Some properties of a macromolecular conjugate of lysozyme prepared by modification with a monomethoxypolyethylene glycol derivative

Hen egg-white lysozyme was modified with a succinyl ester derivative of monomethoxypolyethylene glycol (mPEG-COONSu), and some properties of the resulting conjugate (mPEG-lysozyme) were studied. The conjugate was prepared by modification of lysozyme with mPEG-COONSu and purified with use of columns of CM-Toyopearl 650M and Sephadex G-75. Analytical data indicated that in the conjugate, 1.05 moles of mPEG with an average molecular weight of 5,000 were covalently attached to the lysozyme molecule. Tryptic peptide analysis of the conjugate showed that Lys 33 in lysozyme is the residue mainly modified with mPEG-COONSu. Covalent attachment of the mPEG-derivative to amino groups greatly increased the thermostability of lysozyme without any conformational change of the protein molecule. mPEG-lysozyme retained full enzyme activity for glycol chitin, but lytic activity for Micrococcus luteus cells in neutral media was 75% of that of native lysozyme and its optimal pH was at pH 5.0. It was also found that the reactivity of lysozyme with anti-lysozyme antibody from BALB/c mice or human lymphocytes was decreased by modification with mPEG-COONSu. From these findings, it was suggested that mPEG-COONSu can be advantageously used for protein tailoring of lysozyme.     

Optimization of microbial specificity in cyclic peptides by modulation of hydrophobicity within a defined structural framework.

In the present study we have utilized the structural framework of the analog GS14K4 (cyclo(VKLd-KVd-YPL KVKLd-YP, where d denotes a d-amino acid)), to examine the role of hydrophobicity in microbial activity and specificity. The hydrophobicity of GS14K4 was systematically altered by residue replacements in the hydrophobic sites of the molecule to produce a series of analogs that were either less or more hydrophobic than the parent compound. Circular dichroism spectroscopy and reversed-phase high performance liquid chromatography analysis showed that the molecules were structurally similar and only differed in overall hydrophobicity. The hydrophobicity of GS14K4 was found to be the midpoint for hemolytic activity, with more hydrophobic analogs exhibiting increased hemolytic activity and less hydrophobic analogs showing decreased hemolytic activity. For antimicrobial activity there were differences between the hydrophobicity requirements against Gram-positive and Gram-negative microorganisms. The hydrophobicity of GS14K4 was sufficient for maximum activity against Gram-negative microorganisms and yeast, with no further increases in activity occurring with increasing hydrophobicity. With Gram-positive microorganisms significant increases in activity with increasing hydrophobicity were seen in three of the six microorganisms tested. A therapeutic index (calculated as a measure of specificity of the peptides for the microorganisms over human erythrocytes) served to define the boundaries of a therapeutic window within which lay the optimum peptide hydrophobicity for each microorganism. The therapeutic window was found to be at a lower hydrophobicity level for Gram-negative microorganisms than for Gram-positive microorganisms, although the limits were more variable for the latter. Our results show that the balance between activity and specificity in the present cyclic peptides can be optimized for each microorganism by systematic modulation of hydrophobicity.     

Dissociation of antimicrobial and hemolytic activities in cyclic peptide diastereomers by systematic alterations in amphipathicity

We have investigated the role of amphipathicity in a homologous series of head-to-tail cyclic antimicrobial peptides in efforts to delineate features resulting in high antimicrobial activity coupled with low hemolytic activity (i.e. a high therapeutic index). The peptide GS14, cyclo(VKLKVd-YPLKVKLd-YP), designed on the basis of gramicidin S (GS), exists in a preformed highly amphipathic beta-sheet conformation and was used as the base compound for this study. Fourteen diastereomers of GS14 were synthesized; each contained a different single enantiomeric substitution within the framework of GS14. The beta-sheet structure of all GS14 diastereomers was disrupted as determined by CD and NMR spectroscopy under aqueous conditions; however, all diastereomers exhibited differential structure inducibility in hydrophobic environments. Because the diastereomers all have the same composition, sequence, and intrinsic hydrophobicity, the amphipathicity of the diastereomers could be ranked based upon retention time from reversed-phase high performance liquid chromatography. There was a clear correlation showing that high amphipathicity resulted in high hemolytic activity and low antimicrobial activity in the diastereomers. The latter may be the result of increased affinity of highly amphipathic peptides to outer membrane components of Gram-negative microorganisms. The diastereomers possessing the most favorable therapeutic indices possessed some of the lowest amphipathicities, although there was a threshold value below which antimicrobial activity decreased. The best diastereomer exhibited 130-fold less hemolytic activity compared with GS14, as well as greatly increased antimicrobial activities, resulting in improvement in therapeutic indices of between 1,000- and 10,000-fold for a number of microorganisms. The therapeutic indices of this peptide were between 16- and 32-fold greater than GS for Gram-negative microorganisms and represents a significant improvement in specificity over GS. Our findings show that a highly amphipathic nature is not desirable in the design of constrained cyclic antimicrobial peptides and that an optimum amphipathicity can be defined by systematic enantiomeric substitutions.     

