Antimicrobial cationic peptides

Abstract

A novel class of cationic peptides having antimicrobial activity is provided. Examples of such peptides includeNH.sub.2 -KWKSFIKKLTTAVKKVLTTGLPALIS-COOH (SEQ ID NO:1)andNH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH. (SEQ ID NO:2)Also provided are methods for inhibiting the growth of bacteria utilizing the peptides of the invention. The peptides are particularly useful for inhibiting endotoxemia in a subject.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to antimicrobial peptides and specifically to a new class of antimicrobial cationic peptides referred to as bactolysins.

2. Description of Related Art

In 1981, the self-promoted uptake hypothesis was first proposed to explain the mechanism of action of polycationic antibiotics in Pseudomonas aeruginosa. According to this hypothesis, polycations interact with sites on the outer membranes of Gram-negative bacteria at which divalent cations cross-bridge adjacent lipopolysaccharide molecules. Due to their higher affinity for these sites, polycations displace the divalent cations and, since the polycations are bulkier than the divalent cations, cause structural perturbations in the outer membrane. These perturbations result in increased outer membrane permeability to compounds such as the .beta.-lactam antibiotic nitrocefin, the eukaryotic non-specific defense protein lysozyme and to hydrophobic substances. By analogy, molecules accessing this pathway are proposed to promote their own uptake.

It has been clearly demonstrated that the outer membranes of Gram-negative bacteria are semipermeable molecular "sieves" which restrict access of antibiotics and host defense molecules to their targets within the bacterial cell. Thus, cations and polycations which access the self-promoted uptake system are, by virtue of their ability to interact with and break down the outer membrane permeability barrier, capable of increasing the susceptibility of Gram-negative pathogenic bacteria to antibiotics and host defense molecules. Hancock and Wong demonstrated that a broad range of such compounds could overcome the permeability barrier and coined the name "permeabilizers" to describe them (Hancock and Wong, Antimicrob. Agents Chemother., 26:48, 1984). While self-promoted uptake and permeabilizers were first described for P. aeruginosa, they have now been described for a variety of Gram-negative bacteria.

Over the past decade, non-specific defense molecules have been described in many animals, including insects and humans. One subset of these molecules have in common the following features: (a) they are small peptides, usually 15-35 amino acids in length, (b) they contain 4 or more positively charged amino acid residues, either lysines or arginines, and (c) they are found in high abundance in the organisms from which they derive. Several of these molecules have been isolated, amino acid sequenced and described in the patent literature (e.g., cecropins: WO8900199, WO 8805826, WO8604356, WO 8805826; defensins: EP 193351, EP 85250, EP 162161, U.S. Pat. No. 4,659,692, WO 8911291). However, only limited amounts of these peptides can be isolated from the host species. For example, Sawyer, et al., (Infect. Immm. 56:693, 1988) isolated 100-200 mg of rabbit neutrophil defensins 1 and 2 from 10.sup.9 primed peritoneal neutrophils or lipopolysaccharide-elicited alveolar macrophages (i.e., the numbers present in a whole animal).

The gene for human defensin has been cloned and sequenced, but no successful expression has been demonstrated, as yet. Furthermore, production of these peptides using peptide synthesis technology produces peptides in limited amounts and is expensive when scaled up or when many variant peptides must be produced. Also, structural analysis is difficult without specific incorporation of .sup.15 N and .sup.13 C tagged amino acids which is prohibitively expensive using amino acid synthesis technology.

There is a need to develop polypeptides having a broad range of potent antimicrobial activity against a plurality of microorganisms, including gram negative bacteria, gram positive bacteria, fungi, protozoa, viruses and the like.

SUMMARY OF THE INVENTION

The present invention provides a novel class of cationic peptides, referred to as bactolysins, which have antimicrobial activity. Two representative peptides are provided and include

(SEQ ID NO:1)MBI 29, NH.sub.2 -KWKSFIKKLTTAVKKVLTTGLPALIS-COOHand (SEQ ID NO:2)MBI 26, NH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH, analogs, derivatives and conservative variations thereof.

The invention also provides a method of inhibiting the growth of bacteria comprising contacting the bacteria with an inhibiting effective amount of a peptide having an amino acid sequence of MBI 29 (SEQ ID NO:1) or MBI 26 (SEQ ID NO:2) alone, or in combination with an antibiotic. Classes of antibiotics which can be used for synergistic therapy with the peptides of the invention include aminoglycoside, penicillin, cephalosporine, fluoroquinolone, carbepenem, tetracycline and macrolide.

In another embodiment, the invention provides a method of inhibiting an endotoxemia or sepsis associated disorder in a subject having or at risk of having such a disorder, comprising administering to the subject a therapeutically effective amount of a peptide of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a three-dimensional schematic representation of the conformational structure of CEME, MBI 29, and MBI 26.

FIG. 2 shows the axial projection of the .alpha.-helical conformation of each peptide in 2D.

FIG. 3 shows an inner membrane permeabilization assay for MBI 26.

FIG. 4 shows an inner membrane permeabilization assay for MBI 29.

FIG. 5a shows tumor necrosis factor (TNF) levels measured 6 hours after the addition of E. coli 0111:B4 LPS and CEME (ME), CEMA (MA), and MBI 29 to macrophage cells. The data is from two separate assays.

FIGS. 5b and 5c show tumor necrosis factor (TNF) levels measured 6 hours after the addition of E. coli Bort LPS and E. coli 0111:B4 LPS and MBI 29 to macrophage cells. The data is from two separate assays.

FIG. 6a shows tumor necrosis factor (TNF) levels measured 6 hours after the addition of E. coli Bort LPS and CEME (ME), CEMA (MA) and MBI 29 to macrophage cells. The data is from two separate assays.

FIG. 6b shows tumor necrosis factor (TNF) levels measured 6 hours after the addition of P. aeruginosa LPS and MBI 26 to RAW macrophage cells.

FIG. 6c shows tumor necrosis factor (TNF) levels measured 6 hours after the addition of E. coli Bort LPS, E. coli 0111:B4 LPS and MBI 26 to RAW macrophage cells.

FIGS. 7a and 7b show RAW cell TNF production after addition of E. coli 0111:B4 LPS and the effect of the addition of MBI 29 and polymyxin B on TNF.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel class of cationic peptides, called bactolysins, which have antimicrobial activity and have the ability to significantly reduce the level of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF). These peptides are useful for inhibiting microbial infection or growth, as well reducing the effects of endotoxemia and are often synergistic with conventional antibiotics and/or lysozyme. In addition, such peptides are useful as antifungal agents, antitumor agents, or antiviral agents.

The term "antimicrobial" as used herein means that the peptides of the present invention inhibit, prevent, or destroy the growth or proliferation of microbes such as bacteria, fungi, viruses or the like. The term "antiviral" as used herein means that the peptides of the present invention inhibit, prevent or destroy the growth or proliferation of viruses or of virally-infected cells. The term "anti-tumor" as used herein means that the peptides of the present invention may be used to inhibit the growth of or destroy tumors. The term "antifungal" as used herein means that the peptides of the present invention may be used to inhibit the growth of or destroy fungi.

In a first embodiment, the invention provides an isolated antimicrobial peptide having an amino acid sequence:

(SEQ ID NO:26)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VLTTGLPALIS-COOH, (SEQ ID NO:27)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VVTTAKPLISS-COOH, (SEQ ID NO:28)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 ILTTGLPALIS-COOH, (SEQ ID NO:29)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GGLLSNIVTSL-COOH,or (SEQ ID NO:30)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GPILANLVSIV-COOH wherein R.sub.1 is a hydrophobic amino acid residue and R.sub.2 is a hydrophilic amino acid residue. Examples of such peptides of the invention include but are not limited to: (SEQ ID NO:1)NH.sub.2 -KWKSFIKKLTTAVKKVLTTGLPALIS-COOH(MBI 29), (SEQ ID NO:2)NH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH(MBI 26), (SEQ ID NO:3)NH.sub.2 -KWKSFIKNLTKGGSKILTTGLPALIS-COOH(MBI 201), (SEQ ID NO:4)NH.sub.2 -KWKKFIKNLTKGGSKILTTGLPALIS-COOH(MBI 202), (SEQ ID NO:5)NH.sub.2 -KWKSFIKNLEKVLKPGGLLSNIVTSL-COOH(MBI 490), (SEQ ID NO:6)NH.sub.2 -KWKSFIKNLEKVLKKGPILANLVSIV-COOH(MBI 491), (SEQ ID NO:7)NH.sub.2 -KWKEFIKKLTTAVKKVLTTGLPALIS-COOH(MBI 492), (SEQ ID NO:8)NH.sub.2 -KWKKFIKELQKVLAPGGLLSNIVTSL-COOH(MBI 493), (SEQ ID NO:9)NH.sub.2 -KWKSFIKKLTSVLKKVVTTALPALIS-COOH(MBI 29A1), (SEQ ID NO:10)NH.sub.2 -KWKSFIKNLTKVLKKVVTTALPALIS-COOH(MBI 29A2), (SEQ ID NO:11)NH.sub.2 -KWKLFKKKGTGAVLTVLTTGLPALIS-COOH(MBI 29A3), (SEQ ID NO:12)NH.sub.2 -KWKSFIKKLTSVLKKVVTTAKPLISS-COOH, (SEQ ID NO:13)NH.sub.2 -KKKSFIKLLTSAKVSVLTTAKPLISS-COOH,and (SEQ ID NO:14)NH.sub.2 -KWKKFIKELQKVLKPGGLLSNIVTSL-COOH, analogs, derivatives and conservative variations thereof, wherein the peptides have antimicrobial activity. The peptides of the invention include SEQ ID NO: 1-14, as well as the broader groups of peptides having hydrophilic and hydrophobic substitutions, and conservative variations thereof.

The sequence KWKSFIKK (SEQ. ID NO:34), as found in the first 8 NH.sub.2 terminal amino acids of peptides in SEQ ID NO:1 and 2, is important for conferring antimicrobial activity. The presence of a positively charged amino acid (lysine or arginine) at positions 11, 14, 15 and 21 are also very important in antimicrobial activity. A further enhancement of antimicrobial activity can be achieved by making conservative changes in the residues designated R.sub.1 and R.sub.2.

In another embodiment, the invention provides an isolated antimicrobial peptide having an amino acid sequence:

NH.sub.2 -KKWWRRR.sub.1 R.sub.1 R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GPALSNV-COOH (SEQ. ID NO:31) wherein R.sub.1 is a hydrophobic amino acid residue and R.sub.2 is a hydrophilic amino acid residue. Examples of such peptides of the invention include but are not limited to: NH.sub.2 -KKWWRRVLSGLKTGPALSVN-COOH, (SEQ ID NO:15)NH.sub.2 KKWWRRVLKGLSSGPALSNV-COOH, (SEQ ID NO:16)NH.sub.2 -KKWWRRALQALKNGPALSNV-COOH, (SEQ ID NO:17) analogs, derivatives and conservative variations thereof, wherein the peptides have antimicrobial activity. The peptides of the invention include SEQ ID NO:15-17, as well as the broader groups of peptides having hydrophilic and hydrophobic substitutions, and conservative variations thereof

In another embodiment, the invention provides an isolated antimicrobial peptide having an amino terminal amino acid sequence:

NH.sub.2 -KKWWRRX. (SEQ. ID NO:32) Preferably, the peptide is from about 20 to about 30 amino acids in length and therefore X is from about 14 to about 24 amino acids. Most preferably the peptide has a sequence of NH.sub.2 -KKWWRRR.sub.1 R.sub.1 R.sub.2 GLKTAGPAIQSVLNK-COOH(SEQ. ID NO:35) wherein R.sub.1 is a hydrophobic amino acid residue and R.sub.2 is a hydrophilic amino acid residue. Examples of such peptides of the invention include but are not limited to: (SEQ ID NO:18)NH.sub.2 -KKWWRRVLSGLKTAGPAIQSVLNK-COOH (MBI 21A1),and (SEQ ID NO:19)NH.sub.2 -KKWWRRALQGLKTAGPAIQSVLNK-COOH (MBI 21A2), analogs, derivatives and conservative variations thereof, wherein the peptides have antimicrobial activity. The peptides of the invention include SEQ ID NO: 18-19, as well as the broader groups of peptides having hydrophilic and hydrophobic substitutions, and conservative variations thereof.

