WO2008104777A2 - Peptide - Google Patents

Abstract

The invention encompasses a novel peptide with antimicrobial activity, particularly but not only against Gram-negative bacteria, and uses thereof. The peptide is non-cyclic and comprises or consists of an antimicrobial domain that adopts an alpha-helix conformation upon interaction with a negatively- charged membrane. The antimicrobial domain has a sequence shown by formula (IV) : X1- X2 -X3 - X4- X5 - X6- X7 - X8-X9 X10-X11-X12-X13-X14-X15-X16 (SEQ ID NO: 7), in which X1, X5, X10 and X13 are each independently K or R; X15 is C; and the remaining residues are as specified herein. An examplar peptide is RPAFRKAAFRVMRACV ('RTA3'; SEQ ID NO: 9).

Description

Peptide

The present invention relates to an agent such as a peptide active against microbes, and uses thereof.

Chronic lung infections are a major cause of morbidity and mortality in cystic fibrosis patients, with Pseudomonas aeruginosa identified as key pathogen. Two major concerns in the treatment of lung infections in cystic fibrosis patients are the emergence of multi-drug resistant pathogens and a lack of new antibiotics directed against the non-fermenting Gram-negative bacteria e.g. P. aeruginosa and Acinetobacter baumannii.

More generally, MRSA and other multi-drug resistant bacteria have been increasing. The recent clinical introduction of linezolid, daptomycin, dalbavancin and tigecycline have strengthened therapeutic regimens against MRSA and Gram-positive organisms. Tigecycline affords activity against some Gram- negative bacteria; however, there are few new drugs addressing the increasing problem of multi-drug resistant Gram-negative bacteria particularly P. aeruginosa and the new "super bug", A. baumannii .

During the last 15-20 years, this increasing problem of antibiotic resistance has focussed attention onto other antimicrobial molecules, for example a broad group of antimicrobial peptides that constitute the first line of defence against invading organisms in higher animals. Two general features of these peptides are that they are amphipathic (adopting conformations that separate polar and non-polar surface to match the polar-non-polar interfacial regions of cell membranes), and are positively charged (promoting interaction with the negatively charged membranes of prokaryotic cells) .

Antimicrobial peptides are effective at low micromolar concentrations against a broad range of micro-organisms including, in many cases, those resistant to traditional antibiotics .

In general, however, therapeutic applications of the known antimicrobial peptides have been hindered by several problems, perhaps the most important being toxicity, cost of production and bioavailability. Continuing advances in peptide synthesis and purification, combined with economies of scale in large- volume syntheses, indicate that the cost of therapeutics based on peptides of relevant size (12-20 amino acid residues) is less of an issue than in the past. Bioavailability problems may be addressed with peptides showing genuine promise (for example by using D-amino acid versions of effective peptides) . Toxicity, however, remains a limiting factor in peptide antimicrobial use. This is illustrated by the observation that antimicrobial peptides are rarely effective in animal studies at doses below 10-20 mg/kg. Since peptides of this class cannot so far be administered orally, this therapeutic dose requires injections of significant volumes of peptide at concentrations of 1 or 2 mg/ml, corresponding to concentrations in the millimolar range. Thus, although the effective dose as determined by MICs may be in the low micromolar range, the dosing methods require that peptides have low eukaryotic cell toxicity at rather high concentrations .

Partly as a result of these considerations, first generation antimicrobials based on peptides derived from animal or bacterial sources have been limited to topical use (for example, pexaganin based on magainin from frog skin) , or are chemically modified to reduce in vivo toxicity (for example, colistin methanosulfonate in which the active form of the peptide is probably the unmethanosulfonated form resulting from loss of side chain protection in vivo) .

We demonstrated previously (see International patent appl . No. PCT/GB2004/000592 published as WO2004/072093) that a strain of Streptococcus mitis from normal flora of bronchial lavages secreted a low molecular weight peptide which had antibacterial activity against Gram-negative bacteria in particular.

The present invention provides inter alia an alternative antimicrobial peptide obtained from Streptococcus mitis, or produced as a synthetic peptide, derivative or analog. Peptides of the invention in one aspect have reduced toxicity compared with many prior art antimicrobial peptides.

According to a first aspect of the present invention, there is provided an isolated peptide having antimicrobial (for example, antibacterial) activity and comprising or consisting of a sequence shown by formula (I):

X¹-(X²)_a-(X³)_b-(X⁴)_c-(X⁵)_d-(X⁶)e-X⁷-(X⁸)f-X⁹-(X¹⁰)g (D (SEQ ID NO: 1)

in which

X¹ and X⁷ are each independently K or R; X⁹ i s C;

X², X³, X⁴, X⁵, X⁶, X⁸, and X¹⁰ are each independently any amino acid residue; and a = 0-12, b = 0-5, c = 0-5, d = 0-5, e = 0-15, f = 1-2, and g = 0-10, provided that the sum of a, b, c, d and e is at least 2.

The peptide of the invention has been shown to be particular effective as a potent antimicrobial agent, as evidenced in the experimental section. Further advantages of the peptide, which has a different structure not deducible from prior art disclosures such WO2004/072093, will be apparent from the description below.

In the sequence of formula (I) :

X² may be an uncharged non-polar amino acid; and/or X³ may be a charged amino acid; and/or X⁴ may be an amino acid with an uncharged polar side chain and/or a charged amino acid; and/or

X⁵ may be an amino acid with an uncharged polar side chain and/or an uncharged non-polar amino acid; and/or X⁶ may be an uncharged non-polar amino acid and/or a charged amino acid; and/or

X⁸ may be an uncharged non-polar amino acid; and/or

X⁹ may be reduced or not; and/or

X¹⁰ may be an uncharged non-polar amino acid.

Thus X² and/or X³ and/or X⁴ and/or X⁵ and/or X⁶ and/or X⁸ and/or

X¹⁰ of formula (I) may be any amino acid residue or alternatively may be an amino acid as indicated above from the groups consisting of: an amino acid with an uncharged polar side chain; a charged amino acid; and an uncharged non-polar amino acid. All combinations of specified amino acids at the given positions are envisaged.

Amino acids with an uncharged polar side chain as defined herein include serine (S), tyrosine (Y), threonine (T), asparagine (N) and glutamine (Q) .

Uncharged non-polar amino acids as defined herein include glycine (G) , alanine (A) , valine (V) , leucine (L) , isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W) and cysteine (C) .

Charged amino acids as defined herein include lysine (K) , arginine (R), histidine (H), aspartic acid (D) and glutamic acid (E) .

As elaborated below, amino acids contained within the peptide of the invention may be modified, for example by dehydration, phosphorylation or glycosylation . In particular, any S or Y residues may be dehydrated. In the sequence of formula (I) : a may be 1-5, preferably 3; and/or b may be 1-3, preferably 1; and/or c may be 1-3, preferably 1; and/or d may be 0-3, preferably 0 or 1; and/or e may be 3-10, preferably 5 or 6; and/or f may be 1-2, preferably 1; and/or g may be 0-2, preferably 1.

In an embodiment:

X² is any amino acid and/or an uncharged non-polar amino acid, with a = 3 (for example, the amino acid sequence PAF or RAF); and

X³ is any amino acid or a charged amino acid, with b = 1 (for example, the amino acid R) ;

X⁴ is any amino acid or an amino acid with an uncharged polar side chain or a charged amino acid, with c = 1 (for example, the amino acids T or K) ; X⁵ is any amino acid and/or an amino acid with an uncharged polar side chain and/or an uncharged non-polar amino acid, with d = 0 or 1 (for example, the amino acid A) ;

X⁶ is any amino acid and/or an uncharged non-polar amino acid and/or a charged amino acid, with e = 5 (for example, the amino acid sequence AFRVM [SEQ ID NO: 2] ) or e = 6 (for example, the amino acid sequence AAFRVM [SEQ ID NO: 3] ) ;

X⁸ is any amino acid and/or an uncharged non-polar amino acid, with f = 1 (for example, the amino acid A); and

X^:o is any amino acid and/or an uncharged non-polar amino acid, with g = 1 (for example, the amino acid I or V) .

The peptide may in certain embodiments comprise deletions with respect to the sequence of formula (I), provided that bactericidal activity conferred by the residues X¹, X⁷ and X⁹ is not removed. The peptide may alternatively comprise or consist of a sequence shown by formula (II) :

X¹-X⁷-(X⁸)_f-X⁹-(X¹⁰)_g (II) (SEQ ID NO: 4)

in which the amino acids X¹, X⁷, X⁸, X⁹ and X¹⁰ are as defined above. An exemplary peptide falling within formula (II) in particular has an amino acid sequence RRACV (SEQ ID NO: 5) .

The peptide may comprise or consist of a sequence shown by formula (III):

X¹-(X²)₃- (X³)l- (X⁴) l- (X⁵)θ or 1- (X⁶)5 or 6^"X^7" < ^ ) I^"*^9" ( X" ) 0 or 1 (IH) (SEQ ID NO: 6) in which

X¹, X⁷ and X⁹ are as defined above;

X² is any amino acid and/or an uncharged non-polar amino acid (for example, the amino acid sequence PAF or RAF) ;

X³ is a charged amino acid (for example, the amino acid R);

X⁴ is any amino acid or an amino acid with an uncharged polar side chain or a charged amino acid (for example, the amino acid

T or K) ; X⁵ is any amino acid or an amino acid with an uncharged polar side chain or an uncharged non-polar amino acid (for example, the amino acid A) , or is absent from the sequence of formula

(HI);

X⁶ is any amino acid and/or an uncharged non-polar amino acid and/or a charged amino acid (for example, the amino acid sequence AFRVM [SEQ ID NO: 2] or AAFRVM [SEQ ID NO: 3] ) ;

X⁸ is any amino acid or an uncharged non-polar amino acid (for example, the amino acid A) ; and

X¹⁰ is any amino acid or an uncharged non-polar amino acid (for example, the amino acid I or V) , or is absent from the sequence of formula ( III ) . According to a further aspect of the invention there is provided a peptide comprising or consisting of an antimicrobial domain shown by formula (IV) :

X1 -X2-X3-X4 — X5-X6^~X7~^"X8^~X9 — Xl0^~Xll~Xl2-Xl3-Xl4 ~Xl5~Xl6 ( IV )

(SEQ ID NO: 7)

in which

Xi, X₅, X₁₀ and X₁₃ are each independently K or R; and X₂ is selected from the group consisting of: A, V, L, I, M, F, T,

W, P, C, Y, H, S, G, K and R; and

X₃ is selected from the group consisting of: A, V, L, I, M, F, T,

W, P, C, Y, H, S and G; and

X₄ is selected from the group consisting of: A, V, L, I, M, F, T, P, C, Y, H, S and G; and

X₆ is selected from the group consisting of: K, R, H, D, E, A and

T; and

X₇ is selected from the group consisting of: A, V, L, I, M, F, T,

W, P, C, Y, H, S, G and Q; and X₈ and X₉ are each independently selected from the group consisting of: A, V, L, I, M, F, T, W, P, C, Y, H, S and G; and

X₁₁, X₁₂, and X₁₄ are each independently selected from the group consisting of: A, V, L, I, M, F, T, W, P, C, Y, S and G; and

X₁₅ is C; and X₁₆ is absent or is selected from the group consisting of: A, V,

L, I, M, F, T, W, P, C, Y, S and G;

and in which the antimicrobial domain contains no more than one amino acid deletion or insertion between residues X₁ and X₅, and/or no more than one amino acid deletion or insertion between residues X₅ and X_1O, and/or no more than one amino acid deletion or insertion between residues X₁₀ and X₁₃, and no more than one amino acid deletion at residue X₁₆, such that any inserted amino acid is selected from the group consisting A, V, L, I, M, F, T, W, P, C, Y, H, S, G, Q, K and R; characterised in that the peptide has antimicrobial activity and is non-cyclic, and that the antimicrobial domain adopts an alpha-helix conformation within or on attachment to a negatively-charged membrane.

In the peptide:

X₂ may be selected from the group consisting of: A, L, I, F, T,

P, Y, S, G, K and R; and/or

X₃ and/or X₈ and/or X₁₄ may be each independently selected from the group consisting of: A, L, I, F, T, W, P, Y, H, S and G; and/or

X₄ may be selected from the group consisting of: A, L, I, F, T,

P, Y, H, S and G; and/or

X₆ may be selected from the group consisting of: K, R and T; and/or

X₇ may be selected from the group consisting of: A, L, I, F, T,

P, Y, H, S, G and Q; and/or

X₉ may be selected from the group consisting of: A, L, I, F, T,

W, P, Y, S and G; and/or Xn and X₁₂ are each independently selected from the group consisting of: V, L, I, M, F and P; and/or

X₁₆ may be absent or may be selected from the group consisting of: V and I.

In certain embodiments of the invention, the antimicrobial domain of the peptide does not include the amino acid W. As demonstrated below, we have found that the amino acid W may increase toxicity by promoting binding of the peptide to neutral bilayer membranes such as those found in eukaryotic organisms. Absence of the amino acid W may therefore reduce toxicity of the peptide in mammals.

In various embodiments of the peptide:

X₂ is selected from the group consisting of: P, K and R; and/or X₃ and/or X₄ and/or X₈ and/or X₉ and/or X₁₄ are each independently selected from the group consisting of: A, F, P and Y; and/or X₆ is selected from the group consisting of: K, R and T; and/or X₇ is selected from the group consisting of: A, F, P, Y and Q; and/or

X_n and Xi₂ are each independently selected from the group consisting of: V and M; and/or

Xi₆ is selected from the group consisting of: V and I.

The peptide may contain at least one free (unbound) C residue thiol group suitable for effecting antimicrobial activity.

The peptide or antimicrobial domain may contain only one C residue (i.e. the amino acid at position X₁₅ as specified in formula [IV] ) . This will facilitate the peptide being non- cyclic. The one C residues may contain a free thiol group to effect antimicrobial activity of the peptide.

In further embodiments of the peptide:

X₂ may be selected from the group consisting of: P, K and R; and/or X₃ and/or X₄ and/or X₈ and/or X₉ and/or X₁₄ may be each independently selected from the group consisting of: A and F; and/or

X₆ may be selected from the group consisting of: K, R and T; and/or X₇ may be selected from the group consisting of: A and Q; and/or

X₁₁ and X₁₂ may be e each independently selected from the group consisting of: V and M; and/or

X₁₆ may be selected from the group consisting of: V and I.

The C-terminal residue of the peptide may be I in order to facilitate high levels of synthetic production of the peptide.

As with other peptide sequence disclosed above, all combinations of specified amino acids at the given positions in formula (IV) are envisaged. The peptide according to all aspects of the invention may have no more than 30 amino acid residues, preferably no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or 9 amino acid residues, for example 15 or 16 amino acid residues.

The peptide may be no more than 3000 Daltons in size, for example up to 2500, 2000 or 1500 Daltons.

The peptide and/or the antimicrobial domain may comprise or consist of one of the following sequences:

(a) KPAFRTQAFRVMKACV (SEQ ID NO: 8; also referred to herein as "RTAl") ;

(b) RPAFRKAAFRVMRACV (SEQ ID NO: 9; also referred to herein as "RTA3") ; (C) RRAFRKAAFRVMRACV (SEQ ID NO: 10; also referred to herein as "RTA4") ;

(d) RPAFRTQAFRVMKACV (SEQ ID NO: 11);

(e) KPAFRTQAFRVMRACV (SEQ ID NO: 12)

(f) RPAFRTQAFRVMRACV (SEQ ID NO: 13) (g) KPAFRKAAFRVMRACV (SEQ ID NO: 14)

(h) RPAFRKAAFRVMKACV (SEQ ID NO: 15)

(i) KPAFRKAAFRVMKACV (SEQ ID NO: 16)

(j) RPAFRKAAFRVMRACV (SEQ ID NO: 17)

(k) RRAFRTAAFRVMRACV (SEQ ID NO: 18) (1) RRAFRKAAFRVMRACI (SEQ ID NO: 19); and

(m) RPAFRKAAFRVMRACI (SEQ ID NO: 20) .

