Patent Application: US-63244905-A

Abstract:
antibacterial peptides and their multimeric analogues , with a wide range of action and low haemolytic activity are described . in particular , the peptide molecules exhibit a high antimicrobial activity against numerous bacterial species , with reduced cytotoxicity and a low haemolysis rate . the molecules of the invention are advantageously usable as therapeutic agents and coadjutants against infections caused by strains that are resistant to common antibiotics .

Description:
the authors of the invention have identified peptide sequences capable of interacting with the bacterial membrane and hence potentially to perform an antibiotic effect according to the mechanism proposed for natural antimicrobial peptides . therefore , the object of the present invention is an antibacterial peptide having one of the following amino acid sequences from the amino to the carboxylic terminal : qekirvrlsa , qakirvrlsa , qkkirvrlsa , kirvrlsa or any derivative thereof , wherein one amino acid residue is replaced by an alanine residue or wherein one positively charged amino acid is replaced by another positively charged amino acid . preferably the peptide has one of the following amino acid sequences from the amino to the carboxylic terminal : akkirvrlsa , qakirvrlsa , qkairvrlsa , qkkarvrlsa , qkkiavrlsa , qkkirarlsa , qkkirvalsa , qkkirvrasa , qkkirvrlaa . more preferably the peptide has the amino acid sequence qkairvrlsa . alternatively the peptide has one of the following amino acid sequence : qrkirvrlsa , qkrirvrlsa , qrrirvrlsa . in an embodiment the peptide is of linear form , preferably multimerised on a skeleton of polyacrylamide , on a skeleton of dextrane units or on a skeleton of ethylene glycol units . in a preferred embodiment the peptide is in the form of multiple antigenic peptides ( map ), having the following formula : in which r is the peptide as claimed in claim 1 - 4 ; x is a trifunctional molecule ; m = 0 or 1 ; n = 0 , if m = 0 ; n = 0 or 1 , if m = 1 . preferably x is an amino acid having at least two functional aminic groups , more preferably x is lysine , ornithine , nor - lysine or amino alanine . the peptides of the invention are used for the preparation of a medicament with antibacterial activity . the person skilled in the art will choose the appropriate form of administration and dosage , selecting suitable dilutants , coadjutants and / or excipients . preferred forms are eyewashes , mouthwashes , solutions for topical use . the peptides of the invention are also used for the preparation of disinfectant and / or detergent products with antibacterial activity . the peptides of the invention are also used as preservatives for the preparation of food products and / or of cosmetic products and / or of homeopathic products . fig1 . antibacterial activity of l1 and m1 on e . coli ( tg1 strain ) compared to non correlated map ( mnc ) used as negative control . the effect on bacterial growth was assessed at various concentrations ( 2 - 0 . 12 mg / ml ). m1 and l1 inhibited significantly e . coli growth while mnc , as expected , exhibited no antibacterial activity . fig2 . antibacterial activity of ( a ) monomeric linear peptides l1 (▪), l4 ( ), l5 ( ) and l6 (□) and ( b ) tetrabranched map4 form m1 (▪), m4 ( ), m5 ( ) and m6 (□). experiments were performed incubating e . coli tg1 cells ( 8 × 10 7 cfu / ml ) with the indicated amounts of peptide . the survival percentage is the number of living colonies with respect to the number of colonies in controls without peptides . fig3 . time - kill kinetics of m6 against e . coli atcc 25922 ( a ) and p . aeruginosa atcc 27853 ( b ). symbols : ♦, growth control ; ▪, 2 × mic concentration ( 16 μg / ml ) for e . coli atcc 25922 and 8 μg / ml for p . aeruginosa atcc 27853 ); δ , 4 × mic ( 32 μg / ml ) for e . coli atcc 25922 and 16 μg / ml for p . aeruginosa atcc 27853 ). fig4 . cytotoxicity of m1 on j774 a . 1 , cho and spo cells . the figure shows the cytotoxicity of the map m1 peptide expressed in terms of percent of survival evaluated on murine macrophage cells ( j774 a . 1 ), murine myeloma ( spo ) and chinese hamster ovary epithelium cells ( cho k1 ) by means of a colorimetric assay ( mtt ). m1 was added to the various cell lines ( 6 × 10 4 cells / well ) at three different concentrations and incubated for 24 hours at 37 ° c . then 100 μl of mtt were added to each well and incubated for 90 min at 37 ° c . the absorbance values at 595 and 650 nm were measured . fig5 . toxicity of m4 (*), m5 ( δ ) and m6 () dendrimeric peptides on ( a ) mouse macrophage cell line j774 . a1 and ( b ) human hacat keratinocytes . cell viability was measured by a calorimetric assay ( mtt ). data points represent means of three replicates . fig6 . stability of m1 peptide in solution . time course of antibacterial activity of m1 on e . coli strain tg1 . map m1 peptide was dissolved in pbs at a concentration of 0 . 