In another embodiment, the invention provides an isolated antimicrobial peptide having an amino terminal amino acid sequence:

NH.sub.2 -KKWWKX. (SEQ ID NO:33) Preferably, the peptide is from about 20 to about 30 amino acids in length and therefore X is from about 14 to about 24 amino acids. Most preferably the peptide has a sequence of (SEQ ID NO:20)NH.sub.2 -KKWWKAQKAVNSGPNALQTLAQ-COOH (SEQ ID NO:21)NH.sub.2 -KKWWKAKKFANSGPNALQTLAQ-COOH,or (SEQ ID NO:22)NH.sub.2 -KKWWKFIKKAVNSGTTGLQTLAS-COOH, analogs, derivatives and conservative variations thereof, wherein the peptides have antimicrobial activity. The peptides of the invention include SEQ ID NO:20-22, as well as the broader groups of peptides having hydrophilic and hydrophobic substitutions, and conservative variations thereof.

Other cationic peptides of the invention include:

(SEQ ID NO:23)NH.sub.2 -KKSFFKKLTSVASSVLS-COOH (MBI 21A14), (SEQ ID NO:24)NH.sub.2 -WKVFKSFIKKASSFAQSVLD-COOH,and (SEQ ID NO:25)NH.sub.2 -KKWRKSFFKQVGSFDNSV-COOH, analogs, derivatives and conservative variations thereof, wherein the peptides have antimicrobial activity.

The sequence of SEQ ID NO:1 contains helix-forming hydrophilic amino acids at residues 4, 8, 10, 11, and 14. Amino acids 6 and 9 are hydrophobic residues (see FIG. 1). The helical nature of the first 10 amino acids is depicted in FIGS. 1 and 2. The cationic charge of the peptide of SEQ ID NO:1 was achieved by placing a lysine at positions 8 and 14, thereby increasing the total positive charge to 6. The carboxy terminus of SEQ ID NO:1 was converted to the methyl ester and the antibacterial activity of this derivative was the same as that of the unmodified peptide. The peptide of SEQ ID NO:1 also has antifungal activity. Amino acid residues 3, 4, 7, 8, 10, 11, 14, and 15 are preferably hydrophilic residues, while amino acid residues 2, 5, 6, 9, 12, and 13 are preferably hydrophobic residues.

SEQ ID NO:1 was modified to increase the alpha helical nature by changing amino acids in the C-terminal tail, thereby resulting in SEQ ID NO:2. Amino acids 11 (neutral hydrophilic) and 13 (hydrophobic) of SEQ ID NO:1 were changed to another neutral hydrophilic and hydrophobic amino acid, respectively, in order to increase alpha helicity in this region. The C-terminal tail (residue 20-26) was modified to include a positive charge and increase alpha helicity. Therefore residues 20, 21, 23, 24 and 25 were changed (See FIGS. 1 and 2). SEQ ID NO:3 was obtained by changing amino acids 8, 11, 12, 13, 14, and 16 of SEQ ID NO:1. The first 15 amino acid residues contain lysine at every fourth residue of the alpha helix. Thus, the lysines at positions 8 and 14 in SEQ ID NO:1 were replaced by asparagine and serine, respectively, and the serine at position 11 was changed to lysine. Further changes were made at positions 12, 13 and 16 to conserve the helical nature. The C-terminal tail was unchanged. SEQ ID NO:4 is identical to SEQ ID NO:3, except for the amino acid at position 4, which incorporates an additional positive charge. SEQ ID NO:5 and SEQ ID NO:6 represent peptides having the same amino acid sequence as SEQ ID NO:3 for the first 9 amino acids. The amino acid at position 10 is glutamic acid. Residues 10, 11, 12, and 13 in both SEQ ID NO:5 and SEQ ID NO:6 are different than SEQ ID NO:1, but remain either hydrophilic or hydrophobic as described above.

Residue 14 in SEQ ID NO:5 is the same as in SEQ ID NO:1. All residues thereafter, 15-26, are completely changed to construct a hydrophobic helical tail, and bears no resemblance to the tail of SEQ ID NO:1. SEQ ID NO:6 contains the same residues as SEQ ID NO:1 at positions 14 and 15. Residues 16-26 have been changed to form a helical tail.

The term "isolated" as used herein refers to a peptide substantially free of proteins, lipids, nucleic acids, for example, with which it is naturally associated. Those of skill in the art can make similar substitutions to achieve peptides with greater antibacterial activity and a broader host range. For example, the invention includes the bactolysin peptides depicted in SEQ ID NO:1-25, as well as analogues or derivatives thereof, as long as the bioactivity of the peptide remains. Minor modifications of the primary amino acid sequence of the peptides of the invention may result in peptides which have substantially equivalent activity as compared to the specific peptides described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the peptides produced by these modifications are included herein as long as the biological activity of the original peptide still exists.

Further, deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity. This can lead to the development of a smaller active molecule which would also have utility. For example, amino or carboxy terminal amino acids which may not be required for biological activity of the particular peptide can be removed. Peptides of the invention include any analog, homolog, mutant, isomer or derivative of the peptides disclosed in the present invention, so long as the bioactivity as described herein is remains. All peptides were synthesized using L amino acids, however, all D forms of the peptides (e.g., see Table 1b, CEMA) can be synthetically produced. In addition, C-terminal derivatives can be produced, such as C-terminal methyl esters, in order to increase the antimicrobial activity of a peptide of the invention.

The peptide of the invention include peptides which are conservative variations of those peptides specifically exemplified herein. The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. Neutral hydrophilic amino acids which can be substituted for one another include asparagine, glutamine, serine and threonine. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Such conservative substitutions are within the definition of the classes of the peptides of the invention with respect to R.sub.1 and R.sub.2. For the peptides of the invention, a preferred conservative variation is substitution of lysine by arginine.

The biological activity of the peptides can be determined by standard methods known to those of skill in the art, such as "minimal inhibitory concentration (MIC)" assay described in the present examples, whereby the lowest concentration at which no change in OD is observed for a given period of time is recorded as MIC. Alternatively, "fractional inhibitory concentration (FIC)" is also useful for determination of synergy between the peptides of the invention, or the peptides in combination with known antibiotics. FICs are performed by checkerboard titrations of peptides in one dimension of a microtiter plate, and of antibiotics in the other dimension, for example. The FIC is calculated by looking at the impact of one antibiotic on the MIC of the other and vice versa. An FIC of one indicates that the influence of the compounds is additive and an FIC of less than one indicates synergy. Preferably, an FIC of less than 0.5 is obtained for synergism. As used herein, FIC can be determined as follows: ##EQU1## Peptides of the invention can be synthesized by such commonly used methods as t-BOC or FMOC protection of alpha-amino groups. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the C terminus of the peptide (See, Coligan, et al, Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9). Peptides of the invention can also be synthesized by the well known solid phase peptide synthesis methods described Merrifield, J. Am. Chem. Soc., 85:2149, 1962), and Stewart and Young, Solid Phase Peptides Synthesis, (Freeman, San Francisco, 1969, pp.27-62), using a copoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer. On completion of chemical synthesis, the peptides can be deprotected and cleaved from the polymer by treatment with liquid HF-10% anisole for about 1/4-1 hours at 0.degree. C. After evaporation of the reagents, the peptides are extracted from the polymer with 1% acetic acid solution which is then lyophilized to yield the crude material. This can normally be purified by such techniques as gel filtration on Sephadex G-15 using 5% acetic acid as a solvent. Lyophilization of appropriate fractions of the column will yield the homogeneous peptide or peptide derivatives, which can then be characterized by such standard techniques as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility, and quantitated by the solid phase Edman degradation.

The invention includes polynucleotides encoding: (SEQ ID NO:26)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VLTTGLPALIS-COOH, (SEQ ID NO:27)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VVTTAKPLISS-COOH, (SEQ ID NO:28)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 ILTTGLPALIS-COOH, (SEQ ID NO:29)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GGLLSNIVTSL-COOH,and (SEQ ID NO:30)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GPILANLVSIV-COOH wherein R.sub.1 is a hydrophobic amino acid residue and R.sub.2 is a hydrophilic amino acid residue. More specifically, the invention also includes an isolated polynucleotide which encodes the MBI 29 peptide of SEQ ID NO: 1, an isolated polynucleotide which encodes the MBI 26 peptide of SEQ ID NO:2, and isolated polynucleotides which encode SEQ ID NO:3-25. In addition, the invention includes polynucleotides which encode analogs, mutants and variants of the peptides of the invention. The term "isolated" as used herein refers to a polynucleotide substantially free of proteins, lipids, nucleic acids, for example, with which it is naturally associated. As used herein, "polynucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides, in the form of a separate fragment or as a component of a larger construct. DNA encoding a peptide of the invention can be assembled from cDNA fragments or from oligonucleotides which provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit. Polynucleotide sequences of the invention include DNA, RNA and cDNA sequences. A polynucleotide sequence can be deduced from the genetic code, however, the degeneracy of the code must be taken into account. Polynucleotides of the invention include sequences which are degenerate as a result of the genetic code. Such polynucleotides are useful for the recombinant production of large quantities of a peptide of interest, such as the peptide of SEQ ID NO: 1-25.

In the present invention, the polynucleotides encoding the cationic peptides of the invention may be inserted into a recombinant "expression vector". The term "expression vector" refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of cationic genetic sequences. Such expression vectors of the invention are preferably plasmids which contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence in the host. The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells. For example, the expression of the peptides of the invention can be placed under control of E. coli chromosomal DNA comprising a lactose or lac operon which mediates lactose utilization by elaborating the enzyme beta-galactosidase. The lac control system can be induced by IPTG. A plasmid can be constructed to contain the lac Iq repressor gene, permitting repression of the lac promoter until IPTG is added. Other promoter systems known in the art include beta lactamase, lambda promoters, the protein A promoter, and the tryptophan promoter systems. While these are the most commonly used, other microbial promoters, both inducible and constitutive, can be utilized as well. The vector contains a replicon site and control sequences which are derived from species compatible with the host cell. In addition, the vector may carry specific gene(s) which are capable of providing phenotypic selection in transformed cells. For example, the beta-lactamase gene confers ampicillin resistance to those transformed cells containing the vector with the beta-lactamase gene.

Transformation of a host cell with the polynucleotide may be carried out by conventional techniques well known to those skilled in the art. For example, where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth and subsequently treated by the CaCl.sub.2 method using procedures well known in the art. Alternatively, Mg.sub.2 Cl or RbCl could be used.

In addition to conventional chemical methods of transformation, the plasmid vectors of the invention may be introduced into a host cell by physical means, such as by electroporation or microinjection. Electroporation allows transfer of the vector by high voltage electric impulse, which creates pores in the plasma membrane of the host and is performed according to methods well known in the art. Additionally, cloned DNA can be introduced into host cells by protoplast fusion, using methods well known in the art.

DNA sequences encoding the cationic peptides can be expressed in vivo by DNA transfer into a suitable host cell. "Host cells" of the invention are those in which a vector can be propagated and its DNA expressed. The term also includes any progeny of the subject host cell. It is understood that not all progeny are identical to the parental cell, since there may be mutations that occur during replication. However, such progeny are included when the terms above are used. Preferred host cells of the invention include E. coli, S. aureus and P. aeruginosa, although other Gram-negative and Gram-positive organisms known in the art can be utilized as long as the expression vectors contain an origin of replication to permit expression in the host.

The cationic peptide polynucleotide sequence used according to the method of the invention can be isolated from an organism or synthesized in the laboratory. Specific DNA sequences encoding the cationic peptide of interest can be obtained by: 1) isolation of a double-stranded DNA sequence from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the cationic peptide of interest; and 3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.

The synthesis of DNA sequences is frequently the method of choice when the entire sequence of amino acid residues of the desired peptide product is known. In the present invention, the synthesis of a DNA sequence has the advantage of allowing the incorporation of codons which are more likely to be recognized by a bacterial host, thereby permitting high level expression without difficulties in translation. In addition, virtually any peptide can be synthesized, including those encoding natural cationic peptides, variants of the same, or synthetic peptides.

