# Patent

## Title
Peptide antimicrobials

## Abstract

Provided are methods and compositions for in vivo display and screening of peptides for antimicrobial activity. The methods can include expressing a random peptide library in a microbial cell culture and identifying clones in which microbial cell growth or survival is affected by the peptide expressed by that clone. Also provided are peptide antimicrobials identified using these methods and compositions.

## Background
CROSS REFERENCES TO RELATED APPLICATIONS
This application is a national phase application of International Patent Application No. PCT/US2011/028733 filed on Mar. 16, 2011, which claims priority to and the benefit of U.S. Provisional Patent Application No. 61/314,269, filed on Mar. 16, 2010, the entire contents of each of which are incorporated by reference herein.


STATEMENT OF GOVERNMENT INTEREST
The invention encompassed by this disclosure was made in part using funds from NIH grant U01AI061192. The government therefore has certain rights in the invention.


FIELD OF THE DISCLOSURE
The present disclosure provides methods of identifying effective antimicrobials and antimicrobials identified using these methods, as well as associated nucleic acid sequences, plasmids and methods of treatment.
BACKGROUND OF THE DISCLOSURE
The search for new antimicrobials is a critical process as many species of bacteria are becoming resistant to multiple antibiotics.
Currently, the most active approach to finding new antimicrobials is the chemical modification of existing antibiotics, many of which are produced by bacteria or fungi. A significant drawback to this approach is that natural antibiotics and resistance to natural antibiotics coevolved, in part to protect the producing organisms.
Some peptide antimicrobials have been identified using standard antimicrobial screening technologies or by generating peptide libraries and screening for peptides that bind to a specific target. Examples of peptide screening technologies are phage display and ribosome display.
While these techniques can be effective at identifying antimicrobial peptides, there are drawbacks associated with their use. For example, the methods often use in vitro screening methods to identify antimicrobials that bind to known targets. This strategy does not identify antimicrobials that are effective based on interaction with an unknown or unchosen target. Moreover, in vitro screening assumes that a target's structure and its interaction with the peptide will be the same in vitro as in vivo. In many cases, however, there are significant differences between in vitro and in vivo conditions. Accordingly, there is room for improvement in techniques and methods used to identify new antimicrobial compounds.
SUMMARY OF THE DISCLOSURE
The present disclosure provides methods of identifying effective peptide antimicrobials that address many drawbacks associated with previously-practiced methods. For example, in the methods disclosed herein, there is no target bias. Thus, any peptide that inhibits bacterial growth for any reason may be identified without prior knowledge of the target. In addition, screens are performed in vivo, so the inhibitory peptides that are identified are active under physiological conditions, providing greater assurance that peptides isolated using this technology will be effective within their target cells. Moreover, expression of the peptides occurs within the cell. Accordingly, peptides do not need to cross the outer membrane to reach their targets during the initial screen.
Peptides identified using these methods are also encompassed within the scope of the present disclosure. Additionally, peptides that share common structural features that produce antimicrobial activity with the particularly identified peptides disclosed herein are described.
Thus, in one aspect, a method is provided for identifying an effective peptide antimicrobial. The method can include the steps of controllably expressing a random peptide library in a microbial host cell within a culture, wherein random peptides are encoded by plasmids comprising a nucleic acid sequence which is under the control of an inducible promoter; and identifying clones in which microbial cell growth or survival is affected by the peptide expressed by that clone.
Various embodiments of the method can include one or more of the following steps or features.
In some embodiments, the method includes growing a microbial cell culture in the absence of an inducer of the inducible promoter before or after the controllable expression.
In some embodiments, the method includes growing the microbial cell culture in the presence of a transcriptional repressor.
In some embodiments, the method includes adding an inducer of the inducible promoter.
In some embodiments, the method includes transforming microbial cells with the plasmids.
In some embodiments, the method includes contacting host cells with an agent (e.g., ampicillin) that negatively selects against replicating cells.
In some embodiments, the method includes identifying the peptide sequence of a clone in which cell growth or survival is affected.
In some embodiments, the method includes constructing the plasmid.
In some embodiments, the nucleic acid sequence of the plasmid includes a NNKn sequence, wherein NNK is a nucleotide triplet that encodes a random amino acid, wherein:
N is A, T, C or G;
K is T or G; and
n is 2-50;
wherein, optionally, n=12 (SEQ ID NO:462).
In some embodiments, the nucleic acid sequence of the plasmid includes a DNK(NNK)n sequence, wherein DNK and NNK are nucleotide triplets that encode a random amino acid, wherein:
N is A, T, C or G;
K is T or G; and
D is A, T, or G; and
n is 1-49;
wherein, optionally, n=11 (SEQ ID NO:463).
In some embodiments, the plasmid encodes a leader peptide.
In some embodiments, the plasmid encodes a leader peptide that targets the random peptides to the periplasm.
In some embodiments, the plasmid encodes an alkaline phosphatase leader peptide.
In some embodiments, the inducible promoter is an arabinose inducible promoter (PBAD).
In some embodiments, the nucleic acid sequence encodes a carrier protein.
In some embodiments, the carrier protein is alkaline phosphatase, and the C terminus of the random peptide is fused to the N terminus of the alkaline phosphatase protein.
In some embodiments, the carrier protein is alkaline phosphatase, and the N terminus of the random peptide is fused to the C terminus of the alkaline phosphatase protein
In some embodiments, the carrier protein is emerald green fluorescent protein, and the C terminus of the random peptide is fused to the N terminus of the emerald green fluorescent protein.
In some embodiments, the carrier protein is emerald green fluorescent protein, and the N terminus of the random peptide is fused to the C terminus of the emerald green protein.
In some embodiments, the host cell is a bacterium (e.g., E. coli).
In another aspect, antimicrobial peptides are provided. The antimicrobial peptides can have the consensus sequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 (SEQ ID NO:465); wherein

    
    
        Xaa1 is a hydrophobic amino acid (in one embodiment, tryptophan);
        Xaa2, is a basic or small amino acid (in one embodiment, selected from glycine or alanine) or a basic amino acid (in one embodiment, arginine);
        Xaa3 is a hydrophobic amino acid (in one embodiment, tryptophan) or a basic amino acid (in one embodiment, arginine);
        Xaa4 is a hydrophobic amino acid (in one embodiment, leucine);
        Xaa5 is a basic amino acid (in one embodiment, arginine or lysine);
        Xaa6 is a basic amino acid (in one embodiment, arginine) or a small amino acid (in one embodiment, alanine or glycine);
        Xaa7 is a hydrophobic amino acid (in one embodiment, selected from leucine or tryptophan) or a basic amino acid;
        Xaa8 is a hydrophobic amino acid (in one embodiment, tryptophan or leucine) or a small amino acid (in one embodiment, alanine or glycine);
        Xaa9 is a basic amino acid (in one embodiment, arginine) or a small amino acid (in one embodiment, glycine);
        Xaa10 is a basic amino acid (in one embodiment, arginine) or a small amino acid (in one embodiment, glycine or alanine);
        Xaa11 is tryptophan or arginine;
        Xaa12 is a basic amino acid (in one embodiment, arginine) or a small amino acid (in one embodiment, glycine).
    
    


In another aspect, antimicrobial peptides are provided which include the amino acid sequence of any one of SEQ ID NOS:7-461, or a sequence 85% homologous thereto (e.g., at least 85% homologous, at least 90% homologous, at least 95% homologous).
In some embodiments, the antimicrobial peptide has an amino acid sequence which is at least 85% homologous to the amino acid sequence: FAWLWSWWRARR (SEQ ID NO:310).
In some embodiments, the antimicrobial peptide has an amino acid sequence which is at least 85% homologous to the amino acid sequence: FMRLLRWWRRMQ (SEQ ID NO:438).
In some embodiments, the antimicrobial peptide has an amino acid sequence which is at least 85% homologous to the amino acid sequence: IRWLARRWRRTF (SEQ ID NO:347).
In another aspect, artificial nucleic acid sequences are provided that encode a peptide having the amino acid sequence of any one of SEQ ID NOS:7-461.
In another aspect, expression vectors (e.g., plasmids) are provided that encode a nucleic acid sequence of a peptide having the amino acid sequence of any one of SEQ ID NOS:7-461.
In another aspect, a vector is provided having a DNA sequence encoding an inducible promoter and a (NNK)n sequence of DNA, where NNK is a nucleotide triplet that encodes a random amino acid, and where N is A, T, C or G, K is T or G, and n is 2-50.
In another aspect, a vector is provided having a DNA sequence encoding an inducible promoter and a DNK(NNK)n sequence of DNA, where NNK is a nucleotide triplet that encodes a random amino acid, wherein N is A, T, C or G, K is T or G, D is A, T, or G; and n is 1-49.