For example, the peptide may comprise or consist of the amino acid sequence RPAFRKAAFRVMRACV ("RTA3"; SEQ ID NO: 9) . Alternatively, the peptide may comprise or consist of the amino acid sequence RRAFRKAAFRVMRACV ("RTA4"; SEQ ID NO: 10).

The peptide and/or antimicrobial domain of the invention may be unstructured, i.e. comprise no particular secondary structure, in solution and form an alpha-helix upon attachment to or insertion into a negatively charged membrane (for example, as found in Gram-negative bacteria) .

The peptide and/or antimicrobial domain may comprise a positively charged amino acid (for example, lysine or arginine) at a position corresponding to the non-polar face of an alpha- helix formed by the peptide and/or antimicrobial domain.

The peptide and/or antimicrobial domain may further not be induced or inducible into a secondary structure (such as an alpha-helix) by neutral ( zwitterionic) lipids such as vesicles composed of neutral lipids, e.g. mammalian membranes.

The structure of the peptide and/or antimicrobial domain may be determined by methods known in the art, such as circular dichroism (CD) spectroscopy (for example as described below in the experimental section) .

In another aspect of the invention, the peptide may be unable to bind to membranes having an external lipid bilayer in which the lipids are exclusively neutral lipids (for example, a membrane as found in eukaryotic organisms) .

We consider that the present peptides may contain anti-microbial properties due in part to their attachment onto, and then possible formation of pores in, negatively charged membranes.

Our biophysical data indicate that peptides of the invention produce a pore size in the Gram-negative bacteria tested that is different to that of colistin suggesting a different target site. The present peptides therefore physically have a different mechanism of action to colistin. This is also supported by in vivo and in vitro studies on colistin resistant isolates where the minimum inhibitory concentrations (MICs) against the peptides do not alter. Our data indicates that the peptides have an avidity in particular for Gram-negative membranes, although antimicrobial activity has also been found against Gram-positive bacteria such as Staphylococcus aureus (see below). The peptide may have a secondary lethal target such as a cytoplasmic target.

The peptide may be unable to form, or only reluctantly form, a dimer and/or multimer, and show higher antimicrobial activity as a monomer. This characteristic is unusual for antimicrobial peptides which are normally more active as dimers or multimers, although the Maximin class of peptides (Lee et al., 2005, FEBS Letters 579, 4443-4448) similarly require a free cysteine thiol for activity. Unlike other classes of antimicrobial peptides, the Maximin class of peptides has not been well characterised in terms of potential therapeutic antibiotics. In one aspect, the present invention excludes any peptide previously identified in the Maximin class of peptides.

The peptide may have L- and D- stereoisomeric forms of which the L-form (or "L-isomer") may have higher antimicrobial activity and enhanced safety over the D-form (or "D-isomer") . The L-form of the peptide thus forms an aspect of the present invention. In vivo studies indicate that the L-form of RTA3, for example, is highly potent with no toxic effects whereas the D-form produced adverse reactions in vivo. Again, this is an unexpected property which differs from most known antimicrobial peptides and most therapeutic peptides generally. It is understood that in certain embodiments, the L-form of the peptide may bind to serum proteins protecting the peptide from proteases and/or any potential toxic reactions to a host (for example, a human or animal patient).

The peptide may be active against multi-drug resistant and/or pan-drug resistant bacteria, for example against Gram-negative multi-drug resistant and/or pan-drug resistant bacteria such as colistin-resistant Pseudomonas aeruginosa . The peptide may also be active against Gram-negative and Gram-positive bacterial species outlined below. The activity against multi-drug resistant and/or pan-drug resistant Gram-negative bacteria further supports the idea of a novel target site of the peptide of the invention. Additionally, at present we have been unable to generate mutants resistant to peptides of the invention, regardless of the strain or genotype of the bacteria.

The peptide (for example, the L-isomer) may have lower toxicity against eukaryotic membranes than magainin. Our evidence shows that peptides of the invention have low toxicity against different mammalian cell lines (such as haemolysin and HeIa) and toxicity was only demonstrated at 200-fold the MIC. This property is unusual among antimicrobial peptides and indicates that the peptides will be particularly advantageous for treatment of microbial (such as bacterial) infections.

The peptide may be non-immunogenic. Other classes of antimicrobial peptides are often isolated from reptiles, amphibians or other organisms, and are immunogenic when used in heterologous organisms. We have been unable to generate antibodies to peptides according to the invention in a rabbit after repeated challenges over a two week period. These data, and see also below, show that the peptides are well tolerated in mammals and will be tolerated well in humans.

We have noted that standard media and in vitro testing does not necessarily reflect accurately the in vivo activity of the peptides. Proteins in the media and/or high-levels of cations and/or other unknown factors may produce falsely high MICs (see below) . Our data indicate that certain peptides of the invention bind in vitro to beta-casein, a common ingredient in standard testing media. In contrast, studies with minimal media indicate that RTA3 MICs, for example, are reduced 10 to 20-fold and are more reflective of the peptides in vivo activity (see below). Thus, specific media may be required to correlate in vitro activity of the peptide with activity in animal studies. The peptide may be bacterially active, for example in a non- static manner, i.e. cause active lysis of bacterial cells. Time- kill curve studies show that the peptides are significantly more cidal than colistin, particularly at higher concentrations. The killing by the peptides has been found in certain embodiments to be time-dependent.

The peptide may bind to high molecular proteins in vivo, for example fibrinogen or fibronectin. This property may assist reducing the toxicity of the peptides and/or protecting the peptides from serum proteases. However, the peptide in one embodiment does not bind serum albumin, which is a distinguishing feature over several other antimicrobial peptides.

We have also found (data not shown) that in one aspect of the invention, human serum does not alter peptide (for example, RTA3) antimicrobial activity but that human plasma doubles MIC (i.e. halves activity). Serum protease inhibitors do not affect this plasma-induced reduction in activity, indicating the peptide in one aspect exhibits about 50% binding to human plasma .

In a further aspect of the invention, one or more amino acid residues of the peptide may be replaced by an amino acid analog.

Amino acid analogs may be defined as any of the amino acid-like compounds that are similar in structure and/or overall shape to one or more of the twenty L-amino acids commonly found in naturally occurring proteins. These twenty L-amino acids are defined and listed in WIPO Standard ST.25 (1998), Appendix 2, Table 3. An amino acid analog may include natural amino acids with modified side chains or backbones. The analogs may share backbone structures, and/or even the most side chain structures of one or more natural amino acids, with the only difference (s) being containing one or more modified groups in the molecule. Such modification may include substitution of an atom (such as N) for a related atom (such as S) , addition of a group (such as methyl, or hydroxyl group, etc.) or an atom (such as Cl or Br, etc.), deletion of a group (supra), substitution of a covalent bond (single bond for double bond, etc.), or combinations thereof. Amino acid analogs may include α-hydroxy acids, and β- amino acids, and can also be referred to as "modified amino acids". Amino acid analogs may either be naturally occurring or unnaturally occurring (e.g. synthesised) . As will be appreciated by those skilled in the art, any structure for which a set of rotamers is known or can be generated can be used as an amino acid analog. The side chains may be in either the (R) or the (S) configuration (or D- or L-configuration) .

Also provided according to the present invention is an antimicrobial peptide comprising no more than 50 amino acid residues, preferably no more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or 9 amino acid residues, and including a (preferably free) Cys residue, in which the peptide disrupts negatively charged membranes upon formation of an alpha-helical conformation, shows antimicrobial activity in monomeric form, has a more active L-isomer than D-isomer, is active against colistin-resistant Pseudomonas aeruginosa, and has lower toxicity against eukaryotic membranes than magainin.

According to a further aspect of the invention, there is provided a variant of the peptide described herein. A "variant" is a sequence of amino acids which differs from the base sequence from which they are derived in that one or more amino acids within the base sequence are substituted for other amino acids. The variant may comprise conservative and/or non- conservative substitutions. Amino acid substitutions may be regarded as "conservative" where an amino acid is replaced with a different amino acid with broadly similar properties. "Non- conservative" substitutions are where amino acids are replaced with amino acids of a different type. Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptide. Suitably variants will be at least 60% identical, more suitably at least 70% identical, yet more suitably at least 80%, such as at least 87.5%, 90%, 93.5%, 95%, 97.5% or even 99% identical to the base sequence .

Sequence identity between amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same amino acid, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids or bases at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.

Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include the Gap program (Needleman & Wunsch, 1970, J. MoI. Biol. 48: 443-453) and the FASTA program (Altschul et al . , 1990, J. MoI. Biol. 215: 403-410). Gap and FASTA are available as part of the Accelrys GCG Package Version 11.1 (Accelrys, Cambridge, UK), formerly known as the GCG Wisconsin Package. The FASTA program can alternatively be accessed publically from the European Bioinformatics Institute (http://www.ebi.ac.uk/fasta) and the University of Virginia (http: //fasta.biotech. Virginia. edu/fasta_www /cgi) . FASTA may be used to search a sequence database with a given sequence or to compare two given sequences (see http: //fasta.bioch .Virginia . edu/fasta_www/cgi/search_frm2. cgi) . Typically, default parameters set by the computer programs should be used when comparing sequences. The default parameters may change depending on the type and length of sequences being compared. A sequence comparison using the FASTA program may use default parameters of Ktup = 2, Scoring matrix = Blosum50, gap = -10 and ext = -2.

According to a further aspect of the present invention there is provided an active fragment of the peptide as defined herein. The expression "fragment" refers to any portion of the given amino acid sequence that has antibacterial activity. Fragments will suitably comprise at least 5 consecutive amino acids from the basic sequence. Alternatively, more than one consecutive amino acid region of the peptide may be joined together to form an active fragment.

An isolated nucleic acid molecule encoding a peptide as defined herein is a further aspect of the invention. The nucleic acid molecule may be a DNA molecule, for example as shown in Fig. 2

(particularly the DNA sequence within SEQ ID NO: 21 encoding "RTAl" [SEQ ID NO: 8] in open reading frame 1 [SEQ ID NO: 24]).

Each of the antimicrobial peptides encoded by the three open reading frames of SEQ ID NO: 21, shown within SEQ ID NOs 22-24 in Fig. 2, and variants of each of these peptides, are within the scope of the present invention.

Also provided is a vector or plasmid which comprises a nucleic acid as defined herein.

Yet further provided is a recombinant cell comprising a nucleic acid as defined herein or a vector or plasmid as defined herein. The invention encompasses a peptide recombinantly produced by expressing in a suitable host organism a nucleic acid sequence as defined herein and exhibiting antimicrobial (for example, antibacterial) activity.

The peptide of the invention may be an isolated peptide (for example, an isolated naturally occurring peptide) , a recombinant peptide and/or a synthetic peptide.

Naturally occurring peptides of the invention may be isolated from the appropriate strain of S. mitis using conventional methods. Such peptides typically are secreted and therefore may be isolated from the supernatant of a culture of the S. mitis.

For example, a strain of S. mitis may be cultured under conventional conditions, such as at 37°C in the presence of a culture medium. After a suitable incubation period, for example from 12-48 hours, samples of the culture supernatant may be removed and desired peptides separated. The supernatant may for example be treated with a commercial protease blocker and sodium azide (0.2%) to prevent any deterioration of the target molecules. The peptides may be concentrated from the supernatant by various methods including ammonium sulphate precipitation, or ultracentrifugation or by using commercially available centricons. Once the proteins and peptides are concentrated, for example to a concentration of from 200-400 ng/ml, mass spectral analysis can be carried out, and the desired peptides identified. All these procedures are well known in the art.

Antimicrobial (such as antibacterial) peptides of the invention may then be identified and isolated or purified (see for example as described in the experimental section below) .

Peptides of the invention may be prepared using chemical methods, for example using a peptide synthesiser. Alternatively, peptides may be prepared using recombinant DNA methods. For example, a nucleic acid encoding a peptide of the invention may be incorporated into an expression vector or plasmids using conventional methods. These may then be used to transform a host cell, which may be a prokaryotic or eukaryotic cell, but is preferably one of the known prokaryotic expression hosts, such as Lactococcus, wherein the cell is not highly susceptible to the effect of the peptide. Peptides of the invention may then be recovered from the culture.

The peptide as described herein may be cation-sensitive. For example, higher concentrations of cations may render the peptide less active.

In another aspect of the invention there is provided a binding agent which binds a peptide as defined herein. The binding agent may, for example, be an antibody or an antibody fragment. Production of binding agents which bind to a known peptide are well known in the art.

In a further aspect of the invention there is provided a composition comprising a peptide as defined herein.

The composition may further comprise at least one antibiotic. The antibiotic of the composition and the peptide may have a synergistic antimicrobial effect.

An antibiotic may generally be defined as a substance, produced by or derived from a micro-organism, which destroys or inhibits the growth of another micro-organism such as a bacterial or fungal organism. In the present context, an antibiotic may be either synthetic, semi-synthetic or naturally occurring. The antibiotic may be a synthetic or semi-synthetic analogue of a naturally occurring antibiotic. The antibiotic used in a composition according to the present invention may be of the "static" or the "cidal" type, i.e. it may serve either to destroy a micro-organism or to inhibit its growth and/or reproduction. It may be selected from the group consisting of: macrolides (for example erythromycin and clarithromycin); ansamycins such as rifampicin; polymyxins (for example polymyxin E) ; cephems, including 1st generation (for example cephalothin and cefazolin) , 2nd generation (for example cefamandole, cefoxitin, cefuroxime, cefonicid, cefmetazole, cefotetan, cefprozil, loracarbef and cefaclor) , 3rd generation (for example cefixime, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefpodoxime and cefetamet) and 4th generation (for example cefepime, cefoselis and cofpirome) cephalosporins and carbapenems (for example imipenem) ; quinolones (for example ciprofloxacin, nalidixic acid, ofloxacin, fleroxacin, norfloxacin, enoxacin, lomefloxacin and cinoxacin) , including fluoroquinolones; lincosamides such as clindamycin and lincomycin; streptogramins (for example quinupristin/dalfopristin) ; folate pathway inhibitors such as sulphonamides (in particular sulphacetamide) and trimethoprim; fosfomycins; glycopeptides (for example teicoplanin and vancomycin); oxazolidinones (for example linezolid); tetracyclines (for example oxytetracycline, minocycline, doxycycline, lymecycline, tetracycline and chlortetracycline) ; other peptides (for example bacitracin); lipopeptides (for example daptomycin) ; anti-tubercular drugs (for example isoniazid, ethambutol, capreomycin, cycloserine, pyrazimamide and aminosalicylic acid); oxazolidinones (for example linezolid); nitrofurans (for example furazolidone); azalides (for example azithromycin); ketolides (for example telithromycin) ; nitroimidazoles (for example metronidazole); phenicols such as chloramphenicol; mupirocin (pseudomonic acid); fusidic acid (a fusidane); aminocyclitols (for example spectinomycin) ; nitrofurantoins; monobactams (such as aztreonam) ; bacitracin; sulfamethoxazole; and mixtures thereof. A composition according to the invention may contain more than one antibiotic.