Cloning of the cDNA of a human neutrophil bactericidal protein. Structural and functional correlations

The bactericidal permeability increasing protein (BPI) is a 50-60-kDa membrane-associated protein isolated from granules of polymorphonuclear leukocytes. A full-length cDNA clone encoding human BPI has been isolated and the derived amino acid sequence reveals a structure that is consistent with previously determined biological properties. BPI may be organized into two domains: the amino-terminal half, previously shown to contain all known antimicrobial activity, contains a large fraction of basic and hydrophilic residues. In contrast, the carboxyl-terminal half contains more acidic than basic residues and includes several potential transmembrane regions which may anchor the holoprotein in the granule membrane. The cytotoxic action of BPI is limited to many species of Gram-negative bacteria; this specificity may be explained by a strong affinity of the very basic aminoterminal half for the negatively charged lipopolysaccharides that are unique to the Gram-negative bacterial envelope. The amino-terminal end of BPI exhibits significant similarity with the sequence of a rabbit lipopolysaccharide-binding protein, suggesting that both molecules share a similar structure for binding lipopolysaccharides.     

Modification of lysozyme with oleoyl chloride for broadening the antimicrobial specificity

Lysozyme from egg white was modified by covalent attachment of an oleyl group to the free amino groups of lysozyme. The aim of the chemical modification was to develop an effective antimicrobial lysozyme derivative against both gram-negative and gram-positive bacteria. Lysozyme with various degrees of modification was obtained by changing oleoyl chloride/lysozyme mass ratio. Lysozyme derivatives evidently exhibited an antimicrobial effect against Escherichia coli (ATCC 29998). The modification slightly changed the antimicrobial effect of lysozyme derivative against Staphylococcus aureus (ATCC 121002). Since there was a positive correlation between the modification degree and the antimicrobial effect against E. coli, it was concluded that the change in antimicrobial behavior was due to an increase in hydrophobicity of the enzyme molecule enabling it to penetrate through the bacterial membrane of E. coli. It was also shown that oleoyl chloride with an MIC value of 10?mg/mL was effective against both E. coli and S. aureus.     

一株分离自口腔的鼠李糖乳杆菌及其特性

目的 分离筛选出在维护口腔微生态平衡方面具有潜在益生特性的乳杆菌菌株。 方法 从健康志愿者的口腔样本中分离乳杆菌,采用生理生化和16S rDNA分子测序进行菌株鉴定,并检测其抑菌能力、凝集能力、表面疏水性以及对溶菌酶耐受性。 结果 筛选出1株鼠李糖乳杆菌LR863,对变形链球菌、戈登链球菌、牙龈卟啉单胞菌、具核梭杆菌和伴放线放线杆菌具有抑菌作用,经蛋白酶处理后其抑菌活性降低。同时鼠李糖乳杆菌LR863有较强的自凝集能力并对上述5株指示菌有共凝集效果,对二甲苯、氯仿和乙酸乙酯的疏水率依次为76.91%、87.46%和41.88%,能耐受2.0 mg/mL浓度的溶菌酶。 结论 鼠李糖乳杆菌LR863具有优良生物学特性,可作为口腔保健产品的候选益生菌株。     

Detoxification of lipopolysaccharide (LPS) by egg white lysozyme

Abstract Recent studies carried out by our group suggest that lysozyme binds to bacterial lipopolysaccharide with a high affinity to produce a complex, and inhibits various biological activities of lipopolysaccharide. Although the basic structure of lipopolysaccharide is independent of the species and strains of Gram-negative bacteria, many structural factors such as O-antigenic polysaccharide, lipid A, substituted groups, and associated molecules, affect the biological activities of lipopolysaccharide. In this study, we prepared lysozyme/lipopolysaccharide complexes using various structures of lipopolysaccharide and compared the activity and physiochemical properties. Native and dansylated lysozyme were found to bind to all tested lipopolysaccharides. The mitogenic activity and TNF production by all tested lipopolysaccharides were significantly reduced by complex formation in vitro. Administration of the complex prepared by various lipopolysaccharides produced significantly less quantities of TNF in the septic shock model. These results suggested that binding of lysozyme to lipopolysaccharide is important for the host both in pathophysiological responses to lipopolysaccharides and in the modification of lipopolysaccharide biological activity.     

