METHODS FOR IDENTIFYING POLYCATIONIC, PEPTIDE-LIKE COMPOUNDS WITH ANTIBACTERIAL ACTIVITY

Abstract: A method is provided for identifying polycationic, peptide-like compounds characterized by antibacterial activity against gram negative bacteria. The method takes advantage of the induction of hyperosomotic stress without leakage of cytoplasmic content. The method involves determining whether the compound has the ability to produce phospholipid exchange betwen the lipid bilayers in conjunction with the induction of the osmotic stress response in the target cell. Determining the induction of the osmotic stress response is effected using a bioluminescent detector organism having a osmY-LUX gene fusion.

Description: TITLE METHODS FOR IDENTIFYING POLYCATIONIC, PEPTIDE-LIKE COMPOUNDS WITH ANTIBACTERIAL ACTIVITY This invention was made with government support under GM29703 awarded by Public Health Service/National Institutes of Health. The government has certain rights in the invention. FIELD OF INVENTION This invention relates to the field of microbiological screening techniques and the use of those techniques to identify certain classes of pharmacologically active compounds. More specifically, the invention provides a method for identifying polycationic antibacterial compounds that select against gram negative bacteria. The present method selects for a class of antimicrobial compounds that promote a direct and rapid exchange of phospholipids and lipids. Such antibiotics are not susceptible to development of resistance by genetic mutation. BACKGROUND Biomedical and healthcare professionals are increasingly concerned about the growing number of pathogenic bacteria that have developed resistance to the most common antibiotics. Antibiotic-resistant strains of Haemophilus influenzae and Streptococcus pneumoniae have been isolated from patients with chronic bronchitis and pneumonia. Methicillin-resistant Staphylococcusis is found more and more commonly in hospital and clinic settings. The increase in resistant bacterial strains prompts a redoubled effort to identify new antibacterials that are less susceptible to inducing tolerance in the target organism. Currently, a variety of screening methods are used to identify new antibacterial candidates. Binding assays (in which a test compound present in the sample is measured for biological activity by binding to an antibody bound to a solid phase) are frequently used to determine drug candidates among compounds. However, binding assays have several drawbacks, not the least of which is lack of sensitivity. The problems associated with binding assays have led to the development of recombinant receptors which may be used in cellular functional assays for high-throughput screening. Functional assays separate agonists from antagonists as well as eliminate compounds that bind to targets but which have no pharmacological effects. High-throughput assay capabilities for maximally informative and sensitive assays would be valuable both in the initial screening of compound libraries and in the iterative optimization processes. One of the key elements in any assay is the choice of reporter system that signals if the assay has identified a "hit". Recently, cell-based functional assay systems have examined the efficacy of bioluminescent reporters. Recombinant bacteria have been developed by fusing the lux structural genes to chemically responsive bacterial promoters and then placing such chimeras in appropriate hosts. These recombinant bacteria are sensor organisms that glow in response to specific stimuli. The use of various stress promoters, including those sensitive to genotoxic stress, is disclosed in WO 94/13831 and WO 96/16187. The disclosures of these patent applications are incorporated by reference herein. WO 94/13831 teaches the use of detector organisms containing a stress promoter- bioluminescent gene fusion to detect various stresses including those sensitive to protein damage (heat shock), DNA damage (genotoxic), oxidative damage, cell membrane damage (osmotic sensitivity), amino acid starvation, carbon starvation, and nitrogen starvation. Identification of unique stress sensors and the more complex characterization of bacterial stress promoters in combination with bacterial bioluminescent reporters invites the development of new screening methods for polycationic antibacterial compounds. One such class of compounds are the antimicrobial peptides. The efficacy of these peptides is linked to specific cell membrane perturbation, which ultimately leads to osmotic stress without nonspecific leakage of protons and other solutes. Throughout this application various publications are referenced by Arabic numerals. Citations for these references are set out in full immediately before the claims. The disclosure of these publications describe more fully the art to which this invention pertains and are incorporated by reference in full. Polycationic antimicrobial peptides and proteins are produced by a wide range of organisms (1),(2). Such agents are of interest because their very existence suggests strategies towards target selectivity, putatively without entry into the cytoplasm. They offer possible evolutionary solutions to the problem of antibiotic resistance. For example, polymyxins produced by Gram-positive Polymyxa spp. are active against gram-negative organisms (3). Similarly, magainins produced by frogs (4), cecropins by insect larvae (5) and defensins from humans (6) do not cause significant damage to organisms that produce them, yet they are active against gram-negative organisms (7). Most of these polycationic antimicrobial peptides interact strongly with the membrane, therefore it has been suggested that the basis for their effect lies in their ability to cause leakage of cytoplasmic contents by channel formation (8) or by a gross change in the membrane organization (9),(10),(1 1),(12),(13),(14). Obviously, this hypothesis does not account for the observed antimicrobial target specificity. Although the existence and the postulated mechanism of action of antimicrobial peptides are known, no effective method is known in the art for the selective screening of compounds with antimicrobial target specificity. SUMMARY OF THE INVENTION The present invention provides a method for identifying polycationic, peptide-like compounds with antibacterial activity comprising: (i) contacting a polycationic peptide-like compound suspected of having antibacterial activity with a detector cell, the detector cell comprising an osmotic stress promoter operably linked to a reporter gene, the reporter gene capable of emitting a detectable signal; and (ii) measuring the change in signal emitted by the detector cell before and after the contacting with the polycationic peptide-like compound in step (i), wherein an increase in signal indicates that the polycationic, peptide-like compound has antibacterial activity. BRIEF DESCRIPTION OF THE FIGURES. BIOLOGICAL DEPOSIT. AND SEQUENCE LISTING Figure 1 is a plot of a fluorescence vs. time in min. It illustrates cecropin B-induced fluorescence as a measure of anionic vesicle aggregation. Figure 2 is a plot showing the total change in the fluorescence emission from dilution of self-quenched pyPM/POPC covesicles as a function of cecropin B, gramicidin A, or NP concentration. Figure 3 is a plot of fluorescence vs. Time. It shows the reaction progress for the dithionite (20 mM) quenching of fluorescence of POPC/DMPM vesicles containing 0.6 mole% NBD-PE (110 uM). Figure 4 is a plot of luminescence vs. time. It illustrates growth profiles for the lac-lux fusion strain TV 1048 of E. coli in the absence (open symbols) or presence (closed symbols) of 0.15 uM cecropin B. The growth was monitored as a change on OD at 600 nm (circles) or as the luminescence increase (squares). Figure 5 is a plot of luminescence vs. time. It illustrates the luminescence response of TV 1048 in the early growth phase of varying concentrations of cecropin. Each aliquot was prepared by dilution of overnight cultured broth with fresh medium followed by shaking 1 hour at 30°C. Luminescence was measured after adding cecropin or CCCP to an aliquot of the culture. Figure 6 is a plot of luminescence vs. time. It illustrates the effect of CCCP on the luminescence response from salicylate sensitive DPD2146 (closed triangles) and hyperosmolar sensitive DPD2170 (squares). Dependence on the OD change for DPD2146 measured after 60 min is expressed as the change with CCCP (L) relative to the change in its absence (L c ). Figure 7 is a plot of luminescence vs. time. It illustrates the effect of cecropin B on the luminescence response from salicylate sensitive DPD2146 (triangle) and hyperosmolar sensitive DPD2170 (square) after exposure for 60 min. Top