We have found that the antibacterial activity of RTA3 (SEQ ID NO: 9) against E. coli NCTC 25922 is synergistically enhanced when combined with rifampicin (64-fold increase in activity compared with calculated additive effect), erythromycin (32-fold synergy) and polymyxin E (16-fold synergy). No synergy was found using β-lactam or aminoglycoside antibiotics. (Data not shown.)

In one aspect of the invention, the composition excludes β- lactam (for example, penicillin, benzylpenicillin, flucloxacillin or oxacillin) or aminoglycoside (gentamicin, tobramycin, neomycin or streptomycin) antibiotics.

The invention further provides a pharmaceutical composition comprising a composition as defined herein in combination with a pharmaceutically acceptable carrier.

Suitable carriers may be solid or liquid carriers as are known in the art.

The compositions (including pharmaceutical compositions) of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions) , for administration by inhalation (for example as a finely divided powder or a liquid aerosol, such as produced using a nebuliser) , for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing. Compositions may comprise other well-known formulation additives such as one or more colouring, sweetening, flavouring, preservative agents, inert diluents, granulating and disintegrating agents, binding agents, lubricating agents, and anti-oxidants . The selection will depend upon the particular form the composition will take, and will be determined by a formulation chemist using the principles set out for example in Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansen; Chairman of Editorial Board) , Pergamon Press 1990.

The invention thus provides a method for treating or reducing the severity of an antimicrobial (for example, antibacterial) infection, including prophylactic treatment, comprising administering to a human or animal in need thereof a therapeutically sufficient amount of peptide (including in the form of a composition or pharmaceutical composition) as defined herein.

Treatments using the peptide may be directed to conditions such as sepsis, soft-tissue infections, skin infections, burns, urinary tract infections (UTIs), abdominal infections (such as gastroenteritis) , pneumonia, meningitis, sexually transmitted diseases, and any other condition comprising or susceptible to microbial (for example, bacterial) infection.

The method of the invention is suited to treatment of a microbial infection which is a bacterial infection caused by one or more Gram-negative bacteria. For example, the infection may be caused by one or more of the bacterial species from the group consisting of Haemophilus influenzae, Pseudomonas aeruginosa, Acinetobacter spp. (including A. baumannii, A. xylosoxidans, and A. genom spp.), Stenotrophomonas maltophilia , other non- fermenting bacteria, any member of the family Enterobacteriaceae (including Enterobacter cloacae, Escherichia coli, Klebsiella pneumonia, Proteus mirabilis and other Proteus spp., Salmonella spp. such as S. enteritidis and S. typhi, and Serratia marcescens) , Neisseria species (including N. gonorrhoeae and N. meningitidis) , Moraxella spp. (such as M. catarrhalis) , Helicobacter spp. (such as H. pylori), Stenotrophomonas spp., Bdellovibrio spp., acetic acid bacteria, Legionella (such as L. pneumophila) and alpha-proteobacteria (for example, Wolbachia) .

The method of the invention is also suited to treatment of a microbial infection which is a bacterial infection caused by one or more Gram-positive bacteria, for example from the group consisting of Bacillus spp., Listeria spp., Staphylococcus spp.

(such as S. aureus), Streptococcus spp. (such as S. pneumonia),

Enterococcus spp., and Clostridium spp.

The bacterial infection may in particular be caused by a multidrug or pan-drug resistant species.

The amount of active ingredient that is combined with one or more carriers to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient.

The size of the dose for therapeutic purposes of the peptides of the invention will vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, and will be determined by a clinician in accordance with normal clinical practice. An antimicrobial

(for example, antibacterial) peptide as described above will generally be administered so that a dose (for example, a daily dose) in the range, for example, 0.5 mg to 75 mg per kg body weight (such as about 25 mg per kg body weight) is received.

In one aspect, the treatment comprising at least two doses. We have found that two doses of the peptide can substantially increase effectiveness.

Alternatively, nucleic acids encoding the peptides of the invention may be administered to a patient in need thereof in such way that the peptides are expressed in vivo. For instance, one or more nucleic acids encoding the peptides may be used to transform suitable vectors such as viral or bacterial vectors, or plasmids, which may then be administered to a patient in need thereof.

Alternatively, recombinant plasmids carrying a nucleic acid encoding a peptide of the invention expressed from a donor organism such as Lactococcus could be administered as a therapeutic agent. Thus, a suitable micro-organism,, preferably a commensal micro-organism which is not adversely affected by the peptides of the invention, such as Lactococcus, is engineered using conventional DNA technology, to express the peptide of the invention, and then utilised as a therapeutic agent.

Such probiotic therapies can be carried out either alone or in combination with conventional antimicrobial therapies.

The strains used will suitably be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition for administration purposes.

In a further aspect of the invention there is provided a peptide, a composition or a pharmaceutical composition as defined herein for use in the treatment of a disease. The disease may, for example, be a microbial (such as bacterial) infection . Use of a peptide, a composition or a pharmaceutical composition as defined herein in the manufacture of a medicament for the treatment of a microbial (for example, bacterial) infection is also encompassed.

Further provided according to the present invention is a disinfectant comprising a peptide as defined herein. For example, the disinfectant may comprise the RTA3 peptide (SEQ ID NO: 9) , any of the other peptides described herein, and/or an active fragment, variant or analogues thereof. The disinfectant may be used to disinfect (or sterile) , or may be used to prevent infection, of a surface or substance against microbial (for example, bacterial) species as defined herein. In one embodiment, the disinfectant may further comprise one or more substances active against microbial (for example, fungal or bacterial) species.

Also provided is a method of disinfection comprising applying to a surface (for example, skin such as hand skin, a table surface, or equipment such as surgical equipment including catheters) or a substance (for example, water) to be disinfected a disinfectant as defined above.

The present invention further provides a method of modifying an amphipathic helical peptide comprising a tryptophan (W) residue to reduce toxicity of the peptide against mammalian cells, comprising the step of substituting or deleting the W residue. The W residue may be substituted with another uncharged non- polar amino acid (as defined above) , or another hydrophobic amino acid (from the group consisting of V, L, I, M, F, W, C, A, Y, H, T, S, G and P) , for example another very hydrophobic amino acid (from the group consisting of V, L, I, M, F, W and C) . Toxicity of the peptide may be quantified using an haemolysis assay (for example, employing erythrocytes) as described below. In one aspect of the invention, the peptide is highly soluble in water, for example soluble at levels of to 50-100 g/1. The peptide may be soluble in water at levels of 1-50 g/1, 5-50 g/1, 10-50 g/1, 20-50 g/1, 30-50 g/1, 40-50 g/1, 1-75 g/1, 5-75 g/1, 10-75g/l, 20-75 g/1, 30-75 g/1, 40-75 g/1, 50-75 g/1, 60-75 g/1, 70-75 g/1, 1-100 g/1, 5-100 g/1, 10-100 g/1, 20-100 g/1, 30-100 g/1, 40-100 g/1, 50-100 g/1, 60-100 g/1, 70-100 g/1, 80-100 g/1 or 90-100 g/1.

As used here, the term "antimicrobial" encompasses an agent which has inhibitory activity against or is biocidal (i.e. lethal) to a microbe (i.e. a micro-organism) such as a bacterium and/or a fungus.

Particular non-limiting examples of the present invention will now be described with reference to the following drawings, in which:

Fig. 1 shows agar plates. In (A), Gram-positive and Gram- negative bacterial growth was detected in whole bronchial lavage specimens from a cystic fibrosis patient. The antibiotic strip (Etest®, AB BIODISK, Solna, Sweden) represents a tobramycin (TM) gradient. P. aeruginosa (in this case a tobramycin resistant isolate) only grows within the inhibition ellipse where tobramycin has inhibited S. mitis. In (B), one half of the brain heart infusion agar plate was swabbed with S. mitis and the other left blank. The circle discs on each side of the plate were pregnated with P. aeruginosa, ATCC27853. The growth of S. mitis has completely inhibited growth of the P. aeruginosa;

Fig. 2 shows the single-strand DNA sequence (SEQ ID NO: 21) of a 380bp fragment isolated from Streptococcus mitis. This genetic element is made up by repetition of a 35bp DNA sequence. Analysis of the translated sequence (given below the nucleotide sequence) reveals three copies of an open reading frame ("ORF"). Subtle changes between the copies are due to changes in the underlined amino acid positions. Note the peptide sequences are encoded in all three possible reading frames (SEQ ID NOs 22-24) with the C-terminal peptide sequence in one reading frame overlapping the N-terminal peptide sequence of the subsequent frame;

Fig. 3 shows PCR assay results detecting the 380 base pair DNA sequence (SEQ ID NO: 21) coding for antimicrobial peptides including RTAl (SEQ ID NO: 8) as shown in Fig. 2. Lane M, molecular weight markers (Hyperladder 1, BIOLINE). An approximately 400-bp product was obtained from a Streptococcus mitis clinical isolate (lanes 1 and 7), and reference strain_ 10712 (lane 2), but not Streptococcus pneumoniae (lane 3) and cystic fibrosis negative for antimicrobial activity clinical isolates (lanes 4, 5, 6, 8 and 9);

Fig. 4 is a Western blot using polyclonal antibody (Pacific Immunology Corp., USA) immunoreactive against the synthetic peptide RTAl (lane 1) and a similarly sized peptide in a Streptococcus mitis cell free supernatant (lane 2);

Fig. 5 shows graphs of in vitro and in vivo properties of RTA peptides .

In (A) , the effect of salt on the bactericidal activities of RTAl (SEQ ID NO: 8; light bars) and RTA3 (SEQ ID NO: 9; dark bars) for P. aeruginosa ATCC 27853 were determined in salt free Luria Bertani media supplemented with increasing concentrations of NaCl up to 150 mM. The bactericidal effects of RTA3 (B) and colistin methanesulfonate (C) were evaluated by exposure of P. aeruginosa reference strain ATCC 27853 to serial dilutions of the peptide and determining viable cell counts at different time points. For example, X2 indicates that this is twice the concentration of the MIC. In (D), the in vivo activities of RTA3 (SEQ ID NO: 9) and colistin methanesulfonate were evaluated in a mouse thigh infection model. 4 mice per sampling point were inoculated with P. aeruginosa ATCC 27853 as described in the methods section and 2 hours and in some cases 4 hours after infection the animals were treated with the peptides. Each point represents the bacterial counts in thigh homogenate from a single animal and the line shows the mean value. S = single dose and D = double dose;

Fig. 6 shows alanine scan mutagenesis used to identify the RTAl side chains critical for antimicrobial activity against Pseudomonas aeruginosa ATCC 27853. Numbers on the x-axis refer to amino acid residues of the RTAl peptide (SEQ ID NO: 8) changed to alanine, while the y-axis values are MIC (in μg/ml) determined for the peptides with these alanine changes. Critical RTAl (SEQ ID NO: 8) residues are those of residues Lys-1, Lys-13, and Cys-15. The alanine scan also revealed that the antibacterial activity was enhanced when Thr and GIn at positions 6 and 7 respectively are replaced with alanine. Modified RTA3 (SEQ ID NO: 9) with Arg residues at positions 1 and 13 and Lys and Ala in positions 6 and 7 respectively showed a four fold improvement in activity;

Fig. 7 shows membrane induced structure and permeability activities of RTAl (SEQ ID NO: 8), RTA3 (SEQ ID NO: 9) and magainin peptides. (A) represents circular dichroism spectra of RTA3 (SEQ ID NO: 9; 150μM) in 10 mM potassium phosphate, pH 7.0 containing 50 nm single unilamellar vesicles composed of egg phosphatidylcholine (PC) (line "1"), and in phosphate buffer containing 50 nm vesicles PC:phosphatidylglycerol (PG) (1:1; molrmol; line "2") vesicles (7.5 mM total lipid concentration) . (B) shows side, and end-on, views of an ideal α-helix having the sequence of RTA3 (SEQ ID NO: 9) and illustrating the partial separation of the hydrophobic ("Y") from the polar uncharged ("G"), and positively charged ("B") amino acids on opposite faces of the helix. (C) shows concentration dependence of carboxyfluorescein release from 100 nm PC:PG vesicles by magainin (line "1"), RTAl (SEQ ID NO: 8; line "3"), and RTA3 (SEQ ID NO: 9; line "2"). (D) shows concentration-dependence of carboxyfluorescein release from PC vesicles by magainin (line "1"), ORFl-I (SEQ ID NO: 25; line "2") and RTAl (SEQ ID NO: 8; line "3"};

Fig. 8 shows helical wheel representations of V₆₈₁ (SEQ ID NO: 26; a prior art peptide; see below and Chen et al . , 2005, J. Biol. Chem. 280: 12316-12329) and RTA3 (SEQ ID NO: 9) oriented according to the likely location in the interfacial region of a phospholipid bilayer membrane. Bulky hydrophobic side chains are indicated by large bold lettering, polar side chains are italicised and positively charged residues are starred. Residues mutated in the Example 2 below are boxed;

Fig. 9 provides graphs showing circular dichroism spectra of RTA3 peptides (SEQ ID NOs 9, 27 and 28) (A) and V₆₈₁ peptides (SEQ ID NOs 26 and 29) (B) in the presence of phospholipids vesicles in 10 mM Tris-HCl, 107 mM NaCl, pH 7.4, 20 ⁰C. Peptide concentrations were 150 μM and total lipid concentrations were 10 mM. For both panels filled symbols denote peptide spectra in the presence PC vesicles and open circles denote peptide spectra in the presence of vesicles composed of PC:PG (50:50, mol:mol). Squares are peptides with non-disrupted helical faces (WRTA3-non-dis [SEQ ID NO: 28] in panel A, and V₆₈₁-non-dis [SEQ ID NO: 26] in panel B) , circles are peptides with disrupted helical faces (WRTA3-dis [SEQ ID NO: 27] in panel A, and V₆₈₁-dis [SEQ ID NO: 29] in panel B) and RTA3-dis (equivalent to RTA3; SEQ ID NO: 9) is denoted by triangles;

Fig. 10 provides graphs showing Trp-2 fluorescence emission spectrum of 2 μM V₆₈₁-dis (SEQ ID NO: 29) on titration with increasing amounts of 100 nm vesicles composed of PC: PG (50:50, mol:mol) (panel A) or 100% PC (panel B). The lipid concentrations increase from zero (bottom spectrum) to 50 μM in panel A and from zero to 300 μM in panel B) ; Fig. 11 provides graphs showing tryptophan fluorescence emission blue shifts resulting from titration of peptides (2 μM) in 10 mM Tris HCl, 10⁷ mM NaCL, pH 7.4 with phospholipid vesicles. Panels A and B are data for RTA3 peptides (SEQ ID NOs 27 and 28) and V₆₈i peptides (SEQ ID NOs 26 and 29), respectively.

Open symbols represent titrations with PC:PG (50:50, mol:mol); closed symbols are 100% PC titrations. Squares are peptides with non-disrupted helical faces WRTA3-non-dis [SEQ ID NO: 28] in panel A, and V₆₈i-non-dis [SEQ ID NO: 26] in panel B) , circles are peptides with disrupted helical faces (WRTA3-dis ^■ [SEQ ID NO: 27] in panel A, and V₆₈₁-dis [SEQ ID NO: 29] in panel B);

Fig. 12 shows graphs of fluorescein phosphatidylethanolamine (FPE) fluorescence enhancement resulting from RTA3 peptides (SEQ ID NOs 9, 27 and 28) binding to PC : PG (50:50, mol:mol) (panel A) or 100% PC vesicles (panel B) .