Antimicrobial peptides derived from goose egg white lysozyme

Peptide fragments possessing antimicrobial activity were obtained by protease digestion of goose egg white lysozyme. Digested peptide purified from RP-HPLC which showed no lysozyme activity exhibited bactericidal activity toward Gram-negative and Gram-positive bacteria. LC/MS–MS and automated Edman degradation revealed the amino acid sequence to be Thr-Ala-Lys-Pro-Glu-Gly-Leu-Ser-Tyr. This sequence corresponds to amino acid positions 20–28, located at the N-terminal outer part of goose lysozyme. The peptide acted on bacterial membrane as shown by scanning electron microscopy. The mechanism of action could be explained from a helical structure that may be formed by the centered Pro residue and the terminal Lys residue after the peptide attaches to a cell membrane. This is the first study to report that a peptide derived from the protease digests of G-type lysozyme possesses antimicrobial activity with broad spectrum activity. Our result is comparative to the previous reports of Chicken lysozyme and T4 phage lysozyme, which showed antimicrobial activity after digestion with protease. These results might contribute to the usage of antimicrobial peptides engineered by genetic or chemical synthesis.     

抗菌肽BF2-A/B对细菌表面特性的影响及与脂质体的相互作用

研究抗菌肽BuforinⅡ的衍生肽BF2-A/B对细菌表面特性的影响,以及与脂质体的作用模式。Zeta电位仪和十六烷萃取法检测发现BF2-A/B作用G-菌和G+菌后,能够提高细胞表面电负性和疏水性。选用卵磷脂和心磷脂制备包裹钙黄绿素的脂质体,模拟细菌胞膜,考察发现BF2-A/B能够引起荧光素从脂质体中泄漏,BF2-B对膜的扰动作用更大,引起的泄漏率比BF2-A高,但它们都不破裂脂质体膜。用FITC标记衍生肽,研究发现加入脂质体后,FITC-肽荧光光谱蓝移,量子产率增大,并且脂质体保护FITC-肽免受丙烯酰胺的荧光淬灭,说明BF2-A/B的N-端插入了脂质体的磷脂双分子层中。     

Coral mucus-associated bacteria: a possible first line of defense

Interactions among microorganisms found in coral mucus can be either symbiotic or competitive. It has been hypothesized that microbial communities found on the surface of coral play a role in coral holobiont defense, possibly through production of antimicrobial substances. Selected microorganisms isolated from the mucus layer of a number of coral species were grown using agar-plating techniques. Screening for antimicrobial substances was performed using overlay and drop techniques, employing several indicator microorganisms. Between 25% and 70% of cultivable mucus-associated bacteria from scleractinian corals demonstrated bioactivity. Higher percentages of activity were evident in mucus-associated cultivable bacteria from massive and solitary corals, as compared with bacteria from branching or soft corals. Isolates related to the genera Vibrio and Pseudoalteromonas demonstrated high activity against both Gram-positive and Gram-negative bacteria. Gram-positive bacteria ( Bacillus, Planomicrobium ) demonstrated lower levels of activity, primarily against other Gram-positive bacteria. In some cases, inhibitory effects were confined to the cell fraction, suggesting the involvement of a cell-bound molecule, sensitive to temperature and most likely proteinaceous in nature. These results demonstrate the existence of microorganisms with antimicrobial activity on the coral surface, possibly acting as a first line of defense to protect the coral host against pathogens.     