panel shows the effect after 60 min exposure on OD at 600 nm for DPD2170. Figure 8 is a time course plot of luminescence vs. time. It illustrates the change in the luminescence response of DPD2170 without any additive, after exposure to 30 uM CCCP, 0.25 uM NP. 0.3 M NaCl, and 0.57 M sucrose. Figure 9 is a graph of luminescence Vs concentration of [PxB](open circles), and colistin (triangles) for the osmY strain, DPD2170. Figure 10 is a graph of luminescence Vs concentration of [PxB](open circles), and colistin (triangles) for the micF strain, DPD2191. Figure 1 1 is a plot of luminescence over time during the growth oϊmicF DPD2191 strain in the NaCl-free LB medium with 0M (circles), 0.1M (triangles), 0.3M (squares) and 0.5M (diamond) NaCl. The following strains were deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC) international depository located at 10801 University Boulevard, Manassas, VA 20110-2209, U.S.A.: TV1061 (ATCC #69315) deposited 13 May 1993 Applicants provide sequence listings in conformity with 37 C.F.R. 1.821-1.825 and Appendices A and B ("Requirements for Application Disclosures Containing Nucleotides and/or Amino Acid Sequences"). SEQ ID NO:l refers to a specific polycationic peptide-like antibacterial known as "polymyxin B" or "PxB" and having the general formula: "Acyl- > Leu-Dab- > Leu-Thr- > Leu-Dab- > Leu-Dab- > Leu-Dab- > D-Phe" where Dab is a,g-diaminobutyric acid. [SEQ ID NO:l]. SEQ ID NO: 2 refers to a specific polycationic peptide antibacterial known as "Cecropin A" having the general formula: KWKLFKKIEKVGENIRDGIIKAGPAVAVVGEATEIAK-NH 2 [SEQ ID NO:2]. SEQ ID NO: 3 refers to a specific polycationic peptide antibacterial known as "Cecropin B" having the general formula: KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL-NH 2 [SEQ ID NO:3]. SEQ ID NO:4 refers to a truncated polymxin B known as "polymyxin B nonapeptide" or "NP" which is devoid of the acyl chain and the first residue and has the general formula: Thr-Dap-Dap-Dap-Phe-Leu-Dap-Dap-Thr [SEQ ID NO: 4]. SEQ ID NO:5 refers to the primer 5'-ACTTAAGGATCCCCCCAAAAATGCAGAATA-3' [SEQ ID NO:5] SEQ ID NO: 6 refers to the primer 5'-GCAGCGAATTCGGGCATCCGGTTGAAATAG-3' [SEQ ID NO: 6]. DETAILED DESCRIPTION OF THE INVENTION Applicants have developed a screening method using a transformed detector cell containing a osmosensitivity bacterial stress promoter linked to bacterial genes responsive for bioluminescence. Applicants have made the unexpected finding that osmosensitive stress promoters are specifically responsive to polycationic peptide-like compounds. This characteristic is suitable for use in the selective screening of compounds for antimicrobial activity. Applicants have shown that the antimicrobial selectivity of a series of compounds monitored with osmo-responsive bacteria correlates well with their ability to mediate exchange of phospholipids. Thus, the biophysical lipid exchange could be used as an independent screen and also as a check on the selectivity. Applicants' mutagenesis studies show that these organisms do not develop genetically stable resistance against the antimicrobials that are selected by these antibacterial criteria. The following definitions are to be used to interpret the claims and specification: The terms "polycationic, peptide-like", "polycationic peptide-like antibacterial" and "antimicrobial peptide-like" refer to a compound that is eationic in nature and has selective toxicity against gram negative bacteria. The toxicity of the compound is produced by a combination of membrane perturbation and the induction of osmotic stress. Examples of such peptides include but are not limited to defensins (isolated from human phagocytes), cecropins (isolated from silkmoth pupae or pig intestin), polymyxins (isolated from the gram positive Polymyxa spp.), apidaecins, isolated from honeybee lymp, melittin isolated from bee venom, bombinin, isolated from toad skin; magainins isolated from frog skin as well as Colistin, and Mastoparan. The term "polycationic" describes a polycationic peptide-like antibacterial and refers to the quality of having multiple positive charges. The term "polymyxin B" or "PxB" refers to a specific polycationic peptide-like antibacterial having the general formula: "Acyl->Leu-Dab->Leu-Thr->Leu-Dab->Leu-Dab->Leu-Dab->D-Phe" where Dab is a,g-diaminobutyric acid. [SEQ ID NO:l] The term "Cecropin A" refers to a specific polycationic peptide antibacterial having the general formula: KWKLFKKIEKVGENIRDGIIKAGPAVAVVGEATEIAK-NH 2 [SEQ ID NO:2] The term "Cecropin B" refers to a specific polycationic peptide antibacterial having the general formula: KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL- NH 2 [SEQ ID NO:3] The term "polymyxin B nonapeptide " or "NP" refers to a truncated polymxin B which is devoid of the acyl chain and the first residue and has the general formula: Thr-Dap-Dap-Dap-Phe-Leu-Dap-Dap-Thr. [SEQ ID NO:4] The term "osmosensitive stress promoter" means any bacteria stress promoter derived from a bacterial structural gene that demonstrates an increase in transcription in response to cellular osmotic stress. Such genes include but are not limited to the osmY gene and the micF gene. The term "osmotic stress" refers to a stress produced on a bacterial cell that results in the induction of an osmosensitive bacterial stress promoter. The term "osmY' refers to a bacterial gene responsive to osmotic stress. The term "micF" refers to a bacterial gene which is known to regulate . expression of E. coli membrane proteins. The term "detector cell" refers to a genetically-engineered bacteria which contains a gene fusion consisting of an osmotic stress responsive promoter fused to a structural reporter gene. The term "reporter gene" means any gene that when operably linked to a suitable promoter generates a detectable signal. Examples of suitable reporter genes include but are not limited to the lacZ gene encoding .beta.-galactosidase, the cat gene encoding chloramphenicol acetyl transferase, the galK gene encoding galactose kinase the gus gene, encoding .beta.-glucosidase, the luc gene encoding insect luciferase, the gfp gene encoding green fluorescent protein, the genes encoding proteins responsible bioluminescence from Renilla sp., and the Lux genes responsible for bioluminescence. The term "operably linked" refers to the fusion of two fragments of DNA in a proper orientation and reading frame to be transcribed into functional RNA. The term "bioluminescence" refers to the phenomenon of light emission from any living organism. The terms "luminescent reporter gene complex" and "lux gene complex" mean any reporter gene(s), the products of which result in light production. Examples include but are not limited to the bacterial lux genes; the luciferase genes (luc), from, for example, the firefly (Photinus pyralis) or click beetle (Pyrophorus plagiophthalamus); or the gene encoding the luciferase from the sea pansy (Renilla reniformis). The term "Lux" refers specifically the lux structural genes which include luxA, luxB, luxC, luxD and luxE and which are responsible for the phenomenon of bacterial bioluminescence. A lux gene complex might include all of the independent lux genes, acting in concert, or any subset of the lux structural genes so long as luxA and luxB are part of the complex. The following table gives corresponding symbols of the single letter and three letter amino acid code as well as all possible codons that will encode a given amino acid: Three-Letter One-Letter Amino Acid Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Asparagine or aspartic acid Asx B Cysteine Cys C Glutamine Gin Q Glutamine acid Glu E Glutamine or glutamic acid Glx Z Glycine Gly G Histidine His H Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Pro line Pro P Serine Ser s Threonine Thr T Tryptophan Trp w Tyrosine Tyr Y Valine Val V The following abbreviations are used: "ANTS" means l-aminonapthalene-3,6,8-trisulfonic acid; "CCCP" means carbonylcyanide w-chlorophenylhydrazone; "DMPM" means l,2-dimyristoylglycero-5«-3-phosphomethanol; "DPX" means N,N'-/?-xylenebis(pyridinium bromide); "MIC" means minimum growth inhibitory concentration; "NBD-PE" means N-(7-nitro-2-l,3- benzoxadiazol-4-yl)dioleoylphosphatidylethanolamine; "NP" means polymyxin B nonapeptide; "PLA2" means phospholipase A 2 from pig pancreas; "POPC" means l-palmitoyl-2-oleoylglycero-._77-3-phosphocholine; "POPC ether" means 1 -hexadecyl-2-octadec-9-enylglycero-._7?