5 mg / ml and bactericide activity was measured 1 , 48 and 72 hours after re - suspension in pbs . fig7 . hplc profiles of linear ( l1 ) and dendrimeric ( m1 ) peptides in serum . ( a ) l1 in serum at 0 h . ( b ) l1 after incubation in serum for 2 h : the peptide is no longer detectable . ( c ) m1 in serum at 0 h . ( d ) m1 after incubation in serum for 24 h : the peptide is still present . the vertical bar indicates peptide retention time ( min ). experiments performed in plasma were comparable . fig8 . stability of m4 , m5 and m6 peptides in solution . time course of antibacterial activity of m4 , m5 and m6 on e . coli strain tg1 . m4 , m5 and m6 peptides were dissolved in pbs at a concentration of 0 . 5 mg / ml and bactericide activity was measured 1 , 48 and 144 hours after re - suspension in pbs . fig9 . effect of m5 and m6 on haemolysis of human erythrocytes . the figures show the haemolytic activity of map m5 and m6 peptides on human erythrocytes evaluated by means of erythrocyte osmotic resistance of parpart method in nacl . the percentage of haemolysis is calculated by means of a calibration curve obtained by incubating erythrocytes with increasing concentrations of nacl . after 30 min of incubation , m5 and m6 ( at the maximum concentration tested ) displayed only a weak haemolytic activity (& lt ; 5 %). after 19 hours of incubation , the haemolysis induced by m6 and m5 at 125 μg / ml is 7 % and 19 %, respectively . the percentage of haemolysis of untreated blood after 19 hours ( control ) is very limited (& lt ; 1 %). fig1 . kinetics of membrane permeabilization of ml - 35 e . coli by m4 (*), m5 ( δ ), m6 () and of untreated cells (□). permeabilization was determined by spectrophotometric recording of hydrolysis of p - nitrophenyl - β - d - galactopyranoside , a substrate for β - galactosidase in the cytosol of bacterial cells . bacteria were treated with 16 μg / ml of dendrimeric peptides . fig1 . binding analysis between map m6 peptide and lps in biacore . the figure shows the sensorgram derived from the binding of lps on map m6 immobilised in the dextrane matrix of the biacore sensorchip . on the y - axis are shown the units of response derived from the binding between lps and m6 as a function of time expressed in seconds ( on the x - axis ) fig1 . gel retardation assay . binding was assayed by the inhibitory effect of peptides on the migration of dna . various amounts of m6 peptide were incubated with 200 ng of e . coli plasmid vector pcep4 at room temperature for 1 h and the reaction mixtures were applied to a 1 % ( w / v ) agarose gel electrophoresis . fig1 . clsm image of tg1 e . coli cells treated with rhodamine - labelled m6 after ( a ) 5 min and ( b ) 240 min of incubation . fig1 . bacterial inner - membrane permeation induced by m6 and visualized by fitc fluorescence . fig1 . detection of membrane - perturbed bacteria using double staining with fitc and pi fluorescent probes . ( a ) m6 at 5 μg / ml and ( b ) m6 at 40 μg / ml . the authors have produced and used a phage library of peptides with random sequence at high variability (˜ 10 10 ), in which each peptide is formed by 10 amino acid residues . the selection of the specific ligands was made by incubating the entire library with a solution of whole cells of e . coli , strain tg1 ( at the od 600 of about 0 . 1 ) in pbs . after 1 hour of incubation , the bacteria were centrifuged and the supernatant was eliminated . several washings with pbs - tween followed by centrifugation and elimination of the supernatant were performed to eliminate all the phages which bind aspecifically to the bacterial surface or which expose peptides with low affinity for the bacterial membrane . a glycine solution ( 0 . 2 m , ph 2 . 2 ) was added to the test tube containing bacteria and specific phages for 10 minutes , in order to determine the detachment of the phages bound to the membrane . after further centrifugation , the supernatant containing the eluted phages was collected . the selected phages were amplified in bacterial cells and used for two more rounds of selection . at the end of the process , the presence of specific phages was verified by elisa assay . dna analysis revealed the predominance of a sequence with potential amphipathic properties and positive net charge : qekirvrlsa ( l1 ). the letters are the acronyms of the aminoacids in accordance with iupac - iub nomenclature . it should be noted that the isolated sequence has the typical pattern of antimicrobic peptides which is characterised by alternating hydrophobic residues and positively charged residues ( k and r ). the peptide in question was synthesised in linear form and in tetrabranched multimeric form map ( multiple antigen peptide ) ( 20 ), in which four identical peptides are bonded to a lysine core ( u . s . pat . no . 