Synergistic action of Galleria mellonella anionic peptide 2 and lysozyme against Gram-negative bacteria

Lysozyme and antimicrobial peptides are key factors of the humoral immune response in insects. In the present work lysozyme and anionic defense peptide (GMAP2) were isolated from the hemolymph of the greater wax moth Galleria mellonella and their antibacterial activity was investigated. Adsorption of G. mellonella lysozyme on the cell surface of Gram-positive and Gram-negative bacteria was demonstrated using immunoblotting with anti-G. mellonella lysozyme antibodies. Lysozyme effectively inhibited the growth of selected Gram-positive bacteria, which was accompanied by serious alterations of the cell surface, as revealed by atomic force microscopy (AFM) imaging. G. mellonella lysozyme used in concentrations found in the hemolymph of naive and immunized larvae, perforated also the Escherichia coli cell membrane and the level of such perforation was considerably increased by GMAP2. GMAP2 used alone did not perforate E. coli cells nor influence lysozyme muramidase activity. However, the peptide induced a decrease in the turgor pressure of the bacterial cell. Moreover, in the samples of bacteria treated with a mixture of lysozyme and GMAP2 the sodium chloride crystals were found, suggesting disturbance of ion transport across the membrane leading to cell disruption. These results clearly indicated the synergistic action of G. mellonella lysozyme and anionic peptide 2 against Gram-negative bacteria. The reported results suggested that, thanks to immune factors constitutively present in hemolymph, G. mellonella larvae are to some extent protected against infection caused by Gram-negative bacteria.     

Mechanisms of action of ostrich beta-defensins against Escherichia coli

To understand their mechanism of antimicrobial activity against Gram-negative bacteria, ostrich beta-defensins, ostricacins-1 and 2 (Osp-1 and Osp-2), were compared with those of sheep myeloid antimicrobial peptide (SMAP)-29 and human neutrophil peptide (HNP)-1, well-characterized sheep alpha-helical and human alpha-defensin peptides, respectively. Fluorescence-based biochemical assays demonstrated that the ostricacins bound lipopolysaccharides and disrupted both outer and cytoplasmic membrane integrity. The ostricacins' permeabilizing ability was weaker than that of SMAP-29, but stronger than HNP-1. As ostricacins have previously shown the ability to inhibit bacterial growth, these peptides were suggested to be bacteriostatic to Gram-negative bacteria, which are caused by the interaction between the peptides and cytoplasmic targets causing the inhibition of DNA, RNA, and protein synthesis as well as enzymatic activities. These findings indicated promising possibilities for the peptides to be used in the development of therapeutic and topical products.     

MODIFICATION OF LYSOZYME WITH OLEOYL CHLORIDE FOR BROADENING THE ANTIMICROBIAL SPECIFICITY

Lysozyme from egg white was modified by covalent attachment of an oleyl group to the free amino groups of lysozyme. The aim of the chemical modification was to develop an effective antimicrobial lysozyme derivative against both gram-negative and gram-positive bacteria. Lysozyme with various degrees of modification was obtained by changing oleoyl chloride/lysozyme mass ratio. Lysozyme derivatives evidently exhibited an antimicrobial effect against Escherichia coli (ATCC 29998). The modification slightly changed the antimicrobial effect of lysozyme derivative against Staphylococcus aureus (ATCC 121002). Since there was a positive correlation between the modification degree and the antimicrobial effect against E. coli, it was concluded that the change in antimicrobial behavior was due to an increase in hydrophobicity of the enzyme molecule enabling it to penetrate through the bacterial membrane of E. coli. It was also shown that oleoyl chloride with an MIC value of 10 mg/mL was effective against both E. coli and S. aureus.     

Binding of lysozyme to phospholipid bilayers: evidence for protein aggregation upon membrane association

Biological functions of lysozyme, including its antimicrobial, antitumor, and immune-modulatory activities have been suggested to be largely determined by the lipid binding properties of this protein. To gain further insight into these interactions on a molecular level the association of lysozyme to liposomes composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-phosphatidylserine, or bovine heart cardiolipin was studied by a combination of fluorescence techniques. The characteristics of the adsorption of lysozyme to lipid bilayers were investigated using fluorescein 5'-isothiocyanate labeled protein, responding to membrane association by a decrease in fluorescence. Upon increasing the content of anionic phospholipids in lipid vesicles, the binding isotherms changed from Langmuir-like to sigmoidal. Using adsorption models based on scaled particle and double-layer theories, this finding was rationalized in terms of self-association of the membrane-bound protein. The extent of quenching of lysozyme tryptophan fluorescence by acrylamide decreased upon membrane binding, revealing a conformational transition for the protein upon its surface association, resulting in a diminished access of the fluorophore to the aqueous phase. Steady-state fluorescence anisotropy of bilayer-incorporated probe 1,6-diphenyl-1,3,5-hexatriene was measured at varying lipid-to-protein molar ratios. Lysozyme was found to increase acyl-chain order for liposomes with the content of acidic phospholipid exceeding 10 mol %. Both electrostatic and hydrophobic protein-lipid interactions can be concluded to modulate the aggregation behavior of lysozyme when bound to lipid bilayers. Modulation of lysozyme aggregation propensity by membrane binding may have important implications for protein fibrillogenesis in vivo. Disruption of membrane integrity by the aggregated protein species is likely to be the mechanism responsible for the cytotoxicity of lysozyme.     