The total vesicle lipid concentration was 65 μM in all cases, and the buffer was 10 mM Tris HCl, 107 mM NaCl, pH 7.4 (20 ⁰C). Circles (WRTA3-dis; SEQ ID NO: 27) and triangles (RTA3-dis; SEQ ID NO: 9) represent peptides with disrupted non-polar helix faces, and squares are WRTA3-non-dis (SEQ ID NO: 28; non- disrupted non-polar helix face) ;

Fig. 13 provides graphs showing FPE fluorescence enhancement resulting from V₆₈₁ peptides (SEQ ID NOs 26 and 29) binding to

PC:PG (50:50, molrmol) (panel A) or 100% PC vesicles (panel B).

The total vesicle lipid concentration was 65 μM in all cases, and the buffer was 10 mM Tris HCl, 107 mM NaCl, pH 7.4 (20 ⁰C).

Circles represent V₆₈₁-dis (SEQ ID NO: 29; disrupted non-polar helix face) , and squares are V₆₈i-non-dis (SEQ ID NO: 26; intact non-polar helix face) ; and

Fig. 14 provides graphs showing peptide-induced carboxyfluorescein (CF) release from 100 nm SUVin 10 mM Tris HCl, 107 mM NaCl pH 7.4. Vesicles (65 μM total lipid concentration) contained 50 mM internal CF (in 10 inM salt-free Tris buffer) .

In this figure open symbols represent RTA3 peptides (SEQ ID NOs 9, 27 and 28) and filled symbols represent V₆₈₁ peptides (SEQ ID NOs 26 and 29). Panel A is CF release data from PC:PG (50:50, mol:mol) vesicles and panel B is dye release from 100% PC vesicles. Squares represent peptides with non-disrupted non- polar helical faces (WRTA3-non-dis [SEQ ID NO: 28] and V₆₈₁-non- dis {SEQ ID NO: 26] ) , circles represent peptides with disrupted non-polar helical faces (WRTA3-dis [SEQ ID NO: 27] and V₆₈₁-dis [SEQ ID NO: 29]) and triangles are data from RTA3 (RTA3-dis [SEQ ID NO: 9] ) . Dotted lines are fits to the equation P=Pmaxcn/ (a+cn) , where P is the percentage of dye release, Pmax is the maximum dye release, c is the peptide concentration, and a is a constant. The values of n are listed in Table 4.

Experimental

Example 1

Summary

We have identified cystic fibrosis sputa from which Gram- positive commensal bacteria, Streptococcus mitis, inhibited growth of the Gram-negative pathogen, P. aeruginosa. We show that S. mitis produces a salt-sensitive antimicrobial peptide (RTAl; SEQ ID NO: 8) expressed from a novel genetic element that is active against P. aeruginosa, Stenotrophomonas maltophilia, and A. baumannii. RTAl was used as a template for the design of peptide analogues with enhanced antimicrobial activity, low salt sensitivity and minimal mammalian toxicity. A modified peptide analogue (RTA3; SEQ ID NO: 9) was particularly active against multi-drug resistant P. aeruginosa and A. baumannii . In a neutropenic mouse infection model, RTA3 was 100-fold more potent in killing P. aeruginosa than colistin methanesulfonate, a polypeptide antibiotic reserved for multi-drug resistant Gram- negative infections. RTA3 (SEQ ID NO: 9) in vivo was also 3- fold, 100-fold and 10-fold more potent than colistin in killing Escherichia coli ATCC25922, a colistin-resistant P. aeruginosa clinical isolate, and a pan resistant pandemic clone of P. aeruginosa, respectively. Biophysical studies on RTA peptides indicate that they have a unique bacterial target site. Our results also highlight commensal bacteria as a novel source of potentially useful therapeutic antimicrobial agents.

Results and Discussion Antimicrobial peptides have been reported to play an important role in the innate respiratory immune system (see for example Zasloff, 2002, Nature 415: 389-395). These include the human β- defensin 1 and 2 (hBD-1 and hBD-2), and cathelicidin LL-37/hCAP- 18 peptides which are up-regulated in respiratory epithelial cells in response to bacterial lipopolysacchyaride (LPS) and inflammatory cytokine activity and secreted into the airway lumen. In earlier studies, the bactericidal activity in normal and cystic fibrosis infected human airway surface fluid against Gram-negative bacteria, such as P. aeruginosa, was primarily attributed to the action of hBD-1. Our studies on direct specimen antimicrobial susceptibility testing using bronchial lavages from cystic fibrosis patients as the test inoculum revealed a significant inhibition of P. aeruginosa by commensal Streptococcus spp (see Fig. 1) . This phenomenon, consistent with observations from a previous report (Gallagher et al . , 1999, Thorax 54: A69-a69) , was seen in approximately 20% of cystic fibrosis samples tested.

Fig. IA illustrates a typical example of Pseudomonas growth only where the Streptococcus has been inhibited - in this case by tobramycin. In our pilot study, 29 of 148 specimens tested (from

3 adults and 26 children) showed that their oropharyngeal flora inhibited Gram-negative pathogens such as P. aeruginosa and S. maltophilia . Normal flora bacteria exhibiting this effect were principally found to be S. mitis as identified by routine microbiological tests, and 16s RNA sequence analysis. In Fig. IB we show that a pure culture of S. initis inhibits growth of P. aeruginosa . These data indicate that the normal flora of some cystic fibrosis patients exhibit a growth suppressive role on key cystic fibrosis pathogens.

To ascertain whether the S. mitis inhibitory activity is due to secreted antimicrobial peptides, aliquots of cell free supernatants were passed through disposable C18 chromatography columns. Fractions were tested against a P. aeruginosa reference strain (ATCC 27853) as outlined in the methods section. We detected anti-Gram-negative activity in cell-free supernatants (MW≤ 3000 Daltons) of clinical and reference strains of S. mitis and S. mutans; however, no activity was detected in S. oralis, S. sanguis, or S. anginosus . All of these Streptococcal species constitute normal oropharyngeal flora in both cystic fibrosis patients and healthy humans.

Antimicrobial peptides were purified by subjecting S. mitis cell free supernatant to gel filtration chromatography, and reverse- phase high-performance liquid chromatography (RP-HPLC) . Fractions demonstrating activity against P. aeruginosa were selected for N-terminal sequencing and tandem mass spectrometry (MS-MS) . One fraction possessing these properties gave an N- terminal sequence TQAFS (SEQ ID NO: 30) by Edman degradation, and an internal sequence VRVV (SEQ ID NO: 31) by MS-MS.

Degenerate oligonucleotides designed from the partial amino acid sequence (TQAFS; SEQ ID NO: 30) were used to amplify PCR products of 1200, 700 and 380 base pairs (bp) . Preliminary sequencing of the 1200bp and 700bp products indicated, as judged by data-base comparisons, that they encode S. mitis proteins. The nucleotide and translated amino acid sequences of the full- length clone of the 380bp product (SEQ ID NO: 21) is given in Fig. 2. This genetic element is made up by repetition of a 35bp DNA sequence. Conceptual translation of the 380bp DNA sequence revealed a number of small open reading frames (16 amino acids) each containing the partial N-terminal sequence TQAFS (SEQ ID NO: 30) deduced from Edman analysis and the RW partial sequence deduced by MS-MS analysis. Remarkably, these open reading frames are copied with high sequence conservation in all three reading frames, with the C-terminal peptide sequence in one reading frame overlapping the N-terminal peptide sequence of the subsequent frame. Additionally, the conserved motif TQAF (SEQ ID NO: 32) is encoded on the complementary strand (3' -5') on the same region of DNA and repeated in the three different reading frames. To our knowledge this genetic arrangement is unique in biology. None of these open reading frames was detected within the S. tnitis genome sequenced thus far and no significant homology (>50%) was found with DNA sequences in Genbank (BlastN) . Peptides encoded by reading frames 1 and 2 were found to have a 44% identity with Uperin 3.1, a peptide with anti- Gram-positive and anti-Gram-negative activity isolated from the Australian floodplain toadlet, Uperoleia inundata .

Primers designed to amplify from the conserved 5' and 3' termini of the 380 bp coding sequence (SEQ ID NO: 21; see Fig. 2) revealed as shown in Fig. 3 that the coding sequence could be detected in a Streptococcus mitis clinical isolate (lanes 1 and

7) and reference strain 10712 (lane 2) but not Streptococcus pneumoniae (lane 3) and cystic fibrosis negative for antimicrobial activity clinical isolates (lanes 4, 5, 6, 8 and

9) . Identity was verified by sequencing the amplified product confirming the expected DNA sequence characteristic of the RTAl

(SEQ ID NO: 8) - like peptides.

We investigated the antimicrobial activity of several synthetic peptides corresponding to the sequence predicted from the open reading frames (Fig. 2) . The peptide having the highest antimicrobial activity against a number of target organisms was a C-amidated version of open reading frame-1, peptide copy-3 (ORF1-3; see SEQ ID NO: 24) . This peptide, with no activity against S. mitis, is now referred to as RTAl (SEQ ID NO: 8). A rabbit antibody raised against RTAl (SEQ ID NO: 8) and used in Western blot analysis to confirm secretion of this antimicrobial peptide from S. mitis and further analyse the cystic fibrosis samples producing this inhibitory phenomena. The expression studies reveal that from cystic fibrosis specimens and culture medium, indicate that RTAl (SEQ ID NO: 8) can be shown to complexed to a larger protein molecule and thus appears as a larger band on Western hybridisation analysis (see Fig. 4). Non- complexed RTAl (SEQ ID NO: 8) appears as a smaller band of approximately 1760 Daltons. Where inhibition of P. aeruginosa could not be demonstrated, RTAl (SEQ ID NO: 8) could not be detected either in a complexed or non-complexed form.

Previously characterised antimicrobial peptides such as hBD-1 and hBD-2, active in human airway, display a salt dependent loss in activity. The salt sensitivity of RTAl was evaluated by assessing minimal bactericidal concentrations (MBC) values against P. aeruginosa at different salt concentrations up to 150 mM NaCl (Fig. 5A). In the absence of salt, RTAl (SEQ ID NO: 8), showed comparable anti-P. aeruginosa activity (MBC was 4 μM or 8 mg/1) to that of colistin. However, this activity was reduced by increased concentrations of salt.

To identify residues essential for antimicrobial activity and decrease the salt sensitivity of RTAl (SEQ ID NO: 8), we used alanine-scan mutagenesis (for which see Cunningham & Wells, 1989, Science 244: 1081-1085; Sahm et al . , 1994, Peptides 15: 1297-1302; and ReidhaarOlson et al . , 1996, Biochemistry 35: 9034-9041), as shown in Fig. 6, and selective amino acid replacement to produce a new modified peptide, RTA3 (SEQ ID NO: 9) . This peptide retains the activity of RTAl (SEQ ID NO: 8) in low salt buffers up to physiological ionic strength (125 mM NaCl) and as for RTAl (SEQ ID NO: 8; see Table 2) shows good activity against several species of Gram-negative bacteria including those isolates demonstrating colistin and polymixin-B resistant isolates (Fig. 5A and Table 1) . RTA3 (SEQ ID NO: 9) and RTA4 (SEQ ID NO: 10), a variant of RTA3 (SEQ ID NO: 9) in which P at position 2 of RTA3 (SEQ ID NO: 9) is mutated to R, are also shown to active activity against various Gram-positive bacteria (Table 1). We also assessed the antimicrobial activity of mixtures of synthetic peptides corresponding to the potential ORF' s of Fig. 2, but these do not show enhanced activity over the activities of individual peptides indicating the absence of synergistic effects (data not shown) .

Table 1: Antimicrobial activity (MIC in μg/mL) of RTA3 (SEQ ID NO: 9), RTA4 (SEQ ID NO: 10) _CM, colistin and/or polymyxin B against various bacteria.

Code Organism Colistin Polymyxin B RTA3¹ RTA4'

ATCC 27853 P. aeruginosa 1 1 16 Nd

ATCC 25922 E. coli 0.125 0.25 32 2(1)

ATCC 13636 S. maltophilia 1 1 16 (8) 8(4)

LAl2 324 Acinetobacter spp. 4 2 8 (8) Nd

NAl2 374 Acinetobacter spp. 4 2 8 (2) Nd

NA37 399 Acinetobacter spp. 4 4 16 (1) Nd

PS-26 3 A. xylosoxidans 8 4 >64 (8) Nd

KS6 290 A. baumanii 8 8 8 (4) Nd

NA45 407 Acinetobacter spp. 16 16 8 (8) Nd

CD99 44 A. genom spp 16 8 16 (2) Nd

EU22 284 Acinetobacter spp. 64 16 32 (4) Nd

KS4 288 A. baumanii 1024 256 16 (4) Nd

PS-23 2 A. xylosoxidans 1024 1024 >64 (2) Nd

CDC2031 P. aeruginosa 0.5 1 16 Nd

PS19 1284 P. aeruginosa 0.5 0.5 16 Nd

PS29 1290 P. aeruginosa 1 0.5 16 (8) Nd

PS76 1312 P. aeruginosa 1 2 16 (4) Nd

CDC2300 P. aeruginosa 2 16 (4) Nd

PS28 1289 P. aeruginosa 8 8 32 (4) Nd

PS-47 290 S. maltophilia 0.25 0.5 16 Nd PS-48 291 S. maltophilia 0.25 0.25 16 Nd

PS-27 289 S. mal tophilia 2 1 8 Nd

PS-IO 284 S. mal tophilia 32 16 64 Nd

Proteus spp. Nd Nd >32 >32

Serratia marcescens Nd Nd >32 >32

Haemophilus influenzae Nd Nd (2) Nd

ATCC 12799 Strep, pneumoniae Nd Nd (2) Nd

ATCC 12793 Staph, aureus Nd Nd 4-Σ 2-4 NCTC 6571 S. aureus Nd Nd 4 ATCC 12981 S. aureus Nd Nd 16 4

The minimum inhibitory concentration (MIC) was determined as described in the method section by AB BIODISK, Sweden against a number of colistin sensitive and resistant isolates. Values in brackets for RTA3 (¹SEQ ID NO: 9) and RTA4 (²SEQ ID NO: 10) are MICs in minimal media. "Nd" is not determined.

Table 2: Bactericidal activities (MIC in μg/ml) of RTAl (SEQ ID NO: 8), RTA3 (SEQ ID NO: 9), RTA4 (SEQ ID NO: 10) and/or colistin methanosulfonate ("CM") against various bacteria

Bacteria (No. of isolates) RTAl CM RTA3² RTA4-

MDR E. coli Nd Nd 4-16(2-8) 8-16(2-4)

MDR Klebsiella pneumonia Nd Nd 8-32(4-8) 4-16(1-4) MDR Enterobacter spp. Nd Nd 8-16(4) 8(2) A. baumanii (10)* 0.5-4 16-32 1-16(0.5-8) 1-4 (2-8) S. maltophilia ATCC 13637 16 16 16(8) 8(4) S. maltophilia (6-8)** 2-8 16-128 8-32(4-16) 4-16(2-16)

P. aeruginosa ATCC 27853 16 16 8(4) 4(2)

P. aeruginosa PAOl 8-16 32 8(4) 2(2)

P. aeruginosa (12-54)*** 4-16 16-64 8-32(2-8) 2-8(1-8) MDR are multiple drug resistant strains. A. baumanii* are clinical isolates resistant to all β-lactam antibiotics and aminoglycosides. S. maltophilia** are clinical isolates resistant to all antibiotics apart from cotrimoxazole . Six strains were tested against RTAl (¹SEQ ID NO: 8) and CM, 8 strains were tested against RTA3 (²SEQ ID NO: 9) and RTA4 (³SEQ ID NO: 10) . P. aeruginosa*** are clinical isolates strains resistant to all antibiotics apart from colistin. Twelve strains were tested against RTAl (SEQ ID NO: 8) and CM, 54 strains were tested against RTA3 (SEQ ID NO: 9) and RTA4 (SEQ ID NO: 10) . Values in brackets are MICs in minimal medium. Nd = not determined.