5 , 229 , 490 ). it has been shown that map multimeric forms , due to the presence of 4 peptides in the same molecule , displayed increased antimicrobial activity . in addition , map multimeric form constitute peptides that are more resistant to the peptidase activity of blood , compared to their homologous linear peptides ( 22 , 23 ), enabling to overcome the bottleneck of the development and in vivo use of new peptide drugs . m1 efficacy showed a drop in activity over time ( fig6 ), once resuspended in solution . mass spectrometry analysis performed on the peptide at various time points indicated that the loss of activity was probably due to amide bond formation between the carboxylic group of the glutamic acid ( e ) in position two and the adjacent aminic group of lysine ( k ), with the elimination of an h 2 o molecule ( not shown ). in order to potentially improve the characteristics of the original sequence qekirvrlsa , three peptides were synthesised , starting from the original sequence and replacing glutamic acid ( e ) with a hydrophobic residue such as alanine ( a ), or with a positively charged residue such as lysine ( k ), and lastly performing a deletion of the first two aminoacids at the amino - terminal end . the sequences of the map peptides thus modified are qakirvrlsa ( m4 ), qkkirvrlsa ( m6 ), kirvrlsa ( m5 ) ( table 1 ). the antimicrobic activity of the peptides in linear form ( l1 , l4 , l5 , l6 ) and in map form ( m1 , m4 , m5 , m6 ) was assayed on the tg1 strain of e . coli . the peptides were incubated at various concentrations ( 2 - 1 - 0 . 5 - 0 . 25 - 0 . 12 mg / ml ) with cells of e . coli ( od 600 = 0 . 2 ) for about 1 hour at 37 ° c . subsequently , the cells were plated on agar at dilution such to allow counting of individual colonies . the antimicrobic activity of the synthesised peptide l1 and m1 is shown fig1 and is compared to a non correlated map peptide ( mnc ) used as negative control . while the non correlated map peptide exhibits no activity on bacterial colony growth , the authors observed that the inhibitory activity of the peptide m1 in dendrimeric form is greater than the one of the linear peptide , l1 . this demonstrates that the efficacy of the antibacterial peptide depends exclusively on its primary sequence . the survival percentage ( number of living colonies with respect to the number of colonies in control conditions without peptide ) after treatment with the original peptide and with the modified peptides was determined ( fig2 ). the authors observed that in dendrimeric map4 form the peptides m1 , m4 , m5 and m6 presented a greater activity than their linear counterparts ( l1 , l4 , l5 and l6 ) ( fig2 a and b ). the modified peptides ( m4 , m5 , m6 ) showed good antibacterial activity . notably , m5 and m6 ( which contain one and two additional positive charges , respectively ) prevented tg1 e . coli colony growth at concentrations down to 6 . 25 μg / ml , whereas m1 and m4 appeared less effective at the same concentrations ( fig2 b ). minimum inhibitory concentrations ( mic ) of m4 , m5 and m6 were determined for the reference strains : s . aureus atcc 25923 , e . coli atcc 25922 , chryseobacterium meningosepticum ccug 4310 and p . aeruginosa atcc 27853 , as well as for a number of recent clinical isolates ( including multidrug - resistant ones ) of various species ( table 2 ). mic is defined as the lowest concentration , in an antibiotic dilution range , that inhibits visible bacterial growth . the importance of mic sensitivity test is based on the principle that in vitro sensitivity provides a predictive indication of the in vivo efficacy of the antibiotic therapy . values are expressed as molar concentration and compared to mic values obtained with commercially available antibiotics such as amikacin , ceftriaxone and levofloxacin ( table 3 ). from these data , it is readily apparent that the values of mic for m4 , m5 and m6 are low ( in the order of 10 − 6 - 10 − 7 m ) whereas the best antimicrobic peptides known in the literature reach mic values of around 10 − 6 m ( 0 . 25 - 4 μg / ml ) ( 25 ). all peptides showed relatively poor activity against s . aureus , appearing to be more active against gram - negative bacteria , with m6 being the most active against all species . m6 presented also a good inhibitory activity against e . coli , klebsiella pneumoniae , enterobacter spp . and p . aeruginosa , including clinical isolates showing a multiple - drug resistance phenotype . a somewhat lower activity was observed against citrobacter freundii and acinetobacter baumanii , and even lower activity against proteus mirabilis , morganella morganii , providencia stuartii , stenotrophomonas maltophilia , burkholderia cepacia , and chryseobacterium meningosepticum ( table 2 ). subsequently , the minimal concentration of the m4 , m5 and m6 peptides able to kill 99 . 9 % of the original bacterial inoculum ( mbc ) was evaluated . the mbc was calculated on strains of e . coli atcc 25922 and p . aeruginosa atcc 27853 and it was found to be equal to the calculated values of mic for the same strains . the equality of the values of mic and mbc provides the indication that m4 , m5 and m6 peptides are bactericidal and not bacteriostatic . time - kill experiments demonstrated that m6 exhibited rapid bactericidal activity against e . coli atcc 25922 and p . aeruginosa atcc 27853 , reducing an inoculum larger than 10 7 cfu by & gt ; 99 . 