When the entire sequence of the desired peptide is not known, the direct synthesis of DNA sequences is not possible and the method of choice is the formation of cDNA sequences. Among the standard procedures for isolating cDNA sequences of interest is the formation of plasmid or phage containing cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned. In those cases where significant portions of the amino acid sequence of the cationic peptide are known, the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single stranded form (Jay, et al., Nuc. Acid Res., 11:2325, 1983).

The invention also provides a method of inhibiting the growth of bacteria comprising contacting the bacteria with an inhibiting effective amount of a peptide of the invention, including:

(SEQ ID NO:26)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VLTTGLPALIS-COOH, (SEQ ID NO:27)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VVTTAKPLISS-COOH, (SEQ ID NO:28)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 ILTTGLPALIS-COOH, (SEQ ID NO:29)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GGLLSNIVTSL-COOH, (SEQ ID NO:30)NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GPILANLVSIV-COOH, (SEQ ID NO:31)NH.sub.2 -KKWWRRR.sub.1 R.sub.1 R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2GPALSNV-COOH,or (SEQ ID NO:35)NH.sub.2 -KKWWRRR.sub.1 R.sub.1 R.sub.2 GLKTAGPAIQSVLNK-COOH wherein R.sub.1 is a hydrophobic amino acid residue and R.sub.2 is a hydrophilic amino acid residue. More specifically, the invention provides a method of inhibiting the growth of bacteria comprising contacting the bacteria with an inhibiting effective amount of a peptide of the invention, such as MBI 26 or MBI 29, (SEQ ID NO:1)NH.sub.2 -KWKSFIKKLTTAVKKVLTTGLPALIS-COOH, (SEQ ID NO:2)NH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH,or (SEQ ID NO:3)NH.sub.2 -KWKSFIKNLTKGGSKILTTGLPALIS-COOH, (SEQ ID NO:4)NH.sub.2 -KWKKFIKNLTKGGSKILTTGLPALIS-COOH, (SEQ ID NO:5)NH.sub.2 -KWKSFIKNLEKVLKPGGLLSNIVTSL-COOH(MBI 490), (SEQ ID NO:6)NH.sub.2 -KWKSFIKNLEKVLKKGPILANLVSIV-COOH(MBI 491), (SEQ ID NO:7)NH.sub.2 -KWKEFIKKLTTAVKKVLTTGLPALIS-COOH(MBI 492), (SEQ ID NO:8)NH.sub.2 -KWKKFIKELQKVLAPGGLLSNIVTSL-COOH(MBI 493), (SEQ ID NO:9)NH.sub.2 -KWKSFIKKLTSVLKKVVTTALPALIS-COOH(MBI 29A1), (SEQ ID NO:10)NH.sub.2 -KWKSFIKNLTKVLKKVVTTALPALIS-COOH(MBI 29A2), (SEQ ID NO:11)NH.sub.2 -KWKLFKKKGTGAVLTVLTTGLPALIS-COOH(MBI 29A3), (SEQ ID NO:12)NH.sub.2 -KWKSFIKKLTSVLKKVVTTAKPLISS-COOH, (SEQ ID NO:13)NH.sub.2 -KKKSFIKLLTSAKVSVLTTAKPLISS-COOH, (SEQ ID NO:14)NH.sub.2 -KWKKFIKELQKVLKPGGLLSNIVTSL-COOH, (SEQ ID NO:15)NH.sub.2 -KKWWRRVLSGLKTGPALSNV-COOH, (SEQ ID NO:16)NH.sub.2 -KKWWRRVLKGLSSGPALSNV-COOH, (SEQ ID NO:17)NH.sub.2 -KKWWRRALQALKNGPALSNV-COOH, (SEQ ID NO:18)NH.sub.2 -KKWWRRVLSGLKTAGPAIQSVLNK-COOH(MBI 21A1), (SEQ ID NO:19)NH.sub.2 -KKWWRRALQGLKTAGPAIQSVLNK-COOH(MBI 21A2), (SEQ ID NO:20)NH.sub.2 -KKWWKAQKAVNSGPNALQTLAQ-COOH (SEQ ID NO:21)NH.sub.2 -KKWWKAKKFANSGPNALQTLAQ-COOH, (SEQ ID NO:22)NH.sub.2 -KKWWKFIKKAVNSGTTGLQTLAS-COOH, (SEQ ID NO:23)NH.sub.2 -KKSFFKKLTSVASSVLS-COOH(MBI 21A14), (SEQ ID NO:24)NH.sub.2 -WKVFKSFIKKASSFAQSVLD-COOH,and (SEQ ID NO:25)NH.sub.2 -KKWRKSFFKQVGSFDNSV-COOH. and analogs, derivatives, or conservative variations thereof. The term "contacting" refers to exposing the bacteria to the peptide so that the peptide can effectively inhibit, kill, or lyse bacteria, bind endotoxin (LPS), or permeabilize gram-negative bacterial outer membranes. Contacting may be in vitro, for example by adding the peptide to a bacterial culture to test for susceptibility of the bacteria to the peptide. Contacting may be in vivo, for example administering the peptide to a subject with a bacterial disorder, such as septic shock. "Inhibiting" or "inhibiting effective amount" refers to the amount of peptide which is required to cause a bacteriostatic or bactericidal effect. Examples of bacteria which may be inhibited include E. coli, P. aeruginosa, E. cloacae, S. typhimurium, and S. aureus.

The method of inhibiting the growth of bacteria may further include the addition of antibiotics for combination or synergistic therapy. The appropriate antibiotic administered will typically depend on the susceptibility of the bacteria such as whether the bacteria is gram negative or gram positive, and will be easily discernable by one of skill in the art. Examples of particular classes of antibiotics useful for synergistic therapy with the peptides of the invention include aminoglycosides (e.g., tobramycin), penicillins (e.g., piperacillin), cephalosporins (e.g., ceftazidime), fluoroquinolones (e.g., ciprofloxacin), carbepenems (e.g., imipenem), tetracyclines and macrolides (e.g., erythromycin and clarithromycin). The peptides and/or analogues or derivatives thereof may be administered to any host, including a human or non-human animal, in an amount effective to inhibit not only growth of a bacterium, but also a virus or fungus. These peptides are useful as antimicrobial agents, antiviral agents, and antifungal agents.

The peptide of the invention can be administered parenterally by injection or by gradual infusion over time. The peptide can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. Preferred methods for delivery of the peptide include orally, by encapsulation in microspheres or proteinoids, by aerosol delivery to the lungs, or transdermally by iontophoresis or transdermal electroporation. Other methods of administration will be known to those skilled in the art.

Preparations for parenteral administration of a peptide of the invention include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

The invention provides a method of treating or ameliorating an endotoxemia or septic shock (sepsis) associated disorder, or one or more of the symptoms of sepsis comprising administering to a subject displaying symptoms of sepsis or at risk for developing sepsis, a therapeutically effective amount of a cationic peptide of the invention, for example, SEQ ID NO:1 or SEQ ID NO:2, or analogs, derivatives, or conservative variations thereof. The term "ameliorate" refers to a decrease or lessening of the symptoms of the disorder being treated. Such symptoms which may be ameliorated include those associated with a transient increase in the blood level of TNF, such as fever, hypotension, neutropenia, leukopenia, thrombocytopenia, disseminated intravascular coagulation, adult respiratory distress syndrome, shock and multiple organ failure. Patients who require such treatment include those at risk for or those suffering from toxemia, such as endotoxemia resulting from a gram-negative bacterial infection, venom poisoning, or hepatic failure, for example. In addition, patients having a gram-positive bacterial, viral or fungal infection may display symptoms of sepsis and may benefit from such a therapeutic method as described herein. Those patients who are more particularly able to benefit from the method of the invention are those suffering from infection by E. coli, Haemophilus influenza B, Neisseria meningitides, staphylococci, or pneumococci. Patients at risk for sepsis include those suffering from gunshot wounds, renal or hepatic failure, trauma, burns, immunocompromised (HIV), hematopoietic neoplasias, multiple myeloma, Castleman's disease or cardiac myxoma.

The term "therapeutically effective amount" as used herein for treatment of endotoxemia refers to the amount of cationic peptide used is of sufficient quantity to decrease the subject's response to LPS and decrease the symptoms of sepsis. The term "therapeutically effective" therefore includes that the amount of cationic peptide sufficient to prevent, and preferably reduce by at least 50%, and more preferably sufficient to reduce by 90%, a clinically significant increase in the plasma level of TNF. The dosage ranges for the administration of cationic peptide are those large enough to produce the desired effect. Generally, the dosage will vary with the age, condition, sex, and extent of the infection with bacteria or other agent as described above, in the patient and can be determined by one skilled in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. In any event, the effectiveness of treatment can be determined by monitoring the level of LPS and TNF in a patient. A decrease in serum LPS and TNF levels should correlate with recovery of the patient.

In addition, patients at risk for or exhibiting the symptoms of sepsis can be treated by the method as described above, further comprising administering, substantially simultaneously with the therapeutic administration of cationic peptide, an inhibitor of TNF, an antibiotic, or both. For example, intervention in the role of TNF in sepsis, either directly or indirectly, such as by use of an anti-TNF antibody and/or a TNF antagonist, can prevent or ameliorate the symptoms of sepsis. Particularly preferred is the use of an anti-TNF antibody as an active ingredient, such as a monoclonal antibody with TNF specificity as described by Tracey, et al. (Nature, 330:662, 1987).

A patient who exhibits the symptoms of sepsis may be treated with an antibiotic in addition to the treatment with cationic peptide. Typical antibiotics include an aminoglycoside, such as gentamicin or a beta-lactam such as penicillin, or cephalosporin. Therefore, a preferred therapeutic method of the invention includes administering a therapeutically effective amount of cationic peptide substantially simultaneously with administration of a bactericidal amount of an antibiotic. Preferably, administration of cationic peptide occurs within about 48 hours and preferably within about 2-8 hours, and most preferably, substantially concurrently with administration of the antibiotic.

The term "bactericidal amount" as used herein refers to an amount sufficient to achieve a bacteria-killing blood concentration in the patient receiving the treatment. The bactericidal amount of antibiotic generally recognized as safe for administration to a human is well known in the art, and as is known in the art, varies with the specific antibiotic and the type of bacterial infection being treated.

Because of the antibiotic, antimicrobial, and antiviral properties of the peptides, they may also be used as preservatives or sterillants of materials susceptible to microbial or viral contamination. The peptides of the invention can be utilized as broad spectrum antimicrobial agents directed toward various specific applications. Such applications include use of the peptides as preservatives in processed foods (organisms including Salmonella, Yersinia, Shigella), either alone or in combination with antibacterial food additives such as lysozymes; as a topical agent (Pseudomonas, Streptococcus) and to kill odor producing microbes (Micrococci). The relative effectiveness of the cationic peptides of the invention for the applications described can be readily determined by one of skill in the art by determining the sensitivity of any organism to one of the peptides.

The following examples are intended to illustrate but not limit the invention. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used.