In time kill kinetic studies, P. aeruginosa (ATCC 27853) at a density of 10⁷ colony forming units per millilitre (CFU per mL) was exposed to various concentrations of RTA3 (SEQ ID NO: 9) and colistin methanesulfonate (colistin was used in the methanesulfonate form in vitro to aid the dosing regimen in our in vivo infection model) and viable colonies were determined during the subsequent 5 hours and after 24 hours (Fig. 5B, C).

Both RTA3 (SEQ ID NO: 9) and colistin methanesulfonate were bactericidal against P. aeruginosa in a concentration-dependent manner. However, RTA3 (SEQ ID NO: 9) was more potent even at one multiple of the minimum inhibitory concentration (1 x MIC) with a 3 to 5 log kill at 3 hours.

Both the selectivity for bacterial cells and the killing mechanism of many positively charged antimicrobial peptides correlate with peptide interactions with lipid membranes. For example, magainin, a 23 amino acid peptide with anti-Gram- positive and anti-Gram-negative activity isolated from the

African clawed frog Xenopus laevis, adopts an amphipathic helical conformation on interaction with negatively charged membranes, and forms pores in these membranes at a concentration lower than that required for perturbation of membranes composed of neutral lipids (Dempsey et al . , 2003, Biochemistry 42: 402- 409) . Like magainin, RTA3 (SEQ ID NO: 9) and RTAl (SEQ ID NO: 8) undergo a transition from unstructured conformation to an amphipathic helical conformation on interaction with negatively charged membranes (Fig. 7A,B); no secondary structure is induced by vesicles composed of neutral ( zwitterionic) lipids (Fig. 7A). RTA3 (SEQ ID NO: 9) perturbs negatively charged membranes with an activity similar to that of magainin (Fig. 7C; the peptide concentrations required to release 50% of total entrapped vesicle contents are near 0.26 μM in each case) . Similarities in the biophysical properties of RTAl (SEQ ID NO: 8) and RTA3 (SEQ ID NO: 9) with those of magainin suggests that an interaction and direct disruption of negatively charged membranes may play an important role in the antimicrobial action of these peptides. The potential haemolytic and mammalian cell toxicity of amphipathic peptides such as magainin and melittin, a 26-residue peptide found in the venom of the honey bee, Apis mellifera, correlate with interactions with neutral lipid membranes that mimic the outer bilayer leaflet of eukaryotic cells. Thus, 50% of total entrapped vesicle contents are released at 2 μM of magainin (Fig. 7A). However, even at 50 μM concentration of RTA3

(SEQ ID NO: 9) there is no detectable dye release from vesicles composed of neutral membranes.

Surprisingly, RTAl (SEQ ID NO: 8) and RTA3 (SEQ ID NO: 9) are quite resistant to homo- or hetero-dimerisation (data not shown) under a range of conditions that readily produce, for example, more active homodimers of magainin which also possesses a cysteine residue in similar position to that of RTA3 (SEQ ID NO: 9) . However, the cysteine residue is an absolute requirement for activity (RTA1-C15S analogue [SEQ ID NO: 33] is inactive, unpublished results) . Such observations have been found in the Maximin class of peptides in which activity is dependent on a free cysteine thiol, rather than, for example, the mammalian cryptdin-sequence-related peptides in which antimicrobial diversity is generated by a spectrum of cysteine-linked homo- and hetero-dimers .

RTAl (SEQ ID NO: 8) and RTA3 (SEQ ID NO: 9) were evaluated for their effects on the integrity of eukaryotic membranes with a standard red blood cell haemolytic assay and an MTT cytotoxicity assay (data not shown) . As expected, the viability of cultured HeLa cells is greatly decreased at low concentrations of magainin (25-50 mg/1) . In contrast, RTA3 (SEQ ID NO: 9) displayed minimum toxicity (negligible HeLa cell toxicity at concentrations below 600 mg/1 and 1000 mg/1, and 6% haemolysis at 10 mg/ml compared to >80% haemolysis at 1 mg/ml with magainin; similarly, negligible cell toxicity was observed at concentrations of RTA3 (SEQ ID NO: 9) below 1000 mg/1 using McCoy, Vero and HPA6 cells) illustrating a marked difference in potential chemotherapeutic index between the two peptides. RTA3 (SEQ ID NO: 9) toxicity was also evaluated in mice (groups of six) by subcutaneous injections with a single dose of 120 mg per kg of body weight. RTA3 (SEQ ID NO: 9) was easily tolerated with no deaths occurring and all mice showing the same presentation as the placebo (sterile buffered saline) (data not shown) .

In separate experiments, acute in vivo toxicity was investigated in groups of four mice which were given 200, 300, 400 or 500 mg/kg of RTA3 (SEQ ID NO: 9) and then observed over 48 h.

Chronic in vivo toxicity was investigated in a group of eight mice which were given twice daily injections of 120 mg/kg RTA3

(SEQ ID NO: 9) for one week. In both experiments, no behavioural changes were observed, and post-mortem examination revealed no macroscopic changes of major organs.

The in vivo activities of RTA3 (SEQ ID NO: 9) and colistin methanesulfonate were evaluated in a neutropenic mouse thigh infection model as described in the methods sections (Fig. 5D). The organisms used in separate infection models were: P. aeruginosa (ATCC 27853) , a highly-resistant P. aeruginosa strain (SPM-I) (sensitive only to colistin) , a colistin resistant

(aminoarabinose modification of the lipid membrane) P. aeruginosa (PS28-1289) and Escherichia coli (ATCC 25922) . The effect of treatment was evaluated by a reduction in thigh homogenate bacterial counts in comparison to untreated mice. For

P. aeruginosa ATCC 27853, single subcutaneous treatments of RTA3

(SEQ ID NO: 9; 30 mg per kg) or colistin methanesulfonate (40 mg per kg) resulted in 100 fold and 10 fold drop in bacterial counts, respectively. However, when the mice were given a second subcutaneous dose, RTA3 (SEQ ID NO: 9) resulted in a 10,000-

100,000 fold reduction in bacterial counts, whereas, colistin methanesulfonate did not result in further reduction in bacterial counts. On the basis of these data, the other bacteria in the infection model were treated with a two dose of either RTA3 (SEQ ID NO: 9; 30 mg per kg) or colistin (40 mg per kg) .

RTA3 (SEQ ID NO: 9) gave 10-fold, 100-fold and 4-fold increased killing over colistin for P. aeruginosa strains SPM-I and PS28-

1289 and E. coli ATCC 27853, respectively. Given the fact that

RTA3 (SEQ ID NO: 9) is considerably more active than colistin in vivo yet has significantly higher MICs there is clearly exist a discrepancy between RTA3 (SEQ ID NO: 9) in vivo and in vitro data. This phenomenon can be, in part, explained by the protein binding properties of RTA3 (SEQ ID NO: 9) , as all antimicrobial testing media contains hydrolyzed protein products which will subsequently quench RTA3 (SEQ ID NO: 9) from the media and prevent it from interacting with the Gram-negative membrane.

The data shown in Fig. 5D have been confirmed in mouse/prostate specific antigen (PSA) peritonitis models, with a protective dose of 20 mg/kg RTA3 (SEQ ID NO: 9; data not shown) .

The potent activity observed in animal models where RTA3 (SEQ ID NO: 9) was delivered subcutaneously indicates that RTA3 (SEQ ID NO: 9) possesses favorable pharmacokinetic properties. This study also demonstrates that in vitro susceptibility testing for certain agents (e.g. colistin) may correlate poorly with in vivo effectiveness. Furthermore, antimicrobial peptides such as colistin are restricted in their clinical use by their level of toxicity (therapeutic index being relatively narrow) .

Using the original peptide, RTAl (SEQ ID NO: 8), as a molecular scaffold we have created an antimicrobial peptide, RTA3 (SEQ ID NO: 9) , which possesses unique biophysical and microbiological properties. The in vivo activity of RTA3 (SEQ ID NO: 9) against highly (multi-drug) resistant and colistin-resistant P. aeruginosa, coupled with its protein binding properties and lack of demonstrable toxicity, suggest that these peptides could play a role in our future arsenal against pathogens such as resistant Gram-negative pathogens. The fact that RTAl (SEQ ID NO: 8) produced from S. mitis also possesses similar but less potent properties offers the possibilities that these commensal bacteria could be used in probiotic programs - not least for patients with cystic fibrosis.

Methods Direct testing of cystic fibrosis specimens. Cystic fibrosis bronchial specimens (sputa, cough swabs, bronchial alveolar lavage and nasal pharyngeal aspirates) were homogenised and then evenly spread over Isosensitest agar (BD, Baltimore, USA) plates and different Etest® antibiotic gradient strips (AB BIODISK, Solna, Sweden) were placed on the dried agar surface. Plates were then examined after 48 hours of incubation at 37 ⁰C for evidence of Gram-negative bacterial growth in regions where Gram-positive bacteria were inhibited by the presence of antibiotic.

Peptide purification and N-terminal sequencing. S. mitis was grown without aeration for 48 hours at 37 ⁰C in Brain Heart Infusion (BHI) media. Cell free supernatant obtained by centrifuging at 17,00Og for 30 minutes was lyophilized, dissolved in 0.2% acetic acid and applied to a 600ml G25 gel filtration column. Fractions (4 x 100 mL) collected after the void volume, were lyophilized and subjected to reversed-phase HPLC on a C18 column (Vydac 218TP54, 4.6 x 250 mm); mobile phase A: 0.1% trifluoroacetic acid (TFA) in H₂O; B: 0.1% TFA in 100% acetonitrile with a linear gradient of 0-60% over 50 minutes (2 mL/min; 225 nm detection) . Fractions were freeze dried, and tested for antimicrobial activity using a broth microdilution assay. N-terminal sequence analysis was carried out at Alta Bioscience (University of Birmingham, UK) .

PCR Procedures. DNA sequence encoding antimicrobial peptides were isolated by random primer PCR. Based on N-terminal sequence analysis (see text), degenerate primers (Qiagen, GmbH, Germany) were designed e.g. primer-A: 5' Biotinylated-NSW RAA NGC YTG NGT 3' (SEQ ID NO: 34; non-coding strand), and used (10 pM) in combination with random flanking primers e.g. Rl: 5' CAG TTC AAG CTT GTC CAG GAA TTC NNN NNN NCG CGT 3' (SEQ ID NO: 35) . R= wobble (A+G) , S= wobble (C+G) , Y= wobble (C+T) , N= any nucleotide. For amplification, PCR (94 ⁰C for 4 minutes, 94°C for 1 minute, 45 ⁰C for 1 minute, 68°C for 3 minutes, cycle step 2 for 39 times, incubate 68 ⁰C for 10 minutes) was performed using AB-gene Expand Hi-fidelity master mix containing Pfu/non-proof- reading Taq polymerases and dNTPs (ABGENE house, Surrey, UK) . 1 μL inocula of S. mitis was used as template. Purified biotinylated products were used as template in a second PCR reaction (same program as stage 1) with 1 μl flanking primer (5' TTC GAA CAG GTC CTT AAG 3' [SEQ ID NO: 36]) and 1 μl primer A. PCR products were TOPO-cloned into the pCR 2.1 TOPO cloning vector (Invitrogen, Carlsbad, CA) and selected in Luria Burtani media supplemented with kanamycin (50 μg/ mL) . Plasmid DNA was isolated using Qiagen mini-prep kit (Qiagen, Inc. Valencia, USA) and sequencing performed by Advanced Biotechnology Centre, Imperial College, London.

For the PCR assay shown in Fig. 3, PCR primers were selected from the conserved 5' and 3^r termini of the 380 bp coding sequence (SEQ ID NO: 21; see Fig. 2). Bacterial suspensions from blood plates were boiled for 5 minutes, centrifuged, and 1 μl of the supernatant used for PCR. For amplification, PCR (94 ⁰C for 4 minutes, 94 ⁰C for 1 minute, 55 ⁰C for 1 minute, 72 ⁰C for 1 minutes, cycle step 2 for 39 times, incubate 72 ⁰C for 10 minutes) was performed using AB-gene Expand Hi-fidelity master mix and dNTPs (ABGENE house, Surrey, UK) . Identity was verified by sequencing the amplified product confirming the expected DNA sequence characteristic of the RTAl like peptides.

Peptide synthesis. Peptides were synthesised by Dr. Graham Bloomberg of the Bristol Centre for Molecular Recognition, purified to greater than 95% by HPLC, and the concentration determined by amino acid analysis (Alta Bioscience, University of Birmingham) . The magainin peptide was magainin-F12W, N22C (Dempsey et al, 2003, supra). Colistin methanesulfonate (sodium) was obtained from Sigma (Poole, UK) .

Antimicrobial activity. RTA3 (SEQ ID NO: 9) , ploymixin B and colistin minimum inhibitory concentrations (MIC) were determined by broth microdilution according to the Clinical and Laboratory Standards Institute (formerly NCCLS; 2003, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, Approved Standard - Sixth Edition. M7-A6. Clinical and Laboratory Standards Institute. Wayne, PA) . 100 μL of 0.5 - I x 10⁶ CFU per mL of the test organism in Mueller Hinton cationadjusted broth (BD, Baltimore, USA) were incubated in 96 well micro-titre plates with serial two- fold dilutions of the antimicrobial agents. Minimum bactericidal concentrations (MBC) were determined by plating out aliquots with no visible growth of bacteria from the MIC micro-titre plates onto Muller Hinton agar plates, and incubating at 37 ⁰C for 24 hours. The bactericidal activities of RTA3 (SEQ ID NO: 9) and colistin methanesulfonate were evaluated by exposing P. aeruginosa ATCC 27853 to serial dilutions of the peptide and determining viable cell counts at defined time points. Western blotting. The Streptococcus mitis strain used in Fig. 4 is a clinical isolate recovered from cystic fibrosis sputa. Lyophilised cell free supernatant was dissolved in 4 M urea, and incubated for 30 minutes at room temperature. Peptides under 3000 Daltons were obtained by size-exclusion chromatography. The urea concentration was reduced by dialysis against deionised water using 500 molecular weight cut off membrane. Peptides were concentrated by freeze drying, separated on a 16% SDS-PAGE by electrophoresis, transferred to PVDF membrane and analysed by ECL Western blotting.

Spectroscopic analysis of membrane binding and perturbation. Circular dichroism (CD) spectra were obtained using a Jobin-Yvon spectrapolarimeter with 0.1 mm pathlength cuvettes as previously described (Dempsey et al . , 2003, supra). Vesicles for CD were prepared by repeated high pressure extrusion of dried equimolar mixtures of egg PC and PG hydrated in 10 mM potassium phosphate buffer, pH 7.0 through 50 run pore diameter filters. Vesicles for dye efflux measurements were made by hydrating lipids in 10 mM Tris HCl, pH 7.4, 1 mM EDTA, 50 mM carboxyfluorescein (CF), high pressure extrusion through 100 nm pore diameter filters, and gel filtration in Tris buffer containing 107 mM NaCl to remove external CF while maintaining equi-osmolarity between internal CF and external NaCl. Dye efflux was measured from the release of fluorescence self-quenching as the trapped CF is diluted into the extravesicular solution. The total lipid concentration in the dye release experiments was 60 μM. The fluorescence excitation and emission wavelengths were 490 and 530 nm, respectively.