9 % in 4 h , at a concentration of 16 μg / ml ( fig3 ). bactericidal activity appeared to be concentration - dependent , especially with p . aeruginosa . due to their low mic values , the peptides could be administered at low doses , improving patient compliance , but also the cost - effect ratio of such therapy . the cytotoxicity of antibacterial map peptides was evaluated on different eukaryotic cell lines by a colorimetric assay ( mtt ). this assay measures the cells &# 39 ; ability to convert a soluble tetrazolium salt into an insoluble precipitate : formazan . the cytotoxicity of m1 was evaluated on murine macrophagic cells ( j774 a . 1 ), murine myeloma cells ( spo ) and chinese hamster ovary epithelium cells ( cho k1 ). as shown fig4 , even at high concentrations ( 1 mg / ml ) m1 cytotoxicity on cho k1 cells and on spo cells is low ( percent survival is 80 - 90 %). by contrast , murine macrophage cells , j774 a1 were found to be more sensitive to m1 ( percent survival ˜ 50 %). the toxicity of m4 , m5 and m6 towards mouse macrophage cells j774 . a1 was also tested by mtt and is shown in fig5 a . treatment of cells overnight with 30 μg / ml of m4 , m5 or m6 , did not substantially affect cell viability , whereas a drop in cell viability was evident after treatment with peptide m4 at concentrations of 250 μg / ml and over , and with peptides m5 and m6 at 125 μg / ml and over . the same dendrimeric peptides showed low toxicity for human keratinocyte hacat cells ( fig5 b ) even when used at high concentration ( 1 mg / ml ). moreover , the effect of m4 , m5 and m6 on the pichia pastoris yeast , strain x33 , was evaluated . the number of colonies of yeast treated with the three antimicrobial peptides did not differ from the negative control suggesting an absence of toxicity of the peptides on yeast ( data not shown ). since the use of peptides as therapeutic agents is severely limited by their in vivo half - life , the stability to human serum protease of the linear peptide l1 and of the map peptides m1 , m4 , m5 and m6 was evaluated . the peptides were incubated at the concentration of 10 mm with plasma and with human serum for 2 and 24 hours ; the samples were subsequently analysed in hplc on column c18 ( see materials and methods ) to evaluate the presence of linear and map peptide not digested by the protease . the authors observed that monomeric peptide l1 was completely degraded within 2 h in serum , whereas the dendrimeric form of the same peptide ( m1 ) was still detected after 24 h in plasma and serum ( fig7 , table 4 ). comparable results were obtained with dendrimeric peptides m4 , m5 and m6 ( table 4 ). the haemolytic activity of m5 and m6 was also evaluated and is represented fig9 . haemolysis of fresh human erythrocytes was determined at peptide concentrations ranging from 1 to 125 μg / ml . at a concentration of 125 μg / ml all dendrimeric peptides showed very poor haemolytic activity ( less than 5 %) after an incubation of 30 min . by contrast , after 19 hours of incubation , the haemolysis induced by m6 and m5 at 125 μg / ml is 7 % and 19 %, respectively . the percentage of haemolysis of untreated blood after 19 hours ( control ) is very limited (& lt ; 1 %). the ability of map peptides to perforate the bacterial membrane was evaluated measuring the activity of cytoplasmatic beta - galactosidase ( 24 ) in supernatants of e . coli strain ml - 35 incubated with the peptide and using p - nitrophenyl - β - d - galactopyranoside ( pnpg ) as a substrate . pnpg is digested by beta - galactosidase , therefore releasing p - nitro - phenolate detectable by spectrophotometric reading at 420 nm ( fig1 ). the permeabilization assays showed that peptides m4 , m5 and m6 permeabilize the bacterial inner membrane , unmasking cytoplasmic β - galactosidase in ml - 35 e . coli permease - negative mutant . the activity of dendrimeric peptides against the inner membrane was evaluated at concentrations of 16 , 32 and 64 μg / ml . all dendrimeric peptides permeabilized bacterial inner membrane at 16 μg / ml ( fig9 ). permeabilization occurred after a lag of less than 1 minute , and the rate of permeabilization depended on peptide concentration ( not shown ). moreover , the ability of the m6 map peptide to bind the bacterial lipopolysaccharide ( lps ) was assayed by plasmon surface resonance in a biacore 1000 instrument ( fig1 ) using a protocol perfected by the authors ( 26 ). the sensorgram shows the rapid binding of m6 to the lps . this experiment suggests that m6 might have a detoxifying activity . in an attempt to clarify the molecular mechanism of action , the authors examined the binding properties on dna exerted by m6 dendrimeric peptide and magainin 2 , an antimicrobial peptide which has a pore - forming activity on the cell membrane . the dna binding abilities of m6 and magainin 2 were examined by analyzing the electrophoretic mobility of dna bands at the various weight ratios of peptides to dna on a 1 % ( w / v ) agarose gel . m6 inhibited the migration of dna above weight ratio of 0 . 