monitoring the reactions at an additional wavelength, we could also follow the kinetics of serum-mediated bacteriolysis. These experiments illustrate the principle of multiple wavelength spectrophotometry and provide examples of its use to monitor and dissect the action of biological agents on a gram-negative bacterium.

Microbicidal/cytotoxic proteins of neutrophils are deficient in two disorders: Chediak-Higashi syndrome and "specific" granule deficiency.

Although several genetic defects are known to impair oxidative microbicidal/cytotoxic mechanisms in human PMN, no deficiencies of PMN granule components that mediate oxygen-independent microbicidal activity have yet been reported. We analyzed PMN from patients with various granulocyte disorders for their content of two azurophil granule constituents, defensins and cathepsin G, that exert microbicidal/cytotoxic activity in vitro, and one component, elastase, that has ancillary microbicidal/cytotoxic activity. PMN from two (of two) patients with specific granule deficiency (SGD) displayed an almost complete deficiency of defensins, which in normal cells constitute greater than 30% of the protein content of azurophil granules. The SGD PMN contained normal or mildly decreased amounts of cathepsin G and elastase. Conversely, the PMN of three (of three) patients with Chediak-Higashi syndrome (CHS) substantially lacked cathepsin G and elastase, but their defensin content was normal or mildly decreased. Both CHS and SGD patients suffer from frequent and severe bacterial infections, and CHS patients frequently develop an atypical lymphoproliferative syndrome. The profound deficiency of PMN components with microbicidal/cytotoxic activity in SGD and CHS may contribute to the clinical manifestations of these disorders.

Mammalian antibiotic peptides

Peptide-based compounds containing four invariant cysteine residues which have been oxidized to obtain two intramolecular disulfide bonds are useful as preservatives and in preventing, treating, or ameliorating viral or microbial infection in animals and plants. These compounds, in one embodiment, are of the formula: A.sub.1 -A.sub.2 -A.sub.3 -A.sub.4 -A.sub.5 -C-A.sub.7 -C-A.sub.9 -A.sub.10 -A11 -A12 -C-A14 -C-A16 -(A17 -A18)(1) (SEQ ID NO:4) and the N-terminal acylated, C-terminal amidated or esterified and the cystine-bridged forms thereof wherein A1, A9, A10 and A11 are basic amino acids; A2 and A3 are small amino acids; A5, A7, A12, A14 and A16 are hydrophobic amino acids; and A4 is a basic or a small amino acid; A17 is not present or, if present, is a small amino acid; A18 is not present or, if present, is a basic amino acid.The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus creating a strong need to develop new antimicrobial agents. These new antibiotics should possess novel mechanisms of action and different cellular targets compared with existing antimicrobials. Recent discoveries and isolations of so-called animal antibiotics, mostly small cationic peptides, which represent a potent branch of natural immunity, offered the possibility to acquire new and effective antibiotics of this provenance. To this date, more than 500 antibiotic peptides have been distinguished and defined. Their antimicrobial properties present new opportunities for their use as antibiotics or for construction of their more effective derivatives, but much research is still required to pave the way to their practical use. This is a survey of substances forming an armamentarium of natural immunity of mammals.

Effects of phorbol diesters and teleocidin on normal human platelets

Teleocidins are newly described indole alkaloid tumor promoters that are structurally distinct from phorbol diesters (PDE). We compared the effects of teleocidin and selected PDE on platelet aggregation, secretion and aspects of arachidonate metabolism. Three tumor-promoting PDE (phorbol myristate acetate (PMA), phorbol dibutyrate (PDBu) and 4-beta-phorbol didecanoate (4-beta-PDD] and a non-tumor promoting PDE (4-alpha-phorbol didecanoate (4-alpha-PDD] were used. Teleocidin and tumor promoting PDE caused platelet aggregation after a delay that was inversely related to tumor promoter concentration and also triggered secretion of alpha- and dense granules and selective release of lysosomal enzymes. Aggregation and its associated 125I-fibrinogen binding to platelets were both inhibited by Na2EDTA. 4-alpha-PDD was ineffective. Analysis of platelet aggregation responses and activation kinetics revealed that PDBu was 11.7 times less potent than teleocidin PMA, or 4-beta-PDD. Neither PDE nor teleocidin stimulated 14C-arachidonate release from normal human platelets, and both aggregated aspirin-treated platelets. These results show that representatives of two structurally distinct classes of tumor promoters, phorbol diesters and indole alkaloids, are potent activators of platelet aggregation, fibrinogen binding, and granule/lysosomal secretion, by a mechanism that bypasses arachidonate release and formation of cyclooxygenase-dependent arachidonate metabolites.

Expression, purification, crystallization and preliminary X-ray analysis of the cathelicidin motif of the protegrin-3 precursor

Numerous precursors of antibacterial peptides with unrelated sequences share a similar prosequence which belongs to the cathelicidin family of proteins. The three-dimensional structure of this cathelicidin motif, which contains two disulfide bonds, has not yet been reported. The cathelicidin motif (ProS) of the protegrin-3 precursor was overexpressed in Escherichia coli as a His-tagged protein. The His(6) tag was removed by thrombin cleavage. ProS was purified to homogeneity and single crystals were obtained by the hanging-drop vapour-diffusion method at pH 3-4. Preliminary X-ray diffraction analysis indicated that these crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 51.42, c = 134.25 A. These crystals diffracted beyond 2.75 A (1.9 A at ESRF) and contain one molecule per asymmetric unit.

Defensins in macrophages

Antimicrobial peptides, formerly thought of as specialized products of certain bacteria and fungi, appear to be widespread in the animal kingdom, and have been found in amphibian skin, insect hemolymph, various mucosal secretions and in professional phagocytes (1). Among professional phagocytes, granulocytes (polymorphonuclear leukocytes and eosinophils) are distinguished by a large storage compartment of up to several thousand cytoplasmic ‘granules’ which have proven to be a very rich and highly concentrated source of antimicrobial peptides (2–4). In contrast, macrophages have a modest storage (primary lysosomal) compartment which is continually discharged into phagolysosomes and replenished by protein synthesis. Characterization of microbicidal proteins has relied heavily on the classical techniques of biochemistry: subcellular fractionation, followed by protein purification, bioassay, and amino acid sequencing.

High- and low-affinity receptors regulate platelet responses to phorbol diesters and teleocidin

We examined binding of 3H-phorbol dibutyrate (3H-PDBu) to gel filtered human platelets (GFP) and discovered that GFP possess two classes of receptors for phorbol diesters (PDE). High-affinity (HA) receptors, approximately 5000/GFP, bound 3H-PDBu with an apparent dissociation constant (KD) of approximately 12 nM. Low-affinity receptors were approximately 5 times more numerous (2.4 X 10(4)/GFP) and had a 10-fold lower affinity for 3H-PDBu (apparent KD = 115 nM). The potencies of phorbol myristate acetate (PMA) and PDBu paralleled their binding affinities to the PDE receptors. Teleocidin (Tel), although structurally distinct from PDE, competed with 3H-PDBu for its HA-receptors (KI Tel = 1.9 nM). Binding of PDE to HA- or LA- receptors was rapid, reversible, saturable and stereospecific. The HA- and LA-receptors modulated different platelet responses. HA-receptors regulated the secretion of beta-thromboglobulin from alpha-granules and the release of N-acetyl-beta-D-hexosaminidases from lysosomes. LA-receptors mediated both platelet aggregation and the release of serotonin from dense granules. This is the first demonstration of two physiologically active classes of PDE/Tel receptors in human platelets, and demonstrates that particular platelet responses may be directed by distinct classes of receptors for specific agonists.