BRIEF DESCRIPTION OF THE DRAWINGS
The figures are not necessarily to scale, emphasis instead generally being placed upon illustrative principles. The figures are to be considered illustrative in all aspects and are not intended to limit the invention, the scope of which is defined only by the claims.
FIGS. 1A and B show an in vivo display overview.
FIG. 2 is a table showing the clone, phenotype, peptide, and growth curve.
FIGS. 3A-E show the peptide sequences of clones according to phenotype: A. static/cidal; B. weakly inhibitory<0.7; C. weakly inhibitory>0.7; D. non-inhibitory; and E. lytic.
FIGS. 4A-M are graphs showing the growth curves of clones of FIG. 2.
FIG. 5 shows the vector used in screening peptides for anti-growth phenotypes in the periplasm. pKan phoA had the phoA gene under the power of the inducible araBAD promoter
FIG. 6 shows the vector of FIG. 5, with peptide.
FIG. 7 shows the sequence of a pKan phoA plasmid.
FIG. 8 is a schematic of a display protein.
FIG. 9 shows induced growth curves for each type of peptide antimicrobial.
FIG. 10 is an overview of an in vivo display (IVD) system for screening and identifying peptide antimicrobials.
FIG. 11 is a flowchart of a cytoplasmic IVD system.
FIG. 12 is a flowchart for characterizing peptide antimicrobials generated using a cytoplasmic IVD system.
FIG. 13 shows plasmid constructs for expressing N-terminal fusion proteins in the cytoplasm.
FIG. 14 shows a plasmid construct for expressing N-terminal fusion proteins in the cytoplasm.
FIG. 15 shows the location of various elements of a plasmid for expressing N-terminal fusion proteins in the cytoplasm.
FIG. 16 shows the sequence of a pBac-EmGHt construct.
FIG. 17 shows a plasmid construct for expressing C-terminal peptides.
FIG. 18 shows the location of various elements of a plasmid for expressing C-terminal fusion proteins.
FIG. 19 shows the sequence of a pBac-EmGH construct.
FIG. 20 is a flowchart of a periplasmic IVD system.
FIG. 21 is a flowchart for characterizing and optimizing bacteriolytic peptide antimicrobials identified using a periplasmic IVD system.
FIG. 22 is an overview of the characterization of the rationally designed EO1 peptide antimicrobial.
FIG. 23 shows a plasmid construct for expressing N-terminal fusion proteins in the periplasm.
FIG. 24 shows the location of various elements of a plasmid for expressing N-terminal fusion proteins in the periplasm.
FIG. 25 shows the sequence of a pKan-PhoA construct.
FIG. 26 shows the shows the observed frequencies of amino acids at each position of the bacteriolytic peptide antimicrobials disclosed herein.
FIG. 27 shows peptide antimicrobials isolated using a cytoplasmic N-terminal bacteriostatic and bactericidal screen.
FIG. 28 shows growth rate reducing peptide antimicrobials isolated using a C-terminal constructs in a bacteriostatic and bactericidal screen.
FIG. 29 shows bacteriostatic and bactericidal peptide antimicrobials identified using C-terminal constructs in a bacteriostatic and bactericidal screen.
FIG. 30 shows bacteriolytic peptide antimicrobials isolated from a periplasmic bacteriolytic screen.
FIG. 31 shows bacteriostatic and bactericidal peptide antimicrobials isolated from a periplasmic bacteriolytic screen.
FIG. 32 shows growth rate reducing peptide antimicrobials isolated from a periplasmic bacteriolytic screen.
FIG. 33 shows bacteriostatic and bactericidal peptide antimicrobials isolated from a periplasmic bacteriostatic and bactericidal screen.
FIG. 34 shows bacteriolytic and growth rate reducing peptide antimicrobials isolated from a periplasmic bacteriostatic and bactericidal screen.
FIG. 35 shows consensus peptides based on bacteriolytic peptide residue distribution.
FIG. 36 shows bacteriostatic and bactericidal peptides isolated from an early onset degenerate peptide screen.
FIG. 37 shows bacteriolytic peptides isolated from an early onset degenerate peptide screen.
FIG. 38 shows non-inhibitory peptides isolated from an early onset degenerate peptide screen.
FIG. 39 shows select peptides with and without proline mutation.
FIG. 40 shows minimum inhibitory concentration of EO1 for various bacterial species.
FIG. 41 shows Alanine scan peptides based on EO1.
FIG. 42 shows EO1 site-directed mutations.
FIG. 43 shows growth curve profiles of bacteria expressing bacteriostatic and bactericidal peptide antimicrobials identified using N-terminal constructs in the cytoplasm.
FIGS. 44A-C show growth curve profiles of bacteria expressing growth rate reducing peptide antimicrobials identified using C-terminal constructs.
FIGS. 45A-D show growth curve profiles of bacteria expressing bacteriostatic and bactericidal peptide antimicrobials identified using a C-terminal expression system.
FIGS. 46A-L show growth curve profiles of bacteria expressing bacteriolytic peptide antimicrobials identified using N-terminal constructs in the periplasm.
FIGS. 47A-C show growth curve profiles of bacteria expressing consensus peptides derived from the initial bacteriolytic isolates of the periplasmic screen.
FIGS. 48A-I show growth curve profiles of bacteria expressing bacteriostatic and bactericidal peptide antimicrobials identified using N-terminal constructs in the periplasm.
FIGS. 49A-F show growth curve profiles of bacteria expressing growth rate reducing peptide antimicrobials identified using N-terminal constructs.
FIGS. 50A-B show growth curve profiles of bacteria expressing bacteriostatic and bactericidal peptide antimicrobials identified using N-terminal constructs in the periplasm.
FIGS. 51A-B show growth curve profiles of bacteria expressing bacteriolytic and growth rate reducing peptide antimicrobials identified using N-terminal constructs in the periplasm.
FIGS. 52A-B show growth curve profiles of bacteria expressing bacteriostatic, growth-rate reducing, and bactericidal degenerate peptides derived from the initial early-onset bacteriolytic isolates of the periplasmic screen.
FIGS. 53A-I show growth curve profiles of bacteria expressing bacteriolytic degenerate peptides derived from the initial early-onset bacteriolytic isolates of the periplasmic screen.
FIGS. 54A-B show growth curve profiles of bacteria for select peptide antimicrobials without proline modifications.
FIG. 55 shows growth curve profiles of bacteria for select peptide antimicrobials with proline modifications expressed as free peptides.
FIGS. 56A-B show growth curve profiles of bacteria expressing EO1 solo (e.g., free) peptide expressed in the cytoplasm and periplasm.
FIG. 57 shows growth curve profiles of bacteria expressing EO1 alanine scan peptides.
FIGS. 58A-B show growth curve profiles of bacteria expressing EO1 site-directed mutations.
FIGS. 59A-H show statistical analyses of the amino acids located at each position of bacteriolytic peptide antimicrobials from the periplasmic screen.