- phosphocholine; "pyPA" means l-hexadecanoyl-2-(l-pyrenedecanoyl)glycero-5«-3- phosphatidic acid; "pyPC" means l-hexadecanoyl-2-(l-pyrenedecanoyl)glycero-.y«-3- phosphocholine; "pyPM" means l-hexadecanoyl-2-(l-pyrenedecanoyl)glycero-5/7-3- phosphomethanol ; "R18" means octadecylrhodamine; "RET" means resonance energy transfer; "Rh-PE" means N-(lissamine rhodamine B sulfonyl)-dioleoyl phosphatidylethanolamine and "Dab" means a,g-diaminobutyric acid. "TLRC" will mean threshold luminescence response concentration. Detector Cells And Nonbioluminescent Strains Detector cells are assembled from host cells and reporter gene fusions. Host cells suitable in the present invention include any cell capable of expression of a suitable reporter gene fusion. Host cells are non-bioluminescent strains. Prokaryotic cells are preferred as host cells and members of the enteric class of bacteria are most preferred. Enteric bacteria are members of the family Enterobacteriaceae and include such members as Escherichia, Salmonella, and Shigella. They are gram-negative straight rods, 0.3-1.0 X 1.0-6.0 mm, motile by peritrichous flagella (except for Tatumella) or nonmotile. They grow in the presence and absence of oxygen and grow well on peptone, meat extract, and (usually) MacConkey 's media. Some grow on D-glucose as the sole source of carbon, whereas others require vitamins and/or mineral(s). They are chemoorganotrophic with respiratory and fermentative metabolism but are not halophilic. Acid and often visible gas is produced during fermentation of D-glucose, other carbohydrates, and polyhydroxyl alcohols. They are oxidase negative and, with the exception of Shigella dysenteriae 0 group 1 and Xenorhabdus nematophilus, catalase positive. Nitrate is reduced to nitrite (except by some strains of Erwinia and Yersina). The G + C content of DNA is 38-60 mol% (T m , Bd). DNAs from species within most genera are at least 20% related to one another and to Escherichia coli, the type species of the family. Notable exceptions are species of Yersina, Proteus, Providenica, Hafnia and Edwardsiella, whose DNAs are 10-20% related to those of species from other genera. Except for Erwinia chrysanthemi, all species tested contain the enterobacterial common antigen (Bergy ' s Manual of Systematic Bacteriology, D. H. Bergy et al., Baltimore: Williams and Wilkins, 1984). Particularly useful in the present invention are E. coli and members of the genus Salmonella and those enterics for which the osmY gene and its upstream promoter region has been identified. Culture Conditions: Typically, cells are grown at 37 °C in an appropriate medium. Preferred growth medium are common defined media such as Vogel-Bonner medium (Davis et al.. Advanced Bacterial Genetics. (1980), Cold Spring Harbor, NY:Cold Spring Harbor Laboratory). Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. In some instances rich or complete media such as NB (Nutrient broth) are used. Suitable pH ranges for bacterial growth are between pH 5.0 to pH 9.0. The range of pH 6.0 to pH 8.0 is preferred as the initial condition. Growth of the bacterial cells in liquid medium allows a uniform population of cells to be stressed at various growth phases such as early log phase, mid log phase, late log arithmic phase, or stationary phase. Screen Development Screen development utilized a variety of polycationic antimicrobial peptides including cecropin A and B, polymxyin B, polymxyin nonapeptide, Colistin, Magainin, and Mastoparan as the primary agents for the examination of the mode of action of this class of peptides and the development of a suitable screen for compounds of similar activity. The instant class of polycationic, antimicrobial peptides and proteins mediate direct exchange of phospholipid between vesicles through stable vesicle-vesicle contacts. The Applicants' subsequent discovery that this class of peptides also induced the osmy gene and micF gene (implicated in the cellular response to hyperosmotic conditions) led to the development of the screen of the present invention. Applicants speculate that the antimicrobial action against Gram-negative organisms is effective because cecropin mediates contacts between the outer layer of the plasma membrane and the inner layer of the outer membrane. As the two membranes are drawn closer together, the periplasmic space is effectively reduced. This effect may be accompanied by an efflux of water that effectively increases the solute concentration and osmolarity in the periplasmic space. Phospholipid exchange through polycationic antimicrobial peptides induced would compromise the phospholipid compositional difference that exists between these two membranes in Gram-negatives. Such exchanges may affect the viability of the organism and stasis. This mechanism is consistent with the observation that these peptides do not induce genetically stable resistance because the primary target is the phospholipid bilayer. Results show that the hyperosmotic response profiles with the fusion strains are parallel to the transcriptional stress response profiles induced by peptides like PxB. A positive transcriptional response was seen only with two fusion strains. The role of the osmY gene is osmoregulation is established, although the function of this outer membrane protein remains to be established (43, 44). The bioluminescence of an E. coli strain containing plasmid pMicFLiixl has been shown to be induced by the redox cycling agent methyl viologen under control of the soxRS regulatory circuit (34). Expression of micF is also known to be induced by binding of the multiple antibiotic stress response regulator mar A (45). Furthermore, rob, a DNA binding protein related to soxS and mar A, is known to bind the promoter region of micF (31 ) and to activate micF transcription in vitro (46). Results in Table 3 establish that induction of micF transcription by PxB, NP, colistin, and cecropin A and B does not require the Sox or Mar regulatory circuits. Therefore, a ole of the Rob protein is implicated in response to the hyperosmotic and the peptide stress. The present invention is further defined in the following Examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. These Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. EXAMPLES GENERAL METHODS General procedures and techniques suitable for use in the following examples may be found in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratory Press (1989). Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples may be found in Manual of Methods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, DC. (1994) or Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology. Second Edition (1989) Sinauer Associates, Inc.. Sunderland, MA. Standard bacterial genetic protocols can be found in Miller, J. H., Experiments in Molecular Genetics. Cold Spring Harbor Laboratory (1972); Miller, J. H., (1992) A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press; Silhavey, T. J., Berman, M. L.. and Enquist, L. W., Experiments with Gen Fusions (1984), Cold Spring Harbor Laboratory, and Davis, R. W., Botstein, D. and Roth, J. R. (1980), A Manual for Genetic Engineering-Advanced Bacterial Genetics, Cold Spring Harbor Laboratory. All reagents and materials used for the growth and maintenance of bacterial cells were obtained from Aldrich Chemicals (Milwaukee, WI), DIFCO Laboratories (Detroit. MI). GIBCO/BRL (Gaithersburg, MD), or Sigma Chemical Company (St. Louis, MO) unless otherwise specified. Strains The recombinant strains TV 1061, TV 1048, DPD2794, DPD2511, DPD2146 and DPD217C were used in the following examples. The construction of TV1061 is fully described in U.S. 5,683,868, the disclosure of which is fully incorporated by reference. TV1048, DPD2191, DPD2192, DPD2193, and DPD219 are described below. DPD2794 (see U.S. 5,683,868) is fully described in PCT/US98/03684 (USSN 60/039,582), the disclosure of which is fully incorporated by reference. DPD2511 is fully described in U.S. 5,683,868. Reagents Sources of reagents and protocols for the vesicle- vesicle exchange studies (15,16,17) are adopted from Applicants' earlier work. Specific conditions are given below. Pyrene-labelled phospholipid (pyPA, pyPM and pyPC), ANTS, and DPX were purchased from Molecular Probes (Eugene, Or). POPC, POPC ether, and NBD-PE were from Avanti. Dithionite (sodium thiosulfite) was from Mallinckrodt Chemical Works (St. Louis, MO). Syringomycine E (18) was provided by Jon Takemoto (Salt Lake City, Utah), Magainin 2 by Magainin, Inc. (Philadelphia, PA) and CCCP by Dr. Heytler (DuPont Co., Wilmington, DE). PxB, bacitracin, gramicidin A, gentamicin, valinomycin, and PxB-agarose were all obtained from Sigma Chemical Co. The purity and identity of these peptides, as well as cecropins and magainins (Bachem Co., Torrence, CA) and polymyxin- nonapeptide (Boehringer, Inc., Manheim, Germany), was confirmed by analytical HPLC, amino acid analysis and fast atom bombardment mass spectrometry (FAB-MS). The concentration of peptides in assay mixtures is expressed as mol % relative to moles of phospholipid in the reaction mixture. All manipulations for the biophysical measurements were carried out in glass containers, and (unless noted otherwise) measurements were done at 23-24 °C and pH 8.0 in 10 mM Tris buffer. Spectroscopic measurements in stirred quartz cuvette were typically carried out at less than 1.5 mole % peptide or lipid/peptide mole ratio R >75. In most assays the mediated phospholipid exchange could be seen in the presence of only a few peptide molecules per vesicle. 