A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action

Recently, we have found that partially unfolded lysozyme exerts broad spectrum antimicrobial action in vitro against Gram-negative and Gram-positive bacteria independent of its catalytic activity. In parallel, an internal peptide (residues 98-112) of hen egg white lysozyme, obtained after digestion with clostripain, possessed broad spectrum antimicrobial action in vitro. This internal peptide is part of a helix-loop-helix domain (87-114 sequence of hen lysozyme) located at the upper lip of the active site cleft of lysozyme. The helix-loop-helix (HLH) structures are known motifs commonly found in membrane-active and DNA-binding proteins. To evaluate the contribution of the HLH peptide to the antimicrobial properties of lysozyme, the HLH sequence and its secondary structure derivatives of chicken and human lysozyme were synthesized and tested for antimicrobial activity against several bacterial strains. We found that the full HLH peptide of both chicken and human lysozymes was potently microbicidal against both Gram-positive and Gram-negative bacteria and the fungus Candida albicans. The N-terminal helix of HLH was specifically bactericidal to Gram-positive bacteria, whereas the C-terminal helix was bactericidal to all tested strains. Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its C-terminal helix domain kill Gram-negative bacteria by crossing the outer membrane via self-promoted uptake and causing damage to the inner membrane through channel formation. The results are discussed in terms of proposed mechanisms for the catalytically independent antimicrobial activity of lysozyme that offer a new strategy for the design of potential antimicrobial drugs in the treatment of infectious diseases.     

Comparison of the membrane interaction and permeabilization by the designed peptide Ac-MB21-NH2 and truncated dermaseptin S3

Ac-MB21-NH(2) (Ac-FASLLGKALKALAKQ-NH(2)) and dermaseptin S3(1-16)-NH(2) (ALWKNMLKGIGKLAGK-NH(2)) are cationic amphipathic peptides with antimicrobial activity against a broad spectrum of microorganisms including various fungi. The interaction of the peptides with liposomes was studied by exploiting the tryptophan fluorescence of F1W-Ac-MB21-NH(2) and dermaseptin S3(1-16)-NH(2). Spectral analysis and the use of quenchers indicate that the tryptophans of both peptides insert more deeply in anionic than in zwitterionic liposomes. Membrane insertion correlates with the formation of an alpha-helical peptide structure. Both peptides permeabilize liposomes composed of anionic, cylindric phospholipids more efficiently than liposomes formed of zwitterionic, conic (phospho)lipids.     

Recent advances for the production and recovery methods of lysozyme

Lysozyme is an antimicrobial peptide with a high enzymatic activity and positive charges. Therefore, it has applications in food and pharmaceutical industries as an antimicrobial agent. Lysozyme is ubiquitous in both animal and plant kingdoms. Currently, egg-white lysozyme is the most commercially available form of lysozyme. The main concerns of egg-white lysozyme are high recovery cost, low activity and most importantly the immunological problems to some people. Therefore, human lysozyme production has gained importance in recent years. Scientists have developed transgenic plants, animals and microorganisms that can produce human lysozyme. Out of these, microbial production has advantages for commercial productions, because high production levels are achievable in a relatively short time. It has been reported that fermentation parameters, such as pH, temperature, aeration, are key factors to increase the effectiveness of the human lysozyme production. Moreover, purification of the lysozyme from the fermentation broth needs to be optimized for the economical production. In conclusion, this review paper covers the mechanism of lysozyme, its sources, production methods and recovery of lysozyme.     