DEFINITIONS
Peptide refers to a polymer comprising less than 50 amino acids and, in particular embodiments, less than 20 amino acids.
Antimicrobial refers to the biological activity of the peptides disclosed herein, including the ability to inhibit microbial growth by lysing (e.g., bacteriolytic), killing without lysing (e.g., bactericidal), and inhibiting (e.g., bacteriostatic) or reducing (e.g., weakly inhibitory or growth rate reducing) the ability of microbes to reproduce.
A bacteriolytic peptide refers to a peptide that causes the host cell to lyse or break open, releasing its inter-cellular components into the growth medium.
A bacteriostatic peptide refers to a peptide that causes the host cell to enter a state of dormancy.
A bactericidal peptide refers to a peptide that causes the irreversible death of the host cell without lysing it.
Growth rate reducing, or weakly inhibitory, peptides refer to a peptide that slows but does completely inhibit reproduction of the host cell.
DETAILED DESCRIPTION
This disclosure relates, in part, to the development of high-throughput methods for creating, screening, and isolating effective peptide antimicrobials using an in vivo display (IVD) system. Using these methods, peptide antimicrobial libraries can be transformed into, propagated, expressed, and screened in vivo under physiologic conditions in a host cell culture. Moreover, the methods disclosed herein also permit identification of toxic peptide antimicrobials that would go undetected using other screening methods due to their toxicity.
The IVD systems disclosed herein are advantageous for a number of reasons. For example, there is no target bias. Thus, any peptide that inhibits bacterial growth for any reason may be identified without prior knowledge of the target. In addition, screens are performed in vivo, so the inhibitory peptides identified using IVD are active under physiological conditions, providing greater assurance that peptides isolated using this technology will be effective under therapeutic conditions. Moreover, expression of the peptides occurs within the cell. Accordingly, peptides do not need to cross the outer membrane to reach their targets during initial rounds of screening.
As shown in FIG. 10, peptide antimicrobials can be expressed in host cells. Host cell cultures are then screened to identify proteins that have a deleterious effect on the cells in which they are expressed. The screens are designed to identify peptides that cause the cells to lyse (e.g., bacteriolytic), kill the cell without lysing it (e.g., bactericidal/bacteriostatic) or reduce the ability of the cells to grow and reproduce (growth rate reducing). Plasmids encoding each of these types of peptides can then be isolated and cloned and the amino acid sequence of each of the inhibitory peptides can be determined by sequencing the peptide encoding portion of each plasmid.
Antimicrobial peptides can be characterized using a variety of in vivo and in vitro assays and this information can be used to rationally design additional peptide antimicrobials. Further optimization can involve changing the amino acid sequence and using peptidomimetic and medicinal chemistry to increase the antimicrobial activity, permeability, bioavailability, etc. of the peptide leads isolated with IVD. In addition, peptide antimicrobials can be externally introduced as free peptides to a microbial cell to test efficacy when the peptides originate from outside the microbial cell, such as would be the case during treatment of an infection.
Bacterial cells are relatively simple compared to eukaryotes and are generally divided into two distinct groups, the Gram positives and the Gram negatives. Gram positive bacteria have a thick cell wall composed of peptidoglycan and a plasma membrane that encloses the cytoplasm. Gram negative bacteria have a thinner layer of peptidoglycan and a plasma membrane that does not fully enclose the cytoplasm. In Gram negative bacteria, however, there is an additional outer membrane and the space between the plasma membrane and the outer membrane is called the periplasm or periplasmic space. This difference in the envelopes of Gram positive and Gram negative bacteria affects the flow of materials into and out of the cell, including antibiotics. Generally, it is more difficult for foreign compounds to enter the cytoplasm of Gram negative bacteria because of the two membranes and the periplasm. The periplasm, however, houses several essential enzymes and is the site of a number of critical microbial processes including those involved in generation of the proton motive force for energy transduction in Gram negatives and is therefore a suitable drug target for developing antimicrobials for the treatment of Gram negative infections.
IVD was developed to identify peptides that inhibit critical processes in the cytoplasm and/or the periplasm and can therefore be used directly as antimicrobials or to design antimicrobials for the treatment of diseases with, for example, Gram-positive and/or Gram-negative etiologies.
Using these methods, numerous peptide antimicrobials were isolated, sequenced, and characterized, as discussed in more detail herein. These peptide antimicrobials demonstrated various antimicrobial activities, including bacteriolytic, bacteriostatic, bactericidal, and growth rate reducing activities. In addition, analysis of peptides identified as having antimicrobial activities revealed features (e.g., a consensus sequence; alpha helices) common among bacteriolytic peptides. Using this information, additional peptide antimicrobials were identified using rational design and using degenerate peptides.
In various embodiments, plasmid vectors can be designed for expressing peptide antimicrobials in host cells. These plasmids can encode one or more of the following features, in no particular order: an inducible promoter, a leader peptide, a putative or potential peptide antimicrobial, a carrier and/or reporter protein, a linker amino acid or linker sequence, a protease recognition sequence, and an isolation tag.
In some embodiments, the plasmid encodes a peptide antimicrobial. The peptide antimicrobial can be encoded by a random sequence, a degenerate sequence, or a rationally designed sequence. During initial screening stages, vast numbers of random and/or degenerate peptide antimicrobials can be expressed as a peptide library.
According to various embodiments, a peptide antimicrobial library can be encoded by a random nucleic acid sequence. Accordingly, in some embodiments, the peptide library can be composed of about 12 amino acids encoded by a randomized sequence of DNA. However, it will be appreciated that any other peptide lengths can be used in accordance with this disclosure, such as, for example, peptide antimicrobials having 2 to 50 amino acids, and any length therebetween. For example, the peptide antimicrobial can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, or 50 amino acids long. In embodiments were a 12-mer is used, the random amino acid sequence can be generated using the polymerase chain reaction in which one primer can contain the sequence (NNK)12. As will be appreciated, the NNK sequence (or DNK(NNK) sequence, as discussed below) can be lengthened or shortened depending on the desired number of amino acids. Use of the NNK sequence allows incorporation of all amino acids but eliminates stop codons, except for UAG. To avoid having a peptide sequence terminated by a UAG stop codon, the random peptide libraries can be expressed in host cells that can read-through UAG stop codons, such as E. coli strains that are supE44.
In some embodiments, a DNK(NNK)11 sequence can be used in plasmid constructs that include a proteolytic cleavage site adjacent the N terminus of the peptide antimicrobial. The DNK codon, where D is A, T, or G, prevents placement of a proline in the first amino acid position of the peptide antimicrobial, as proline can interfere with proper proteolytic cleavage. Examples of proteolytic cleavage sites include, for example, expression products having a leader peptide cleavage site or a TEV protease cleavage site.
Degenerate nucleic acid sequences also can be used to generate random peptides having conserved features. In some embodiments, the degenerate sequence is WTBNNKYKGCTGNNKAGNYGGTGGCGTSGTN NKNNK-3′ (SEQ ID NO:464), using standard IUPAC nomenclature for DNA nucleotides.
In some embodiments, peptide antimicrobials can be expressed under the transcriptional regulation of an inducible promoter, thereby allowing peptide expression to be turned on and off. Using an inducible promoter allows transformed host cell cultures to propagate in the absence of peptide antimicrobials. Then, at a desired time, peptide expression can be induced to initiate the screening process. An inducible promoter reduces the likelihood that highly toxic peptide antimicrobials will prematurely kill their host cell, which would result in elimination of valuable peptides from a screen. Non-limiting examples of inducible promoters include, but are not limited to, those induced by the presence of a small molecule (e.g., arabinose (PBAD), IPTG, galactose, tetracycline, abscisic acid), by metals or metal ions (e.g., copper, zinc, cadmium, nickel), and by environmental factors (e.g., heat, cold, stress). The arabinose inducible promoter is particularly advantageous because the promoter has multiple levels of control. For example, in the absence of arabinose, downstream sequences typically show minimal expression. Adding a catabolite, such as glucose, can further silence expression and permit propagation of plasmids encoding highly toxic peptide antimicrobials (e.g., EO1).
In various embodiments, premature peptide expression also can be minimized by using a low copy number plasmid. Suitable low copy number plasmids include, for example, pCC1BACTM (Epicentre; Madison, Wis.). In addition, a low copy number plasmid can be transformed into a host cell strain (e.g., EPI300TM and EPI301TM; Epicentre; Madison, Wis.) that, upon induction, increases plasmid copy numbers, which results in greater peptide expression. Beneficially, and in some embodiments, the gene that regulates plasmid copy number can be under the control of the same inducible promoter that controls peptide antimicrobial expression. Thus, a low copy number plasmid can be used in combination with an inducible promoter to tightly regulate expression of peptide antimicrobials using a single inducer, both to inhibit premature expression and to boost expression upon induction.
In some embodiments, plasmid vectors can encode, and expression products include, a suitable leader peptide or signal sequence for targeting peptide antimicrobials. For example, a leader peptide can be used to target peptide antimicrobials to the periplasm. In some embodiments, an alkaline phosphatase leader peptide can be inserted 5′ to a peptide antimicrobial, which would result in the peptide being transported into the periplasmic space. The PhoA leader peptide would be cleaved following translocation to the periplasm, leaving the peptide antimicrobial exposed at the N-terminus.
In some embodiments, peptide antimicrobials can be expressed as fusion proteins—with or without a linker peptide—with a carrier and/or reporter protein (or peptide). Carrier and/or reporter proteins can serve several purposes, including improved stability by protecting against premature peptide degradation by intracellular machinery. In addition, carrier and/or reporter proteins can act as indicators of peptide expression and/or localization. For periplasmic IVD, the carrier and/or reporter can be a protein that is active in the periplasmic space (e.g., PhoA). For cytoplasmic IVD, the carrier and/or reporter can be a protein that resides and/or is active in the cytoplasm (e.g., emerald green fluorescent protein (EmGFP)). Moreover, expression of the peptide fusions may be monitored following induction where reporter proteins are used. For example, EmGFP is fluorescent and may be monitored in growing cultures by measuring fluorescence, and 5-Bromo-4-chloro-3-indoxylphosphate (X-Phos) is a colorimetric substrate for PhoA that may be added to growth media to follow alkaline phosphatase activity in host cells.
As stated, in some embodiments, peptide antimicrobials can be fused to a carrier and/or reporter protein using a linker. The linker can be, for example, a peptide linker of any suitable length, such as, for example, between 1 and 20 amino acids, and any length therebetween.
In some embodiments, peptide antimicrobials can be fused—directly, or indirectly via a linker—to the N terminus or the C terminus of a carrier and/or reporter.
In various embodiments, plasmid vectors can encode, and expression products can include, elements or tags to facilitate isolation of the expressed peptides and/or peptide-target complexes. Non-limiting examples of tags include polyhistidine (His)6, chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), FLAG, myc, and hemagglutinin (HA).
In some embodiments, peptide antimicrobials can be expressed in host cells as free peptides. In some embodiments, free peptide antimicrobials destined for the cytoplasm can be expressed without a leader peptide and without a carrier and/or reporter. In some embodiments, peptide antimicrobials can be expressed as a fusion with a carrier and/or reporter, which fusion incorporates a proteolytic site (e.g., a TEV protease recognition sequence) between the peptide antimicrobial and the carrier and/or reporter. In the presence of a suitable protease (e.g., TEV protease), the peptide antimicrobial is cleaved from the carrier and/or reporter protein, resulting in a free peptide antimicrobial. In yet other embodiments, a peptide antimicrobial can be expressed with a leader peptide that directs the peptide antimicrobial to the periplasm. Upon transportation to the periplasm, the leader peptide is removed, for example, when it crosses the membrane into the periplasm, resulting in a free peptide antimicrobial in the periplasm.
As stated previously, the IVD systems disclosed herein are advantageous for a number of reasons including that there is no target bias and that screens are performed in vivo. Because expression of the peptides occurs within the cell, peptides do not need to cross the outer membrane to reach their targets during the initial screen.
In some embodiments, peptides can be expressed using a plasmid, pKan phoA, that expresses Alkaline phosphatase (PhoA) under the control of an inducible promoter. PhoA is a natural E. coli protein that, due to its leader peptide, is translocated into the periplasm. PhoA is active after it has been transported across the membrane into the periplasm, and therefore can be used as a reporter of successful transport to the periplasm. The fact that PhoA can only fold correctly and function only if it is translocated to the periplasm makes it both a suitable vehicle and reporter for the periplasmic screens disclosed herein. A stretch of DNA encoding a random peptide can be inserted 3′ to the DNA sequence encoding the leader peptide and 5′ or 3′ to the PhoA gene. For example, in various embodiments, the random peptide sequence is (NNK)12, where N represents any nucleotide and K represents either guanine or thymine. NNK is a nucleotide triplet that encodes a random amino acid while reducing the probability of coding for a stop codon.
In addition to the periplasmic IVD system described above, a cytoplasmic expression system is also provided in which the peptide fusion construct is expressed and resides in the cytoplasm. In some embodiments, peptides can be expressed using a plasmid, pBAC-EmGH or pBAC-EmGHt, that expresses the Emerald Green Fluorescent protein (EmGFP) under the control of an inducible promoter. GFP is not a natural E. coli protein. It is fluorescent, highly stable, and localized to the cytoplasm in E. coli and therefore can be used as a reporter of successful cytoplasmic expression. A stretch of DNA encoding a random peptide can be inserted 3′ or 5′ to the EmGFP gene. For example, in various embodiments, the random peptide sequence is (NNK)12, where N represents any nucleotide and K represents either guanine or thymine. NNK is a nucleotide triplet that encodes a random amino acid while reducing the probability of coding for a stop codon.
There are at least three avenues for finding new antimicrobial leads: the peptide itself, a peptidomimetic, or a small molecule retaining the active chemical groups of the peptide.
In certain embodiments, a library of 12-mer random peptide sequences is displayed between the leader sequence and the N-terminus of alkaline phosphatase (PhoA) containing a histidine tag at the C-terminus. The display protein serves as a reporter for expression and translocation to the periplasm and facilitates purification of peptide-target complexes. The PhoA leader peptide is cleaved following translocation to the periplasm leaving the random peptide library exposed at the N-terminus. In one set of experiments, a library of 30 million clones was constructed and transformed into the phoA strain, DH5α. A high-throughput screen for inhibitory peptides yielded 28 peptides of varying phenotypes. Both bacteriostatic and bacteriolytic peptides were identified using these described embodiments.
Identification of peptide leads is the first step in the process of developing new antimicrobials. The screening methods disclosed herein are designed to provide many new hits. Approximation of a phage display screen against the entire genome, RNome, and proteome of E. coli within an in vivo environment, is underway. This screen will not only elucidate novel leads for antibiotics, but also targets that can be used in other subsequent in vitro screens. mRNA or phage display will then permit the screening of much more complex peptide libraries. The results will also give insight into the microbiology of E. coli. 
In some embodiments, peptide antimicrobials are administered to microbial cells, as opposed to being expressed in vivo, to confirm their antimicrobial effects when not produced within the cell. A minimum inhibitory concentration (MIC) for a peptide antimicrobial can be determined and, if desired, compared against other known antimicrobial agents and antibiotics.
Peptide antimicrobials disclosed herein can be synthesized using methods well known in the art. For example, peptides can be synthesized chemically, such as by Fmoc or t-Boc synthesis. Peptides also can be synthesized biologically by generating a nucleic acid that encodes the peptide of interest. A nucleic acid encoding a particular peptide can be constructed based on the amino acid sequence of the peptide. This nucleic acid can be incorporated (e.g., ligated) into a plasmid or other vector for transforming and expressing in a host cell. The host cell can be prokaryotic (e.g., E. coli) or eukaryotic (e.g., chinese hamster ovary). Alternatively, a cell-free system can be used.
In various embodiments, the peptide antimicrobials disclosed herein (e.g., SEQ ID NOS:7-461) and their encoding nucleic acids are artificially made and therefore they are not naturally occurring.
Peptide antimicrobials can be isolated from other biologic compounds. In addition, peptide antimicrobials can be substantially free of impurities. Substantially free of impurities means at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 99% or 100% free of impurities.
These peptide antimicrobials are useful to treat microbial infections alone or in combination with other antimicrobial agents, such as, for example, other peptide antimicrobials and/or antibiotics.
In accordance with various embodiments, a protocol for high-throughput bacteriolytic peptide screening can include one or more of the following steps:

    
    
        1. Transforming random peptide library in plasmids into the appropriate host strain.
        2. Washing the host cells in growth medium that represses expression of the peptide library.
        3. Resuspending cells in growth medium that represses expression of the peptide library.
        4. Growing culture to early log phase.
        5. Washing cells in growth medium.
        6. Resuspending in growth medium containing inducer(s) of peptide library transcription (and protease if appropriate).
        7. Incubating to allow expression of the peptide library.
        8. Pelleting cells.
        9. Precipitating DNA from supernatent and recover by centrifugation.
    