0.001%) or R >5000, and the peptide-induced effect was linear to > 1 mole % or R < 100. Significant nonlinearity and other functional changes (e.g., fusion, leakage, flip-flop) were typically noted at > 2 mol %. Vesicle Preparation Covesicles of DMPM containing 30 mole % POPC alone, or with indicated amount of the fluorescent probe (NBD-PE, Rh-PE, pyPM, or pyPC) were prepared by codispersion of a mixture of the lipid solutions in CHCI3/CH 3 OH (2:1 v/v); the vacuum dried film was hydrated and then sonicated above the gel-fluid transition temperature until a clear dispersion was obtained, typically 2-4 minutes in a bath type sonicator (Lab Supplies, Hickesville, NY, Gl 12SPIT). After 30 minutes of annealing, vesicles were stable for more than 5 hours without a significant change in any of the properties that were measured. For the ANTS/DPX fusion assay the vesicles contained 10 mM Tris pH 8.0 and either (i) 25 mM ANTS, (ii) 90 mM DPX, or (iii) 12.5 mM ANTS and 45 mM DPX. The vesicles were separated from unencapsulated material on Sephadex G-25 (Pharmacia) column equilibrated with 10 mM Tris, pH 8.0. To calculate the lipid concentration after the passage of the vesicles through the column, vesicles of the same composition, but doped with 0.1 % Rh-PE as a marker and without encapsulated material, were filtered. The amount of lipid recovered from this filtration was calculated according to the Rh-PE fluorescence intensity and taking the sample dilution into account. By this procedure, one calculates a 77+10 % lipid recovery and a dilution factor of 4+0.4 (means of 6 different samples). These vesicles were used within 10 hours. Typically, the regenerated G-25 column was reused 4-5 times. Asymmetrically Labelled POPC/DMPM/NBD-PE Vesicles PC/PM (30:70) covesicles containing NBD-PE (0.6 mol %) were incubated in 0.2 mL of 10 mM Tris/1 mM EGTA buffer with dithionite, to selectively reduce the NBD-PE present in the outer monolayer to a nonfluorescent derivative. After 10 minutes, the reaction mixture was diluted with more buffer to a final volume of 1.5 mL with the final concentration of 54.4 υM lipid and 2.4 mM dithionite. At this point, all the NBD-PE in the outer monolayer had reacted with dithionite. Vesicles were used immediately for the lipid mixing assay. Light Scattering. The 90° light-scattering measurements were carried out on SLM-Aminco AB2 with excitation and emissions set at 360 nm with slit widths of 1 nm. Other conditions were as for the fluorescence measurements. Typically, results are expressed as the change in the intensity on an arbitrary scale above that of vesicles alone. Fluorescence Measurements These measurements were carried out on an SLM-Aminco AB2 spectrofluorimeter equipped for magnetic stirring. Spectral manipulations were carried out with the software provided with the instrument. Typically, the slit- widths were kept at 4 nm each and the sensitivity (PMT voltage) was set for the buffer blank to 1 % for the Raman peak corresponding to the same excitation wavelength. The scattering and the PyPM exchange assays described above were also carried out by rapid mixing by Milliflow stopped-flow mixer (Spectronic Instruments) attached to SLM-Aminco AB2 spectrofluorimeter. (i) Lipid Transfer Assay The exchange of lipid between vesicles on the addition of peptide was assessed by monitoring transfer of pyrene-labelled phospholipid from co-vesicles of pyPM POPC (70:30) or pyPA/POPC to a 125-fold excess of unlabelled phospholipid vesicles of POPC/DMPM and DMPM, respectively, as acceptors. Transfer of octadecylrhodamine (R18) from vesicles containing 6% probe to unlabelled vesicles was also measured with a similar protocol. (ii) ANTS/DPX Fusion Assay Dequenching of coencapsulated ANTS and DPX fluorescence by dilution of the probes was measured to assess leakage of aqueous contents (19). Excitation was set at 360 nm, and emission was monitored at 530 nm as a function of time. The scale was calibrated with the fluorescence of the 1 : 1 mixture of ANTS- and DPX-loaded vesicles taken as 100% leakage; the fluorescence of the same concentration of vesicles containing co-encapsulated ANTS and DPX was taken as 0%) leakage. The fluorescence of the lysed vesicles containing both ANTS and DPX (using 2.2 mM deoxycholate) is the same as the fluorescence of the mixture of vesicles with ANTS and vesicles with DPX (1:1 ratio). din Accessibility of Phospholipid in POPC/DMPM Covesicles to Dithionite Reaction of small sonicated vesicles containing NBD-PE with dithionite selectively eliminated the fluorescence signal derived from NBD-PE present in the outer leaflet by a reduction reaction (20),(21 ). An aliquot of POPC/DMPM/NBD- PE covesicles was added to 1.5 mL of 10 mM Tris/1 mM EGTA buffer (pH 8.0) saturated with nitrogen, with constant stirring (lipid concentration 1 10 uM). When a stable baseline was achieved (usually less than 60 seconds after the addition), the reaction was started by adding dithionite from a stock solution to a final concentration of 20 mM. The time-dependent decrease in the fluorescence at 535 nm was recorded for 800 seconds (resolution 1 second). Excitation wavelength was set at 460 nm with both slit widths at 4 nm. Stock solutions of freshly prepared 1.44 M dithionite in 0.5 M Na 2 CO3 (pH 1 1) buffer saturated with nitrogen gas, were stored at 0 °C and used within 1 h. Civ) Inner Monolayer Mixing by Resonance Energy Transfer (RET) Asymmetrically labelled NBD-PE vesicles, where the NBD groups in the outer monolayer were chemically quenched by reaction with dithionite, were used to monitor mixing of phospholipid in the inner monolayer. A 1 :1 mixture of dithionite pre-treated POPC/DMPM/NBD-PE covesicles with POPC/DMPM/Rh- PE covesicles was used in the RET experiments. With excitation at 460 nm, the fluorescence emission from rhodamine was monitored at 592 nm, where the contribution of NBD fluorescence is minimum. The change in fluorescence was calculated as [F-F 0 ]/[F max -F 0 ], with F 0 and F corresponding to the fluorescence intensities before and after adding the peptides, and F max as the fluorescence after total mixing of inner monolayer lipids, measured with covesicles containing 0.3 mole% of each of the probes at the same total bulk lipid concentration after dithionite reaction. Media. Fusion Strains and Growth Conditions Each of the E. coli strains used in this study contains a plasmid-borne genetic fusion of one of several promoter regions of E. coli stress responsive genes to a bioluminescent luxCDABE operon. Transcription initiated at each promoter region drives expression of the lux genes resulting in a bioluminescent phenotype. Therefore, increased transcription initiation due to stress responsive regulation of gene expression leads to increased transcription of the lux reporter and hence increased bioluminescence. Several luminescence responsive strains are used in this study (Table 1). Plasmid pMicFLuxl containing a fusion of the micF promoter region of the Vibrio βscheri luxCDABE operon in the parental plasmid pUCD615 (30) was made by PCR amplification according to a previously described method (22) using the primers: 5'-ACTTAAGGATCCCCCCAAAAATGCAGAATA-3 * [SEQ ID NO:5] and 5'-AGCAGCGAATTCGGGCATCCGGTTGAAATAG-3' [SEQ ID NO: 6]. The amplified product contains 244 base pairs upstream of the start site on the micF RNA and the entire transcribed region. A series of E. coli strains that are isogenic with the exception of chromosomal mutations in the genes encoding certain regulatory proteins were used to test their effects on micF expression. E. coli strains DPD2191, DPD2192, DPD2193 and DPD2194 were made by transformation using plasmid pMICFLuxl into E. coli strains GC4468 (F-Clac4169 rpsL) (31), N7840 (F-Δlac4l69 rpsL Δ(mar sad) 1738) (32), BW829 (F-Δlac4169 rpsL Δsox-8::cat) (33), and RA4468 (F-Δlac4169 rpsL robr.kan) (31), respectively. The other E. coli strains (Table 1 ) containing genetic fusions in the V. fischeri luxCDABE reporter in parental plasmid pUCD615 have been described. These are TV 1061 containing grpE'-luxCDABE (22), DPD2794 containing recA '-luxCDABE (23), DPD2511 containing katG '-luxCDABE (34), and TV 1048 containing lac'-luxCDABE (35). In addition, several strains containing genetic fusion to the Photorhabdus luminescens luxCDABE in parental plasmid