WO1999045152A1 - Methodes d'identification de composes polycationiques, de type peptidique, a action antibacterienne - Google Patents

Methodes d'identification de composes polycationiques, de type peptidique, a action antibacterienne Download PDF

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WO1999045152A1
WO1999045152A1 PCT/US1999/004795 US9904795W WO9945152A1 WO 1999045152 A1 WO1999045152 A1 WO 1999045152A1 US 9904795 W US9904795 W US 9904795W WO 9945152 A1 WO9945152 A1 WO 9945152A1
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peptide
polycationic
cecropin
vesicles
luminescence
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PCT/US1999/004795
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WO1999045152A8 (fr
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Tina K. Van Dyk
Yolanda Cajal
Mehendra Kumar Jain
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E.I. Du Pont De Nemours And Company
University Of Delaware
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Priority to IL13793499A priority Critical patent/IL137934A0/xx
Priority to AU28950/99A priority patent/AU2895099A/en
Priority to JP2000534683A priority patent/JP2002505120A/ja
Priority to CA002321931A priority patent/CA2321931A1/fr
Priority to EP99909832A priority patent/EP1060268A2/fr
Publication of WO1999045152A1 publication Critical patent/WO1999045152A1/fr
Publication of WO1999045152A8 publication Critical patent/WO1999045152A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

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  • 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.
  • 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
  • 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.
  • 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.
  • 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).
  • 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.
  • FIG. 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 o ⁇ micF
  • ATCC American Type Culture Collection
  • SEQ ID NO:l refers to a specific polycationic peptide-like antibacterial known as "polymyxin B" or "PxB” and having the general formula:
  • SEQ ID NO: 2 refers to a specific polycationic peptide antibacterial known as "Cecropin A" having the general formula:
  • 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:
  • SEQ ID NO:5 refers to the primer
  • 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.
  • polycationic, peptide-like refers 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.
  • peptides examples 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.
  • polycationic describes a polycationic peptide-like antibacterial and refers to the quality of having multiple positive charges.
  • polymyxin B or "PxB” refers to a specific polycationic peptide-like antibacterial having the general formula:
  • Cropin A refers to a specific polycationic peptide antibacterial having the general formula: KWKLFKKIEKVGENIRDGIIKAGPAVAVVGEATEIAK-NH 2 [SEQ ID NO:2]
  • Cropin B refers to a specific polycationic peptide antibacterial having the general formula: KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL- NH 2 [SEQ ID NO:3]
  • 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]
  • 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.
  • osmotic stress refers to a stress produced on a bacterial cell that results in the induction of an osmosensitive bacterial stress promoter.
  • osmY' refers to a bacterial gene responsive to osmotic stress.
  • micF refers to a bacterial gene which is known to regulate . expression of E. coli membrane proteins.
  • reporter 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.
  • reporter gene means any gene that when operably linked to a suitable promoter generates a detectable signal.
  • 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.
  • operably linked refers to the fusion of two fragments of DNA in a proper orientation and reading frame to be transcribed into functional RNA.
  • bioluminescence refers to the phenomenon of light emission from any living organism.
  • luminaireescent 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:
  • ANTS means l-aminonapthalene-3,6,8-trisulfonic acid
  • CCCP 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 polymyxin B nonapeptide
  • PKA2 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
  • Dab means a,g-diaminobutyric acid.
  • TRC threshold luminescence response concentration
  • 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
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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
  • 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.
  • 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 ⁇ M 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.
  • 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.
  • 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.
  • 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).
  • lipid concentration 1 10 uM lipid concentration 1 10 uM
  • Excitation wavelength was set at 460 nm with both slit widths at 4 nm.
  • Civ Inner Monolayer Mixing by Resonance Energy Transfer (RET)
  • RET Resonance Energy Transfer
  • 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.
  • Plasmid pMicFLuxl containing a fusion of the micF promoter region of the Vibrio ⁇ scheri 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.
  • 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- ⁇ lac4l69 rpsL ⁇ (mar sad) 1738) (32), BW829 (F- ⁇ lac4169 rpsL ⁇ sox-8::cat) (33), and RA4468 (F- ⁇ lac4169 rpsL robr.kan) (31), respectively.
  • 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).
  • several strains containing genetic fusion to the Photorhabdus luminescens luxCDABE in parental plasmid pD ⁇ W201 (36) reporter were used.
  • Strain DPD2220 was constructed by transformation of pDEW221 with the osmY' -luxCDABE fusion into strain RA4468 with the chromosomal ro ⁇ -mutation.
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 2 illustrates the concentration dependence of the magnitudes of PyPM exchange induced by three peptides as a function of cecropin B (circle), gramicidin A (closed triangles), or NP (open triangles) concentration.
  • 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).
  • 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.
  • 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.
  • 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.
  • FIG. 4 The growth inhibitory effect of cecropin B on the lac-lux fusion strain TV 1048 is shown in Figure 4.
  • 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).
  • 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.
  • 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.
  • 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 ).
  • 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.
  • DPD2146 responded to the stress induced by internal acidification (25) and this corroborates with the proton translocation function of CCCP.
  • 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 ).
  • 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
  • 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 demonstrates the effect of a variety of polycationic antibacterials on strains containing osmY and micF gene fusions.
  • DPD2191, DPD2192, DPD2193, DPD2194 and DPD2220 were grown according to protocols described above and exposed to a variety of polycationic antibacterials, at the concentrations listed in Table 2.
  • 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 ⁇ M NP, if presence as the second component.
  • 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.
  • 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.
  • antimicrobial agents includes the osmY transcriptional response.
  • 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.
  • gentamicin (39) and bactracin (40) with a membrane target for their antibiotic effects did not cause the osmY response.
  • mastoparan 17 gentamicin, valinomycin, gramicidin A, and bacitracin caused no growth inhibition, at least up to 20 ⁇ m in absence of any other additive.
  • 10 ⁇ m 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.
  • 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.'??)