    


In accordance with various embodiments, a protocol for high-throughput bacteriostatic and bactericidal peptide screening can include one or more of the following steps:

    
    
        1. Transforming random peptide library in plasmids into the appropriate host strain.
        2. Washing the cells in growth medium that represses expression of the peptide library.
        3. Resuspending cells in growth medium that represses expression of the peptide library.
        4. Growing culture to early log phase.
        5. Washing cells in growth medium.
        6. Resuspending in growth medium containing inducer(s) of peptide library transcription (and protease if appropriate).
        7. Incubating to allow expression of the peptide library.
        8. Adding ampicillin or other negative selection agent to the culture and incubate until lysis.
        9. Pelleting cells and discard supernatant.
        10. Washing cell pellet.
        11. Isolating plasmid DNA from pelleted cells.
    
    


Confirmation of Efficacy Protocol. This protocol applies equally to all plasmid DNA preparations from the three above screens (bacteriolytic, bacteriostatic, and bactericidal). In various embodiments, the protocol can include one or more of the following steps:

    
    
        1. Transforming cells with plasmids isolated from the bacteriolytic, or bactericidal and bacteriostatic screens.
        2. Plating on growth medium that represses expression of the peptides.
        3. Growing until colonies form.
        4. Replicating onto growth medium containing inducer(s) and medium without inducer(s).
        5. Incubating until colonies form on the plate without inducer.
        6. Selecting colonies that grow in the absence of inducer only.
    
    


In various embodiments, one or more peptide antimicrobials disclosed herein may be used to treat and/or prevent a microbial (e.g., bacterial) infection in a subject (e.g., a human or other animal, including, without limitation, research and veterinary animals). A peptide antimicrobial may be administered alone, in combination with one or more additional peptide antimicrobials, in combination with an antibiotic or other medication, and/or in combination with any suitable pharmaceutically acceptable carrier, excipient, binder, filler, buffer, solvent, moisturizer, surfactant, or preservative. Techniques for formulating drugs are well known in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.
The peptide antimicrobials disclosed herein may be useful for treating diseases caused by Gram positive and Gram negative bacteria, including but not limited to E. coli, P. aeruginosa, E. faecalis, S. aureus, B. subtilis, S. typhi, P. aeruginosa, S. pyogenes, S. aureus, M. tuberculosis, K. pneumoniae, and S. marcescens. 
Peptide antimicrobials may be administered in any therapeutically effective dosage. Dosages will vary depending on the dosage form, route of administration, and peptide.
The following Examples are included to demonstrate particular embodiments of the disclosure. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Example 1
General Materials and Methods
Referring to FIGS. 1A and 1B, transformants were recovered for several hours before the culture was split and entered the two pathways of the screen. FIG. 1A shows a bactericidal/lytic screen; arabinose was added to induce peptide production. Lytic peptides cause cells to release lytic peptide plasmid into the supernatant. Resuspension with ampicillin caused non-inhibitory and bacteriostatic cells to lyse, leaving bactericidal cells intact. FIG. 1B shows a bacteriostatic screen.
Isolation of non-lytic, bactericidal peptides (FIG. 1A). Arabinose and ampicillin were added to lyse non-inhibitory, lytic, and cidal peptide-containing cells. To isolate peptides that kill host cells without causing lysis, cultures were grown to mid-log phase, induced by the addition of 0.2% L-arabinose and further incubated for a period of time sufficient to allow expression of the fusion protein to reach maximal levels (1 hr). Cells were recovered by centrifugation, washed, resuspended and grown to mid-log phase in nonselective medium (LB+0.4% glucose) and subjected to negative selection as described above. Cells from this selection were recovered by centrifugation and used to purify plasmid. EPI300 was transformed with the resulting plasmid and screened by replicating on selective medium. Clones with bactericidal peptides were retained for further analysis.
Isolation of bacteriostatic and weakly inhibitory peptides (FIG. 1B). Negative selection was used to isolate peptides that inhibit growth but are not lethal. Cultures were grown to early-log phase, induced by the addition of 0.2% L-arabinose and further incubated for a period of time sufficient to allow expression of the fusion protein to reach maximal levels (1 hr). Ampicillin (300 μg/ml) was added to the culture and the incubation was continued until the OD600 of the culture stops decreasing. Unlysed cells are recovered by centrifugation, washed, incubated in non-inducing medium (LB+0.4% glucose) overnight and the negative selection procedure repeated. At each stage of the selection, a sample of the recovered cells were plated on nonselective medium and replicated onto selective medium (containing 0.2% L-arabinose) to identify transformants expressing bacteriostatic or weakly inhibitory fusion peptides. Clones with confirmed peptides were retained for further analysis.
Example 2
Construction and Screening of a Random Peptide Library
To construct the random peptide libraries, random codons encoding twelve amino acids were incorporated into a DNA cassette containing the appropriate restriction sequences. The cassette was ligated at either the N-terminus or C-terminus of the display proteins. The amino acids were encoded by the sequence NNK (N=equal representation of A, T, C and G and K=T or G). The resulting ligated plasmids were then transformed into E. coli DH5, plated under nonselective conditions, and sequenced to confirm randomness.
To make the random plasmid library, additional transformations are performed. The transformants were pooled, after which plasmids encoding the random peptide sequences were purified. To isolate inhibitory peptides, E. coli EPI 300 was transformed with the peptide libraries and screened to identify bacteriolytic, bactericidal, and bacteriostatic peptides, and peptides that specifically inhibit ribosome function using a combination of positive and negative selection strategies.
Both the C and N-terminal peptide screens were done in the E. coli strain, EPI300, for the ability to induce a higher copy number of plasmid and initiate protein expression. Vector was prepared by cutting with BsrGI-HindIII (C-terminal) or NotI-NcoI (N-terminal) and then ligated to a cassette in a 1:2 molar ratio. The random peptide libraries were constructed by inserting a cassette containing the random dodecamer library, in the appropriate vector. FIG. 5 illustrates a vector used in screening peptides for anti-growth phenotypes in the periplasm. FIG. 6 illustrates pKan phoA had the phoA gene under the power of the inducible araBAD promoter
Diversity of the library can be confirmed for example by sequencing 100 transformants in 10 reactions, pooling 10 transformants per reaction. Base bands were expected to be in roughly equal amounts at the randomized positions, except the third position, or K, which was expected to have a 50/50 chance of being G or T.
A library of 1 billion clones was constructed, representing only a fraction of the potential amino acid sequences encoded by (NNK)12. When a single cell E. coli bacilli is transformed with one member of this library, it can be induced to express a peptide-PhoA fusion. The disclosed random 12 NNK triplets form a random nucleotide sequence 36 bases long. The random triplets give rise to a random 12 amino acid long peptide following translation. The peptide-PhoA fusion also has a C-terminal (Histidine)6 tag for in vitro purification of the fusion and any potential targets with which it is bound.
Upon expression, the leader peptide of PhoA directs its translocation to the periplasm by directing the translating ribosome to the plasma membrane. The leader peptide and the attached random peptide-PhoA fusion are then translocated through the plasma membrane into the periplasm. At this point the leader peptide is cleaved by a periplasmic peptidase, exposing the random peptide at the N-terminus of the fusion and allowing it to interact with any molecule in the periplasmic environment. The basis of the screens disclosed herein is to extract any random peptide that is able to affect the growth of E. coli by binding to and interfering with, preventing, or inhibiting the function of a molecule present in the periplasm.
To undertake this task on a high-throughput scale, a portion of the random peptide library encoded on pKan phoA plasmids was transformed into E. coli DH5α, a phoA strain. The cells were allowed to recover in SOC broth and were then transferred to a batch of LB+kanamycin to select for the plasmid; glucose was used to repress any expression of the peptide-PhoA fusion to prevent a particularly lethal peptide from prematurely removing itself from the screen by killing its host early. The cells were grown to mid-log phase; here they are carrying out normal cellular processes at a high rate and the cell density was high enough to recover plasmid DNA. At this point one of three screens were begun, to isolate growth-affecting peptides. Each screen utilized the phenotype of the peptide's expression to remove it from the background of peptides that were non-inhibitory.
As stated, bacteriolytic peptides caused the host cell to lyse or break open, releasing its inter-cellular components into the growth medium. Arabinose was added to a batch of mid-log phase cells to induce the expression of the peptide-PhoA fusion. If a particular peptide was able to cause cell lysis, the plasmid that encoded it would be released into the medium along with all other cellular components. The plasmid DNA could then be recovered using isopropanol precipitation.
As stated, bacteriostatic peptides induced the cell to enter a state of dormancy. While the cell could no longer replicate, if the peptide were to be removed from the cell it could resume growth and replication. Thus, peptides were removed by removing the inducer of its expression, arabinose, and by adding a repressor, glucose, in its stead. Arabinose was initially added to a batch of transformed cells. After a period of time any bacteriostatic peptides were presumed to have stopped their host cells from replicating. At this point it was necessary to remove any background cells or cells that are actively replicating in the presence of their peptides.
Ampicillin, an antibiotic that causes cell lysis only if a cell is actively replicating, is added to the medium. Once lysis of the culture was complete (cell density reaches a minimum) the cells were washed to remove arabinose and ampicillin, and re-suspended in LB broth with glucose to repress any peptide-PhoA fusion expression. This step selected for cells that were not actively replicating in the presence of their peptides, but upon the repression of expression were able to replicate once again. This procedure was repeated two more times to enrich for cells containing plasmids that encoded for bacteriostatic peptides versus ones with non-inhibitory peptides. After this procedure was finished, the plasmid could be extracted from the surviving cells and further analyzed.
As stated previously, bactericidal peptides cause the irreversible death of their host cells. Arabinose was added to a batch culture of mid-log phase cells as above. After a period of induction the cells were washed to remove the arabinose. The cells were re-suspended in LB broth with glucose to repress any expression of the fusion protein. At this point, cells containing plasmids encoding for bactericidal peptides died and could no longer replicate. Ampicillin was added to the broth to lyse all remaining cells in the culture. The bactericidal peptide-containing cells were still intact, so they could be removed from the lysed cells and their plasmids by centrifugation and washing steps to reduce plasmid contamination containing non-inhibitory peptides. The dead cells could be chemically lysed to purify their plasmids.
Numerous periplasmic screens have been performed for the identification of bacteriolytic peptides, encompassing more than 90 million clones. Selected clones have been sequenced and confirmed by induced growth curves, confirming that the disclosed bacteriolytic screen enriches plasmid preparations for peptides that elicit cell lysis. For example, an isolated clone is grown in fresh LB medium to early log phase. Arabinose is added to induced peptide-PhoA fusion expression, and the peptide's effect on growth is observed by measuring the cell density of the culture. Many clones of differing growth phenotypes were analyzed. Bacteriolytic peptides have predominated the extracted clones, which is to be expected given that the bacteriolytic screen was used. Peptides were recovered that had either a bacteriostatic or bactericidal effect and others that have a weakly inhibitory effect as they reduce the rate of E. coli's growth considerably and may find use as precursors to actual peptide hits.
Example 3
Characterization of Inhibitory Peptides Discovered Using In Vivo Display
The affinity tags present on the carrier proteins allow the identification of targets of select inhibitory peptides. Each isolated peptide's intracellular target may be identified using purification techniques. First, the clone will be grown in a medium supplemented with isotopically labeled uridine, thymine and methionine. After induction, the culture is centrifuged to collect the cells and lysed. The target-peptide-fusion protein is then purified by IMAC via the (His)6 tag on the fusion protein. Once the macromolecular nature of the target is identified, mass spectrometry may be used to specifically identify proteins, or nucleic acid sequencing for RNA and DNA.
Synthetic production of selected peptides may then be used for further characterization. Identified target may be purified and used to perform binding assays using any of the following methods: ELISA, surface plasmon resonance, isothermal titration calorimetry, equilibrium dialysis, electrospray mass spectrometry or fluorescence polarization depending on what is suitable for the target.
The spectrum or range of species that the peptide is able to affect was determined by synthesizing the peptide coupled with one of several motifs increasing permeability and adding it to cultures at various concentrations containing bacterial or fungal species of increasing difference. MIC's (minimum inhibitory concentrations) or IC50's were calculated by subjecting select species to the synthetic peptide including E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Klebsiella pneumoniae, Serratia marcescens using the broth dilution method. Growth curves were performed and compared to species exposed to a non-lethal peptide, simultaneously.
Cellular macromolecules may be isotopically labeled in vivo and the target isolated in pull-down experiments in which the target-peptide-fusion protein complex is purified using immobilized metal affinity chromatography (IMAC). The targets may be further characterized by enzymatic digestion. Specific identification of the targets is accomplished using mass spectrometry for protein targets or nucleic acid sequencing for nucleic acid targets. Binding studies may be performed using a variety of methods including but not limited to ELISA, NMR, surface plasmon resonance, isothermal titration calorimetry, equilibrium dialysis, electrospray mass spectrometry, and fluorescence polarization, depending on the nature of the target.
Specific peptides may be selected for further development. Factors affecting which peptides are chosen for further development include the nature of the target, antimicrobial activity, binding constant and solubility. Synthetic peptidomimetic libraries may be synthesized that incorporate the key structural and chemical motifs identified in this study and these libraries are screened against the peptide targets to identify compounds with increased therapeutic potential. These lead compounds may then be subjected to a variety of medicinal chemistry techniques as appropriate, to increase solubility, increase target specificity and decrease toxicity.
Example 4
Peptides Isolated and Sequenced
Peptides isolated using periplasmic bacteriolytic screen are shown in FIG. 2. Phenotype definitions: Bacteriolytic: OD600 of culture decreases in value, correlating with a reduction in the concentration of cells. Bactriostatic/bactericidal: OD600 of culture flatlines, correlating with a complete stop in the growth of the cells and constant cell density. Weakly inhibitory OD600<0.7: Replication is slowed but not stopped. Final OD600 as culture enters stationary phase is less than 0.7. Weakly inhibitory OD600>0.7: Similar to the above, only final OD600 exceeds 0.7. Peptides have only a minor effect on growth. Non-inhibitory: No growth effect was observed versus the expression of PhoA with no peptide at its N-terminus.
Peptide sequences in single letter amino acid code are shown in FIGS. 3A-E.
Five phenotypes were isolated as shown in FIG. 3: (i) Bacteriolytic; (ii) Bacteriostatic/bactercidal; (iii) Weakly inhibitory OD600<0.7; (iv) Weakly inhibitory OD600>0.7 and (v) Non-inhibitory.
After replica plating, clones were grown in LB+30 μg/ml kanamycin. When OD600 ̃0.15, arabinose was added to a final concentration of 0.2%. The OD600 of the culture was then measured every 30 minutes to see the effect of peptide-PhoA fusion expression on the cell. The growth curve results are shown in FIG. 4 A-M.
Example 5
Protocols for High-Throughput Peptide Screening
A. A protocol for High-throughput bacteriolytic peptide screening was as follows:

    1. Mix 1 μg of pKan phoA library with 30 μls of DH5α electrocompetent cells.
    2. Electroporate mixture at 1.44 kV.
    3. Move mixture to 1 ml of SOC broth+0.2% glucose (w/v).
    4. Repeat steps 1-3 nine times.
    5. Incubate at 37° C. for 1 hr.
    6. Centrifuge cells at 7,000 rpm for 5 mins.
    7. Pour off supernatant.
    8. Gently re-suspend cells in 10 mls pre-warmed LB broth+0.2% glucose.
    9. Centrifuge cells at 7,000 rpm for 5 mins.
    10. Pour off supernatant.
    11. Gently re-suspend cells in 10 mls pre-warmed LB broth+0.2% glucose.
    12. Add suspension to 500 mls of LB+30 μg/ml kanamycin+0.2% glucose.
    13. Incubate at 37° C. until culture's OD600 ̃0.4, centrifuge culture at 7,000 rpm for 5 mins.
    14. Pour off supernatant.
    15. Gently re-suspend cells in 25 mls of pre-warmed LB.
    16. Centrifuge at 7,000 rpm for 5 mins.
    17. Pour off supernatant.
    18. Gently re-suspend cells in 25 mls of pre-warmed LB.
    19. Add suspension to 500 mls of LB+30 μg/ml kanamycin+0.2% arabinose.
    20. Incubate at 37° C. for 2 hrs.
    21. Remove 21 mls of the culture and place in an oakridge tube.
    22. Centrifuge at 13,000 rpm for 10 mins.
    23. Pour supernatant into a new oakridge tube and centrifuge at 13,000 rpm for 10 mins.
    24. Pour supernatant into a new oakridge tube along with 12.6 mls room temperature isopropanol, 2.1 mls 3M Na Acetate pH 5.2, and 105 μls of 10 μg/ml salmon testes DNA.
    25. Centrifuge at 11,000 rpm for 30 mins.
    26. Pour off supernatant.
    27. Add 5 mls of 70% ethanol, centrifuge at 11,000 rpm for 15 mins.
    28. Pour off supernatant.
    29. Re-suspend DNA pellet in 500 μls dH2O.
    30. Store at −20° C.


B. A protocol for High-throughput bacteriostatic peptide screening was as follows:

    1. Mix 1 μg of pKan phoA library with 30 μls of DH5α electrocompetent cells.
    2. Electroporate mixture at 1.44 kV.
    3. Move mixture to 1 ml of SOC broth+0.2% glucose (w/v).
    4. Repeat steps 1-3 nine times.
    5. Incubate at 37° C. for 1 hr.
    6. Centrifuge cells at 7,000 rpm for 5 mins.
    7. Pour off supernatant.
    8. Gently re-suspend cells in 10 mls pre-warmed LB broth+0.2% glucose.
    9. Centrifuge cells at 7,000 rpm for 5 mins.
    10. Pour off supernatant.
    11. Gently re-suspend cells in 10 mls pre-warmed LB broth+0.2% glucose.
    12. Add suspension to 500 mls of LB+30 μg/ml kanamycin+0.2% glucose.
    13. Incubate at 37° C. until culture's OD600 ̃0.4, centrifuge culture at 7,000 rpm for 5 mins.
    14. Pour off supernatant.
    15. Gently re-suspend cells in 25 mls of pre-warmed LB.
    16. Centrifuge at 7,000 rpm for 5 mins.
    17. Pour off supernatant.
    18. Gently re-suspend cells in 25 mls of pre-warmed LB.
    19. Add suspension to 500 mls of LB+30 μg/ml kanamycin+0.2% arabinose.
    20. Incubate at 37° C. for 2 hrs.
    21. Add 500 μls of 300 mg/ml ampicillin to the culture.
    22. Continue to incubate for another hour.
    23. Centrifuge culture at 7,000 rpm for 10 mins.
    24. Pour of supernatant.
    25. Gently re-suspend in 25 mls of HN buffer.
    26. Centrifuge at 7,000 rpm for 10 mins.
    27. Pour off supernatant.
    28. Repeat steps 18-20.
    29. Gently re-suspend in 25 mls LB.
    30. Add to 500 mls of LB++30 μg/ml kanamycin+0.2% glucose.
    31. Incubate at 37° C. overnight.
    32. Use 1 ml of overnight culture to inoculate 500 mls of LB+30 μg/ml kanamycin+0.2% glucose.
    33. Repeat steps 13-25.
    34. Repeat steps 13-24.
    35. Perform a plasmid preparation of the cells.