2 ( fig1 ) while magainin 2 did not suppress the migration of dna until the weight ratio of 5 . this result indicates that m6 binds to dna at least over 25 times tightly than magainin 2 . clsm experiments showed that rhodamine - labelled m6 is able to enter the cells within 5 minutes and tends to cluster in discrete patches , often situated at the cell poles , instead of distributing evenly inside the bacteria ( fig1 ). moreover , there are no significant differences between e . coli images taken after 5 ( fig1 a ) or 240 min ( fig1 b ) of incubation with 20 μg / ml m6 . to further visualize the membrane - perturbing activity of m6 , the authors used fitc , a low molecular - mass ( 389 . 4 da ) green fluorescent probe . fitc was unable to cross the cytoplasmic membrane of control intact cells . indeed , when e . coli tg1 cells were incubated with the probe without pretreatment with the peptide , no appreciable fluorescent signal was discerned ( data not shown ). in contrast , fitc was readily accumulated in bacteria after their exposure to 20 μg / ml m6 , suggesting that m6 increases the permeability of the bacterial membrane as assessed by clsm analysis ( fig1 ). the results obtained with the double fitc - pi staining approach are illustrated in fig1 . e . coli cells were incubated respectively with 5 μg / ml ( fig1 a ), and 40 μg / ml of m6 ( fig1 b ). the authors observed that microbial cells treated with the highest peptide concentration display an increased membrane permeability to both fitc and pi ( fig1 b ). the lowest concentration of m6 lead to a limited alteration of bacterial membrane ( fig1 a ). surprisingly , the membrane remained almost impermeable to the smaller dye ( fitc , 389 . 4 da ) but was permeable to the larger dye ( pi , 668 . 4 da ). this finding could be explained by electrostatic interactions of the dye with the bacterial outer membrane : fitc in solution is negatively charged while pi has two positive charges that can promote its uptake . all treated bacteria maintain a typical “ stick ” shape without losing their nucleic acids content , as manifested by their clear , intense red fluorescence due to propidium iodine binding to dna . in order to identify the critical residues responsible for the antibacterial activity of m6 , the sequence of m6 was subjected to “ alanine scanning ”. “ alanine scanning ” is a procedure in which every amino acid of the peptide in question is sequentially replaced by an alanine . a mini - library in map form of 9 peptides was thereby synthesised ( table 5 ). table 5 . m6 sequence and sequences of the peptides derived from “ alanine scanning ” of m6 for each map peptide , mic was then calculated on three reference strains : e . coli atcc 25922 , p . aeruginosa atcc 27853 ( gram negative ) and attc25923 ( gram positive ) ( table 6 ). mic values obtained for the m6 derivative peptides show that the replacement of alanine with any hydrophobic residue led to a significant increase in mic reflecting (?) a diminished antimicrobic activity . from the mini - library , the peptide m33 was identified as particularly active against the gram negative bacteria , e . coli atcc 25922 , and p . aeruginosa atcc 27853 with mic values , expressed in molarity , of 1 . 5 × 10 − 6 m for both strains . lastly , the effect of replacing the lysines of the m6 peptide with another positively charged aminoacid , arginine ( r ) was evaluated . arginine has a more distributed positive charge than lysine , due to the presence of the guanidinium group . the primary amine of lysine and the guanidinium group of arginine appear to interact differently with the bacterial phospholipids ( 27 ). for this purpose , 3 peptides in map form were synthesised ( table 7 ). for each peptides , mic was calculated on three reference strains : e . coli atcc 25922 , p . aeruginosa atcc 27853 ( gram negative ) and s . aureus attc25923 ( gram positive ). mic values obtained from replacing m6 lysines with the arginines show that the replacement of lysine in position 2 with an arginine does not influence the antimicrobial activity of map ( table 8 ). from this mini - library , the petide m28 was identified as particularly active against the gram negative bacteria e . coli atcc 25922 and p . aeruginosa atcc 27853 with mic values , expressed in molarity , respectively of 3 . 8 × 10 − 7 and 7 . 6 × 10 − 7 m . in one example , the tetrabranched map peptides with the amino acid sequence : qakirvrlsa , kirvrlsa , qkkirvrlsa are used individually in a bacterial colony growth inhibition test . the test is conducted by incubating different concentrations of map peptides with e . coli ( strain tg1 ) and plating bacterial cells on agar at a dilution such to allow for individual colonies counting . the following day , the number of colonies grown after treatment with the three map peptides is compared . the map peptides with sequence kirvrlsa and qkkirvrlsa exhibit a bactericidal activity on tg1 cells down to a concentration of 6 . 