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Abstract

Cette invention a trait à une méthode d'identification de composés polycationiques, de type peptidique, se caractérisant par leur action antibactérienne à l'encontre de bactéries Gram négatives. Cette méthode tire parti de l'induction d'une contrainte hyperosmotique sans perte de contenu cytoplasmique. Il est déterminé, dans le cadre de cette méthode si le composé est apte à assurer une échange phospholipidique entre les bi-couches lipidiques concurremment avec l'induction de la réponse de contrainte osmotique dans la cellule cible. La détermination de l'induction de la réponse de contrainte osmotique se fait à l'aide d'un organisme détecteur bioluminescent possédant une fusion de gènes osmY-LUX.
PCT/US1999/004795 1998-03-06 1999-03-05 Methodes d'identification de composes polycationiques, de type peptidique, a action antibacterienne WO1999045152A1 (fr)

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IL13793499A IL137934A0 (en) 1998-03-06 1999-03-05 Methods for identifying polycationic, peptide-like compounds with antibacterial activity
AU28950/99A AU2895099A (en) 1998-03-06 1999-03-05 Methods for identifying polycationic, peptide-like compounds with antibacterial activity
JP2000534683A JP2002505120A (ja) 1998-03-06 1999-03-05 抗菌活性を有するポリカチオン性ペプチド様化合物を同定する方法
CA002321931A CA2321931A1 (fr) 1998-03-06 1999-03-05 Methodes d'identification de composes polycationiques, de type peptidique, a action antibacterienne
EP99909832A EP1060268A2 (fr) 1998-03-06 1999-03-05 Methodes d'identification de composes polycationiques, de type peptidique, a action antibacterienne

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006066588A2 (fr) 2004-12-23 2006-06-29 Novozymes A/S Sequences de commande reagissant a des amp et leurs utilisations
WO2006096063A1 (fr) * 2005-03-11 2006-09-14 Stichting Voor De Technische Wetenschappen Construction et methode pour cribler, selectionner et identifier des facteurs de stress
CN110684098A (zh) * 2019-10-09 2020-01-14 江苏医药职业学院 制备东方火腹蟾蜍抗菌肽bombinin的方法及其应用

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WO1994005810A1 (fr) * 1992-08-28 1994-03-17 Trustees Of Tufts College Recherches d'operon resistant a plusieurs antibiotiques
WO1994013831A1 (fr) * 1992-12-04 1994-06-23 E.I. Du Pont De Nemours And Company Procede tres sensible destine a la detection d'agressions de l'environnement
WO1994017208A1 (fr) * 1993-01-21 1994-08-04 President And Fellows Of Harvard College Methodes et trousses de diagnostic faisant appel aux promoteurs de stress des mammiferes pour determiner la toxicite d'un compose
WO1996016187A1 (fr) * 1994-11-23 1996-05-30 E.I. Du Pont De Nemours And Company Reactif bacterien bioluminescent lyophilise servant a la detection d'agents toxiques

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EP0168933A2 (fr) * 1984-06-05 1986-01-22 AMERSHAM INTERNATIONAL plc Détection et/ou identification de micro-organismes dans un échantillon par bioluminescence ou par d'autres marqueurs exogènes introduits génétiquement
WO1994005810A1 (fr) * 1992-08-28 1994-03-17 Trustees Of Tufts College Recherches d'operon resistant a plusieurs antibiotiques
WO1994013831A1 (fr) * 1992-12-04 1994-06-23 E.I. Du Pont De Nemours And Company Procede tres sensible destine a la detection d'agressions de l'environnement
WO1994017208A1 (fr) * 1993-01-21 1994-08-04 President And Fellows Of Harvard College Methodes et trousses de diagnostic faisant appel aux promoteurs de stress des mammiferes pour determiner la toxicite d'un compose
WO1996016187A1 (fr) * 1994-11-23 1996-05-30 E.I. Du Pont De Nemours And Company Reactif bacterien bioluminescent lyophilise servant a la detection d'agents toxiques

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JOON-TAEK OH ET AL: "Osmotic stress in viable Escherichia coli as the basis for the antibiotic response by polymyxin B", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 246, 29 May 1998 (1998-05-29), pages 619 - 23, XP002107635 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006066588A2 (fr) 2004-12-23 2006-06-29 Novozymes A/S Sequences de commande reagissant a des amp et leurs utilisations
WO2006066588A3 (fr) * 2004-12-23 2006-08-24 Novozymes As Sequences de commande reagissant a des amp et leurs utilisations
WO2006096063A1 (fr) * 2005-03-11 2006-09-14 Stichting Voor De Technische Wetenschappen Construction et methode pour cribler, selectionner et identifier des facteurs de stress
CN110684098A (zh) * 2019-10-09 2020-01-14 江苏医药职业学院 制备东方火腹蟾蜍抗菌肽bombinin的方法及其应用

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