C. A protocol for High-throughput bactericidal peptide screening was as follows:

    1. Mix 1 μg of pKan phoA library with 30 μls of DH5α electrocompetent cells.
    2. Electroporate mixture at 1.44 kV.
    3. Move mixture to 1 ml of SOC broth+0.2% glucose (w/v).
    4. Repeat steps 1-3 nine times.
    5. Incubate at 37° C. for 1 hr.
    6. Centrifuge cells at 7,000 rpm for 5 mins.
    7. Pour off supernatant.
    8. Gently re-suspend cells in 10 mls pre-warmed LB broth+0.2% glucose.
    9. Centrifuge cells at 7,000 rpm for 5 mins.
    10. Pour off supernatant.
    11. Gently re-suspend cells in 10 mls pre-warmed LB broth+0.2% glucose.
    12. Add suspension to 500 mls of LB+30 μg/ml kanamycin+0.2% glucose.
    13. Incubate at 37° C. until culture's OD600 ̃0.4, centrifuge culture at 7,000 rpm for 5 mins.
    14. Pour off supernatant.
    15. Gently re-suspend cells in 25 mls of pre-warmed LB.
    16. Centrifuge at 7,000 rpm for 5 mins.
    17. Pour off supernatant.
    18. Gently re-suspend cells in 25 mls of pre-warmed LB.
    19. Add suspension to 500 mls of LB+30 μg/ml kanamycin+0.2% arabinose.
    20. Incubate at 37° C. for 2 hrs.
    21. Centrifuge culture at 7,000 rpm for 10 mins.
    22. Pour of supernatant.
    23. Gently re-suspend cells in 25 mls of pre-warmed LB.
    24. Centrifuge at 7,000 rpm for 10 mins.
    25. Pour off supernatant.
    26. Gently re-suspend cells in 25 mls of pre-warmed LB.
    27. Add suspension to 500 mls LB+30 μg/ml kanamycin+0.2% glucose+300 μg/ml ampicillin.
    28. Incubate at 37° C. for 3 hrs.
    29. Centrifuge culture at 7,000 rpm for 10 mins.
    30. Pour off supernatant.
    31. Gently re-suspend pellet in 50 mls of dH2O.
    32. Centrifuge at 7,000 rpm for 10 mins.
    33. Pour off supernatant.
    34. Gently re-suspend pellet in 50 mls of dH2O.
    35. Centrifuge at 7,000 rpm for 10 mins.
    36. Pour of supernatant.
    37. Perform a plasmid preparation on the remaining cells.


Confirmation of Efficacy Protocol
This protocol applies equally to all plasmid DNA preparations from the three above screens (bacteriolytic, bacteriostatic, and bactericidal).

    7. Mix 1 μl of a plasmid DNA preparation from one of the screens with 30 μls of DH5α electrocompetent cells.
    8. Electroporate mixture at 1.44 kV.
    9. Move mixture to 1 ml of SOC broth+0.2% glucose (w/v).
    10. Repeat steps 1-3 nine times.
    11. Incubate at 37° C. for 1 hr.
    12. Spread onto a series of LB+30 μg/ml kanamycin+0.2% glucose plates.
    13. Incubate overnight at 37° C.
    14. Replica plate from these plates first onto an LB+30 μg/ml kanamycin+0.2% arabinose and then to an LB+30 μg/ml kanamycin+0.2% glucose plate.
    15. Incubate plates at 37° C. for 8 hrs.
    16. Select colonies that did not grow on the arabinose plate for further study.


Example 6
Polynucleotide Sequences
The numbers refer to the nucleotide position in SEQ ID NO:1, which is the DNA sequence of a pKan phoA plasmid.
151-177 araC promoter
276-303 araBAD promoter
358-420 phoA peptide leader
421-435 Linker sequence
436-1785 phoA
1786-1803 His tag
1819-1862 T1 terminator
1994-2023 T2 terminator
2838-3383 pACYC ori
3977-4792 neo
5065-5943 araC
SEQ ID NO:2 and 3 shown below are two complimentary strands of DNA containing the NNK 12-mer. SEQ ID NO:2 contains a BsrGI sticky end, and SEQ ID NO:3 contains a KasI sticky end.







(SEQ ID NO: 2)


GTA CAC CCC TGT GAC AAA AGC CDN KNN KNN KNN KNN


 


KNN KNN KNN KNN KNN KNN KNN KGA AGG CGG CG


 


(SEQ ID NO: 3)


TG GGG ACA CTG TTT TCG GHN MNN MNN MNN MNN MNN


 