25 μg / ml . in an additional example , the minimum inhibitory concentration ( mic ) of the tetrabranched map peptides having the sequence : qakirvrlsa , kirvrlsa , qkkirvrlsa was calculated on different gram negative bacterial strains . the mic values of kirvrlsa and qkkirvrlsa , expressed in molarity , are in the order of 10 − 6 - 10 − 7 m for the gram negative bacteria e . coli atcc 25922 and p . aeruginosa atcc 27853 . in an additional example , the minimum inhibitory concentration ( mic ) of the tetrabranched map peptides having the sequence : qakirvrlsa , kirvrlsa , qkkirvrlsa was calculated on different gram positive bacterial strains , such as s . aureus attc25923 . the values of mic computed for the three map peptides are in the order of 10 − 5 m . in another example , the minimal concentration able to kill 99 . 9 % of the micro - organisms ( mbc ) of the tetrabranched map peptides having the sequence : qakirvrlsa , kirvrlsa , qkkirvrlsa , was evaluated . the mbcs were calculated on strains of e . coli atcc 25922 and p . aeruginosa atcc 27853 and were found to be equal to the corresponding mic values for the same strains . in a further example , the haemolytic activity on human erythrocytes of the tetrabranched map having the sequence : kirvrlsa , qkkirvrlsa was calculated . the percentage of haemolysis is calculated using the parpart method by means of a calibration curve obtained incubating the erythrocytes with increasing concentrations of nacl . at a concentration of 125 μg / ml qkkirvrlsa and kirvrlsa showed very poor haemolytic activity ( less than 5 %) after an incubation of 30 min . by contrast , after 19 hours of incubation , the haemolysis induced by qkkirvrlsa and kirvrlsa at 125 μg / ml is 7 % and 19 %, respectively . in another example , the tetrabranched map peptides having the sequence : qakirvrlsa , kirvrlsa , qkkirvrlsa are tested in an in vitro assay , in which their cytotoxicity on murine macrophage j774 a . 1 cells and on human hacat keratinocytes is determined by a colorimetric assay ( mtt ). as the concentration of map peptides increases , the vitality of j774 a . 1 cells decreases , whilst human hacat keratinocytes are particularly resistant to the peptides even when administered at a concentration of 1 mg / ml . in a further example , the map peptide m6 ( sequence qkkirvrlsa ) demonstrated that it effectively binds the bacterial lipopolysaccharide when it is passed on a sensorchip of a biacore instrument , previously sensitised with the same map peptide m6 . in an additional example , the map peptides derived from “ alanine scanning ”, conducted on the sequence of m6 peptide ( table 6 ) are each one used to calculate their minimum inhibitory concentration ( mic ) on the bacterial strains e . coli atcc 25922 , p . aeruginosa atcc 27853 and s . aureus attc25923 . alanine scanning by replacing sequentially every amino acid of m6 with an alanine , allows to identify the critical residues responsible for bactericidal activity of the peptide . from this mini - library , a peptide was identified ( m33 ) which proved to be particularly active against the gram negative bacteria e . coli atcc 25922 and p . aeruginosa atcc 27853 with mic values of 1 . 5 × 10 − 6 m for both strains ( table 6 ). in an additional example , map peptides obtained by replacing the lysines ( k ) with arginines ( r ) of the map peptide m6 ( table 7 ) are each used to calculate their minimum inhibitory concentration ( mic ) on the bacterial strains e . coli atcc 25922 , p . aeruginosa atcc 27853 and s . aureus attc25923 . from this mini - library , a peptide was identified ( m28 ) which proved to be particularly active against the gram negative bacteria e . coli atcc 25922 and p . aeruginosa atcc 27853 with mic values of 3 . 8 × 10 − 7 and 7 . 6 × 10 − 7 m , respectively ( table 8 ). the peptides able to have an antibacterial effect were selected using a phage library of random peptides of 10 mer , following standard protocols for the use of these libraries . the peptides were selected by means of three pannings . 1 ml of cells of e . coli strain tg1 at the od 600 = 0 . 1 ( about 0 . 8 × 10 7 cells ) was centrifuged at 17000 × g for 3 min . the pellet was re - suspended in 1 ml of pbs and incubated under slow agitation for about 10 14 phages for 60 minutes at ambient temperature . cells and phages were recovered after a centrifugation at 17000 × g for 3 min . the supernatant was aspirated and the pellet washed 10 times with pbs - tween 0 . 1 % to remove the phages not bound in the first selection round and washed with pbs - tween 0 . 5 % in the subsequent rounds . the cells with the phages attached were centrifuged at 17000 × g for 3 min and the pellet was re - suspended in 1 ml of elution buffer [ 0 . 2 m glycine - hcl ( ph 2 . 2 )] leaving under slow agitation for about 5 minutes at ambient temperature . the sample was centrifuged as done previously and the supernatant transferred into an eppendorf tube and neutralised with 150 μl of 1m tris - hcl ( ph 9 . 1 ). 100 μl of eluted phage were used to infect 10 ml of e . coli tg1 in exponential growth phase for 30 min at 37 ° c . after the infection , the bacteria were centrifuged for 10 minutes at 3300 × g , re - suspended in 1 ml of 2 × ty ( descrivere ) and plated on agar containing ampicillin ( 100 μg / ml )- glucose ( 1 %). after overnight incubation ( o . n .) at 30 ° c ., the colonies were recovered from the plate by adding 5 - 10 ml of 2 × ty in such a way as to obtain an homogeneous suspension . 