Neutropenic mouse thigh model. P. aeruginosa ATCC 27853 was grown overnight at 37 °C in Mueller Hinton broth and on the following morning subcultured and incubated for 4 h at 37 ⁰C. The inocula were adjusted to ~5.0 x 10⁷ CFU per mL, washed and re- suspended in phosphate buffered saline. Female BALB/c mice, 7-8 weeks of age, were made neutropenic by intraperitoneal injection of cyclophosphamide (Cytoxan; Mead Johnson) at 150 mg/kg of body weight on days -4 and -1. On day 0, the mice were infected by intramuscular injection of 0.1 mL of bacterial inoculum into the right thigh. Mice were treated with 0.2 mL of RTA3 (SEQ ID NO: 9; 27 mg per kg) or colistin methanosuplhonate (40 mg per kg) by subcutaneous injection between shoulders 2 hours after infection and in some cases a second dose was administered 4 hours post infection. 10 hours post infection thighs were removed aseptically and homogenized in 10 mL of ice-cold phosphate- buffered saline. Serial 10-fold dilutions of the homogenized material were plated on Mueller-Hinton agar, and the colonies were counted. The change in bacterial counts was determined by subtracting the bacterial counts in the treatment groups from the bacterial counts in the untreated controls at the start of therapy.

Example 2

Summary- Further to the work described in Example 1, we show here how the RTA3 peptide (SEQ ID NO: 9) conforms to the positively-charged, amphipathic helical peptide motif, but has a positively charged amino acid (Arg5) on the non-polar face that forms when the peptide structures upon membrane binding. We consider that disruption of the hydrophobic face with a positively-charged residue plays a role in minimizing eukaryotic cell toxicity, and in the present example tested this using a mutant with an Arg5- >Leu5 substitution. The greatly enhanced toxicity in the mutant peptide correlated with its ability to bind and adopt helical conformations upon interacting with neutral membranes; the wild type peptide RTA3 (SEQ ID NO: 9) did not bind to neutral membranes (binding constant reduced by at least 1000-fold) . Spectroscopic analysis indicates that disruption of the hydrophobic face of the parent peptide is accommodated in negatively charged membranes without partial peptide unfolding. These observations apply generally to amphipathic helical peptides of this class since we obtained similar results with a peptide and mutant pair (Chen et al., 2005, supra) having similar structural properties. In contrast to previous interpretations, we demonstrate that these peptides simply do not bind well to membranes (like those of eukaryotes) with exclusively neutral lipids in their external bilayer leaflet. We highlight a significant role for tryptophan in promoting binding of amphipathic helical peptides to neutral bilayers, augmenting the arsenal of strategies to reduce mammalian toxicity in antimicrobial peptides.

.Results

The initial experiments described below were designed to compare the membrane binding properties of two sets of peptides, each set comprising a peptide (s) having the non-polar helix face disrupted by a positively charged R or K residue (RTA3-dis, equivalent to RTA3 [SEQ ID NO: 9]; WRTA3-dis [or RTA3-F4W; SEQ ID NO: 27] and V₆₈₁-dis [or V₆₈₁-V13K; SEQ ID NO: 29), and the other of the set having a non-disrupted helix face (WRTA3-non- dis [or RTA3-F4W,R5L; SEQ ID NO: 28] and V₆₈₁-non-dis [SEQ ID NO: 26) . We have employed the "dis"/"non-dis" designation here to simplify the terminology of these peptides, particularly since the "wild type" RTA3 peptide (RTA3-dis; SEQ ID NO: 26) has a disrupted ("dis") non-polar helix face, whereas it is the "mutant" V₆₈₁ peptide (V₆₈₁-V13K or V₆₈₁-dis; SEQ ID NO: 29) that has the disrupted ("dis") non-polar helix face. We synthesised F4W-variants of the RTA peptides (WRTA3-dis and WRTA3-non-dis; SEQ ID NOs 27 and 28, respectively) in which the Phe-4 residue was replaced with a tryptophan, for two reasons. First, in order to obtain accurate peptide concentrations that are important for quantitative interpretation of circular dichroism (CD) data. Secondly, the presence of a tryptophan residue allows quantitative analysis of peptide binding to lipid vesicles through the effects of binding on tryptophan fluorescence. V₆₈₁ (SEQ ID NO: 26) already contains a tryptophan (W2) in its "native" sequence.

The minimum inhibitory concentrations against P. aeruginosa and the haemolytic activities of the peptides are given in Table 3. We present haemolysis data at low concentrations, and very high concentrations that are relevant for in vivo considerations as described above.

Table 3: Biological activities of studied peptides.

MIC haemolytic activity (%)

(μg/ml) μM 80 μM 2.5 mM

RTA3 8 4 2 6

(RTA3-dis; SEQ ID NO: 9)

RTA3-F4W 4 2 4 21

(WRTA3-dis; SEQ ID NO: 27)

RTA3-F4W,R5L 8 4 80 100

(WRTA3-non-dis; SEQ ID NO: 28)

V₆₈₁-V13K 8 2.5 5 66 (V₆₈₁-dis; SEQ ID NO: 29)

V₆₈₁ 8 2.5 100 100

(V₆₈₁-non-dis; SEQ ID NO: 26)

Peptides with disrupted non-polar helical faces have greatly reduced haemolytic activity while retaining anti-Pseudomonas activity similar to that of the equivalent peptide having a non- disrupted non-polar helix face. The latter peptides have very high haemolytic activity. These observations are generally consistent with the results on V₆₈₁ (SEQ ID NO: 26) and V₆₈₁-V13K

(SEQ ID NO: 29) previously described by Chen et al. (2005, supra) . Two important observations that we explore in the following sections are the extremely low haemolytic activity of wild-type RTA3 (SEQ ID NO: 9) at very high concentrations, and the significantly enhanced haemolytic activity of the analogue of RTA3 (SEQ ID NO: 9) in which the Phe-4 residue is replaced with a tryptophan (see SEQ ID NO: 27) .

Each of -the peptides used in this example is unstructured in buffer A as indicated by a "random coil" CD spectrum (not shown) . Figure 9a shows that both WRTA3-dis (SEQ ID NO: 27) and WRTA3-non-dis (SEQ ID NO: 28) adopt helical structure upon binding to membrane vesicles composed of 50%PC and 50% PG in buffer A. The helical content, calculated using the formalism of Luo and Baldwin (Luo & Baldwin, 1997, Biochemistry 36: 8413- 8421; see also Dempsey et al . , 2005, Biochemistry 44: 775-781) is around 78% in each case, and is very similar to the maximum helical content induced by these peptides in 40% TFE (in buffer A; not shown) , which generally induces a maximum helix content in peptides with moderate to high helix forming potential. The high concentrations of peptide (150 μM) and lipid (10 mM) required for adequate spectroscopic CD data is expected to promote peptide binding to lipid, and for the strongly binding peptides the CD signal should be representative of the conformation of the peptide in its membrane-bound state. The equivalence of the helix content in membrane-bound forms of WRTA3-dis (SEQ ID NO: 27) and WRTA3-non-dis (SEQ ID NO: 28) indicates that the presence of an Arg residue on the hydrophobic face of the otherwise amphipathic helix doesn't detectably disrupt helical conformations when WRTA3-dis (SEQ ID NO: 27) binds to a negatively charged membrane (50% PC:PG).

WRTA3-dis (SEQ ID NO: 27) adopts minimal helical structure (around 10%) when incubated at very high concentrations with vesicles composed exclusively of PC (Figure 9a) . However if the Arg-5 residue in WRTA3-dis (SEQ ID NO: 27) is replaced with a leucine (in WRTA3-non-dis; SEQ ID NO: 28), a helical conformation (82%) is induced, presumably as a result of membrane binding, that is essentially equivalent to that induced by interaction of both WRTA3-dis (SEQ ID NO: 27) and WRTA3-non- dis (SEQ ID NO: 28) with negatively-charged membrane vesicles

(50% PC:50% PG). The small but measurable helical content of

WRTA3-dis (SEQ ID NO: 27) is interesting in view of the complete absence of helical content in RTA3-dis ("wild-type" RTA3; (SEQ ID NO: 9) when incubated with high concentrations of vesicles composed of neutral lipid (Figure 9a) . This observation indicates that the apparently conservative replacement of the Phe-4 residue of RTA3-dis (SEQ ID NO: 9) with a Trp results in measurable differences in the interaction of RTA3-dis (SEQ ID NO: 9) and WRTA3-dis (SEQ ID NO: 27) with neutral membranes.

Similar observations were made with the wild-type V₆₈₁-non-dis (SEQ ID NO: 26) and V₆₈₁-dis mutant (V₆₈₁-V13K; SEQ ID NO: 29) that has the disrupted non-polar helix face (Figure 9b). Each of the peptides has a high helical content induced upon incubation with negatively-charged membranes, but only the V₆₈i-non-dis wild-type peptide (SEQ ID NO: 26) , having an intact non-polar helix face (Figure 8), has a high helix content induced upon interaction with membranes composed of neutral lipids. The. V₆₈₁-dis peptide (SEQ ID NO: 29) has slightly reduced helical content when bound to negatively charged membranes (68%) compared to the wild type V₆₈₁-non-dis (SEQ ID NO: 26; 81%) indicating that helical conformations are somewhat disrupted upon binding at the membrane interface. However, since 81% helix corresponds to around 21 amino acid residues in V₆₈₁ (SEQ ID NO: 26) and 68% corresponds to 17-18 amino acid residues, the V13K mutation does not result in large scale unfolding of helical conformations to release the K13 residue from the non-polar helix face.

The sample restrictions in CD spectroscopy require that conformational analyses that relate to membrane binding can only be done under conditions of very high lipid and peptide concentrations. In addition, the possibility that the absence or limited amount of helical conformations in the presence of lipid vesicles results from binding without structure formation cannot be formally ruled out from CD data alone. We therefore performed two separate series of experiments to assess peptide binding directly, and under conditions that more closely match the peptide concentrations used in antimicrobial assays. These experiments allow an assessment of relative membrane binding affinities of the peptides, and additionally can establish whether binding is cooperative or exhibits "hyperbolic" behaviour. These are each of interest with respect to the mechanisms of the membrane properties of the peptides. Since RTA3 (SEQ ID NO: 9) does not contain a Trp residue we used F4W "mutants" (SEQ ID NOs 27 and 28) to assess binding to phospholipids vesicles based on binding-induced perturbation of Trp fluorescence.

The sequestering of the Trp indole group in an environment of reduced polarity upon membrane binding results in a blue shift in the fluorescence excitation maximum, as illustrated by the emission spectrum of the Trp2 residue of V₆₈₁-dis (SEQ ID NO: 29) when titrated with increasing concentrations of lipid in the form of vesicles composed of 50% PC : PG (Figure 10a). Titrating the same peptide with neutral lipid vesicles (100% PC) results in very small lipid-dependent blue shifts (Figure 10b) . Figure 11 illustrates the lipid concentration-dependent fluorescence blue shifts of each of the tryptophan-containing peptides. Very similar behaviour was observed for the RTA3 (SEQ ID NO: 9; Figure Ha) and V₆₈₁ (SEQ ID NO: 26; Figure lib) series of peptides. In each case disruption of the non-polar helix face [with an Arg (WRTA3-dis; SEQ ID NO: 27) or Lys (V₆₈₁-dis; (SEQ ID NO: 29) ] had only a minor effect on peptide binding to negatively-charged vesicles, whereas binding to neutral vesicles was greatly suppressed. An intact non-polar helix face (WRTA3- non-dis [SEQ ID NO: 28], V₆₈₁-non-dis [SEQ ID NO: 26]) resulted in strong binding to neutral lipid vesicles, although the reduced maximum blue shift upon saturation indicates that the tryptophan indole in each peptide may adopt a shallower membrane insertion compared to the binding to negatively charged vesicles. Likewise the reduced W4 blue shift of WRTA3-dis (SEQ ID NO: 27), compared to WRTA3-non-dis (SEQ ID NO: 28), on binding to negatively charged membranes may also indicate a location of Trp-4 nearer the membrane surface for the former peptide compared to the latter.

The very small structure-formation in WRTA3-dis (SEQ ID NO: 27; Figure 9a) and V₆₈₁-dis (SEQ ID NO: 29; Figure 9b) upon incubation with vesicles composed of neutral lipids, and the very small perturbation of Trp-fluorescence induced by neutral lipid vesicles (Figures 11a, b) support the conclusion that these peptides bind very poorly to membranes lacking a negative surface charge. However these experiments do not formally rule out the possibility that the peptides might bind with neither structure formation nor burial of the respective Trp residues in an apolar environment. In addition, the tryptophan fluorescence data does not address the difference in structuring of RTA3-dis (SEQ ID NO: 9) and WRTA3-dis (SEQ ID NO: 27) upon incubation with neutral vesicles (Figure lla) . To address these points we made a series of experiments in which vesicles "doped" with a small concentration (0.2 mole%) of FPE were used to assess membrane binding upon titrating with increasing concentrations of peptide. FPE is very sensitive to the surface charge of the membrane, through charge effects on the pKa of the carboxylate of the fluorescein moiety localized in the head group region of the bilayer. Reduction of the negative membrane surface charge resulting from positively-charged peptide binding to negatively- charged vesicles (50% PC:50%PG), or enhancement of the positive surface charge by positively-charged peptide binding to neutral membranes, each results in small pK_a shifts promoting deprotonation of the fluorescein carboxylate, and a fluorescence enhancement (Wall et al., 1995, MoJ. Membr. Biol. 12: 183-192). These effects do not require burial of any region of the peptide into the membrane, and can therefore assess binding in any form, even if this involves superficial interaction of positively charged side chains with negatively charged membrane lipids. This technique is particularly suited to the highly positively charged antimicrobial peptides, and is a useful way of assessing cooperativity in peptide binding under conditions that don't involve extremely high peptide : lipid ratios at the initial parts (low lipid concentration) of the binding curves determined by titrating a fixed concentration of peptide with increasing lipid concentrations .

Figures 13a and 13b illustrate the fluorescence enhancement of FPE resulting from titrating negatively charged vesicles or neutral vesicles, respectively, with increasing concentrations of RTA3 peptides (SEQ ID NOs 9, 26 and 27) . All of the peptides bound with high affinity to negatively charged vesicles, with the binding data for RTA3-dis (SEQ ID NO: 9) and WRTA3-dis (SEQ ID NO: 27) being virtually superimposed. WRTA3-non-dis (SEQ ID NO: 28) binds marginally less strongly and saturates at a maximum fluorescence enhancement that is around 15% smaller than that of RTA3-dis (SEQ ID NO: 9) and WRTA3-dis (SEQ ID NO: 27) . Each of these is consistent with the reduced positive charge of the latter peptide ( +5) compared to RTA3-dis (SEQ ID NO: 9) and WRTA3-dis (SEQ ID NO: 27; +6), since enhanced positive charge should both promote binding to negatively charged vesicles, and will make a stronger contribution to reduction of the negative surface charge density of vesicles composed of 50% negatively charged lipids.