MNN MNN MNN MNN MNN MNN MCT TCC GCC GCC GCG






Example 7
Identification of Antimicrobial Peptides Using a Cytoplasmic System
To avoid bias due to placement of the peptide, two cytoplasmic libraries were constructed; one in which the peptide sequence was fused to the N-terminus of EmGFP and the other in which it was fused to the C-terminus of EmGFP.
The cytoplasmic, N-terminus system (see FIG. 11, left side). To isolate N-terminal inhibitory peptides, the construct pBac-EmGHt was created. The key elements of this plasmid and its sequence are shown FIGS. 14, 15, and 16.
To identify antimicrobial N-terminal peptides, E. coli strain EPI301 (an amber suppressor mutant of EPI300 (Epicentre Inc.; Madison, Wis.) containing plasmids pRK603 and pZS4int-tetR (FIG. 13) were transformed with the pBac-EmGHt random library, grown to early log phase and the TEV protease gene was induced by adding anhydrotetracycline (ATC) to a final concentration of 250 ng/ml of culture and the incubation was continued until mid-log phase. At mid-log phase, the peptide fusion construct was induced by adding L-arabinose to a final concentration of 0.2% of culture (w/v), and incubation was continued for 1.5 hrs. In screens to identify bacteriolytic peptides, the culture was centrifuged and plasmid DNA was precipitated from the supernatant and used to transform E. coli EPI301 and the transformants were screened on solid medium with or without inducers. No bacteriolytic peptides were identified in the N-terminal cytoplasmic screen. To identify bacteriostatic or bacteriocidal peptides, negative selection was performed in which ampicillin (Cf=500 μg/ml) was added to the mid-log phase cultures to lyse any bacteria that were actively dividing. The non-dividing (dead or static) cells were recovered by centrifugation and their plasmids were isolated and used to transform E. coli EPI301. Transformants harboring inhibitory peptides were identified by replica plating on solid media with and without inducers and the plasmids were sequenced to identify the amino acid sequences of the inhibitory peptides. A list of the peptides identified in this screen is provided in FIG. 27.
The cytoplasmic, C-terminus system (see FIG. 11, right side). To isolate N-terminal inhibitory peptides, the construct pBac-EmGH was created. The key elements of this plasmid are shown in FIGS. 17, 18, and 19.
To identify antimicrobial C-terminal peptides, E. coli EPI301 cells were transformed with the pBac-EmGH random library, grown to mid log phase and L-arabinose was added to a final concentration of 0.2% of culture (w/v) and the incubation was continued for 1.5 hrs. In screens to identify bacteriolytic peptides, the culture was centrifuged and plasmid DNA was precipitated from the supernatant and used to transform E. coli EPI301 and the transformants were screened on solid medium with or without inducers. No bacteriolytic peptides were identified in the C-terminal cytoplasmic screen. To identify bacteriostatic or bacteriocidal peptides, negative selection was performed in which ampicillin (Cf=500 μg/ml) was added to the mid-log phase cultures to lyse any bacteria that were actively dividing and the non-dividing (dead or static) cells were recovered by centrifugation, their plasmids were isolated and used to transform E. coli EPI301. Transformants harboring inhibitory peptides were identified by replica plating on solid medium with and without inducers and the plasmids were sequenced to identify the amino acid sequence of the inhibitory peptides. Some of these peptides slowed bacterial replication significantly without completely stopping it and were classified as “weakly inhibitory” (see FIG. 28). Other peptides either strongly blocked replication of or killed the cells and were classified as bacteriostatic or bacteriocidal (see FIG. 29).
Example 8
Characterization of Inhibitory Cytoplasmic Peptides
Each of the clones isolated in the cytoplasmic screens were characterized to determine their effects on cellular growth and to identify the amino acid sequences of the peptides (see FIG. 12).
Growth curve induction profiles (see FIG. 12, left side). Pure cultures of E. coli EPI301 harboring each of the plasmids encoding inhibitory peptides were grown overnight, used to inoculate LB medium and growth of the cultures was monitored by measuring the optical density of the culture at λ=600 nm (OD600). The resulting cultures were grown to early-, mid- or late-log phase and either ATC and L-arabinose (N-terminal peptides) or L-arabinose (C-terminal peptides) were added and the OD600 of each culture was followed. The phenotypes of the N-terminal peptides were consistent with peptides that strongly inhibited growth of the culture without lysing the cells (see FIG. 43). The C-terminal peptides all inhibited growth of the cultures but showed some differences in growth response following induction. Some of the C-terminal peptides lysed the cells while others only slowed the growth rate (see FIGS. 44A-C) and others blocked the growth of the culture (FIGS. 45A-D).
Sequence analysis (FIG. 12, right side). The amino acid sequence of each of the inhibitory peptides isolated in the cytoplasmic screen was determined by sequencing the region on the plasmids containing the randomized peptide sequence. The amino acid sequences of the N-terminal peptides is shown in FIG. 27 and the amino acid sequences of the C-terminal weakly inhibitory and bacteriolytic peptides are shown in FIG. 28 and the amino acid sequences of the strongly inhibitory peptides are shown in FIG. 29.
Example 9
Identification of Antimicrobial Peptides Using the Periplasmic System
The procedure for isolation of inhibitory peptides using the periplasmic system is illustrated in FIG. 20. Because the periplasmic fusion protein must be synthesized in the cytoplasm and pass through the plasma membrane into the periplasm, the random peptides were placed at the N-terminus of the carrier protein, PhoA. To isolate N-terminal inhibitory peptides, the construct pKan-PhoA (see FIGS. 23, 24, and 25) was created.
To identify antimicrobial periplasmic peptides, E. coli DH5α cells were transformed with the pKan-PhoA random library, grown to mid-log phase and L-arabinose was added to a final concentration of 0.2% w/v of culture and the incubation was continued for 1.5 hrs. In screens to identify bacteriolytic peptides, the culture was centrifuged and plasmid DNA was precipitated from the supernatant, used to transform E. coli DH5α and the transformants were screened on solid medium with or without inducers. To identify bacteriostatic or bacteriocidal peptides, negative selection was performed in which ampicillin (Cf=500 μg/ml) was added to the induced mid-log phase cultures to lyse any bacteria that were actively dividing and the non-dividing (dead or static) cells were recovered by centrifugation, their plasmids were isolated and used to transform E. coli DH5α. Transformants harboring inhibitory peptides were identified by replica plating on solid media with and without inducers and the plasmids were sequenced to identify the amino acid sequence of the inhibitory peptides.
The periplasmic bacteriolytic screen produced inhibitory peptides falling into all three phenotypic categories: bacteriostatic, bacteriocidal/bacteriostatic and weakly inhibitory (FIG. 20 left side). The bacteriolytic clones were sequenced (FIG. 30) and growth curves were performed on each (FIGS. 46A-L). The amino acid sequences of the bacteriolytic isolates were analyzed to identify common sequence motifs that contribute to their antimicrobial activity and these data were used to construct additional peptides in an effort to optimize their antimicrobial activity (see FIG. 21). The bacteriostatic/bacteriocidal peptides and the weakly inhibitory peptides were sequenced (FIGS. 31 and 32) and growth curves were performed (FIGS. 47A-C and 48A-I). The periplasmic screen for bacteriostatic and bacteriocidal also produced peptides that fell into each of the three phenotypic categories (FIG. 20, right side). Each of the isolates was sequenced (FIGS. 33 and 34) and growth curves were performed (FIGS. 49A-F and 50A-B).
Example 10
Further Characterization and Optimization of Bacteriolytic Peptides
To optimize the antimicrobial activity of the lytic peptides isolated from the periplasmic screen (FIG. 20), the bacteriolytic amino acid sequences were statistically analyzed to identify common motifs or other sequence elements and characteristics that contributed to or detracted from their antimicrobial activity and new peptides were designed, constructed and analyzed (see FIG. 21). The statistical analyses for these peptides are shown in the file labeled Statistics I.
In one set of mutants, common features of the lytic peptides were combined and six new peptides were constructed containing these features and analyzed (FIG. 21, left side). The results of these analyses are shown in FIG. 35 and FIGS. 51A-B. One of the peptides, EO1, showed significantly enhanced antimicrobial activity over the original isolates and was chosen for further study (see FIG. 22).
In another set of experiments, the common features identified in our analysis were used to construct a constrained library of peptide sequences in which variations on each of the elements identified as potentially important for antimicrobial activity were included (FIG. 21, right side). In another set of experiments, the common features identified in our analysis were used to construct a constrained library of peptide sequences in which variations on each of the elements identified as potentially important for antimicrobial activity were included (FIG. 21, right side). The degenerate sequence used to make the constrained peptide library based on analysis of early onset peptide sequences was 5′-WTBNNKYKGCTGNNKAGNYGGTGGCGTS GTNNKNNK-3′ (SEQ ID NO: 464), using standard IUPAC nomenclature for DNA nucleotides. “Early onset” refers to peptides listed in FIG. 30 that caused lysis of the cells within one hour of induction. Early onset suggests that lower concentrations of the peptide are required which in turn suggests that the early onset peptides are active at lower concentrations than late onset peptides. The design of the degenerate peptides is derived from the sequences of the early onset peptides in FIG. 30 in an effort to combine characteristics that enhance lysis.
A total of 120 randomly selected clones were screened by growth curve analysis and categorized by their resulting phenotypes. The phenotypes of the isolates are bacteriostatic (FIG. 36, FIGS. 52A-B), weakly inhibitory (FIG. 36, FIGS. 52A-B), bacteriolytic (FIG. 37, FIGS. 53A-I), and non-inhibitory (FIG. 38). The pattern of amino acids in the bacteriolytic peptides and non-inhibitory peptides was also statistically analyzed and these data are located in the files Statistics II and Statistics III respectively. These analyses suggested that alpha helices may be important for antimicrobial activity in some of the peptides and that placement of a proline residue within the alpha helical sequence of an inhibitory peptide might therefore block its antimicrobial activity. To test this hypothesis, an alanine was replaced with a proline in the inhibitory peptide, EOD037, to produce EOD037 Pro+ and a leucine was substituted for the proline in the non-inhibitory peptide, EOD084, to produce EOD084 Pro− and all four of the peptides were analyzed (FIG. 39, FIGS. 54A-B). The results show that in these peptides, the presence of a proline residue does result in loss of antimicrobial activity suggesting that the presence of an alpha helical segment is important.
Because the described antimicrobial measurements to date used the peptides expressed as protein fusions, whether the free peptide (not attached to the carrier) would also show antimicrobial activity was evaluated. For this two different sets of experiments were performed. First, special expression vectors that do not contain the carrier protein but produce the peptides in cells as free peptides were created. One vector expresses the free peptides in the cytoplasm and the other vector express the free peptide in the periplasm. These systems were used to test several different peptides and the results of these data are labeled “free peptide” in the tables and charts. EOD084 Pro− retained its antimicrobial activity when expressed in the periplasm in the absence of PhoA (FIG. 39, FIG. 55).
Example 11
Further Characterization of the EO1 Peptide
Several peptides were selected to determine if they retain their antimicrobial activity when added as a purified peptide to growing bacterial cultures. For this, the peptides were synthesized, diluted in growth medium, added to freshly inoculated bacterial cultures, incubated and the lowest concentration of each peptide that completely inhibited the growth of the culture was recorded as its the minimal inhibitory concentration (MIC). These data are provided in the tables containing the lists of peptides and their analyses. For EO1, its MIC against several other bacterial strains were measured and these data show that the EO1 peptide inhibits a broad spectrum of bacteria (Gram positive and Gram negative) at concentrations that are comparable to the MICs of common antibiotics such as chloramphenicol and ampicillin, (FIG. 40).
EO1 was also tested to determine if it retained its antimicrobial activity as a free peptide. The free peptide data for peptide EO1 are shown in FIG. 35 and FIGS. 56A-B. These data show that EO1 retains its antimicrobial activity when expressed as a free peptide in the periplasm but not when it is expressed as a free peptide in the cytoplasm.
Alanine scanning was performed in which substitution of a single alanine for each of the amino acids in EO1 was constructed and analyzed in vivo to determine if substitution of any single amino acid would affect its antimicrobial activity (FIG. 41, FIG. 57). The data show that only replacement of the leucine at position 4 of the peptide significantly affects its antimicrobial activity.
A series of site-directed mutants of EO1 were constructed in an effort to increase its solubility in water but most of these resulted in significant decreases in antimicrobial activity (FIG. 42, Chart FIGS. 58A-B).
Example 12
Consensus Peptide from all Lytic Peptides of the Periplasmic-Bacteriolytic Peptide Screen
The following consensus peptide is based on 85 peptides isolated from the periplasmic bacteriolytic peptide screen (FIG. 30) that were shown to exhibit the bacteriolytic growth phenotype (FIGS. 46A-L). More detailed analysis including positional selection against particular amino acids or groups of amino acids shown in FIG. 59.
Distribution of amino acid residues and groups by position in linear peptide. There is a general bias against acidic amino acids and proline residues. The following data are presented in a linear peptide format proceeding from the N-terminal amino acid to its covalently attached neighboring peptide. However, these peptides fit (there are outliers) to an amphipathic α-helix model where neighboring amino acids are determined by the rotation of the helix. Referring to FIG. 26, a graphical representation of the model is shown. Raw numbers supporting these observations are provided simply to illustrate the model. The raw data were statistically analyzed to determine if the amino acid distributions at each position were random or not and to identify preferences for specific amino acids or groups of amino acids using Chi-squared, and binomial analyses (see FIGS. 59A-H). Since there are 20 amino acids, the random occurrence of any given amino acid is approximately 1/20—this varies slightly depending on the codon because of the NNK sequence randomization used in the initial library construction. Also, if a preference for one type of amino acid is observed for any given position, a corresponding decrease in the other types of amino acids also will be seen at that position. A greater than expected decrease in another category of amino acid would be notable. Thus, in some instances both a preference for one type of amino acid and a selection against another type was seen, whereas in other instances either a selection for or against a particular type of amino acid was seen.
Position 1.
There is a strong preference for hydrophobic amino acids with a specific preference for tryptophan. Out of the 85 peptides analyzed, 75 had a hydrophobic amino acid, 30 of which were tryptophan. Note: In the number one position of the randomized peptide sequence, the randomized codon used for the initial periplasmic screen was DNK instead of NNK to avoid placement of prolines in the first position.
Position 2.
There is a preference for basic and small (glycine, alanine) amino acids and a strong selection against hydrophobic residues (only 6 hydrophobic amino acids were present but we would have expected 35 hydrophobic residues at this position by random chance. Out of 85 peptides analyzed, 27 had a basic amino acid, 21 of which were arginine, 25 had a small amino acid.
Position 3.
There is a preference for hydrophobic amino acids and arginine, a basic amino acid. Out of 85 peptides, 50 have a hydrophobic amino acid, 24 being tryptophan, and 19 have a basic residue, 17 being arginine.
Position 4.
There is a strong preference for hydrophobic residues with a preference for leucine. Out of 85 peptides, 77 have hydrophobic amino acids, 36 of which have leucine.
Position 5.
There is a strong preference for basic amino acids with a preference for arginine and lysine. Out of 85 peptides, 54 have a basic amino acid, 38 of which are arginine and 15 are lysine.
Position 6.
There is a strong preference for basic amino acids, especially arginine and a minor preference for small amino acids as well as a minor selection against hydrophobic amino acids. Out of 85 peptides, 48 have a basic residue, of which 39 are arginine, and 15 of the remaining amino acids are alanine or glycine.
Position 7.
There is a preference for hydrophobic amino acids, split between leucine and tryptophan, a minor preference for basic amino acids, and hydrophilic amino acids are selected against. Out of 85 peptides, 58 have a hydrophobic amino acid, 22 being tryptophan and 22 being leucine, 18 of the remaining 27 amino acids are basic.
Position 8.
There is a strong preference for hydrophobic amino acids tryptophan or leucine with a minor preference for small amino acids. Out of 85 peptides, 63 have a hydrophobic residue, 24 are tryptophan and 20 are leucine, 11 of the remaining 22 are either alanine or glycine.
Position 9.
There is a preference for basic amino acids, overrepresented by arginine with a slight bias towards small residues, particularly glycine. Out of 85 peptides, 37 have a basic amino acid, 31 of which are arginine, and 13 peptides have a glycine residue.
Position 10.
There is a preference for basic amino acids, overrepresented by arginine with a bias towards small amino acids. Out of 85 peptides, 35 have a basic residue, 29 of which are arginine, and 19 peptides have a small amino acid.
Position 11.
There appears to be a strong preference for tryptophan and arginine that drives the distribution here. Out of 85 peptides, 18 are arginine and 15 have a tryptophan.
Position 12.
There is a preference for basic residues, particularly arginine with a slight preference for glycine. Out of 85 peptides 36 have a basic amino acid of which 25 are arginine and 17 peptides have a small amino acid of which 13 are glycine.
Accordingly, in various embodiments, a consensus amino acid sequence for a 12mer peptide antimicrobial can be Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 (SEQ ID NO:465); wherein

    
    
        Xaa1 is a hydrophobic amino acid (in one embodiment, tryptophan);
        Xaa2, is a basic or small amino acid (in one embodiment, selected from glycine or alanine) or a basic amino acid (in one embodiment, arginine);
        Xaa3 is a hydrophobic amino acid (in one embodiment, tryptophan) or a basic amino acid (in one embodiment, arginine);
        Xaa4 is a hydrophobic amino acid (in one embodiment, leucine);
        Xaa5 is a basic amino acid (in one embodiment, arginine or lysine);
        Xaa6 is a basic amino acid (in one embodiment, arginine) or a small amino acid (in one embodiment, alanine or glycine);
        Xaa7 is a hydrophobic amino acid (in one embodiment, selected from leucine or tryptophan) or a basic amino acid;
        Xaa8 is a hydrophobic amino acid (in one embodiment, tryptophan or leucine) or a small amino acid (in one embodiment, alanine or glycine);
        Xaa9 is a basic amino acid (in one embodiment, arginine) or a small amino acid (in one embodiment, glycine);
        Xaa10 is a basic amino acid (in one embodiment, arginine) or a small amino acid (in one embodiment, glycine or alanine);
        Xaa11 is tryptophan or arginine;
        Xaa12 is a basic amino acid (in one embodiment, arginine) or a small amino acid (in one embodiment, glycine).
    