EXAMPLE 1

MIC VALUES FOR CATIONIC PEPTIDES

The minimum inhibitory concentrations of (MIC) of CEME, CEMA, the first 8 amino acid residues of

CEME (NH.sub.2 -KWKLFKKIGIGAVLKVLTTGLPALIS-COOH; SEQ ID NO:1 with changes at residues 4, 6, 8-11, 14) and CEMA (NH.sub.2 -KWKLFKKIGIGAVLKVLTTGLPALKLTK-COOH; SEQ NO:1 with changes at residues 4, 6, 8-11, 14, and 25-28), (Piers, K. and Hancock, R, Molec. Microbiology, 12(6), 1994) 20 carboxy terminal amino acids of melittin (MA), 8 amino terminal amino acids from cecropin, and the peptides shown in SEQ ID NO:1, MBI 29 and SEQ ID NO:2, MBI 26, were determined for a number of different bacteria (Table 1a and 1b). Briefly, cells were grown overnight at 37.degree. C. in LB-S (Luria broth without any salt supplement) and diluted one in 10,000 in the same medium to give concentrations of about 10.sup.4 to 10.sup.5 CFU/ml. The broth dilutions were set up in a 96 well microtiter plate by putting 200 .mu.l of LB-S containing the initial concentration of antibiotic or compound in column 1 and 100 .mu.l of the same medium in columns 2-12. The compounds were diluted by taking 100 .mu.l of broth from column 1 and mixing it with column 2, resulting in a one in two dilution. This was continued to column 10. Finally, 10 .mu.l of bacteria were pipetted into columns 1-11, and the plates incubated overnight at 37.degree. C. The next day the plates were scored for growth in the wells, and the MIC determined. TABLE 1a__________________________________________________________________________MINIMUM INHIBITORY CONCENTRATION(MIC) VALUES FOR CATIONIC PEPTIDES__________________________________________________________________________STRAIN CEME CEMA MBI 29 MBI 26 CE-8 MA-20__________________________________________________________________________S. aureus 32 .gtoreq.64 20 .gtoreq.64 .gtoreq.64 .gtoreq.64(K147)S. aureus 24 .gtoreq.64 18 .gtoreq.64 .gtoreq.64 .gtoreq.64(SAP0017)S. epidermis 16 .gtoreq.64 10 .gtoreq.64 .gtoreq.64 .gtoreq.64E. coli 5 10 3 3 .gtoreq.64 .gtoreq.64(UB1005)P. aeruginosa 8 26 5 11 .gtoreq.64 .gtoreq.64(H187)Candida albicans .gtoreq.64 .gtoreq.64 40 .gtoreq.64 .gtoreq.64 .gtoreq.64(CAN105)__________________________________________________________________________Additional MICs were determined for MBI-26 and MBI-29 and MBI 29and MBI21 derivatives as noted for the following strains:Strain MBI 26 MBI 29__________________________________________________________________________P. aeruginosa 4 4(H103)E. coli. Bort 2 6E. coli 0111:B4 2 2X maltophilia 2 3A. calcoaceticus 2 4E. cloacae 2 2K. pneumoniae 8 16__________________________________________________________________________ E. coli 0111:B4 S. epiPeptide .mu.g/ml E. faecalis B. Subtilis H103 0017 SAP K147 C610__________________________________________________________________________MBI29A1 4 64 16 16 16 16 16 8MBI29A2 8 64 8 16 16 8 16 8MBI29A3 8 >128 16 16 16 32 32 8MBI21A1 16 >128 >128 64 128 >28 >128 16MBI21A2 32 >128 >28 128 >128 >128 >128 16MBI21A14 >128 >128 >128 >128 >128 >128 >128 128__________________________________________________________________________ In a separate set of experiments, the following MIC values were obtained: TABLE lb__________________________________________________________________________MINIMUM INHIBITORY CONCENTRATION(MIC) VALUES FOR CATIONIC PEPTIDESSTRAIN CEMA CEMA.sup.me MBI 29 MBI 29.sup.me MBI 26D MBI 26L__________________________________________________________________________S. aureus .gtoreq.64 .gtoreq.64 32 16 .gtoreq.64 .gtoreq.64(K147)S. aureus .gtoreq.64 .gtoreq.64 32 16 .gtoreq.64 .gtoreq.64(SAP0017)S. epidermis 43 43 16 8 8-16 32P. aeruginosa 32 53 16 8 16 32(H187)E. coli 13 32 4 4 4-8 4(UB1005)E. faecalis .gtoreq.64 .gtoreq.64 32 .gtoreq.64 .gtoreq.64 .gtoreq.64Candida albicans .gtoreq.64 .gtoreq.64 32 32 .gtoreq.64 .gtoreq.64(CAN105)__________________________________________________________________________ The results show that both MBI 29 (SEQ ID NO:1) and MBI 26 (SEQ ID NO:2) are effective antimicrobial agents for a variety of gram positive and gram negative bacteria. In addition, all peptides were effective against Candida albicans. Modification of the peptides, such as methyl ester modification, or L to D amino acids provided a broader class of antimicrobial agents.

Agarose Dilution Susceptibility Testing

The following is a novel agarose-based solid phase assay system to measure antimicrobial activity of the cationic peptides of the invention, for example, either alone or in combination with a conventional antibiotic. Standard MIC methods, as described above or this method can be used to determine MICs of the peptides described herein.

A) Preparing plates

1) Unsupplemented Mueller-Hinton agarose (i.e. no Ca.sup.2+ or Mg.sup.2+ added) was used. (Unsupplemented broth is used with agar because agar contains divalent cations).

Prepared as follows: 22 g/L Mueller-Hinton powder (BBI, #11443) 15 g/L agarose (Sigma, A-0576) QS to 1 L with sdH.sub.2 O Heat to boiling and boil 1 min Adjust pH to 7.3 with NaOH or KOH (adjust the meter to the correct temperature--note agarose solidifies at <40.degree. C.) Autoclave for 10 min at 121.degree. C. After the agarose has been autoclaved, it is allowed to cool to 40-45.degree. C. in a water bath before aseptically adding antimicrobial solutions, and pouring the plates.

2) Appropriate dilutions of antimicrobial solutions are added to molten test agarose that has been allowed to equilibrate in a water bath to 40-45.degree. C. In general, a scheme in which 1 part of antimicrobial solution is added to 9 parts of liquid agarose is used (i.e. 2 mL of drug to 18 mL of agar for each plate; 1 mL of each drug in the combination if testing synergy).

3) The agarose and antimicrobial solution are mixed throughly by gentle inversion about 10.times. to avoid bubbles and the mixture poured into petri-dishes on a level surface to result in an agar depth of 3-4 mm.

4) The plates are poured as quickly as possible after mixing to prevent cooling and partial solidification.

5) The agarose is allowed to solidify at room temperature and the plates are either used immediately or stored in sealed plastic bags at 4.degree. C. Storage time can vary but plates should be used within a week.

6) Plates stored at 4.degree. C. should be allowed to equilibrate at room temperature before use. The surface should be dry before inoculating the plates. If necessary, plates can be placed in the fume hood for 30 min with lids ajar to hasten drying of agarose surface.

B) Inoculum Preparation

1) To standardize the inoculum density, a 0.5 McFarland standard can be prepared as follows:

A 0.5 mL aliquot of 0.48 m BaCl.sub.2 is added to 99.5 mL of 0.36 N H.sub.2 SO.sub.4 (1% v/v) with constant stirring to maintain a suspension. The correct density should be verified using a spectrophotometer with a 1 cm light path and matched cuvettes to determine the absorbance. The absorbance at 630 nm should be 0.08 to 0.10. The suspension should be transferred to 10 mL aliquot into screwcap tubes of the same size as those used in growing or diluting the bacterial inoculum. These tubes should be tightly sealed and stored in the dark at room temperature. The turbidity standard should be rigorously agitated on a vortex mixer prior to use. If large particles appear, the standard should be replaced. Replacement should occur every month (use a new standard tubes each time, and when ten are used up, make a new batch).

2) 3 to 5 morphologically identical colonies are selected from a TSA plate (the top of each is touched with a sterile loop) and transferred to a tube of 5 mL TSB.

3) The broth culture is incubated at 37.degree. C. with shaking until it achieved or exceeds the turbidity of the standard (2 to 6 hours)--approx. 1 to 2.times.10.sup.8 CFU/mL.

4) The turbidity can be adjusted with broth to obtain a turbidity equal to the 0.5 standard. Use adequate lighting and hold up a white card with black lines behind the cultures to assist in the comparison.

5) Cultures adjusted to the standard are diluted 1/10 in sterile broth to obtain a density of approximately 10.sup.7 CFU/mL. Most inoculum replicators deposit approximately 1 to 2 .mu.L on the agarose surface. The final inoculum on the agarose will then be approximately 10.sup.4 CFU in a spot of 5 to 8 mm in diameter on the agarose. The adjusted suspensions should be used for final inoculation within 15 min of preparation.

The tubes containing the adjusted and diluted bacterial suspensions should be arranged in order on a rack. An aliquot of each well-mixed suspension is placed into the corresponding well in the replicator inoculum block. The agarose plates are marked for orientation of the inoculum spots. A growth control plate (no antimicrobial) is inoculated first and then, starting with the lowest concentration, the plates continuing the different antimicrobial concentration are inoculated (Optional--A second growth control plate is inoculated last to ensure that there was no contamination or significant antimicrobial carryover during the inoculation).

Optional--a sample of each inoculum is streaked on a suitable agar plate and incubated

6) The inoculated plates are allowed to stand at room temperature until the moisture in the inoculum spots have been absorbed into the agarose. The plates are inverted and incubated at 35.degree. C. for 24 hours, 48 hours.

7) The plates should be placed on a dark, nonreflecting surface to determine the end points. The MIC is recorded as the lowest concentration of antimicrobial agent that completely inhibits growth, disregarding a single-colony or a faint haze caused by the inoculum.

8) If two or more colonies persist in concentrations of the agent beyond an obvious end point, or if there is no growth at lower concentrations, the culture purity is checked and test possibly repeated.

9) After 48 hours a replica plating tool can be used to transfer cells onto a fresh TSA or BHI plate and growth is read after 24 hrs.

EXAMPLE 2

EFFECT OF FORMULATION ON BACTERICIDAL ACTIVITY

Effect of formulation on bactericidal activity and MIC was determined using the test solutions listed below. The results show that citrate and related formulations reduced the MIC (Table 2). Similar effects were seen with acetate formulated peptide (Tables 2 and 3).

TABLE 2______________________________________EFFECT OF CITRATE, ASCORBIC ACID AND DEXTROSE ONMBI 26 (10 MG/ML) KILLING OF P. AERUGINOSA Survivors at Time (min.)Test Solution Peptide 0 15 30 60______________________________________Citrate Control 1.1 .times. 10.sup.7 1.1 .times. 10.sup.7 1.2 .times. 10.sup.7 1.4 .times. 10.sup.7(0.129M) + MBI 26 1.1 .times. 10.sup.7 0 0 0Citrate Control 1.3 .times. 10.sup.7 1.2 .times. 10.sup.7 1.7 .times. 10.sup.7 1.7 .times. 10.sup.7(0.129M) + MBI 26 1.3 .times. 10.sup.7 0 0 0+ Ascorbicacid(100 mM)Citrate- Control 1.2 .times. 10.sup.7 5.8 .times. 10.sup.6 6.9 .times. 10.sup.6Dextrose + MBI 26 9 .times. 10.sup.6 20 0 0Citrate- Control 7.8 .times. 10.sup.6 6.5 .times. 10.sup.7 1.5 .times. 10.sup.7 1.1 .times. 10.sup.7PhosphateDextrose + MBI 26 0 0 0Dextran Control 2.3 .times. 10.sup.7 2.2 .times. 10.sup.7 1.4 .times. 10.sup.7 2 .times. 10.sup.7 + MBI 26 1.9 .times. 10.sup.7 1.2 .times. 10.sup.6 1 .times. 10.sup.6 8.5 .times. 10.sup.5 (20 .mu.g)______________________________________ TABLE 3______________________________________EFFECT OF CITRATE ADDITION TO CATION ADJUSTEDMEDIA ON MINIMAL INHIBITORY CONCENTRATIONS MIC (.mu.g/mL)Strain CAMHB CAMHB + Citrate______________________________________E. coli (UB 1005) 4 2E. coli (111: B4) 8 2E. coli (Bort) 16 8S. typhimurium (C610) 8 2E. facaelis (C625) 16 >32A. calcoacet. (C402) 8 4A. calcoacet (C403) 1 1E. cloacae (C601) 8 4E. cloacae (C602) 8 4E. cloacae (C603) 4 2P. aeruginosa (H103) 16 2______________________________________ The media used in this experiment was Cation Adjusted Muller Hinton (CAMHB), and CAMHB + 8 mM citrate. MBI 26 was prepared in sterile distilled water.