Consistent with the tryptophan fluorescence binding data, neutral vesicles strongly distinguish between peptides having intact and disrupted non-polar helix faces (Figure 12b). The binding of WRTA3-non-dis (SEQ ID NO: 28) to PC vesicles is nearly as strong as binding to PC : PG vesicles, whereas WRTA3-dis (SEQ ID NO: 27) binds very weakly. Reverting WRTA3-dis (SEQ ID NO: 27) back to the wild-type peptide (RTA3-dis; SEQ ID NO: 9) virtually abolishes binding to neutral vesicles. In fact the wild type peptide with a perturbed non-polar helix face and lacking a tryptophan residue has a binding constant for neutral membranes reduced more than 1000-fold compared to its interaction with negatively charged membranes. This observation explains the difference in structuring between RTA3-dis (SEQ ID NO: 9) and WRTA3-dis (SEQ ID NO: 27) on incubating with very high concentrations of neutral vesicles (Figure 9a) .

The FPE vesicle binding data for the V_68x peptides (SEQ ID NOs 26 and 29; Figure 13) are generally consistent with those described for the RTA3 peptides (SEQ ID NOs 9, 26 and 27; Figure 12) and for V₆₈₁ peptides (SEQ ID NOs 26 and 29) binding measured using tryptophan fluorescence (Figure lib) . We were only able to determine binding of wild-type V₅₈₁ (V₆₈₁-non-dis; SEQ ID NO: 26) to PC: PG vesicles at moderate peptide concentrations since interaction of the peptide with these vesicles at higher concentrations induced light scattering due to peptide-induced vesicle aggregation or fusion. However the data demonstrate rather similar binding of V₆₈₁-non-dis (SEQ ID NO: 26) and V₆₈₁-dis (SEQ ID NO: 29) to negatively charged vesicles (Figure 13a) . Likewise the FPE binding data confirm that the minimal structuring of V₆₈₁-dis (SEQ ID NO: 29) upon incubation with neutral lipid vesicles (Figure 9b) and the limited effect of neutral vesicles on the fluorescence emission of the Trp2 residue (Figure lib) is due to low binding affinity of this peptide to neutral membranes.

For all of the peptides studied, no evidence of cooperative binding (either positive or negative cooperativity) was apparent, since in each case linear variations in fluorescence enhancement with increasing peptide concentrations was observed at low peptide concentrations. How do the membrane binding properties of the peptides relate to their effects on the structural integrity of phospholipids membranes? We tested the ability of the peptides to release carboxyfluorescein (CF) trapped at high concentrations within lOOnm SUV composed of either 50% PC : PG or 100% PC under conditions (65 μM total lipid, high salt buffer) equivalent to those used for the FPE binding experiments of Figure 13. All of the data is compiled in Figure 14.

Each of the 5 peptides releases internal CF from negatively charged vesicles at low peptide concentrations, either through pore formation or generalised disruption of the lipid membrane bilayer organisation (Figure 14a; notice that the designation of the peptides is different in Figure 14, with open and closed symbols referring to RTA3 peptides [SEQ ID NOs 9, 27 and 28] and Vεβi peptides [SEQ ID NOs 26 and 29] , respectively) . The membrane lytic activity generally relates to the positive charge density of the peptides with the highly charged (5 or 6 positive charges per 16 amino acids) RTA3 peptides (SEQ ID NOs 9, 27 and 28) being particularly active with half-maximal dye release occurring at concentrations near 0.1 μM. The less highly charged V₆₈i peptides (SEQ ID NOs 26 and 29) were half-maximally active at concentrations of around 0.3 μM (V_68:-dis [SEQ ID NO: 29]; 7 positive charges per 26 residues) and 0.5 μM (V₆₈₁-non-dis [SEQ ID NO: 26]; 6 positive charges), the relative activity again corresponding to the positive charge density. The relationship between activity and peptide concentration was sigmoidal in negatively charged vesicles with apparent "cooperativity" (see Table 4 and following section) in the range of 1.8 -2.3.

Peptides having non-disrupted non-polar helix faces (WRTA3-non- dis [SEQ ID NO: 29] and V₆₈₁-non-dis [SEQ ID NO: 26]) had very high activity against neutral lipid vesicles with half-maximal activities in the range 0.3-0.5 μM (Figure 14b). Consistent with the membrane binding data of Figures 11-13, peptides with non- polar helix faces disrupted with a positively charged residue had very low activity against neutral vesicles. Wild-type RTA3 (RTA3-dis; SEQ ID NO: 9) was particularly ineffective against neutral vesicles with barely detectable dye release (1-2%) at a concentration of 50 μM. This corresponds to a ratio of effectiveness in dye release from negatively charged vesicles over neutral vesicles of around 10, 000-fold fold, a factor that results from a combination of the greatly reduced binding of RTA3 (SEQ ID NO: 9) to neutral membranes (near 1000-fold) and the apparent "cooperativity" in peptide-induced membrane perturbation.

The membrane-induced helix formation, membrane binding and membrane perturbation data for the 5 peptides studies is summarised in Table 4. In all experiments, peptides having non- polar helix surface disrupted with a positively charged Lys (V₆₈₁-V13K; SEQ ID NO: 29), or Arg residue (RTA3 [SEQ ID NO: 9], RTA3-F4W [SEQ ID NO: 27]) showed limited binding to neutral lipid vesicles coupled with very low activity in disrupting vesicles composed of neutral lipids. On the other hand these peptides retain strong binding to negatively charged vesicles, and perturb these membranes (either via non-specific membrane perturbation or "pore" formation) at low concentrations. This general observation is entirely consistent with the relative activities of the peptides on eukaryotic cells (erythrocyte haemolysis) and bacterial cells (MICs), respectively Table .3). We have also shown that negligible haemolysis occurs with sheep, horse and human erythrocytes at concentrations of RTA3 (SEQ ID NO: 9) of less than 800 mg/1.

We have tabulated the data in Table 4 in terms of the lipid or peptide concentration required to elicit half-maximal binding or half-maximal dye release. This is appropriate for the dye release data since the dose-response curves (Figure 14) in PC: PG are sigmoidal with near second-order dependence on peptide concentration (n in equation 2 of between 1.8 - 2.3) for each of the peptides studied (Table 4). The binding data fitted simple hyperbolic binding isotherms in all cases, indicating that any cooperativity in peptide-induced dye release in PCrPG vesicles must arise from the properties of the membrane bound peptides, rather than in the binding process itself. Previous studies used peptide dimerisation to confirm that apparent cooperativity in peptide-induced dye leakage from vesicles was likely due to self-association of membrane bound peptides. The resistance of RTA3 peptides to disulfide-dimerisation precluded a similar analysis here, and we cannot rule out the possibility that the apparent cooperativity in peptide-induced dye release results from other factors. For example, bilayer perturbation resulting from partitioning of peptide into the membrane interfacial region may increase as the square of the peptide concentration independent of any self-association phenomena.

The binding and dye-release data allow a semi-quantitative analysis of the peptide-membrane interaction. In negatively charged vesicles, half-maximal saturation of 2 μM peptide binding occurs at a lipid concentration around 10-15 μM (Table 4), suggesting that a peptide "binding site" constitutes no more than around 10-15 lipids (assuming that in a "peptide-saturated" vesicle, lipids in both the inner and outer bilayer leaflet become accessible to peptide) .

Table 4: Summary of peptide interactions with membranes.

membrane-induced structure membrane binding bilayer disruption

CD (% helix) ¹ Trp fluorescence² FPE fluorescence³ CF-dye release⁴

PC PC: PG PC(μM) PC:PG(μM) PC(μM) PC:PG(μM) PC(μM) PC:PG(μM) (n)⁵

RTA3⁶ <5 >75 — - >3000 3.2 735 0.068 (2.2)

RTA3-F4W⁷ 10 78 792 15 196 3.2 30 0.073 (1.8)

RTA3-F4W,R5L⁸ 78 82 23 11 2.2 4.3 0.11 0.065 (2.0)

10 V₆₈₁ ⁹ 82 81 17 7 2.0 1.7 0.37 0.51 (2.3)

V₆₈₁-V13K¹⁰ 12 68 1720 14 125 1.4 63 0.27 (2.2) OO

¹I peptide helix; 150 μM peptide, 10 μM total lipid (50 nm SUV) in 1OmM Tris, 107 mM NaCl, pH 7.4

(buffer A) 15 ²lipid concentration (as 100 nm SUV) inducing 50% peptide binding; 2 μM peptide concentration in buffer A

³peptide concentration inducing H maximal FPE fluorescence shift in FPE-doped 100 ran SUV (65 μM total lipid) in buffer A

⁴peptide concentration required for H maximal CF dye release from 100 nm SUV (65 μM total lipid) in 20 buffer A

⁵value of n in the sigmoidal fit of dye release data (see legend to Figure 14)

⁶SEQ ID NO: 9; ⁷SEQ ID NO: 27; ⁸SEQ ID NO: 28; ⁹SEQ ID NO: 26; ¹⁰SEQ ID NO: 29.

Peptide-induced dye release requires far fewer bound peptides than those required to saturate the membrane. RTA3 peptides (SEQ ID NOs 9, 27 and 28) cause half-maximal dye release at peptide- lipid ratios near 1:1000 (peptide : lipid; molrmol), supporting the interpretation that an event requiring a local peptide associated state underlies the membrane perturbation through which trapped CF is released. Interestingly, RTA3 peptides (SEQ ID NOs 9, 27 and 28) are significantly more effective than the V₆₈₁ peptides (SEQ ID NOs 26 and 29) in this regard, requiring between 4-7-fold less peptide for half-maximal dye release, despite the observation that the V₆₈₁ peptides (SEQ ID NOs 26 and 29) bind PC: PG membranes slightly more effectively than the RTA3 peptides (SEQ ID NOs 9, 27 and 28). In other words, the enhanced activity of RTA3 peptides (SEQ ID NOs 9, 27 and 28) in - disrupting negatively charged membranes (Figure 14; Table 4) is not due to an enhanced binding constant, and we conclude that membrane-bound forms of RTA3 peptides (SEQ ID NOs 9, 27 and 28) are somewhat more effective in inducing the membrane perturbation that underlies dye release than are the membrane- bound forms of V₆₈i peptides (SEQ ID NOs 26 and 29) .

Discussion

This example illustrates in part that vesicle bilayer membranes composed either of neutral lipids or mixed PG: PC membranes can be surprisingly good analogs for eukaryotic and bacterial cell membranes, respectively. The general correspondence between the effects of the peptides on vesicles (Figure 14; Table 4), and on bacteria or erythrocytes (Table 4), provides strong evidence that interaction with the membranes of target cells plays an important role in their mechanisms of antimicrobial action. A detailed analysis of vesicle binding, membrane-induced structure formation, vesicle membrane perturbation and activity measurements against target bacteria and erythrocytes yields a consistent interpretation of the effects of disruption of the amphipathic structure of positively charged antimicrobial helical peptides by inserting a positively charged amino acid onto the non-polar helix face. As previously described in Chen et al. (2005, supra), this modification can result in greatly reduced eukaryotic cell toxicity while retaining high antimicrobial activity, a conclusion that is reinforced by similar observations with RTA3 (SEQ ID NO: 9) and the designed sequence variants (Table 3) . It was previously suggested that the reduction of activity against eukaryotic membranes upon disruption of the non-polar helix face (with a Lys in V₆₈i-V13K; SEQ ID NO: 29) resulted from the inability of the peptide to insert deeply into the cell membrane to form peptide "pores". Our results demonstrate that the loss of membrane disruption of neutral membranes, including those of eukaryotes, has a simpler explanation: the peptides simply do not bind to membranes without negative surface charge.

This conclusion is generally consistent with our understanding of the interaction of amphipathic helical peptide with the interfacial region of bilayer membranes. For neutral membranes, binding is dominated by the interaction of the amphipathic peptide structure with the complementary membrane interfacial region. Amphipathic helix formation is crucial for this process since it allows the polar peptide backbone to substitute intramolecular (helical) hydrogen bonding for the loss of solvation energy as the peptide buries within the interfacial region of the bilayer. If the amphipathic structure is perturbed, this essential contribution to binding is lost. If the peptide helix partially unwinds to remove the positively charged amino acid from the non-polar helix face, then the amphipathic nature of the peptide that dominates the favourable binding energy is again attenuated. Thus non-amphipathic peptides cannot easily bind in the interfacial region of neutral bilayer membranes. The results shown in this example demonstrate that a single positively charged amino acid (Lys or Arg) on the non-polar face of an interfacially-bound amphipathic helical peptide is sufficient for very large attenuation of binding to neutral phospholipids bilayer membranes, a result that is consistent with the very high energetic barriers to the burial of a positively charged residue in the non-polar regions of a membrane .

On the other hand, binding to negatively charged membranes has contributions both from the complementary nature of helical amphipathic peptides and the interfacial region of the bilayer, and complementary electrostatics. As indicated by the CD data of Figure 9, the negatively charged membrane surface may also provide complementary negative charges for the positively charged amino acids disrupting the non-polar helix face. This seems to be required to explain the observation of the retention of virtually unperturbed helical content of RTA3 (SEQ ID NO: 9) and RTA3-F4W (SEQ ID NO: 27), and the relatively small perturbation of helical structure in V₆₈₁-V13K (SEQ ID NO: 29), on binding to negatively charged membranes. Observations of RTA3 (SEQ ID NO: 9) - induced vesicle fusion, together with molecular dynamics simulations suggest that the accommodation of the Arg residue on the non-polar helix surface of RTA3 (SEQ ID NO: 9) may be achieved by tilting of the peptide helix at the membrane interface that allows the Arg5 side chain to reach the solvated regions of the interface (not shown) . However the accommodation of the Lysl3 residue on the non-polar helix face of V₆₈i-V13K (SEQ ID NO: 29) upon binding to negatively charged membranes is unlikely to be achieved by helix tilting since the positively charged residue lies near the centre of the relatively long peptide helix, and an unrealistically high degree of helix tilt would be required for the Lys residue to "reach" the highly hydrated regions of the membrane interface. In the case of V₆₈₁- V13K (SEQ ID NO: 29), lipid head group phosphates might provide "neutralizing" charges for the positive K13 charge.

One unexpected finding is the observation that substitution of

Phe4 of RTA3 (SEQ ID NO: 9) with Trp significantly enhances binding to neutral membranes. This can be explained in terms of the very high interfacial propensity of Trp compared to all the other amino acids, even though Phe is a more "hydrophobic" amino acid side chain. This observation provides evidence that the removal of Trp residues from amphipathic helical antimicrobial peptides might be an additional general strategy for reducing eukaryotic cell toxicity. The absence of a tryptophan, combined with the presence of an arginine residue on the non-polar helix face, results in a peptide with extraordinarily low binding to neutral lipid membranes and this seems to underlie the extremely low eukaryotic cell toxicity in RTA3 (SEQ ID NO: 9) , despite the retention of very high affinity for binding to negatively charged membranes (Table 4) and strong antimicrobial activity (Table 3). These specific sequence characteristics are a product of evolutionary design within a commensal organism (Streptococcus mitis) , and highlight the potential of commensals as a source of novel antimicrobials having low eukaryotic cell toxicity.

Methods

Peptide synthesis, purification and characterization. The peptides listed in Table 1 were synthesised as in Example 1 using standard Fmoc solid phase synthesis. The peptides were purified by HPLC, and confirmed to be at least 97% pure by analytical HPLC and to have the predicted m/e ratio by mass spectrometry. Phospholipids were from Lipid Products (Nutfield, U.K.), carboxyfluorescein (CF) was from Sigma (Poole, U.K.) and fluorescein-phosphatidylethanolamine (FPE) was from Avanti .