pDΕW201 (36) reporter were used. E. coli strains DPD2146 containing inaA' -luxCDABE (37) and DPD2170 containing osmY' -luxCDABE (37) have been described. Strain DPD2220 was constructed by transformation of pDEW221 with the osmY' -luxCDABE fusion into strain RA4468 with the chromosomal roό-mutation. Table 1 Properties of the Stress Sensitive Strains of E. coli Gene Type Inducer A grpEV* TV1061 Protein folding Ethanol (0.5M) B recAV* DPD2794 DNA damage Mitomycin C (0.3 mM) C katG'/* DPD2511 Oxidative H202 (15 mM) D inaA'/* DPD2146 Proton leakage Salicylate (1 mM), CCCP (15 μM) E lac/* TV 1048 Limited carbon source F osmY'/* DPD2170 Hyperosmotic Sucrose of NaCl (0.5 M) G micF/* DPD2191 Superoxide Methyl viologen H micF/mar- DPD2192 Superoxide Methyl viologen I micF/sox- DPD2193 Superoxide Methyl viologen J micF/rob- DPD2194 Superoxide Methyl viologen K osmY/rob- DPD2220 Superoxide Methyl viologen The plasmid, pLacLux, in TV 1048 was constructed by ligation of 232 base pair Pvu II to Eco RI fragment of pUC 19 (26) into Sma I and EcoRI digested pUCD615 (27). The plasmid was placed by CaCl 2 -mediated transformation into E. coli strains RFM443 (28) to yield strain TV1048. Antibiotic selection appropriate for each plasmid was used in the growth medium for all experiments. Typically, growth and measurements were carried out at 30 °C with shaking at 200 rpm in LB medium containing kanamycin monosulfate or ampicillin (50 ug/mL) to maintain the plasmid. The OD (Spectronix 2000, Bausch & Lomb) at 600 nm of the growth medium was measured after 5 to 15-fold dilution in the medium at the indicated intervals. When ODgoo reached 0.2, 5 mL culture broth was transferred to a 25 mL flask, followed by the addition of appropriate concentration of the inducer (Table 1), or other controls. The inducer solutions were pre-filtered through a 0.2 urn filter. Luminescence of the culture broth was monitored without dilution by transferring 0.2 mL aliquot to 1.5 mL polyethylene tubes (Turner Design) on a Model 20e Turner Design luminometer pre-set at a constant sensitivity. These signal values are expressed as L/L c - 1 , where L is the observed luminescence under a given set of conditions for the variable, and L c is the control luminescence without the variable. Independent controls also showed that the cells were undisturbed and viable after the luminescence measurements. EXAMPLE 1 CECROPIN-INDUCED EXCHANGE OF PHOSPHOLIPID BETWEEN VESICLES Example 1 demonstrates that cecropin A and B mediate a direct exchange of phospholipid between vesicles. Such an exchange is seen with a wide variety of proteins and peptides including CCCP, NP, gramicidin A (results not shown). Peptide-mediated contact through which direct vesicle-vesicle exchange of phospholipid occurs can be explained mechanistically (15,16) by an apposition of vesicles. Cecropin causes aggregation of vesicles monitored as an increase in the 90° light scattering of POPC/DMPM covesicles or of anionic vesicles of other compositions. The increase in the scattering is rapid and virtually complete in less than 60 seconds (results not shown). The magnitude of the change in scattering induced by cecropin depends on its mole fraction in vesicles. Qualitatively similar scattering change was seen with vesicles containing 15 to 100 mol % of other anionic phospholipid in POPC or DMPC. This suggests that stable aggregate formation by cecropin B occurs through ionic interactions. NP and gramicidin A at 1 mol % did not induce aggregation of vesicles, and these peptides were also inactive in the phospholipid exchange and osmotic stress assays described below. These results showed that the polycationic character of the peptide is a necessary but not sufficient condition for inducing aggregation of anionic vesicles. Cecropin-B mediated phospholipid exchange between vesicles is shown by results in Figure 1. Figure 1 illustrates the time-course of the cecropin B-induced increase in the fluorescence at 396 nm (ex. 346 nm) of self-quenched pyrene label present as (full line) pyPM/POPC (7:3) covesicles diluted with an 100-fold excess of DMPM/POPC (7:3) covesicles; (dotted line) pyPC vesicles diluted with lOOx excess of DMPC vesicles; (dashed line) pyPA vesicles diluted with lOOx excess of DMPA vesicles. In the case of pyPC 0.7 mole % cecropin B was added only once and a slow increase in the signal is was observed that saturates in >500 seconds. In the other two cases the cecropin induced change is was rapid and successive additions gave additional signal. In this assay, the exchange was readily monitored as an increase in the fluorescence intensity due to the dilution of self-quenched pyPM by exchange with unlabelled phospholipid from vesicles in contact. For example, a rapid increase in the monomer emission intensity occured on the addition of cecropin to pyPM vesicles mixed with an 125-fold excess of unlabelled POPC/DMPM covesicles. Phospholipid compositional selectivity of the contact formation or of exchange through the contact was measured with three other probes: pyPC, pyPA and R18. The time course of the change in the emission signal due to the dilution of self-quenched pyPA (Figure 1) or RI 8 in DMPM vesicles (results not shown) was virtually the same as with pyPM. In both cases the exchange was rapid, and sequential addition of more cecropin induced a further rapid increase in the fluorescence. These results show that cecropin-contacts in anionic vesicles do not dissociate on the time scale of a few minutes to form new contacts with other vesicles, and, further, that the cecropin B mediated exchange does not have a significant specificity for the probes. The concentration dependence of the magnitudes of PyPM exchange induced by three peptides is shown in Figure 2. Figure 2 illustrates the total change in the fluorescence emission from dilution of self-quenched pyPM/POPC (7:3) covesicles (as in Figure 1) as a function of cecropin B (circle), gramicidin A (closed triangles), or NP (open triangles) concentration. As indicated in Figure 2, Gramicidin A and NP were inert in this assay. This was also the case in the aggregation assay. The extent of the increase in the pyPM fluorescence induced by cecropin B was detectable at very low peptide concentrations (~ 0.05 mole %), and the change was linear up to at least 1.5 mole %>. More complex time-dependent changes (not shown here) at > 2 mole % cecropin were attributed to gross changes in the bilayer organization which lead to a phase change, fusion and leakage of contents (see Example 2). Controls also showed that physical forces, such as osmotic stress or amphiphile additives that affect the curvature of vesicles, do not influence the peptide- mediated rapid exchange of phospholipid. EXAMPLE 2 MIXING OF OUTER MONOLAYER PHOSPHOLIPIDS OCCURS WITHOUT LEAKAGE. FLIP-FLOP, OR FUSION One of the most sensitive assays for quantifying the outer layer lipid accessible from the bulk aqueous phase is based on the dithionite-induced quenching of fluorescence from NBD-PE codispersed in vesicles (20), (Figure 3). This assay also monitors leakage and other disruptive changes in vesicles. Figure 3 illustrates the reaction progress for the dithionite (20 mM) quenching of fluorescence of POPC/DMPM vesicles containing 0.6 mole % NBD-PE (110 uM). The fluorescence loss is due to the chemical reaction of NBD-PE with dithionite. (a) Although only one progress curve with cecropin B (1 mole %) is shown with NP, magainin 2 or without any peptide are virtually indistinguishable. (b) Progress curve with 1 mol % polylysine shows modification of all the probes. A similar progress curve is seen when 1 mol % deoxycholate or other detergents make vesicles leaky. Addition of dithionite to POPC/DMPM/NBD-PE covesicles with or without cecropin B (or NP, results not shown), results in a partial decrease (-60% of the total) of the fluorescence from NBD-PE. By this criterion cecropin or PxB- nonapeptide do not make vesicles leaky to dithionite. On the other hand as shown in Figure 3, polylysine or deoxycholate induce 100%) quenching presumably due to leakage or disruption of vesicles. The time course of the quenching of NBD-PE by dithionite has two components: the rapid phase is complete in less than 100 seconds, beyond which a slow decrease in the fluorescence continues for more than an hour. The rapid change was due to the reduction of the readily exposed lipid present in the outer monolayer of the vesicles. The residual fluorescence from the inner monolayer was modified only if vesicles became leaky and dithionite enters the inner aqueous compartment, or if the transbilayer exchange of NBD-PE is induced. Data from several controls support the conclusion that the slower phase of the fluorescence decrease is due to permeabilization of