EXAMPLE 3

INNER MEMBRANE PERMEABILIZATION

Inner membrane permeability was determined by measurement in E. coli ML 35 of .beta.-galactosidase activity using ONPG as a substrate. Normally ONPG cannot pass across the cytoplasmic membrane to permit hydrolysis by the cytoplasmic enzyme .beta.-galactosidase. However, cationic peptides, by permeabilizing the inner membrane, unmasks this enzyme leading to color development as ONP is released. MBI 26 and MBI 29 were tested with the same conditions as shown in FIG. 3 and 4 (4 ug MBI 26 or MBI 29 alone, .rect-solid.; +100mM NaCl, .quadrature.; +100 uM CCCP, .rect-solid.; +5 mM Mg, .rect-solid..sup..box-solid..box-solid. .rect-solid.). While 100 mM NaCl had little effect on MBI 26 permeabilization, 5 mM Mg++ had a large inhibitory effect. MBI 29 permeabilization of the inner membrane was the least affected by any of the conditions especially Mg.sup.++ as seen in FIG. 4. Addition of citrate increased the lag time before the peptides were able to permeabilize the membranes. When the experiments were executed in cation adjusted media (CAMHB), the lag time was increased up to 30 minutes. The data in Table 4 shows the rate and lag time for inner membrane permeabilization of MBI 26, MBI 29 and peptides 490, 491, 492 and 493 (SEQ ID NO:2, 1, and 5-8, respectively).

TABLE 4______________________________________INNER MEMBRANE PERMEABILIZIATION Rate of HydrolysisPeptide Lag Time (Sec.) (.DELTA.AU min.sup.-1)______________________________________MBI 26 (SEQ ID NO:2) 210 0.081MBI 29 (SEQ ID NO:1) 66 0.21490 (SEQ ID NO:5) 156 0.081491 (SEQ ID NO:6) 152 0.14492 (SEQ ID NO:7) 68 0.23493 (SEQ ID NO:8) 136 0.14______________________________________

EXAMPLE 4

INHIBITION OF LPS-MEDIATED TNF INDUCTION

IN MACROPHAGES BY CATIONIC PEPTIDES

The effect of cationic peptides,

CEME (KWKLFKKIGIGAVLKVLTTGLPALIS (SEQ ID NO:36)) and CEMA (KWKLFKKIGIGAVLKVLTTGLPALKLTK (SEQ ID NO:37)) and the peptides of the invention, MBI 29, NH.sub.2 -KWKSFIKKLTTAVKKVLTTGLPALIS-COOH (SEQ ID NO:1) and MBI 26, NH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH (SEQ ID NO:2), on LPS-induced TNF in macrophages was examined. RAW 264.7 macrophage cells were grown by seeding 10.sup.6 cells into a 162 cm.sup.2 cell culture flask and incubated at 37.degree. C., 5% CO.sub.2 for 1 week. RAW cell media [(Dulbecco's Modified Eagle Medium with Hepes buffer 450 ml; 2.4 mM L-glutamine 3 ml (400 mM); Pen/Strep 3 ml (10.sup.4 U/ml of Pen, 1 mg/ml strep); and 10% heat inactivated fetal bovine serum (FBS) 50 ml)] was then completely removed from flasks. 10 mls of cell dissociation solution (Sigma) was added to each flask and incubated at 37.degree. C. for 10 minutes. Cells were removed from flasks, diluted in RAW cell media and centrifuged for 6 minutes. The cell pellet was resuspended in 5 ml of media/flask used. 100 .mu.l cell suspension was removed and added to 400 .mu.l of trypan blue and cells were counted using a hemocytometer. The cell suspension was diluted to 1.times.10.sup.6 cells/ml and 1 ml of suspension was added per well of a 24 well plate. The 24 well plates were incubated at 37.degree. C., 5% CO.sub.2 overnight for use in the assay.

After an overnight incubation, the media was aspirated from all the wells. 100 .mu.l of Lipopolysaccharide (LPS) was added at 100 ng/100 .mu.l. CEME, CEMA or MBI 29 was added at the desired concentration/100 .mu.l to specified wells. RAW cell media was added to all the wells so they all had a final volume of 1 ml. The plates were then incubated for six hours at 37.degree. C., 5% CO.sub.2. The supernatant was then removed from the wells and stored overnight at 4.degree. C. For those wells in which whole bacteria were added directly to the wells, the supernatant was centrifuged in 0.2 .mu.m filter eppendorf tubes for 5 minutes.

The supernatants were then used in cell cytotoxic L929 assay. The samples were transferred to 96 well plates. 50 .mu.l of TNF media was added to all the wells in all the plates except to those wells in the first row. 10 .mu.l of murine TNF standard (20 ng/ml) and 90 .mu.l of TNF media was added in duplicate to the plate and diluted 1:2 down the plate to the second to last row. Test samples (75 .mu.l), comprising the supernatants from the RAW cell assays, were added to separate rows in duplicate and diluted 1:3 to the second to last rows.

TNF-sensitive L929 mouse fibroblast cells were grown by seeding 10.sup.6 cells into a 162 cm.sup.2 cell culture flask and left to grow for 1 week. L929 cells were removed from the flask with 10 mls of trypsin-EDTA/flask and incubated 3-5 minutes. Cell suspension was diluted and centrifuged for 6 minutes. The pellet was resuspended in 5 mls of fresh L929 media/flask and counted (same as RAW cells). Cell suspension was diluted to 10.sup.6 cells/ml. 100 .mu.l was used to inoculate each well of the 96 well plates with the supernatants. (L929 Growth Media was the same as RAW cell media except instead of FBS, 50 mls of 10% heat inactivated horse serum was utilized; TNF Assay Media was the same as RAW cell media except 4 .mu.g/ml Actinomycin D was added.)

The plates were incubated at 37.degree. C. at 5% CO.sub.2 for 2 days. The media was then aspirated and replaced with 100 .mu.l of the dye MTT (0.5 mg/ml) in modified Eagle Medium without phenol red. The plates were then incubated at 37.degree. C. at 5% CO.sub.2 for 3 hours. The dye was then removed and replaced with 100 .mu.l of absolute ethanol. The plates were left at room temperature for 10-15 minutes to dissolve the formazan dye crystals.

The plates were read at 570 nm in a ELISA plate reader with 690 nm reference filter. One unit of TNF activity is defined as the amount required to kill 50% of the L929 cells. The TNF level in Units per ml therefore was the reciprocal of the dilution which led to a 50% killing of L929 cells.

FIGS. 5 and 6 show levels of TNF (U/ml) after a 6 hour treatment with increasing amounts (0, 5, 10, 20, 30, 40 or 50 .mu.g) of either CEME (ME), CEMA (MA), MBI 26 (SEQ ID NO:2) or MBI 29 (SEQ ID NO:1) peptide and 100 ng of LPS. TNF levels were measured six hours after the addition of E. coli 0111:B4 or E. coli Bort LPS (FIG. 5a, 5b, 5c and FIG. 6a and 6c as labeled). P. aeruginosa LPS was also added in FIG. 6b. The data shows the results of several separate experiments and indicate that both peptides efficiently reduce the level of LPS-induced TNF in the culture with three distinct LPS samples at concentrations of peptides as low as 5 .mu.g/ml.

In addition, the peptides of SEQ ID NO: 5-8 also bound effectively to LPS with high affinity based on Dansyl polymyxin displacement assays (Table 5).

TABLE 5______________________________________Peptide I.sub.50 (mM) I.sub.max (%)______________________________________MBI 26 (SEQ ID NO:2) 1.8 38MBI 29 (SEQ ID NO:1) 2.8 40490 (SEQ ID NO:5) 4.9 40491 (SEQ ID NO:6) 4.5 24492 (SEQ ID NO:7) 5.4 48493 (SEQ ID NO:8) 3.8 56______________________________________

EXAMPLE 5

CATIONIC PEPTIDE REDUCTION OF LPS-INDUCED TNF

In order to determine how rapidly the cationic peptides reduced LPS-induced TNF production, E. coli 0111:B4 was added at time 0 to RAW macrophages. MBI 29 or Polymyxin B was added at time 0, 30 and 60 minutes. Levels of TNF were measured after 6 hours. The results are shown in FIG. 7a and 7b(PMB, Polymyxin B; 29, MBI 29 (SEQ ID NO:1)). The results show that MBI 29 inhibited TNF induction by LPS in a similar manner to polymyxin B. Furthermore, MBI 29 was effective at reducing the ability of LPS to induce TNF in RAW cell lines even when added 60 minutes after the addition of LPS. MBI 29 demonstrated a distinct and reproducible advantage over polymyxin B when added 60 minutes after the addition of LPS. To confirm that MBI 29 was acting on LPS rather than directly upon macrophage cell lines, 20 .mu.g of MBI 29 was added to RAW cells and incubated for 60 minutes prior to aspiration of the medium and washing the cells 3 times with HBSS (Hanks Buffered Salt Solution). Addition of 10 ng or 100 ng of LPS to the washed RAW cells resulted in a high level of TNF induction (14,000-20,000 Units of TNF per ml), suggesting that the MBI 29 had not permanently depressed the ability of RAW cells to induce TNF in response to LPS addition. In contrast, the aspirated medium containing MBI 29 could depress the ability of fresh RAW cells to induce TNF in response to 10 ng or 100 ng of E. coli LPS by 98.5% and 75% respectively. Up to 50 .mu.g of MBI 29 caused no apparent decrease in RAW cell viability as judged by Trypan blue exclusion.

EXAMPLE 6

PROTECTION FROM LETHAL LPS ENDOTOXICITY IN A MOUSE ENDOTOXIC SHOCK MODEL

The ability of MBI 29 and MBI 26 to protect against LPS-induced endotoxemia is assessed in vivo. Mice (8-10 weeks old) are injected intraperitoneally with 20 .mu.g D-galactosamine (Dgal) to sensitize them to LPS according to the method of Galanos (Galanos, et al., Proc. Natl. Acad Sci., USA, 76:5939-5943, 1979), followed by 0, 50, 100, or 200 .mu.g MBI 29 or MBI 26 in 100 .mu.l. Immediately afterwards LPS (10 or 20 .mu.g) in 100 .mu.l is injected. The mice are observed at 24 and 48 hours after injections and survivors noted. MBI 26 was shown to be non-toxic up to 30.6 mg/kg in mice.

To demonstrate that survival is associated with a reduction in TNF levels, 10 .mu.g of LPS and 20 mg of Dgal are injected at time 0. Thirty minutes later, the mice are sacrificed and the blood is taken and centrifuged to separate the serum which was used in the cell cytotoxic L929 assay. The results suggest that the bactolysins have potential in therapy against endotoxin-associated disorders.

EXAMPLE 7

CHARACTERIZATION OF DERIVATIVE PEPTIDES

Peptides 490-493 (SEQ ID NO:5-8) were found to possess comparable anti microbial activity to MBI 26 and MBI 29 (Table 6). These peptides do not lyse Red Blood Cells at concentration below 55 .mu.g/mL.

TABLE 6______________________________________MIC (.mu.g/mL) RBC LysisPeptide E. coli (UB 1005) P. aeruginosa (H187) (.mu.g/mL)______________________________________MBI 26 1.1 4.5 >55MBI 29 0.5 1.0 >55490 1 8 >55491 2 8 >55492 2.4 4.8 >55493 5 10 >55______________________________________ .alpha. Helical Content of Cationic Peptides by CD Spectra The .alpha. helical content of the peptides were measured in buffer, liposomes and 20% Hexafluoroisopropanol (HFPIP). The liposomes constituted of POPC alone or POPC and POPC and 30% POPG (Table 7) TABLE 7______________________________________% .alpha. helicity POPC POPC/POPGPeptide Buffer Liposomes Liposomes 20% HFIP______________________________________MBI 26 0 0 8 70MBI 29 0 16 8 50490 0 29 27 41491 0 0 15 44492 0 42 19 48493 0 18 20 42______________________________________

EXAMPLE 8

SYNERGISM BETWEEN CATIONIC PEPTIDES AND ANTIBIOTICS

Synergy between the peptides of the invention and conventional antibiotics from several different classes was determined by MIC and FIC values. The following Tables 8-47 show the MIC and FIC values for MBI 26 and MBI 29 with different bacteria (A. calcoaceticus, P. aeruginosa, E. cloacae and X. maltophila) and different classes of antibiotics. Synergism is identified as an FIC of about .ltoreq.0.5.