    


Example 13
Further analysis includes target identification utilizing the (His)6 tag to purify and collect the target molecule. Some peptides of various phenotypes have shown that they do not have a protein target. Microscopy is used to determine these peptides' effects on membrane perturbance.

    1. A method for identifying an effective peptide antimicrobial, said method comprising:
    
        controllably expressing a random peptide library in one or more microbial cell culture(s), wherein random peptides are encoded by plasmids comprising a nucleic acid sequence which is under the control of an inducible promoter; and
        identifying microbial cell culture(s) in which microbial cell growth or survival is affected by the peptide expressed by that culture.
    
    
    2. The method of embodiment 1, comprising growing the one or more microbial cell culture(s) in the absence of an inducer of the inducible promoter before and/or after said controllable expression.
    3. The method of embodiment 2, comprising growing the one or more microbial cell culture(s) in the presence of a transcriptional repressor.
    4. The method of embodiment 1, comprising adding an inducer of the inducible promoter.
    5. The method of embodiment 1, comprising transforming the microbial cells with the plasmids.
    6. The method of embodiment 1, comprising contacting the one or more microbial cell culture(s) with an agent that negatively selects against replicating cells.
    7. The method of embodiment 6, wherein the agent is ampicillin.
    8. The method of embodiment 1, 2, 3, or 4 comprising identifying the peptide sequence of a microbial cell culture in which cell growth or survival is affected.
    9. The method of embodiment 1, comprising constructing the plasmid.
    10. The method of embodiment 1, wherein the nucleic acid sequence comprises a NNKn sequence, wherein NNK is a nucleotide triplet that encodes a random amino acid, wherein:
    
        N is A, T, C or G;
        K is T or G; and
        n is 2-50;
    
    


wherein, optionally, n=12 (SEQ ID NO:462).

    11. The method of embodiment 1, wherein the nucleic acid sequence comprises a DNK(NNK)n sequence, wherein DNK and NNK are nucleotide triplets that encode a random amino acid, wherein:
    
        N is A, T, C or G;
        K is T or G; and
        D is A, T, or G; and
        n is 1-49;
    
    


wherein, optionally, n=11 (SEQ ID NO:463).

    12. The method of any one of embodiments 1-11, wherein the plasmid encodes a leader peptide.
    13. The method of embodiment 12, wherein the leader peptide targets the random peptides to the periplasm of gram negative microbial host cells.
    14. The method of embodiment 12, wherein the leader peptide is an alkaline phosphatase leader peptide.
    15. The method of any one of embodiments 1-11, wherein the inducible promoter is an arabinose inducible promoter (PBAD).
    16. The method of any one of embodiments 1-11, wherein the nucleic acid sequence encodes a carrier protein.
    17. The method of embodiment 16, wherein the carrier protein is alkaline phosphatase, and wherein the C terminus of the random peptide is fused to the N terminus of the alkaline phosphatase protein.
    18. The method of embodiment 16, wherein the carrier protein is alkaline phosphatase, and wherein the N terminus of the random peptide is fused to the C terminus of the alkaline phosphatase protein.
    19. The method of embodiment 16, wherein the carrier protein is emerald green fluorescent protein, and wherein the C terminus of the random peptide is fused to the N terminus of the emerald green fluorescent protein.
    20. The method of embodiment 16, wherein the carrier protein is emerald green fluorescent protein, and wherein the N terminus of the random peptide is fused to the C terminus of the emerald green fluorescent protein.
    21. The method of any one of embodiments 1-11, wherein the host cell is a bacterium.
    22. The method of embodiment 21, wherein the bacterium is E. coli. 
    23. An antimicrobial peptide comprising the sequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 (SEQ ID NO:465); wherein
    
        Xaa1 is a hydrophobic amino acid;
        Xaa2, is a basic and/or small amino acid;
        Xaa3 is a hydrophobic amino acid or a basic amino acid;
        Xaa4 is a hydrophobic amino acid;
        Xaa5 is a basic amino acid;
        Xaa6 is a basic amino acid and/or a small amino acid;
        Xaa7 is a hydrophobic amino acid or a basic amino acid;
        Xaa8 is a hydrophobic amino acid or a small amino acid;
        Xaa9 is a basic amino acid and/or a small amino acid;
        Xaa10 is a basic amino acid and/or a small amino acid;
        Xaa11 is tryptophan or arginine;
        Xaa12 is a basic amino acid and/or a small amino acid.
    
    
    24. An antimicrobial peptide comprising the amino acid sequence of any one of SEQ ID NOS:7-461.
    25. The antimicrobial peptide of embodiment 24, comprising an amino acid sequence which is at least 85% homologous to the amino acid sequence:











(SEQ ID NO: 310)



FAWLWSWWRARR.






    26. The antimicrobial peptide of embodiment 25, comprising an amino acid sequence which is at least 85% homologous to the amino acid sequence:











(SEQ ID NO: 438)



FMRLLRWWRRMQ.






    27. The antimicrobial peptide of embodiment 26, comprising an amino acid sequence which is at least 85% homologous to the amino acid sequence:











(SEQ ID NO: 347)



IRWLARRWRRTF.






    28. An artificial nucleic acid sequence encoding a peptide comprising the amino acid sequence of any one of SEQ ID NOS:7-461.
    29. An expression vector comprising the artificial nucleotide sequence of claim 28.
    30. A vector comprising a DNA sequence encoding an inducible promoter and a (NNK)n sequence of DNA, wherein NNK is a nucleotide triplet that encodes a random amino acid, wherein N is A, T, C or G, K is T or G, and n is 2-50.
    31. A vector comprising a DNA sequence encoding an inducible promoter and a DNK(NNK)n sequence of DNA, wherein NNK is a nucleotide triplet that encodes a random amino acid, wherein N is A, T, C or G, K is T or G, D is A, T, or G; and n is 1-49.
    32. An antimicrobial peptide of embodiment 23 comprising the sequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Xaa12 (SEQ ID NO:465); wherein
    
        Xaa1 is tryptophan;
        Xaa2 is glycine, alanine, or arginine;
        Xaa3 is tryptophan, or arginine;
        Xaa4 is leucine;
        Xaa5 is arginine or lysine;
        Xaa6 is arginine, alanine or glycine;
        Xaa7 is leucine or tryptophan;
        Xaa8 is tryptophan, leucine, alanine or glycine;
        Xaa9 is arginine or glycine;
        Xaa10 is arginine, glycine or alanine;
        Xaa11 is tryptophan or arginine;
        Xaa12 is arginine or glycine.
    
    


Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Specific embodiments disclosed herein may be further limited in the claims using consisting of or and consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.
In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

## Claims

1. A method for isolating an antimicrobial peptide comprising:
obtaining a microbial cell culture comprising microbial cells transformed with a plasmid wherein the microbial cells are transformed with different plasmids, each plasmid comprising a nucleic acid sequence encoding a random peptide wherein expression of the random peptide is under the control of an inducible arabinose promoter, wherein the nucleic acid sequence encoding the random peptide comprises a DNK(NNK)n sequence, wherein DNK and NNK are nucleotide triplets that encode a random amino acid, wherein:
N is A, T, C or G;
K is T or G; and
D is A, T, or G; and
n is 10-49;
culturing the microbial cell culture under a condition that represses expression of the random peptides;
controllably expressing a random peptide library by (i) removing the condition that represses expression from the microbial cell culture; and (ii) adding arabinose to the microbial cell culture, wherein the nucleic acid sequence of the plasmid further encodes a leader peptide and wherein following controllable expression, the leader peptide is at the N-terminus of the random peptide, wherein the microbial cell is a Gram-negative microbial cell comprising a periplasm and wherein the leader peptide targets the random peptide to the periplasm of the Gram negative microbial cell, wherein the leader peptide is an alkaline phosphatase leader peptide;
evaluating the microbial cell culture for evidence of an antimicrobial bacteriolytic, bacteriostatic, or bacteriocidal phenotype and if an antimicrobial phenotype is present;
isolating a nucleic acid sequence encoding a peptide leading to the antimicrobial phenotype.

2. The method of claim 1 wherein n=11.

3. A method of claim 1 wherein the evaluating includes detection of a plasmid in supernatant following centrifugation of the microbial cell culture and wherein the antimicrobial phenotype is bacteriolytic.

4. A method of claim 1 further comprising:
adding a negative selection agent to the microbial cell culture; and
removing arabinose and the negative selection agent;
wherein the evaluating includes detection of replicating microbial cells following removal of the arabinose and the negative selection agent and wherein the antimicrobial phenotype is bacteriostatic.

5. A method of claim 4 wherein the negative selection agent is ampicillin.

6. A method of claim 4 further comprising enriching a population of microbial cells expressing a bacteriostatic peptide by:
isolating the replicating microbial cells of claim 4;
culturing the isolated replicating cells under a condition that represses expression of the isolated cells' encoded peptides;
controllably expressing the isolated cells' encoded peptides by (i) removing the condition that represses expression from the microbial cell culture; and (ii) adding arabinose to the microbial cell culture;
adding a negative selection agent to the microbial cell culture; and
removing arabinose and the negative selection agent;
thereby enriching the population of microbial cells expressing a bacteriostatic peptide.

7. A method of claim 4 wherein the detection occurs based on examination of growth colonies.

8. A method of claim 1 further comprising:
removing arabinose; and
adding a negative selection agent;
wherein the evaluating includes centrifuging the microbial cell culture and removing non-lysed cells from a pellet and wherein the antimicrobial phenotype is bacteriocidal.

9. A method of claim 8 wherein the negative selection agent is ampicillin.