vesicles to dithionite leading to the modification of the probe in the inner monolayer. For example, if the vesicles were disrupted with deoxycholate prior to addition of dithionite, all the labeled lipid reacted in less than 100 seconds, thus showing that the reaction with exposed NBD-PE lipid is very fast. Another possibility, that dithionite is exhausted in the cuvette over the time period of the reaction, was ruled out by adding a fresh aliquot of dithionite at the end of the reaction. In this case, only a slight increase in the slower rate was observed, but not a rapid decrease in fluorescence as would be expected if there was more NBD-PE readily available for reaction. The possibility of leakage of aqueous contents induced by peptides was monitored by the ANTS/DPX fusion assay (19), with some modifications (16). In the dithionite assay, Applicants could not detect leakage in the presence of cecropin. To confirm this, and to examine the possibility of mixing of aqueous contents of vesicles by fusion under Applicants ' assay conditions, vesicles containing both ANTS and DPX were incubated with the peptides. Levels at 1 mole % NP or cecropin showed no leakage, whereas a time-dependent dequenching of ANTS fluorescence indicative of leakage of probes was seen at >3% > cecropin or fusion induced with CaCl 2 . These results at higher mole fractions cecropin B were comparable to the recently reported effects of cecropin A (29). Inner monolayer of vesicles do not mix in the presence of less than 1 mole % cecropin B. This was shown with asymmetrically labelled covesicles of POPC/DMPM with NBD-PE only in the inner monolayer (see GENERAL METHODS). Adding cecropin to an equimolar mixture of asymmetric NBD- vesicle with Rh-PE vesicles did not produce a resonance energy transfer signal. This was expected only if the inner monolayer of vesicles in contact do not mix. In short, biophysical results with probes show that the phospholipid exchange occurs through vesicle- vesicle contacts formed by cecropin B. Results at hand also rule out fusion and solubilization of vesicles as the basis for the intervesicle exchange of phospholipid at low mole fraction (<1 mole %>) of cecropin. Since peptide contacts through which the exchange occurs are essentially irreversible. Applicants rule out the possibility that hemi-fusion of vesicles has occurred, however this possibility does not influence the phospholipid exchange. EXAMPLE 3 POLYMYXIN AND CECROPIN B INDUCES HYPER-OSMOTIC STRESS IN E. COLI Example 3 illustrates that the high selectivity of cecropin B-induced phospholipid exchange correlates well with its ability to induce osmotic stress in E. coli. Results in Table 1 show that cecropin-induced luminescence response has the same profile as that induced by hyperosmolar NaCl or sucrose. Specificity for the cecropin-induced stress is shown by a positive luminescence signal only with the strain DPD2170 containing the osmotic stress promoter osmY, but not with any of the other stresses. Also, the pattern of effects induced by CCCP was quite different which rules out effects associated with leakage. Key details and controls of this study are described below. The growth inhibitory effect of cecropin B on the lac-lux fusion strain TV 1048 is shown in Figure 4. In Figure 4 the growth profile for strain TV1048 of E. coli in the absence of 0.15 uM cecropin B is shown by open symbols and the profile in the presence of 0.15 uM cecropin B is shown by closed symbols. Gramicidin A (20 uM) or NP (100 uM) did not have any effect on the growth curve for TV 1048 monitored with either of these methods (results not shown). In this strain the lux operon was coupled to the promoter for the lac operon. Therefore, a luminescence response was observed under normal growth conditions under modest carbon starvation. In the presence of 0.15 uM cecropin B the magnitude of the luminescence response and OD change was smaller at any given point in time. The effect of cecropin B on the growth, measured as a change in OD and summarized below in the upper panel of Figure 7, shows that the minimum inhibitory concentration (MIC) in the log phase of growth is 0.1 uM. As the stationary growth phase approaches, the luminescence response decreased without a significant change in the OD. The stress response requires de novo mRNA synthesis, and thus a decrease in luminescence suggests a dramatic decrease in the new transcription in the stationary phase. Therefore, for measurements for stress response as described below were carried out in the log phase of growth. All fusion strains in Table 1 were derived from the same parent, therefore their growth characteristics, monitored as a change in OD, were virtually identical under identical conditions. On the other hand, luminescence from each strain with a specific stress promoter was generally nonexistent or considerably lower during the normal growth phase, and the luminescence response increased only if stress was induced by the specific inducer. CCCP showed a distinct growth inhibitory effect on TV 1048, however as summarized in Table 1 and shown below for certain strains, there were qualitatively distinct differences in the effect of cecropin B and CCCP. The short terms effects shown in Figure 5 are instructive. Figure 5 illustrates the short-time luminescence response of TV 1048 in the early growth phase of varying concentrations of cecropin B (circles) 0.07, (square) 0.3, (triangle) 0.6, and (inverted triangles) 1.0 uM cecropin B; or (diamond) 5 or (hexagon) 30 uM CCCP added at time zero. The luminescence response was expressed as the change with CCCP (L) relative to the change in its absence (L c ). The luminescence of TV 1048 in the log growth phase decreased rapidly on the addition of CCCP at 3xMIC, although the effect was negligible below the MIC of 10 uM (top panel in Figure 6). This reduction in luminescence is expected if the ATP levels required for the luminescence reaction are lowered due to a depletion of the proton gradient. The cecropin B-induced response at MIC was virtually none; however, more significantly even at 1 OxMIC the luminescence decrease was modest and slow. These observations are consistent with other results described below that show that unlike CCCP, cecropin B does not induce stress arising from the proton leakage. Two strains were chosen to analyze the effect of the stresses induced by cecropin, CCCP, and hyperosmolarity. The CCCP concentration-dependence of the luminescence response from two strains is compared in Figure 6. Figure 6 illustrates the effect of CCCP on the luminescence response from salicylate sensitive DPD2146 (closed triangles), and hyperosmolar sensitive DPD2170 (squares). Dependence of the OD change for DPD2146 is shown in the top panel. The luminescence response measured after 60 minutes is expressed as the change with CCCP (L) relative to the change in its absence (L c ). Since the change in OD induced by these three strains was virtually identical, only the OD change in TV 1048 is shown here. Based on the change in OD, MIC for CCCP was 10 uM. The luminescence response from the osmolarity sensitive DPD2170 in the presence of CCCP was a monotonic decrease above the MIC, as was also the case with all other strains except DPD2146. The decrease in luminescence seen at higher concentrations of CCCP is attributed to the leakage of protons in all strains, as expected on the basis of reduced ATP levels available for the luminescence response. The luminescence response to CCCP from the DPD2146 strain increased below the MIC and then decreased above the MIC. Note that DPD2146 responded to the stress induced by internal acidification (25) and this corroborates with the proton translocation function of CCCP. On the other hand, the DPD2170 strain, which responded to osmotic stress does not respond to CCCP. The cecropin B concentration dependence of the luminescence response from DPD2170 and DPD2146 is compared in Figure 7, and only the results with cecropin A are shown for DPD2170. Figure 7 illustrates the effect of [cecropin] on the luminescence response from salicylate sensitive DPD2146 (closed • triangles), and hyperosmolar sensitive DPD2170 (squares). Dependence of the OD change for DPD2170 is shown in the top panel. The luminescence response measured after 60 minutes is expressed as the change with CCCP (L) relative to the change in its absence (L c ). Here the hyperosmolarity sensitive DPD2170 with osmY-lux fusion responded with the luminescence increase below the MIC, which shows that cecropin B induced the stress the end result of which was comparable to the hyperosmolarity stress. A control in Figure 8 showed that both NaCl and sucrose induced luminescence in this osmY-lux fusion strain. Figure 8 showed the time course of