Reconstitution of a porcine submaxillary gland beta-D-galactoside alpha 2----3 sialyltransferase into liposomes

Form A of the beta-D-galactoside alpha 2----3 sialyltransferase from porcine submaxillary glands was incorporated into liposomes. Incorporation was achieved by gel filtration of the enzyme in the presence of octylglucoside-phospholipid micelles. As detergent was removed during gel filtration, liposomes (average diameter, 370 A) with bound enzyme were formed and emerged unretarded from the column. The recovery of enzyme activity in the liposomes was about 40% of the initial activity starting with as little as 9 micrograms of transferase. Chromatography on Sepharose CL6B and sucrose density gradient centrifugation confirmed the association of enzyme with liposomes. In contrast to Form A, Form B of the sialyltransferase, which lacks the proposed lipid-binding domain of Form A, cannot be incorporated into liposomes. Form A of the transferase was also incorporated into liposomes composed of phosphatidylcholine, cholesterol, and a mixture of phospholipids from the membranes of the Golgi apparatus from porcine submaxillary glands. Although the transferase was distributed about equally on the internal and external surface of the phosphatidylcholine liposomes, most of the transferase was on the external surface in liposomes containing cholesterol (72%) or in liposomes containing Golgi apparatus phospholipids (88%). The enzyme bound to phosphatidylcholine liposomes was shown by kinetic analysis to have the same activity as that found in the presence of activity-stimulating detergents such as Triton X-100. Enzyme incorporated into cholesterol-containing liposomes had the same activity. In contrast, enzyme bound to liposomes formed from the Golgi apparatus mixed phospholipids had a lower activity, but one similar to that of the transferase in Golgi apparatus membranes. These studies suggest that the composition of a biological membrane may well influence the orientation of the transferase in the membrane as well as modulate its enzymic activity.     

Purification of novel peptide antibiotics from human milk

A strategy was established for the identification of novel antimicrobial peptides from human milk. For the generation of bioactive peptides human milk was acidified and proteolyzed with pepsin simulating the digest in infants stomachs. Separation of proteins and resulting fragments was performed by means of reversed-phase chromatography detecting the antimicrobial activity of each fraction using a sensitive radial diffusion assay. In order to avoid the purification of the known abundant antimicrobial milk protein lysozyme, it was identified in HPLC fractions by its enzymatic activity and by matrix-assisted laser desorption ionization–mass spectrometry (MALDI–MS). On condition that lysozyme was not detectable and antibacterial activity of HPLC fractions was caused by a peptide, which was confirmed by proteolytic cleavage leading to a loss of activity, further purification was performed by consecutive chromatographic steps guided by the antibacterial assay. Using this strategy, an as yet unknown casein fragment exhibiting antimicrobial activity was purified in addition to antimicrobial lactoferrin fragments. The new antimicrobial peptide resembles a proteolytic fragment of human casein-κ (residues 63–117) and inhibits the growth of Gram-positive, Gram-negative bacteria, and yeasts. Our results confirm that antimicrobially-active peptides are liberated from human milk proteins during proteolytic hydrolysis and may play an important role in the host defense system of the newborn.     

Antimicrobial activity of native and synthetic surfactant protein B peptides

Surfactant protein B (SP-B) is secreted into the airspaces with surfactant phospholipids where it reduces surface tension and prevents alveolar collapse at end expiration. SP-B is a member of the saposin-like family of proteins, several of which have antimicrobial properties. SP-B lyses negatively charged liposomes and was previously reported to inhibit the growth of Escherichia coli in vitro; however, a separate study indicated that elevated levels of SP-B in the airspaces of transgenic mice did not confer resistance to infection. The goal of this study was to assess the antimicrobial properties of native SP-B and synthetic peptides derived from the native peptide. Native SP-B aggregated and killed clinical isolates of Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and group B streptococcus by increasing membrane permeability; however, SP-B also lysed RBC, indicating that the membranolytic activity was not selective for bacteria. Both the antimicrobial and hemolytic activities of native SP-B were inhibited by surfactant phospholipids, suggesting that endogenous SP-B may not play a significant role in alveolar host defense. Synthetic peptides derived from native SP-B were effective at killing both Gram-positive and Gram-negative bacteria at low peptide concentrations (0.15-5.0 microM). The SP-B derivatives selectively lysed bacterial membranes and were more resistant to inhibition by phospholipids; furthermore, helix 1 (residues 7-22) retained significant antimicrobial activity in the presence of native surfactant. These results suggest that the role of endogenous SP-B in host defense may be limited; however, synthetic peptides derived from SP-B may be useful in the treatment of bacterial pneumonias.