TABLE 8______________________________________Synergy of MBI-26 + Ciprofloxacin against A. calcoaceticusIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Ciprofloxacin and Ciprofloxacin______________________________________1 ATCC 23055 128 0.5 0.512 23 32 0.5 0.753 27 64 0.5 0.754 28 16 8 0.535 8220 64 2 0.56 8221 64 1 0.57 8222 64 1 0.58 8223 64 2 0.389 8224 64 2 0.510 8225 64 2 0.3811 8226 64 2 0.38______________________________________ TABLE 9______________________________________Synergy of MBI-29 + Ciprofloxacin against A. calcoaceticusIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Ciprofloxacin and Ciprofloxacin______________________________________1 ATCC 23055 32 0.5 0.512 23 32 0.5 0.753 27 32 8 0.634 28 32 8 0.635 8220 16 2 0.566 8221 16 0.5 17 8222 16 2 0.758 8223 16 2 0.759 8224 16 1 110 8225 16 2 0.7511 8226 16 2 0.75______________________________________ TABLE 10______________________________________Synergy of MBI-26 + Imipenem against A. calcoaceticusIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Imipenem and Imipenem______________________________________1 ATCC 23055 128 2 1.02 23 64 512 0.133 27 32 512 0.144 28 32 16 0.255 8220 32 2 0.536 8221 64 1 1.017 8222 64 4 0.318 8223 32 2 0.759 8224 64 2 0.6310 8225 64 2 0.6311 8226 64 4 0.5______________________________________ TABLE 11______________________________________Synergy of MBI-29 + Imipenem against A. calcoaceticusIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Imipenem and Imipenem______________________________________1 ATCC 23055 64 2 12 23 32 512 0.133 27 8 512 0.54 28 32 8 0.55 8220 8 2 16 8221 8 1 1.037 8222 32 2 0.758 8223 16 2 19 8224 32 2 0.5110 8225 32 2 0.5111 8226 32 2 1______________________________________ TABLE 12______________________________________Synergy of MBI-29 + Tobramycin against A. calcoaceticusIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Tobramycin and Tobramycin______________________________________1 ATCC 23055 64 4 0.312 23 32 16 ND3 27 32 1 1.014 28 32 128 0.265 8220 64 4 0.56 8221 64 4 0.57 8222 64 4 0.58 8223 64 4 0.59 8224 64 4 0.510 8225 64 4 0.511 8226 64 4 0.5______________________________________ TABLE 13______________________________________Synergy of MBI-29 + Tobramycin against A. calcoaceticusIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Tobramycin and Tobramycin______________________________________1 ATCC 23055 32 2 0.752 23 4 4 1.253 27 8 1 1.034 28 32 128 0.265 8220 32 4 0.56 8221 32 4 0.57 8222 32 4 0.58 8223 32 4 0.59 8224 32 4 0.510 8225 32 4 0.511 8226 32 4 0.5______________________________________ TABLE 14______________________________________Synergy of MBI-29 + Piperacillin against A. calcoaceticusIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Piperacillin and Piperacillin______________________________________1 ATCC 23055 64 8 0.752 23 32 1024 0.263 27 32 16 0.384 28 16 4 1.035 8220 64 16 0.386 8221 64 16 0.387 8222 64 32 0.258 8223 64 128 0.139 8224 64 256 0.1610 8225 64 32 0.2511 8226 64 32 0.25______________________________________ TABLE 15______________________________________Synergy of MBI-29 + Piperacillin against A. calcoaceticusIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Piperacillin and Piperacillin______________________________________1 ATCC 23055 32 8 12 23 16 1024 0.283 27 16 16 0.54 28 8 4 1.035 8220 32 16 0.56 8221 32 16 0.387 8222 32 32 0.258 8223 32 32 0.259 8224 32 128 0.1910 8225 32 32 0.2511 8226 32 32 0.25______________________________________ TABLE 16______________________________________Synergy of MBI-26 + Ceftazidime against A. calcoaceticusIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Ceftazidime and Ceftazidime______________________________________1 ATCC 23055 128 2 1.012 23 64 128 0.253 27 32 32 0.384 28 32 16 0.385 8220 32 16 0.386 8221 64 8 0.257 8222 64 16 0.58 8223 32 16 0.259 8224 64 64 0.2510 8225 64 32 0.3111 8226 64 64 0.38______________________________________ TABLE 17______________________________________Synergy of MBI-29 + Ceftazidime against A. calcoaceticusIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Ceftazidime and Ceftazidime______________________________________1 ATCC 23055 64 4 0.632 23 32 128 0.263 27 8 4 1.54 28 32 16 0.635 8220 8 8 1.036 8221 16 8 17 8222 32 16 0.388 8223 16 16 0.759 8224 32 64 0.2510 8225 32 8 0.511 8226 32 16 0.38______________________________________ TABLE 18______________________________________Synergy of MBI-29 + Ciprofloxacin against E. cloacaeIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Ciprofloxacin and Ciprofloxacin______________________________________12 ATCC 13047 64 0.25 1.0113 37-1 256 1 0.2614 938-2 64 1 0.3815 B5546 64 1 0.3816 R4148 64 2 0.3817 R4148-2 64 2 0.3818 3077 64 0.5 0.7519 3356 128 0.25 120 14269 256 1 0.2821 14661 512 1 0.2822 18801 32 1 0.38______________________________________ TABLE 19______________________________________Synergy of MBI-29 + Ciprofloxacin against E. cloacaeIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Ciprofloxacin and Ciprofloxacin______________________________________12 ATCC 13047 32 0.25 1.0113 37-1 32 1 114 938-2 32 1 0.515 B5546 32 1 0.516 R4148 32 2 0.6317 R4148-2 16 2 0.7518 3077 32 1 0.7519 3356 32 0.25 1.0120 14269 32 1 0.7521 14661 32 1 0.7522 18801 32 1 0.75______________________________________ TABLE 20______________________________________Synergy of MBI-26 + Imipenem against E. cloacaeIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FICNumber Strain Number MBI-26 Imipenem MBI-26 and Imipenem______________________________________12 ATCC 13047 16 1 113 37-1 64 2 0.7514 938-2 64 4 0.7515 B5546 64 2 116 R4148 64 4 0.7517 R4148-2 32 2 118 3077 64 4 0.7519 3356 64 4 0.7520 14269 64 4 0.5121 14661 64 2 1.0122 18801 64 2 1.01______________________________________ TABLE 21______________________________________Synergy of MBI-29 + Imipenem against E. cloacaeIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FICNumber Strain Number MBI-269 Imipenem MBI-29 and Imipenem______________________________________12 ATCC 13047 64 2 113 37-1 128 2 114 938-2 32 2 1.515 B5546 32 2 0.7516 R4148 64 4 0.5617 R4148-2 32 2 0.7518 3077 32 2 119 3356 32 2 1.520 14269 128 2 121 14661 64 2 122 18801 64 2 1______________________________________ TABLE 22______________________________________Synergy of MBI-26 + Tobramycin against E. cloacaeIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Tobramycin and Tobramycin______________________________________12 ATCC 13047 64 1 1.0113 37-1 64 1 1.0114 938-2 64 2 1.0115 B5546 32 2 0.5116 R4148 32 1 1.0617 R4148-2 64 1 ND18 3077 64 1 1.0619 3356 64 1 1.0120 14269 64 1 1.0121 14661 64 1 1.0122 18801 32 1 1.01______________________________________ TABLE 23______________________________________Synergy of MBI-29 + Tobramycin against E. cloacaeIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Tobramycin and Tobramycin______________________________________12 ATCC 13047 32 1 113 37-1 32 1 114 938-2 16 1 1.0115 B5546 32 1 1.0116 R4148 16 1 1.0317 R4148-2 16 1 1.0118 3077 32 1 1.0119 3356 32 1 1.0120 14269 16 1 1.0121 14661 32 1 1.0122 18801 16 1 1.01______________________________________ TABLE 24______________________________________Synergy of MBI-26 + Piperacillin against E. cloacaeIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Piperacillin and Piperacillin______________________________________12 ATCC 13047 64 8 0.7513 37-1 64 4 1.0114 938-2 64 8 0.5115 B5546 64 8 0.5616 R4148 64 1024 0.1317 R4148-2 64 1024 0.2518 3077 64 8 0.5619 3356 64 64 0.2520 14269 512 1024 0.0321 14661 64 16 0.522 18801 32 4 1.01______________________________________ TABLE 25______________________________________Synergy of MBI-29 + Piperacillin against E. cloacaeIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Piperacillin and Piperacillin______________________________________12 ATCC 13047 32 8 0.7513 37-1 32 4 1.0114 938-2 32 4 1.0115 B5546 32 8 0.6316 R4148 32 1024 0.2817 R4148-2 32 1024 0.2818 3077 32 4 1.0119 3356 32 64 0.3120 14269 128 1024 0.0721 14661 32 8 1.0122 18801 16 4 1.01______________________________________ TABLE 26______________________________________Synergy of MBI-26 + Ceftazidime against E. cloacaeIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Ceftazidime and Ceftazidime______________________________________12 ATCC 13047 16 4 0.7513 37-1 256 2 114 938-2 64 2 1.0115 B5546 64 2 1.0116 R4148 64 512 0.2517 R4148-2 32 512 3118 3077 64 2 1.0119 3356 128 128 0.1920 14269 512 512 0.0321 14661 512 512 0.0322 18801 256 2 1______________________________________ TABLE 27______________________________________Synergy of MBI-29 + Ceftazidime against E. cloacaeIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Ceftazidime and Ceftazidime______________________________________12 ATCC 13047 64 4 0.5313 37-1 128 2 114 938-2 32 2 1.0115 B5546 32 2 1.0116 R4148 64 512 0.1617 R4148-2 32 512 0.2818 3077 32 2 1.0119 3356 32 128 0.320 14269 128 512 0.0921 14661 128 512 0.0922 18801 128 2 1______________________________________ TABLE 28______________________________________Synergy of MBI-26 + Ciprofloxacin against P. aeruginosaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Ciprofloxacin and Ciprofloxacin______________________________________23 ATCC 27853 64 1 0.5624 34 64 1 0.5325 923-1 64 0.25 1.0126 3101 64 0.25 1.0127 14644 256 32 ND28 15036 256 16 129 15545 64 0.25 1.0130 B3999-1 64 0.25 1.0131 U7688-1 64 0.25 1.0132 W2897-1 64 0.25 1.0133 W5483-2 64 0.25 1.01______________________________________ TABLE 29______________________________________Synergy of MBI-29 + Ciprofloxacin against P. aeruginosaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Ciprofloxacin and Ciprofloxacin______________________________________23 ATCC 27853 32 1 0.7524 34 32 1 0.7525 923-1 32 0.25 1.0126 3101 32 0.25 1.0127 14644 128 32 ND28 15036 128 32 ND29 15545 32 0.25 0.6330 B3999-1 32 0.25 1.0131 U7688-1 32 0.25 1.0132 W2897-1 32 0.25 1.0133 W5483-2 32 0.25 1.01______________________________________ TABLE 30______________________________________Synergy of MBI-26 + Imipenem against P. aeruginosaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Imipenem and Imipenem______________________________________23 ATCC 27853 64 8 0.524 34 128 512 0.3825 923-1 64 4 1.0126 3101 64 8 0.7527 14644 64 16 0.5128 15036 64 16 129 15545 64 128 ND30 B3999-1 64 128 ND31 U7688-1 64 16 132 W2897-1 64 128 0.2533 W5483-2 64 64 ND______________________________________ TABLE 31______________________________________Synergy of MBI-29 + Imipenem against P. aeruginosaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Imipenem and Imipenem______________________________________23 ATCC 27853 64 4 0.7524 34 32 512 0.1625 923-1 64 2 1.1326 3101 32 4 0.7527 14644 64 8 128 15036 64 8 129 15545 32 256 0.6330 B3999-1 64 256 131 U7688-1 64 256 0.7532 W2897-1 32 256 0.7533 W5483-2 64 256 0.5______________________________________ TABLE 32______________________________________Synergy of MBI-26 + Tobramycin against P. aeruginosaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Tobramycin and Tobramycin______________________________________23 ATCC 27853 64 1 1.0124 34 128 128 0.525 923-1 64 1 1.0126 3101 64 1 1.0127 14644 64 2 0.5328 15036 64 1 1.0129 15545 64 2 130 B3999-1 64 2 0.5131 U7688-1 64 1 1.0132 W2897-1 64 1 1.0133 W5483-2 64 1 1.01______________________________________ TABLE 33______________________________________Synergy of MBI-29 + Tobramycin against P. aeruginosaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Tobramycin and Tobramycin______________________________________23 ATCC 27853 32 1 1.0124 34 16 64 0.525 923-1 32 1 1.0126 3101 32 1 1.0127 14644 32 1 1.0128 15036 32 1 1.0129 15545 32 2 0.7530 B3999-1 32 1 1.0131 U7688-1 32 2 0.5632 W2897-1 32 1 1.0133 W5483-2 32 2 0.51______________________________________ TABLE 34______________________________________Synergy of MBI-26 + Piperacillin against P. aeruginosaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Piperacillin and Piperacillin______________________________________23 ATCC 27853 128 256 0.1924 34 128 1024 0.525 923-1 64 64 0.526 3101 64 64 0.6327 14644 64 512 ND28 15036 256 1024 0.0929 15545 256 512 0.1430 B3999-1 256 512 0.2531 U7688-1 256 512 ND32 W2897-1 64 64 133 W5483-2 256 128 1______________________________________ TABLE 35______________________________________Synergy of MBI-29 + Piperacillin against P. aeruginosaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Piperacillin and Piperacillin______________________________________23 ATCC 27853 64 128 ND24 34 32 1024 0.2825 923-1 32 32 0.526 3101 32 128 0.3127 14644 128 1024 ND28 15036 128 1024 0.0929 15545 64 512 0.1630 B3999-1 128 256 0.1231 U7688-1 32 256 0.532 W2897-1 32 32 0.7533 W5483-2 128 512 0.19______________________________________ TABLE 36______________________________________Synergy of MBI-26 + Ceftazidime against P. aeruginosaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Ceftazidime and Ceftazidime______________________________________23 ATCC 27853 128 32 0.3124 34 128 32 0.7525 923-1 128 4 1.0126 3101 128 8 0.5627 14644 256 128 128 15036 256 2 229 15545 256 8 0.5330 B3999-1 256 16 1.0131 U7688-1 256 32 0.2532 W2897-1 256 32 0.2633 W5483-2 256 8 1.01______________________________________ TABLE 37______________________________________Synergy of MBI-29 + Ceftazidime against P. aeruginosaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Ceftazidime and Ceftazidime______________________________________23 ATCC 27853 64 32 0.3824 34 32 64 0.3125 923-1 64 4 0.5626 3101 64 8 0.5627 14644 128 256 128 15036 128 4 0.5129 15545 64 8 0.530 B3999-1 128 8 0.3131 U7688-1 128 8 0.6332 W2897-1 64 4 0.5133 W5483-2 128 8 0.56______________________________________ TABLE 38______________________________________Synergy of MBI-26 + Ciprofloxacin against X. maltophila Lowest FICIsolate MIC (.mu.g .multidot. mL.sup.-1) MBI-26 andNumber Strain Number MBI-26 Ciprofloxacin Ciprofloxacin______________________________________34 ATCC 13637 64 0.5 0.6335 6 64 4 0.3836 16 64 8 137 3095 64 16 138 7901 256 32 ND39 B3110.1 64 2 2.0140 R1302-1 128 4 141 R4230 64 2 142 TF29-1 128 8 ND43 TF29-2 128 8 144 W6896-2 256 4 0.1345 W11291 64 2 2.01______________________________________ TABLE 39______________________________________Synergy of MBI-29 + Ciprofloxacin against X. maltophila Lowest FICIsolate MIC (.mu.g .multidot. mL.sup.-1) MBI-29 andNumber Strain Number MBI-29 Ciprofloxacin Ciprofloxacin______________________________________34 ATCC 13637 32 0.5 1.0135 6 32 2 0.3836 16 32 8 ND37 3095 64 16 0.538 7901 64 32 0.7539 B3110.1 32 2 1.0140 R1302-1 32 4 0.5641 R4230 16 4 0.542 TF29-1 16 8 143 TF29-2 16 8 0.5344 W6896-2 16 8 145 W11291 32 4 0.75______________________________________ TABLE 40______________________________________Synergy of MBI-26 + Imipenem against X. maltophilaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Imipenem and Imipenem______________________________________34 ATCC 13637 64 512 0.1335 6 128 512 0.1636 16 256 512 ND37 3095 512 512 ND38 7901 256 512 ND39 B3110.1 256 512 ND40 R1302-1 128 512 ND41 R4230 64 512 ND42 TF29-1 128 512 ND43 TF29-2 128 512 ND44 W6896-2 256 512 0.2545 W11291 512 512 ND______________________________________ TABLE 41______________________________________Synergy of MBI-29 + Imipenem against X. maltophilaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Imipenem and Imipenem______________________________________34 ATCC 13637 32 512 0.1935 6 32 512 0.2536 16 32 512 0.337 3095 64 512 ND38 7901 64 512 0.3839 R3110.1 32 512 ND40 R1302-1 64 512 0.2541 R4230 32 512 0.542 TF29-1 16 512 0.5643 TF29-2 16 512 0.5644 W6896-2 64 512 0.3845 W11291 64 512 0.38______________________________________ TABLE 42______________________________________Synergy of MBI-26 + Tobramycin against X. maltophilaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Tobramycin and Tobramycin______________________________________34 ATCC 13637 64 32 0.1635 6 64 4 0.2536 16 256 128 0.6337 3095 32 32 0.538 7901 32 32 1.0139 B3110.1 256 32 0.6340 R1302-1 128 32 0.6341 R4230 64 16 0.3842 TF29-1 32 2 1.0143 TF29-2 32 2 1.0144 W6896-2 32 16 0.545 W11291 512 128 ND______________________________________ TABLE 43______________________________________Synergy of MBI-29 + Tobramycin against X. maltophilaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Tobramycin and Tobramycin______________________________________34 ATCC 13637 8 8 0.6335 6 16 4 0.7536 16 32 128 0.3137 3095 64 32 0.1638 7901 64 32 0.2839 B3110.1 32 16 0.5640 R1302-1 64 32 0.5341 R4230 16 16 0.5642 TF29-1 8 1 1.0343 TF29-2 16 1 1.544 W6896-2 16 8 0.6345 W11291 64 128 ND______________________________________ TABLE 44______________________________________Synergy of MBI-26 + Piperacillin against X. maltophilaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Piperacillin and Piperacillin______________________________________34 ATCC 13637 64 1024 0.1435 6 64 512 ND36 16 256 1024 ND37 3095 64 512 ND38 7901 64 512 ND39 B3110.1 64 512 ND40 R1302-1 64 512 ND41 R4230 64 1024 0.542 TF29-1 128 1024 0.243 TF29-2 128 1024 0.544 W6896-2 32 1024 0.645 W11291 64 512 ND______________________________________ TABLE 45______________________________________Synergy of MBI-29 + Piperacillin against X. maltophilaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Piperacillin and Piperacillin______________________________________34 ATCC 13637 32 1024 0.1935 6 32 64 0.1936 16 32 1024 0.3837 3095 64 1024 0.3838 7901 64 1024 0.2839 B3110.1 32 1024 0.6340 R1302-1 64 1024 0.2541 R4230 32 1024 0.342 TF29-1 32 1024 0.243 TF29-2 32 1024 0.2844 W6896-2 64 1024 0.1345 W11291 64 1024 0.31______________________________________ TABLE 46______________________________________Synergy of MBI-26 + Ceftazidime against X. maltophilaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-26Number Strain Number MBI-26 Ceftazidime and Ceftazidime______________________________________34 ATCC 13637 64 64 0.2835 6 128 64 0.2536 16 256 32 0.5137 3095 512 512 ND38 7901 256 128 139 B3110.1 256 64 1.1340 R1302-1 128 16 141 R4230 64 4 142 TF29-1 128 4 143 TF29-2 128 4 1.0144 W6896-2 256 64 145 W11291 512 128 1______________________________________ TABLE 47______________________________________Synergy of MBI-29 + Ceftazidime against X. maltophilaIsolate MIC (.mu.g .multidot. mL.sup.-1) Lowest FIC MBI-29Number Strain Number MBI-29 Ceftazidime and Ceftazidime______________________________________34 ATCC 13637 32 64 0.2835 6 32 32 0.3136 16 32 16 0.6337 3095 64 512 ND38 7901 64 256 0.3139 B3110.1 32 128 0.7540 R1302-1 64 32 0.2541 R4230 32 8 0.542 TF29-1 16 4 143 TF29-2 16 2 1.0144 W6896-2 64 64 0.545 W11291 64 256 0.28______________________________________ Note: MBI 26 (1.25 .mu.g/mL) also showed synergy with Lysozyme (>64.times. reduction in MIC), Novobiocin (>4.times. reduction in MIC), Nalidixic acid (2.times. reduction in MIC) and carbenicillin (2.times. reduction in MIC).

Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 37- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- Lys Trp Lys Ser Phe Ile Lys Lys Leu Thr Th - #r Ala Val Lys Lys Val# 15- Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser# 25- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- Lys Trp Lys Ser Phe Ile Lys Lys Leu Thr Se - #r Ala Ala Lys Lys Val# 15- Val Thr Thr Ala Lys Pro Leu Ile Ser Ser# 25- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:- Lys Trp Lys Ser Phe Ile Lys Asn Leu Thr Ly - #s Gly Gly Ser Lys Ile# 15- Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser# 25- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:- Lys Trp Lys Lys Phe Ile Lys Asn Leu Thr Ly - #s Gly Gly Ser Lys Ile# 15- Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser# 25- (2) INFORMATION FOR SEQ ID NO:5:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:- Lys Trp Lys Ser Phe Ile Lys Asn Leu Glu Ly - #s Val Leu Lys Pro Gly# 15- Gly Leu Leu Ser Asn Ile Val Thr Ser Leu# 25- (2) INFORMATION FOR SEQ ID NO:6:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:- Lys Trp Lys Ser Phe Ile Lys Asn Leu Glu Ly - #s Val Leu Lys Lys Gly# 15- Pro Ile Leu Ala Asn Leu Val Ser Ile Val# 25- (2) INFORMATION FOR SEQ ID NO:7:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:- Lys Trp Lys Glu Phe Ile Lys Lys Leu Thr Th - #r Ala Val Lys Lys Val# 15- Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser# 25- (2) INFORMATION FOR SEQ ID NO:8:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:- Lys Trp Lys Lys Phe Ile Lys Glu Leu Gln Ly - #s Val Leu Ala Pro Gly# 15- Gly Leu Leu Ser Asn Ile Val Thr Ser Leu# 25- (2) INFORMATION FOR SEQ ID NO:9:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:- Lys Trp Lys Ser Phe Ile Lys Lys Leu Thr Se - #r Val Leu Lys Lys Val# 15- Val Thr Thr Ala Leu Pro Ala Leu Ile Ser# 25- (2) INFORMATION FOR SEQ ID NO:10:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:- Lys Trp Lys Ser Phe Ile Lys Asn Leu Thr Ly - #s Val Leu Lys Lys Val# 15- Val Thr Thr Ala Leu Pro Ala Leu Ile Ser# 25- (2) INFORMATION FOR SEQ ID NO:11:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:- Lys Trp Lys Leu Phe Lys Lys Lys Gly Thr Gl - #y Ala Val Leu Thr Val# 15- Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser# 25- (2) INFORMATION FOR SEQ ID NO:12:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:- Lys Trp Lys Ser Phe Ile Lys Lys Leu Thr Se - #r Val Leu Lys Lys Val# 15- Val Thr Thr Ala Lys Pro Leu Ile Ser Ser# 25- (2) INFORMATION FOR SEQ ID NO:13:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:- Lys Lys Lys Ser Phe Ile Lys Leu Leu Thr Se - #r Ala Lys Val Ser Val# 15- Leu Thr Thr Ala Lys Pro Leu Ile Ser Ser# 25- (2) INFORMATION FOR SEQ ID NO:14:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:- Lys Trp Lys Lys Phe Ile Lys Glu Leu Gln Ly - #s Val Leu Lys Pro Gly# 15- Gly Leu Leu Ser Asn Ile Val Thr Ser Leu# 25- (2) INFORMATION FOR SEQ ID NO:15:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 20 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:- Lys Lys Trp Trp Arg Arg Val Leu Ser Gly Le - #u Lys Thr Gly Pro Ala# 15- Leu Ser Asn Val 20- (2) INFORMATION FOR SEQ ID NO:16:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 20 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:- Lys Lys Trp Trp Arg Arg Val Leu Lys Gly Le - #u Ser Ser Gly Pro Ala# 15- Leu Ser Asn Val 20- (2) INFORMATION FOR SEQ ID NO:17:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 20 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:- Lys Lys Trp Trp Arg Arg Ala Leu Gln Ala Le - #u Lys Asn Gly Pro Ala# 15- Leu Ser Asn Val 20- (2) INFORMATION FOR SEQ ID NO:18:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 24 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:- Lys Lys Trp Trp Arg Arg Val Leu Ser Gly Le - #u Lys Thr Ala Gly Pro# 15- Ala Ile Gln Ser Val Leu Asn Lys 20- (2) INFORMATION FOR SEQ ID NO:19:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 24 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:- Lys Lys Trp Trp Arg Arg Ala Leu Gln Gly Le - #u Lys Thr Ala Gly Pro# 15- Ala Ile Gln Ser Val Leu Asn Lys 20- (2) INFORMATION FOR SEQ ID NO:20:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 22 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:- Lys Lys Trp Trp Lys Ala Gln Lys Ala Val As - #n Ser Gly Pro Asn Ala# 15- Leu Gln Thr Leu Ala Gln 20- (2) INFORMATION FOR SEQ ID NO:21:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 22 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:- Lys Lys Trp Trp Lys Ala Lys Lys Phe Ala As - #n Ser Gly Pro Asn Ala# 15- Leu Gln Thr Leu Ala Gln 20- (2) INFORMATION FOR SEQ ID NO:22:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 23 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:- Lys Lys Trp Trp Lys Phe Ile Lys Lys Ala Va - #l Asn Ser Gly Thr Thr# 15- Gly Leu Gln Thr Leu Ala Ser 20- (2) INFORMATION FOR SEQ ID NO:23:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 17 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:- Lys Lys Ser Phe Phe Lys Lys Leu Thr Ser Va - #l Ala Ser Ser Val Leu# 15- Ser- (2) INFORMATION FOR SEQ ID NO:24:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 20 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:- Trp Lys Val Phe Lys Ser Phe Ile Lys Lys Al - #a Ser Ser Phe Ala Gln# 15- Ser Val Leu Asp 20- (2) INFORMATION FOR SEQ ID NO:25:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 18 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:- Lys Lys Trp Arg Lys Ser Phe Phe Lys Gln Va - #l Gly Ser Phe Asp Asn# 15- Ser Val- (2) INFORMATION FOR SEQ ID NO:26:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE:#8, 10, 11, 14 and 15N: 4, 7,#where Xaa at positionsORMATION:#8, 10, 11, 14 and 15 are selected from argin - #ine, lysine, g - #lutamic acid, aspartic acid, glutamine,#serine, histidine and threonine#9, 12 and 13 LOCATION: 5, 6,#where Xaa at positionsORMATION:#9, 12 and 13 are selected from isoleucine, valine, l - #eucine, alanine, cysteine, glycine, phenylalanin - #e, proline, tryptophan, tyrosine,#and methioninenorleucine- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:- Lys Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Val# 15- Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser# 25- (2) INFORMATION FOR SEQ ID NO:27:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE:#8, 10, 11, 14 and 15N: 4, 7,#where Xaa at positionsORMATION:#8, 10, 11, 14 and 15 are selected from argin - #ine, lysine, g - #lutamic acid, aspartic acid, glutamine,#serine, histidine and threonine#9, 12 and 13 LOCATION: 5, 6,#where Xaa at positionsORMATION:#9, 12 and 13 are selected from isoleucine, valine, l - #eucine, alanine, cysteine, glycine, phenylalanin - #e, proline, tryptophan, tyrosine,#and methioninenorleucine- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:- Lys Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Val# 15- Val Thr Thr Ala Lys Pro Leu Ile Ser Ser# 25- (2) INFORMATION FOR SEQ ID NO:28:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE:#8, 10, 11, 14 and 15N: 4, 7,#where Xaa at positionsORMATION:#8, 10, 11, 14 and 15 are selected from argin - #ine, lysine, g - #lutamic acid, aspartic acid, glutamine,#serine, histidine and threonine#9, 12 and 13 LOCATION: 5, 6,#where Xaa at positionsORMATION:#9, 12 and 13 are selected from isoleucine, valine, l - #eucine, alanine, cysteine, glycine, phenylalanin - #e, proline, tryptophan, tyrosine,#and methioninenorleucine- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:- Lys Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Ile# 15- Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser# 25- (2) INFORMATION FOR SEQ ID NO:29:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE:#8, 10, 11, 14 and 15N: 4, 7,#where Xaa at positionsORMATION:#8, 10, 11, 14 and 15 are selected from argin - #ine, lysine, g - #lutamic acid, aspartic acid, glutamine,#serine, histidine and threonine#9, 12 and 13 LOCATION: 5, 6,#where Xaa at positionsORMATION:#9, 12 and 13 are selected from isoleucine, valine, l - #eucine, alanine, cysteine, glycine, phenylalanin - #e, proline, tryptophan, tyrosine,#and methioninenorleucine- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:- Lys Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Gly# 15- Gly Leu Leu Ser Asn Ile Val Thr Ser Leu# 25- (2) INFORMATION FOR SEQ ID NO:30:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE:#8, 10, 11, 14 and 15N: 4, 7,#where Xaa at positionsORMATION:#8, 10, 11, 14 and 15 are selected from argin - #ine, lysine, g - #lutamic acid, aspartic acid, glutamine,#serine, histidine and threonine#9, 12 and 13 LOCATION: 5, 6,#where Xaa at positionsORMATION:#9, 12 and 13 are selected from isoleucine, valine, l - #eucine, alanine, cysteine, glycine, phenylalanin - #e, proline, tryptophan, tyrosine,#and methioninenorleucine- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:- Lys Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Gly# 15- Pro Ile Leu Ala Asn Leu Val Ser Ile Val# 25- (2) INFORMATION FOR SEQ ID NO:31:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 20 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE:#10 and 11(B) LOCATION: 7, 8,#where Xaa at positions 7, 8, 10 and 11 are selec - #ted from isoleucine, valine, leucine,alanine,#glycine, phenylalanine, proline, tryptophan,#norleucine and methionine#and 13 (B) LOCATION: 9, 12,#where Xaa at positions 9, 12, and 13 are selec - #ted from arginine, lysine, glutamic acid,#acid, glutamine, asparagine, serine,#and threonine histidine- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:- Lys Lys Trp Trp Arg Arg Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Gly Pro Ala# 15- Leu Ser Asn Val 20- (2) INFORMATION FOR SEQ ID NO:32:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 7 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (B) LOCATION: 7...7#where Xaa at position 7 is from about 14- - #24 amino acid residues selected from#valine, leucine, alanine, cysteine,#phenylalanine, proline, tryptophan, tyrosine,#methionine, arginine, lysine, glutamic acid, asp - #artic acid, glutamine, asparagine, serine,#and threonine histidine- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:- Lys Lys Trp Trp Arg Arg Xaa 1 5- (2) INFORMATION FOR SEQ ID NO:33:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 6 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (B) LOCATION: 6...6#where Xaa at position 6 is from about 14- - #24 amino acid residues selected from#valine, leucine, alanine, cysteine,#phenylalanine, proline, tryptophan, tyrosine,#methionine, arginine, lysine, glutamic acid, asp - #artic acid, glutamine, asparagine, serine,#and threonine histidine- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:- Lys Lys Trp Trp Lys Xaa 1 5- (2) INFORMATION FOR SEQ ID NO:34:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 8 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:- Lys Trp Lys Ser Phe Ile Lys Lys 1 5- (2) INFORMATION FOR SEQ ID NO:35:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 24 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (B) LOCATION: 6#where Xaa at position 6 is selected from argi - #nine, lysine, glutamic acid, aspartic acid,#asparagine, serine, histidine and threonine#5 (B) LOCATION: 4 and#where Xaa at positions 4 and 5 are#from isoleucine, valine, leucine, alanine,#glycine, phenylalanine, proline, tryptophan,#norleucine and methionine- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:- Lys Lys Trp Trp Arg Arg Xaa Xaa Xaa Gly Le - #u Lys Thr Ala Gly Pro# 15- Ala Ile Gln Ser Val Leu Asn Lys 20- (2) INFORMATION FOR SEQ ID NO:36:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 26 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:- Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gl - #y Ala Val Leu Lys Val# 15- Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser# 25- (2) INFORMATION FOR SEQ ID NO:37:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 28 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:- Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gl - #y Ala Val Leu Lys Val# 15- Leu Thr Thr Gly Leu Pro Ala Leu Lys Lys Th - #r Lys# 25__________________________________________________________________________