the change in the luminescence response of DPD2170 (circles) without any additive, (squares) after exposure to 30 uM CCCP, (open triangles) 0.25 uM NP, (solid triangles) 0.3 M NaCl, and (diamond) 0.57 M sucrose. As expected on the basis of proton gradient depletion and loss of ATP, no response was seen by CCCP above its MIC. NP had no significant effect, whereas hyperosmolar sucrose or NaCl induced an increase in luminescence. In short, results summarized in Table 1 show that the sub-inhibitory concentration of cecropin B induced a luminescence response only in one strain, that which contains the osmY-lux fusion. This was also the only strain that responded to the stress induced by hyperosmolar NaCl or sucrose. Results with sucrose clearly ruled out a possible ionic effect of NaCl and cationic cecropin B on an ion-translocation site, however the osmotic stress by cecropin was quite specific because other strains that responded to the specific stresses, dod not respond to cecropin. Comparable results (not shown) were obtained with cecropin A under the conditions of Figures 2 and 7. EXAMPLE 4 INDUCTION OF osmY ANO micF BY CECROPIN. POLYMXYIN. POLYMXYIN. COLISTIN. MAGAININ. AND MASTOPARAN Example 4 demonstrates the effect of a variety of polycationic antibacterials on strains containing osmY and micF gene fusions. The strains TV1061, DPD2794, DPD2511, DPD2146, TV1048, DPD2170, DPD2191, DPD2192, DPD2193, DPD2194 and DPD2220 (Table 1) were grown according to protocols described above and exposed to a variety of polycationic antibacterials, at the concentrations listed in Table 2. Table 2 Luminescence Response from the Stress Sensitive Strains of E. coli (it from Table 1) to Peptides and Other Additives at PC (Table 2) Strain Peptide #A B C D E F G H I J K l PxB + + + + + 2 NP + + + + 3 Colistin + + + + Colistin + NP + + + + 5 Cecropin A + + + + Cecropin B + + + + Maginin 1 + - 8 Magainin 1 + NP WS - 9 Magainin 2 + - 10 Magainin 2 + NP WS - 11 Mastoparan X WS - 12 Mastoparan X + NP + + + + 13 Mastoparan 17+ NP 14 Gramicidin A + NP 16 Valinomycin + NP 17 Gentamicin + NP 18 Bacitracin + NP Strain Peptide A B C D E F G H I J K 19 CCCP - + - - - - WS 20 Polylysine 21 Protamine 22 NaCl . .. .. .. . + + + + WS 23 Sucrose - - + + + + WS Strains A-K (Table 1). More than 2x increase in the luminescence is indicated by +. A lesser increase is indicated as a weak signal (ws). ^ Of the various strains several peptides are summarized in Table 3. Of the various strains tested, the [PxB] dependent increase in the luminescence was observed only with osmY-lux strain DPD2170 (Figure 9) and micF-lux strain, DPD2191 (Figure 10) strains; with all other strains only a monotonic decrease in the luminescence was seen above the MIC. The parameters TLRC (threshold luminescence response concentration), PLC (peak luminescence concentration), and PI (peak luminescence intensity) derived from such plots (Figure 9) for several peptides are summarized in Table 3. TABLE 3 Concentrations (μM) for 10% Inhibition of Growth of DPD2170 (MIC) Luminescence (TLRC, PC, P j ) 1 and the Fluorescence Change (FCp) Parameters # Peptide TLRC PC (PI) MIC FC 14 1 Polymyxin B (PxB) 0.1 0.25 (7.6) 0.2 0.9 2 PxB-nonapeptide (NP) 30 50 (2) 40 10.2 3 Colistin 0.1 0.3 (7.9) 0.15 1.9 4 Colistin + NP 0.07 0.3 (6.4) 0.15 5 Cecropin A 0.07 0.2 (3.5) 0.1 0.3 6 Cecropin B 0.035 0.13 (4) 0.1 0.2 7 Magainin 1 5 20 (3) 12 7.8 8 Magainin 1 + NP 5 30 (0.4) 10 9 Magainin 2 3 10 (6) 5 9.1 10 Magainin 2 + NP 5 20 (0.3) 15 11 Mastoparan X 8 20 (0.4) 12 0.72 12 Mastoparan X + NP 3 20 (1) 4 1.5 13 Mastoparan 17 + NP * * * 14 Gramicidin A + NP * * 1 16 Valinomycin + NP * * 0.7 17 Gentamicin + NP * * < 5 18 Bacitracin + NP * * 19 CCCP * * 30 ! See Figure 9 for the definition of parameters: TLC, threshold luminescence response concentration; PC, peak luminescence response concentration; PI, peak luminescence intensity. All measured parameters have an uncertainty of 20% on the same culture: FCj 2 , is the reciprocal of the signal (as the % of the maximum change in the fluorescence) induced by 1 mole % peptide in the pyPG exchange assay. Based on the repeated runs the uncertainty in these parameters is 30%. 10 μM NP, if presence as the second component. Although growth inhibition is seen with most agents in Table 2, the osmY response is induced only by certain peptides (#1-12) in actively growing osmY DPD2170 strain. The MIC values at 60 minutes for the fusion strains are identical to that for the parent strain (38), and the value (Figure 9) is in the range of TLC form osmY OPO2170 which contains the osmotic stress promoter. The peak luminescence intensity (PI) induced by the various peptides are different. According to Figure 9, PI values depend on two opposing factors: with increasing PxB concentration the luminescence increase depends specifically on the magnitude of the stress, whereas the decrease at higher concentrations depends on the magnitude of the nonspecific legal response, such as a decrease in the number of viable cells. If efficacies of peptides for the two effects are different, the PI values will vary. Results in Table 3 show that colistin, cecropins, magainins, as well as. mastoparan X and CCCP are growth inhibitors. However, not all antimicrobial agents includes the osmY transcriptional response. For example, antimicrobial ionophores, gramicidin A or valinomycin alones (results not shown) or with NP, did not have any effect on the luminescence response from DPD2170, although they inhibited growth only in the presence of NP. Also, gentamicin (39) and bactracin (40) with a membrane target for their antibiotic effects, did not cause the osmY response. In contrast, mastoparan 17, gentamicin, valinomycin, gramicidin A, and bacitracin caused no growth inhibition, at least up to 20 μm in absence of any other additive. Note that 10 μm NP, the polymyxin nonapeptide which showed a very weak growth inhibition, induced growth inhibition by gramicidin, valinomycin and gentamicin by permitting their leakage into the periplasmic space by the disrpution of the LPS layer of the outer membrane (41, 42). The Transcriptional Response is Specific to osmY and micF Fusions. Not only the luminescence response from osmY DVO2170 is induced only with some of the antimicrobial peptides, but results in Table 2 show that such peptides do not induce transcription associated with stresses associated with oxidative damage, depletion of proton gradient, or changes in the macromolecular synthesis. The transcriptional response from several fusion strains, that respond to certain stresses (Table 1), is compared in Table 2. Remarkably, only the osm and micF transcription is induced, not only by peptides #1-12, but also by hyperosmotic NaCl and sucrose. Also, these stresses do not induce transcriptional response from stains that respond to macromolecular synthesis, or deplete the proton gradient or oxidative damage. Hyperosmolar NaCl and Sucrose Induce micF -response. DPD2191 strain shows a difference in the luminescence response to hyperosmotic and hypoosmotic stress. As shown in Figure 11, an increase in luminescence in seen with hyperosmotic NaCl, but not with hypoosmotic stress. Note that a modest growth inhibition is seen with both hypo- and hyperosmolar NaCl. These results are similar to those induced by NaCl and sucrose in o^ 7DPD2170 strain (35, 38), and suggest that the luminescence response of DPD2191 from the induction oϊ micF is primarily due to an increase in the osmolarity, as is the case with osmY (43). REFERENCES ( 1 ) Hoffman, J. A. ( 1995) Corr. Opinion. Immunolo. 7, 4-10 (2) Saberwal, G., & Nagaraj, R. (1994) Biochim. Biophys. Acta 1 197, 109-131 (3) Storm, D. R., Rosenthal, K. S., Swanson, P. E. (1977) Ann. Rev. Biochem. 46, 723-763 (4) Zasloff, M. (1987) Proc. Natl. Acad. Sci U. S. 84, 5449-5453 (5) Hultmark, D., Steirer, Rasmuson, T., & Boman, H. G. (1980) Eur. J. Biochem. 