100 ml of 2 × ty - ampicillin ( 100 μg / ml )- glucose ( 1 %) were inoculated with 100 μl of a bacterial suspension until obtaining an od 600 = 0 . 4 - 0 . 5 , 10 ml of culture were drawn and infected with 100 μl of the phage helper vcs . m13 (& gt ; 10 11 transforming unit ( tu )/ ml ). the infected bacteria were centrifuged at 3300 × g for 10 min , the recovered pellet was then re - suspended in 100 ml of 2 × ty - ampicillin ( 100 μl / ml )- kanamycin ( 25 μg / ml ) and agitated over night at 30 ° c . the phages were purified and concentrated for precipitation with peg / nacl ( 20 % polyethylene glycol 6000 - 2 . 5 m nacl ) and re - suspended in 2 ml of pbs . the eluted phages were recovered , amplified and used for two more selection cycles . at the end of the process , the presence of specific phages for the bacterial surface was verified by elisa assay . the solid phase synthesis of the linear peptides was conducted by means of syro multisyntech ( wittenbochum , d ) peptide synthesiser , using a resin of p -( 2 , 4 - dimethoxyphenyl - fmoc - aminomethyl )- phenoxyacetamidonorleucyl -( 4 - methylbenzydryl - amine ) ( rink - mbha ) and the chemistry of fluorenylmethoxycarbonyl ( fmoc ). the de - protection reaction was obtained by adding 40 % of piperidine in n - methylpyrrolidone and , for the attack reaction , n - hydroxybenzotriazole esters of f - moc - aminoacids prepared in situ were used for the conjugation reaction . the peptides were detached from the resin and simultaneously de - protected using a trifluoroacetic acid / thioanisole / ethanedithiol / water mixture ( 93 / 2 / 3 / 2 ) for 3 hours at ambient temperature . the peptides were purified by means of reverse phase hplc on a vydac c18 semi - preparative column using a 30 min gradient of buffer b from 0 % to 100 % ( buffer a : 0 . 1 % trifluoroacetic acid / water ; buffer b : 0 . 1 % trifluoroacetic acid / methanol ). the synthesis of the multiple tetraramified antigenic peptides ( map ) was achieved by a solid phase procedure on wang fmoc 4 - k 2 - k - a resin , using fmoc chemistry . the map peptides were separated from the support using standard techniques and purified by means of reverse phase hplc . the peptides were checked by mass spectrometry . antimicrobic tests were conducted incubating for 75 min at 37 ° c ., 25 μl of e . coli at the od 600 of 0 . 2 with 25 μl of map peptide dissolved in pbs at the various concentrations . the different incubations were further diluted 1 : 1000 in 2 × ty medium and 100 μl were plated on solid 2 × ty medium . the plates were left overnight at 30 ° c . and the individual grown colonies were counted and compared with a control , not treated with map peptide . reference strains ( escherichia coli atcc 25922 , pseudomonas aeruginosa atcc 27853 , staphylococus aureus atcc 25923 and chryseobacterium meningosepticum ccug 4310 ) and several recent clinical isolates ( including multidrug - resistant ones ) of various species ( table 2 ) were used for conventional susceptibility testing experiments . minimum inhibitory concentration ( mic ) was determined by a standard microdilution assay as recommended by the national committee for clinical laboratory standards ( nccls ) using cation - supplemented mueller - hinton ( mh ) broth ( oxoid ltd . basingstoke , uk ) and a bacterial inoculum of 5 × 10 4 cfu per well , in a final volume of 100 μl . results were recorded by visual inspection after 24 h of incubation at 37 ° c . minimum bactericidal concentration ( mbc ), defined as the concentration at which ≧ 9 . 9 % of the bacterial inoculum is killed , was determined as recommended by the nccls after mic testing . the mbc is defined as the minimal concentration of antibiotic able to kill 99 . 9 % of the micro - organisms of the original inoculation of the species in question . the mbc was determined as recommended by the national committee for clinical laboratory standards ( nccls ) on strains of e . coli atcc 25922 and p . aeruginosa atcc 27853 . assay of bactericidal activity in time - kill experiments was carried out as follows . the peptide was added , at the desired concentration , to exponentially growing cultures of the test strain in mh broth containing a total inoculum of 5 × 10 7 cfu ( 1 × 10 7 cfu / ml ) at 37 ° c . samples were drawn at different times and suitable dilutions were plated on mh agar to score the residual number of cfu . a culture without peptide was always grown in parallel as control . for cytotoxicity tests , different cell lines were used : murine myeloma cells spo , hamster ovary epithelium cells cho k1 , murine macrophage cells j774 a . 1 and human keratinocytes hacat . the cells were plated in medium in rpmi 1640 ( spo and cho k1 ) and dmem ( j774 a . 1 and hacat ) with antibiotics and bovine foetal serum at 10 %, in 96 - well plates at the concentration of 6 × 10 4 ( spo , cho k1 and j774 a . 