Biological activities. Minimum inhibitory concentrations (MIC) of the peptides were determined as described in Example 1 above. Haemolytic activity was determined by incubating 10% (v/v) suspension of horse erythrocytes with peptides. Erythrocytes were rinsed in 10 mM phosphate buffered saline, pH 7.2, by repeated centrifugation and re-suspension (3 minutes at 3000 x g) . Erythrocytes were incubated at room temperature for 1 hour in either deionised water (fully haemolysed control), phosphate buffered saline (PBS), or with peptide in PBS. After centrifugation at 10,000 g for 5 minutes, the supernatant was separated from the pellet and the absorbance measured at 570 nm. Absorbance of the suspension treated with deionised water defined complete haemolysis.

Preparation of lipid vesicles. All experiments were performed at room temperature. Small unilamellar vesicles (100 nm diameter) were used for all spectroscopic measurements except for circular dichroism (CD) spectroscopy for which smaller (50 nm) vesicles were used to minimize light scattering effects. Lipids [either 100% egg PC or 50%PC:50%PG] were dried from chloroform:methanol solution and pumped under high vacuum overnight to remove traces of solvent. Lipids were hydrated at a concentration of 10 mg/ml in 10 mM Tris HCl, pH 7.4 containing either 107 mM NaCl (buffer A) , or, for the CF-dye-release experiments, 50 mM CF. Vesicles doped with FPE were prepared similarly except that 0.5 mol% of FPE in methanol was added to the lipids in organic solvent before drying. Hydrated lipids were extruded 10 times through two 100 nm or 50 nm pore membranes, using a Lipex Biomolecular extruder (Vancouver, Canada) . Vesicles for CD and peptide binding, monitored using either tryptophan fluorescence or FPE fluorescence, were used directly. Vesicles for CF-dye-release measurements were used after gel filtration on a Sephadex G-15 column with buffer A as the mobile phase, to remove non-trapped CF. Thus in all experiments, interaction of the peptide with vesicles was determined in the same buffer (buffer A) .

Fluorescence spectroscopy. Fluorescence measurements were made using a SPEX Fluoromax fluorimeter. Peptide solutions were made in plastic tubes or cuvettes to minimize loss of peptide at low concentrations due to binding to glass surfaces. For the measurement of vesicle-induced changes in the emission spectra of tryptophan in Trp-containing peptides, a 2 μM peptide solution was incubated in buffer A, and aliquots of vesicles suspension were added to give total lipid concentrations in the range 0 to 300 μM total lipid. Tryptophan fluorescence was excited at 280 nm, and the emission spectrum was measured between 300 and 450 nm in 1 nm increments with 1 s signal averaging. Binding data were fitted to a simple hyperbolic function to obtain estimates of the maximum fluorescence emission blue shift (Δλmax) and the concentration of lipid at which the lipid-induced blue shift was half-maximal.

Peptide binding to FPE-labelled vesicles was measured^' by adding aliquots of peptide to a suspension of vesicles at 65 μM total lipid concentration in buffer A. FPE emission was measured at 520 nm (excitation at 490 nm) . The experiments were made by adding successive aliquots of peptide to a single vesicle sample. Control experiments showed that the same FPE fluorescence enhancement was obtained by adding a single large aliquot of peptide or the same amount of peptide in successive small aliquots; the latter method facilitates analysis of peptide binding at low peptide concentrations allowing an assessment of the extent to which binding is cooperative (Dempsey et al . , 2003, supra).

Peptide-induced dye release from vesicles loaded with CF was measured from the loss of CF self-quenching as the dye dilutes into the extravesicular medium. Experiments were done with the same lipid concentration (65 μM) as the FPE binding measurements and in buffer A so that data from the different experiments can be interpreted in a consistent manner. CF emission was measured at 520 nm (excitation at 490 nm) . The fluorescence resulting from 100% release of encapsulated CF was determined by adding 10 μl of 20% Triton-X100. To ensure rapid mixing of peptide and vesicles, and to avoid high local concentrations of peptide, 1 ml of a peptide solution at double the post-mix concentration was rapidly ejected from an Eppendorf pipette into 1 ml of a vesicle suspension at 130 μM concentration (65 μM post mix) to initiate binding and dye release. The fluorescence emission intensity was measured 3 minutes after mixing CF-loaded vesicles with peptide. Circular dichroism spectroscopy. Circular dichroism (CD) spectra were obtained at 20 ⁰C using a Jobin-Yvon CD6 spectrapolarimeter . All samples were made in buffer A. Spectra of peptides in solution were measured in 1 mm or 2 mm quartz cuvettes. Spectra in the presence of vesicles were measured in 0.1 mm path length cuvettes to minimize light scattering contributions. All spectra are averages of 5 (vesicle-free solutions) or 9-11 scans (peptides plus vesicles) with appropriate peptide-free blank spectra subtracted and were zeroed at 260 nm before plotting without smoothing. Peptide helix content was calculated from the ellipticity at 222 nm (Θ222) (Dempsey et al . , 2005, supra) using parameters determined by Luo and Baldwin (Luo & Baldwin, 1997, supra) .

Although the present invention has been described with reference to preferred or exemplary embodiments, those skilled in the art will recognize that various modifications and variations to the same can be accomplished without departing from the spirit and scope of the present invention and that such modifications are clearly contemplated herein. No limitation with respect to the specific embodiments disclosed herein and set forth in the appended claims is intended nor should any be inferred.

All documents cited herein are incorporated by reference in their entirety.

Claims

Claims

1. A peptide comprising or consisting of an antimicrobial domain shown by formula (IV) :

(SEQ ID NO: 7)

in which Xi, X₅, Xio and Xi₃ are each independently K or R; and

X₂ is selected from the group consisting of: A, V, L, I, M, F, T,

W, P, C, Y, H, S, G, K and R; and

X₃ is selected from the group consisting of: A, V, L, I, M, F, T,

W, P, C, Y, H, S and G; and X₄ is selected from the group consisting of: A, V, L, I, M, F, T,

P, C, Y, H, S and G; and

X₆ is selected from the group consisting of: K, R, H, D, E, A and

T; and

X₇ is selected from the group consisting of: A, V, L, I, M, F, T, W, P, C, Y, H, S, G and Q; and

Xe and X₉ are each independently selected from the group consisting of: A, V, L, I, M, F, T, W, P, C, Y, H, S and G; and

Xii_/ Xi_2r and X₁₄ are each independently selected from the group consisting of: A, V, L, I, M, F, T, W, P, C, Y, S and G; and Xi₅ is C; and

X₁₆ is absent or is selected from the group consisting of: A, V,

L, I, M, F, T, W, P, C, Y, S and G;

and in which the antimicrobial domain contains no more than one amino acid deletion or insertion between residues X₁ and X₅, and/or no more than one amino acid deletion or insertion between residues X₅ and X₁₀, and/or no more than one amino acid deletion or insertion between residues X₁₀ and X₁₃, and no more than one amino acid deletion at residue X₁₆, such that any inserted amino acid is selected from the group consisting A, V, L, I, M, F, T,

W, P, C, Y, H, S, G, Q, K and R; characterised in that the peptide has antimicrobial activity and is non-cyclic, and that the antimicrobial domain adopts an alpha-helix conformation within a negatively-charged membrane.

2. The peptide according to claim 1, in which

X₂ is selected from the group consisting of: A, L, I, F, T, P, Y,

S, G, K and R; and/or

X₃ and/or X₈ and/or X₁₄ are each independently selected from the group consisting of: A, L, I, F, T, W, P, Y, H, S and G; and/or

X₄ is selected from the group consisting of: A, L, I, F, T, P, Y,

H, S and G; and/or

X₆ is selected from the group consisting of: K, R and T; and/or

X₇ is selected from the group consisting of: A, L, I, F, T, P, Y, H, S, G and Q; and/or

X₉ is selected from the group consisting of: A, L, I, F, T, W, P,

Y, S and G; and/or

X₁₁ and X₁₂ are each independently selected from the group consisting of: V, L, I, M, F and P; and/or X₁₆ is absent or is selected from the group consisting of: V and

I.

3. The peptide according to either of claim 1 or claim 2, in which antimicrobial domain does not include the amino acid W.

4. The peptide according to any preceding claim, in which

X₂ is selected from the group consisting of: P, K and R; and/or

X₃ and/or X₄ and/or X₈ and/or X₉ and/or X₁₄ are each independently selected from the group consisting of: A, F, P and Y; and/or X₆ is selected from the group consisting of: K, R and T; and/or

X₇ is selected from the group consisting of: A, F, P, Y and Q; and/or

X₁₁ and X₁₂ are each independently selected from the group consisting of: V and M; and/or X₁₆ is absent or selected from the group consisting of: V and I.

5. The peptide according to any of the preceding claims, in which the antimicrobial domain contains only one C residue (at position Xi₅) .

6. The peptide according to any of the preceding claims, in which

X₂ is selected from the group consisting of: P, K and R; and/or X₃ and/or X₄ and/or X₈ and/or X₉ and/or X_i4 are each independently selected from the group consisting of: A and F; and/or X₆ is selected from the group consisting of: K, R and T; and/or X₇ is selected from the group consisting of: A and Q; and/or X₁₁ and X₁₂ are each independently selected from the group consisting of: V and M; and/or X₁₆ is absent or selected from the group consisting of: A and I.

7. The peptide according to any preceding claim, having no more than 30 amino acid residues, for example no more than 20 or 16 amino acid residues.

8. The peptide according to any preceding claim, in which the antimicrobial domain has one of the following sequences:

(a) KPAFRTQAFRVMKACV ("RTAl"; SEQ ID NO: 8 ) ;

(b) RPAFRKAAFRVMRACV ("RTA3"; SEQ ID NO: 9);

(c) RRAFRKAAFRVMRACV ("RTA4"; SEQ ID NO: 10); (d) RPAFRTQAFRVMKACV (SEQ ID NO: 11)

(e) KPAFRTQAFRVMRACV (SEQ ID NO: 12)

(f) RPAFRTQAFRVMRACV (SEQ ID NO: 13)

(g) KPAFRKAAFRVMRACV (SEQ ID NO: 14) (h) RPAFRKAAFRVMKACV (SEQ ID NO: 15) (i) KPAFRKAAFRVMKACV (SEQ ID NO: 16) (j) RPAFRKAAFRVMRACV (SEQ ID NO: 17) (k) RRAFRTAAFRVMRACV (SEQ ID NO: 18)

(1) RRAFRKAAFRVMRACI (SEQ ID NO: 19) and (m) RPAFRKAAFRVMRACI (SEQ ID NO: 20)

9. The peptide according to claim 8, comprising or consisting of the amino acid sequence RPAFRKAAFRVMRACV ("RTA3"; SEQ ID NO:

9) -

10. The peptide according to claim 8, comprising or consisting of the amino acid sequence RRAFRKAAFRVMRACV ("RTA4"; SEQ ID NO:

10) .

11. The peptide according to any preceding claim, in which the peptide is unable to form a dimer and/or multimer.

12. The peptide according to any preceding claim, in an L-form.

13. The peptide according to any preceding claim, in which the peptide is active against multi-drug resistant and/or pan-drug resistant bacteria, for example against Gram-negative multi-drug resistant and/or pan-drug resistant bacteria such as colistin- resistant Pseudomonas aeruginosa .

14. The peptide according to any preceding claim, in which the peptide has lower toxicity against eukaryotic membranes than magainin.

15. The peptide according to any preceding claim, in which the peptide is bacterially active, for example in a non-static manner .

16. The peptide according to any preceding claim, in which the peptide binds to high molecular proteins in vivo.

17. The peptide according to any preceding claim, in which the peptide does not bind serum albumin.

18. The peptide according to any preceding claim, in which one or more amino acid residues are replaced by amino acid analogs.

19. An isolated nucleic acid molecule encoding a peptide as defined in any of claims 1-18.

20. A vector or plasmid which comprises a nucleic acid as defined in claim 19.

21. A recombinant cell comprising a nucleic acid as defined in claim 19 or a vector or plasmid as defined in claim 20.

22. A peptide recombinantly produced by expressing in a suitable host organism a nucleic acid sequence as defined in claim 19 and exhibiting anti-microbial activity.

23. A binding agent which binds a peptide as defined in any of claims 1-18 or 22.

^■ 24. A composition comprising a peptide as defined in any of claims 1-18 or 22.

25. The composition according to claim 24, further comprising at least one antibiotic.

26. The composition according to claim 25, in which the antibiotic is selected from the group consisting of: macrolides, ansamycins, polymyxins, cephems, quinolines, lincosamides, folate pathway inhibitors, fosfomycins, glycopeptides, oxazolidinones, tetracyclines, other antimicrobial peptides, lipopeptides, anti-tubercular drugs, nitrofurans, azalides, ketolides, nitroimidazoles, phenicols, mupirocin, fusidic acid, aminocyclitols, nitrofurantoins, monobactams, bacitracin, sulfamethoxazole, and mixtures thereof.

27. A pharmaceutical composition comprising the composition of any of claims 24-26, in combination with a pharmaceutically acceptable carrier.

28. A method for treating or reducing the severity of a microbial infection, comprising administering to a human or animal in need thereof a therapeutically sufficient amount of peptide as defined in any of claims 1-18 or 22, the composition as defined in any of claims 24-26, or the pharmaceutical composition as defined in claim 27.

29. The method according to claim 25, comprising at least two doses .

30. The method according to either of claim 28 or claim 29, in which the microbial infection is a bacterial infection caused by one or more Gram-negative bacteria.

31. The method according to claim 30, in which the bacterial infection is caused by one or more of the bacterial species from the group consisting of Haemophilus influenzae, Pseudomonas aeruginosa, Acinetobacter spp. (including A. baumannii, A. xylosoxidans, and A. genom spp.), Stenotrophomonas maltophilia , other non-fermenting bacteria, any member of the family Enterobacteriaceae (including Enterobacter cloacae, Escherichia coli, Klebsiella pneumonia, Proteus mirabilis and other Proteus spp., Salmonella spp. such as S. enteritidis and S. typhi, and Serratia marcescens) , Neisseria species (including N. gonorrhoeae and N. meningitidis) , Moraxella spp. (such as M. catarrhalis) , Helicobacter spp. (such as H. pylori), Stenotrophomonas spp., Bdellovibrio spp., acetic acid bacteria, Legionella (such as L. pneumophila) and alpha-proteobacteria (for example, Wolbachia).

32. The method according to either of claim 28 or claim 29, in which the microbial infection is a bacterial infection caused by one or more Gram-positive bacteria.

33. The method according to claim 31, in which the bacterial infection is caused by one or more of the bacterial species from the group consisting of Bacillus spp., Listeria spp., Staphylococcus spp. (such as S. aureus), Streptococcus spp. (such as S. pneumonia) , Enterococcus spp., and Clostridium spp.

34. The method according to any of claims 30-33, in which the bacterial infection is caused by a multi-drug or pan-drug resistant species.

35. A peptide as defined in any of claims 1-18 or 22, a composition as defined in any of claims 24-26, or a pharmaceutical composition as defined in claim 27, for use in the treatment of a disease, for example a microbial (such as bacterial) infection.

36. Use of a peptide as defined in any of claims 1-18 or 22, a composition as defined in any of claims 24-26, or a pharmaceutical composition as defined in claim 27, in the manufacture of a medicament for the treatment of a microbial

(for example, bacterial) infection.

37. A disinfectant comprising a peptide as defined in any of claims 1-18 or 22, a composition as defined in any of claims 24- 26, or a pharmaceutical composition as defined in claim 27.

38. A method of disinfection comprising applying to a surface to be disinfected a disinfectant as defined in claim 37.

39. A peptide substantially as described herein with reference to the accompanying drawings.