106, 7-16 (6) Lehrer, R. I. (1993) Ann. Rev. Immunology 11, 105-. (7) Gazit, E., Boman, A., Boman, H. G., & Shai, Y. (1995) Biochemistry 34, 11479-11488 (8) Pratt, W. B., & Fekety, R. (1986) The Antimicrobial Drugs, Oxford University Press, pp. 252-261 (9) Duclohier, H., Molle, G., & Spach, G. ( 1989) Biophys. J. 56, 1017- 1021 (10) Vaara, M. ( 1992) Microbiol. Rev. 56, 395-411 (11) Cornut, I., Thiaudiera, E., & Dufourcq, j. (1993) in The Amphipathic _- Heli (Epand, R., Ed.) pp. 173-219. CRC Press, Boca Raton, FL. Dixon, R. A., & Chopra, I. (1986) Antimicrobial Chemotherapy 18, 557-563. (12) Wieprecht, T., Dathe, M., Epand, R. M., Beyermann, M., Krause, E., Maloy, W. L., MacDonald, D. L., & Bienert, M. (1997) Biochemistry 36, 12869-12880 (13) Lee, J., & Lentz, B. R. (1997) Biochemistry 36, 6251-6259 (14) Pecheur, E., Hoekstra, D., Sainte-Marie, J., Maurin, L., Bienvenue, A., Philippot, j. R. (1997) Biochemistry 36, 3773-81 (15) Cajal, Y., Rogers, J., Berg, O. G., & Jain, M. K. (1996a) Biochemistry 35, 299-308 (16) Cajal, Y., Ghanta, J., Easwaran, K., Surolia, A., & Jain, M. K. (1996b) Biochemistry 35, 5684-5695 (17) Cajal, M. K., & Jain, M. K. (1997) Biochemistry 36, 3882-3893 (18) Feigin, A. M., Takemoto, J. Y., Wangspa, R., Teeter, J. H., & Brand, J. G. (1996) J. Membrane Biol. 149, 41-47 (19) Ellens, H., Bentz, J., & Szoka, F. C. (1985) Biochemistry 24, 3099-3106 (20) Mclntyre, J. C, & Sleight, R. G. (1991) Biochemistry 30, 1 1819-1 1827 (21) Hoekstra, D.. Buist-Arkema, R., Klappe, K., and Reutelingsperger, C. P. M. (1993) Biochemistry 32, 14194-14202 (22) Van Dyk, T. K., Belkin, S., Vollmer, A. C, Smulski, D. R., Reed, T. R., & LaRossa, R. A. (1994a). In Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects, A. K. Cambell, L. J. Kricka & P. E. Stanley (eds.). John Willow & Sons., Chichester. pp. 147-150 (23) Vollmer, A. C, Belkin, S., Smulski, D. R., Van Dyk, T. K., & LaRossa. R. A. (1997) Appl. Environ. Microbiol 63, 2566-2571 (24) Belkin, S., Smulski, D. R., Dadon, S., Vollmer, A. C, Van Dyk, T. K., & LaRossa, R. A. A. (1994). Appl. Environ. Microbiol. 60, 1414-1420 (25) Van Dyk, T. K., Ayers, B. L., Morgan, R. W., & LaRossa, R. A. (1998a), submitted (26) Yanisch-Perron, C, Vieira, J., & Messing, J. (1985) Gene 33, 103-119 (27) Rogowsky, M., Phoenix, P., Menzel, R., Masse, E., Liu, L. F., & Crouch, R. J. (1995) J. Bacteriol 169, 5101-5112 (28) Drolet, M., Phoenix, P., Menzel, R., Masse. E., Liu, L. F., & Crouch, R. j. (1995) Proc. Natl. Acad. Sci USA 92, 3526-3530 (29) Silvestro, L., Gupta, K., Weiser, j. N., Axelsen, P. H. (1997) Biochemistry 36, 11452-60 (30) Rogowsky, P. M., T. j. Close, J. A. Chimera, J. J. Shaw, and C. I. Kado. 1987. Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58. J. Baceteriol. 169:5101-5112 (31) Ariza, R. R., Z. Li, N. Ringstad, and B. Demple. 1995. Activation of multiple antibiotic resistance and binding of stress-inducible promoters by Escherichia coli Rob protein, j. Baceriol. 177:1655-1661 (32) Rosner, J. L., and J. L. Slonczewski. 1994. Dual regulation of inaA by the multiple antibiotic resistance (Mar) and superoxide (soxRS) stress response systems of Escherichia coli. J. Bacteriol. 176:6262-6269 (33) Tsaneva, I. R., and B. Weiss. 1990. soxR, a locus governing a superoxide response regulon in Escherichia coli. J. Bacteriol. 172:4197-4205 (34) Belkin, S., A. C. Vollmer, T. K. Van Dyk, D. R. Smulski, T. R. Reed and R. A. LaRossa. 1994. Oxidative and DNA damaging agents induce luminescence in E. coli harboring lux fusions to stress promoters. P. 509-512. In A. K. Campbell, L. J. Kricka and P. E. Stanley (Ed.) Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects. John Wiley & Sons Ltd., Chichester. NY (35) Oh., J.-T., Y. Cajal, P. S. Dhurjati, T. K. Van Dyk, and M. K. Jain. 1998b. Cecropin induces hyperosmotic stress response in viable Escherichia coli through membrane contacts in the periplasmic space. Submitted. (36) Van Dyk, T. K. and R. A. Rosson. 1998. Photohabdus luminescens luxCDABE promoter probe vectors. 102:85-95. In R. A. LaRossa (ed.) Methods in Molecular Biology: Bioluminscencent Protocals. Humana Press Inc., Clifton, NJ (37) Van Dyk, T. K., B. L. Ayers, R. W. Morgan and R. A. LaRossa. 1998. Constricted flux through the branched-chain amino acid biosynthetic enzyme acetolactate synthase triggers elevated expression of genes regulated by rpoS and internal acidification. J. Bacteriol. 180 785-792 • (38) Oh, J.-T., T. K. Van Dyk, Y. Cajal, P. S. Dhurjati, M. Sasser, and M. K. Jain. 1998a. Osmotic Stress in Viable Escherichia coli as the basis for the antibiotic response by polymyxin B. Biochem. Biophys. Res. Comm. 246:619-623 (39) Kadurugamuwa, J. L., A. J. Clark, and T. J. Beveridge. 1993. Surface action of gentamicin on Pseudomonas aeruginosa. J. Bacteriol. 175:5798-5805 (40) Pollock, T. J., L. Thorne, M. Yamazaki, M. J. Mikolajczak, and R. W. Armentrout. 1994. Mechanism of bacitracin resistance in gram-negative bacteria that synthesize exopolysaccharides. J. Bacteriol. 186:6629-6237 (41) Kubesch, R., J. Boggs, L. Luciano, G. Maass, and B. Tummler. 1987. Interaction of polymyxin B nonapeptide with anionic phospholipid. Biochemistry 26:2139-2149 (42) Vaara, M., and T. Vaara. 1983. Sensitization of Gram-negative bacteria to antibiotics and complement by a nontoxic oligopeptide. Nature 303:526-588 (43) Yim, H. H., and M. Villarejo. 1992. osmY, a new hperosomotically inducible gene, encodes a periplasmic protein in Escherichia coli. J. Bacteriol. 174:3637-3644 (44) Yim, H. H., R. L. Brems, and M. Villarejo. 1994. Molecular characterization of the promoter of osmY, an rpoS-dependent gene. J. Bacteriol. 176:100-107 (45) Jair, J. W., R. G. Martin, J. L. Rosner, N. Fujita, A. Ishihama, and R. E. Wolf, Jr. 1995. Purification and regulatory properties of MarA protein, a transcriptional activator of Escherichia coli multiple antibiotic and superoxide resistance promoters. J. Bacteriol. 177:7100-7104 (46) Jair, J. W., X. Yu, K. Skarstand, B. Thony, N. Fujita, A. Ishihama, and R. E. Wolf. 1996. Transcriptional activation of promoters of the superoxide and multiple antibiotic resistance regulon by Rob, a binding protein of the Escherichia coli origin of chromosomal replication. J. Bacteriol. 178:2507-2513 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM A. The indications made below relate to the microorganism referred to in the description on page , line 17 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | | Name of depositary institution AMERICAN TYPE CULTURE COLLECTION Address of depositary institution (including postal code and country) 10801 University Blvd. Manassas, Virginia 20110-2209 USA Date of deposit Accession Number 13 May 1993 ATCC 69315 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet Q In respect of those designations in which a European patent is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample. (Rule 28(4) EPC) D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if th indications are not for all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the International Bureau later (specify the general nature o " the indications e g . "Accession Number of Deposit") For receiving Office use only For international Bureau use onlv I L liis sheet was received with the international application I I This sheet was received by the International Bureau on f_O US 05 ftlAR1999 6s.03.'??) Λuti W Authorized officer Tw a s rtecaF-tfvrhHaarrrrccαα I orm PCT/RO/134 (July 1992)

Claims: WHAT IS CLAIMED IS: 1. A method for identifying polycationic, peptide-like compounds with antibacterial activity comprising: (i) contacting a polycationic, peptide-like compound with a detector cell, the detector cell comprising an osmotic stress promoter operably linked to a reporter gene; and (ii) measuring a change in signal emitted by the detector cell before and after the contacting with the polycationic, peptide-like compound in step (i), wherein an increase in signal indicates that the polycationic, peptide-like compound has antibacterial activity. 2. The method of Claim 1 wherein the reporter gene is selected from the group consisting of the lacZ gene encoding .beta.-galactosidase, the cat gene encoding chloramphenicol acetyl transferase. the galK gene encoding galactose kinase, the gus gene encoding .beta.-glucosidase. the luc gene encoding insect luciferase. the gjp gene encoding green fluorescent protein, the genes encoding proteins responsible for bioluminescence from Renilla sp. , and the Lux genes responsible for bioluminescence. 3. The method of Claim 1 wherein the osmotic stress promoter is selected from the group consisting of the osm Y promoter and the micF promoter. 4. The method of Claim 1 wherein the detector cell comprises: (i) the osmotic stress promoter operably linked to a reporter gene; and (ii) an enteric bacteria. 5. The method of Claim 4 wherein the enteric bacteria is selected from the group of genera consisting of Escherichia and Salmonella. 6. The method of Claim 1 wherein the polycationic, peptide-like compound with antibacterial activity has an activity similar to compounds selected from the group consisting of defensin, cecropin, polymyxin, apidaecins, melittin, bombinin, Colistin, and Mastoparan and magainin and combinations thereof.

Inventors: CAJAL YOLANDA, JAIN MEHENDRA KUMAR, VAN DYK TINA K, CAJAL, YOLANDA, JAIN, MEHENDRA, KUMAR, VAN DYK, TINA, K.
Applicants: CAJAL YOLANDA, DU PONT, DYK TINA K VAN, JAIN MEHENDRA KUMAR, UNIV DELAWARE, CAJAL, YOLANDA, E.I. DU PONT DE NEMOURS AND COMPANY, JAIN, MEHENDRA, KUMAR, UNIVERSITY OF DELAWARE, VAN DYK, TINA, K.
Application year: 1999
Main classification: C12Q1
IPC codes: C12N15, C12Q1