1 ) and 3 × 10 4 ( hacat ). peptides , previously filtered with a 0 . 2 μm filter disk ( whatman ), were added at various concentrations to the different cell lines and left in incubation over night at 37 ° c . cell viability was determined adding the mtt tetrazolium salt at the concentration of 0 . 5 mg / ml and incubating for 90 min . the cells were solubilised with a solution at ph 4 . 5 containing sds 10 % and dimethylformamide 45 % and read at the dual wavelength of 595 / 650 nm with a plate reader . effect of qakirvrlsa ( m4 ), kirvrlsa ( m5 ) and qkkirvrlsa ( m6 ) on the pichia pastoris yeast strain x33 to a volume of 50 μl of culture of pichia pastoris grown 24 hour at 30 ° c . in ypd ( yeast extract / peptone / dextrose ) medium , 50 μl of map peptides ( 2 mg / ml ) were added and left in incubation 150 min at 37 ° c . subsequently , 50 μl of each incubation were plated on ypd solid medium and it was allowed to grow for 48 hours at 30 ° c . the number of colonies grown was compared with a control , where the yeast was not treated with the map . the various peptides in map form and the linear peptide ( l1 ) were dissolved in h 2 o at the concentration of 10 mm and incubated with 10 μl of plasma and human serum for 2 and 24 hours at 37 ° c . to each sample were added 150 μl of methanol to block the proteolytic reaction ; each sample was then centrifuged at 13 , 000 rpm for 2 min and to the supernatant were added 0 . 75 ml of 0 . 1 % trifluoroacetic acid . the samples were analysed in reverse phase hplc on a vydac c18 semi - preparative column using a 30 min gradient of buffer b from 20 % to 95 % ( buffer a : 0 . 1 % trifluoroacetic acid / water ; buffer b : 0 . 1 % trifluoroacetic acid / methanol ), to evaluate the presence of linear and map peptide after the proteolytic treatment . the haemolytic activity of the kirvrlsa ( m5 ) and qkkirvrlsa ( m6 ) peptides was evaluated by the parpart erythrocyte osmotic resistance assay in nacl . the percentage of haemolysis was calculated by means of a calibration curve obtained incubating the erythrocytes with increasing concentrations of nacl and measuring the absorbance increase , due to haemolysis , at 540 nm . 0 . 9 % nacl solutions containing the map peptides at different concentrations were then prepared , whereto was added human blood in the ratio of 1 : 100 ( v / v ). the samples were left at ambient temperature for 30 min and 19 hours ; subsequently , a portion was drawn for each incubation , centrifuged at 1500 rpm for 5 min and the absorbance of the super was measured with the spectrophotometer at 540 nm . the ability of the qakirvrlsa ( m4 ), kirvrlsa ( m5 ) and qkkirvrlsa ( m6 ) map peptides to perforate the bacterial membrane was evaluated measuring the activity of cytoplasmatic beta - galactosidase using as a substrate p - nitrophenyl - β - d - galactopyranoside ( pnpg ), which , digested by the beta - galactosidase , frees the p - nitro - phenolate detectable by spectrophotometric reading at 420 nm . in order to do this , e . coli cells of the strain ml - 35 were used : they constitutively produce beta - galactosidase and their lactose transporter is deactivated . the bacterial cells were drawn during the logarithmic growth phase ( od 600 = 0 . 4 - 0 . 5 ) and re - suspended in phosphate buffer 10 mm containing nacl 100 mm ( ph 7 . 4 ) and 1 . 5 mm pnpg . at time zero , the peptide in map form was added at the final concentration of 16 , 32 and 64 μg / ml and the absorbance change was measured at 420 nm . gel - retardation experiments were performed by mixing 200 ng of the e . coli plasmid vector pcep4 ( invitrogen ) with increasing amounts of m6 peptide in 20 μl of binding buffer ( 5 % glycerol , 10 mm tris - hcl ( ph 8 . 0 ), 1 mm edta , 1 mm dtt , 20 mm kcl and 50 μg / ml bsa ). the reaction mixtures were incubated at room temperature for 1 h . subsequently , 4 μl of native loading buffer was added ( 40 % saccarose , 0 . 25 % bromophenol blue ) and an aliquot of 12 μl was applied to a 1 % agarose gel electrophoresis in 1 mm tris borate - edta buffer . tg1 e . coli cells were grown overnight in 2 × ty . after dilution 1 : 10 in cell medium , 5 × 1 ml aliquots were prepared , washed two times with 10 mm sodium phosphate buffer ( pbs ) ph 7 . 4 and incubated in 200 μl of a tetramethylrhodamine ( tmr ) labelled peptide solution ( 20 μg / ml in pbs ) for 5 min at 37 ° c . after washing with pbs , each aliquot of the cells were resuspended in 200 μl of pbs and kept in the dark at 37 ° c . respectively for 2 , 30 , 60 , 120 , 240 min . the cells were then mounted in a glass slide and observed with a bio - rad mrc600 laser scanning confocal microscope ( clsm ). fluorescent images were obtained with a 568 nm bandpass filter for excitation of tmr . software merging of images was carried out by using a comos software . a double - staining method was developed to visualize , with two marker at the same time , the membrane perturbating activity induced by m6 on bacteria . the following fluorochromes were used : ( i ) the propidium iodide ( pi ), a dna - 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