Claims

1. An isolated polynucleotide which encodes a peptide selected from the group consisting of:

(SEQ ID NO:1)NH.sub.2 -KWKSFIKKLTTAVKKVLTTGLPALIS-COOH, (SEQ ID NO:2)NH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH, (SEQ ID NO:3)NH.sub.2 -KWKSFIKNLTKGGSKILTTGLPALIS-COOH, (SEQ ID NO:4)NH.sub.2 -KWKKFIKNLTKGGSKILTTGLPALIS-COOH, (SEQ ID NO:5)NH.sub.2 -KWKSFIKNLEKVLKPGGLLSNIVTSL-COOH, (SEQ ID NO:6)NH.sub.2 -KWKSFIKNLEKVLKKGPILANLVSIV-COOH, (SEQ ID NO:7)NH.sub.2 -KWKEFIKKLTTAVKKVLTTGLPALIS-COOH, (SEQ ID NO:8)NH.sub.2 -KWKKFIKELQKVLAPGGLLSNIVTSL-COOH, (SEQ ID NO:9)NH.sub.2 -KWKSFIKKLTSVLKKVVTTALPALIS-COOH, (SEQ ID NO:10)NH.sub.2 -KWKSFIKNLTKVLKKVVTTALPALIS-COOH, (SEQ ID NO:11)NH.sub.2 -KWKLFKKKGTGAVLTVLTTGLPALIS-COOH, (SEQ ID NO:12)NH.sub.2 -KWKSFIKKLTSVLKKVVTTAKPLISS-COOH, (SEQ ID NO:13)NH.sub.2 -KKKSFIKLLTSAKVSVLTTAKPLISS-COOH, (SEQ ID NO:14)NH.sub.2 -KWKKFIKELQKVLKPGGLLSNIVTSL-COOH, (SEQ ID NO:15)NH.sub.2 -KKWWRRVLSGLKTGPALSNV-COOH, (SEQ ID NO:16)NH.sub.2 -KKWWRRVLKGLSSGPALSNV-COOH, (SEQ ID NO:17)NH.sub.2 -KKWWRRALQALKNGPALSNV-COOH, (SEQ ID NO:18)NH.sub.2 -KKWWRRVLSGLKTAGPAIQSVLNK-COOH, (SEQ ID NO:19)NH.sub.2 -KKWWRRALQGLKTAGPAIQSVLNK-COOH, (SEQ ID NO:20)NH.sub.2 -KKWWKAQKAVNSGPNALQTLAQ-COOH, (SEQ ID NO:21)NH.sub.2 -KKWWKAKKFANSGPNALQTLAQ-COOH,and (SEQ ID NO:22)NH.sub.2 -KKWWKFIKKAVNSGTTGLQTLAS-COOH.

2. An isolated polynucleotide which encodes a peptide selected from the group consisting of:

NH.sub.2 -KKSFFKKLTSVASSVLS-COOH, (SEQ ID NO:23)NH.sub.2 -WKVFKSFIKKASSFAQSVLD-COOH, (SEQ ID NO:24),andNH.sub.2 -KKWRKSFFKQVGSFDNSV-COOH. (SEQ ID NO:25).