WO2014102782A1

WO2014102782A1 - Compositions and methods of killing bacteria

Info

Publication number: WO2014102782A1
Application number: PCT/IL2013/051062
Authority: WO
Inventors: Ehud Qimron; Ruth KIRO
Original assignee: Ramot At Tel-Aviv University Ltd
Priority date: 2012-12-24
Filing date: 2013-12-24
Publication date: 2014-07-03

Abstract

Uses, compositiona and methods of treating an infection in a subject are disclosed. The invention provides a Gp0.4 T7 bacteriophage protein and uses thereof in the treatment of infectious disease and for inhibiting bacterial growth.

Description

COMPOSITIONS AND METHODS OF KILLING BACTERIA

FIELD OF THE INVENTION

The present invention, relates to compositions and methods of killing microbes. More particularly, the invention provides a bacteriophage protein as well as methods, compositions and kits using a bacteriophage protein for inhibiting bacterial growth.

REFERENCES

1. Watve MG, (2001) How many antibiotics are produced by the genus Streptomyces Arch Microbiol.176(5):386-90.

2. Riley and Wertz, (2002) Bacteriocins: evolution, ecology, and application Annu Rev Microbiol. 56: 117-37.

3. Studier FW (1972) Bacteriophage T7. Science 176(33):367-376.

4. Qimron U, Marintcheva B, Tabor S, Richardson CC (2006) Genomewide screens for Escherichia coli genes affecting growth of T7 bacteriophage. Proc Natl Acad Sci USA 103(50): 19039- 19044.

5. Barrangou R, et al. (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315(5819): 1709- 1712.

6. Gazit E (2007) Use of biomolecular templates for the fabrication of metal nanowires. FEBS J 21A{2)- 17-322.

7. Erickson HP (1995) FtsZ, a prokaryotic homolog of tubulin? Cell 80(3):367-370.

8. Zhou H, Lutkenhaus J (2005) MinC mutants deficient in MinD- and DicB-mediated cell division inhibition due to loss of interaction with MinD, DicB, or a septal component. Bacteriol 187(8):2846-2857.

9. Conter A, Bouche JP, Dassain M (1996) Identification of a new inhibitor of essential division gene ftsZ as the kil gene of defective prophage Rac. J Bacteriol 178(17):5100-5104.

10. Greer H (1975) The kil gene of bacteriophage lambda. Virology 66(2):589-604.

11. Rigaut G, et al. (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17(10): 1030-1032.

12. Keseler IM, et al. (2011) EcoCyc: a comprehensive database of Escherichia coli biology. Nucleic Acids Res 39(Database issue):D583-590.

13. Dai K, Mukherjee A, Xu Y, Lutkenhaus J (1994) Mutations ftsZ that confer resistance to SulA affect the interaction of FtsZ with GTP. Bacteriol 176(1): 130-136. 14. Chen Y, Erickson HP (2011) Conformational changes of FtsZ reported by tryptophan mutants. Biochemistry 550:4675-4684.

15. Chen Y, Milam SL, Erickson HP (2012) SulA inhibtis assembly of FtsZ by a simple sequestration mechanism. Biochemistry 51:3100-3109.

16. Baba T, et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection. Mol Syst Biol 2:0008.

17. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97(12):6640-6645

18. Lin-Chao S, Chen W-T, Wong T-T (1992) High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II. Mol Microbiol 6(22):3385-3393.

19. Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Bacteriol 177(14):4121-4130.

20. Molineux Π (2005) The Bacteriophages, eds Abedon ST, Calendar RL (Oxford Univ Press, Oxford), pp 275-299.

21. Kitagawa M, et al. (2005) Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): Unique resources for biological research. DNA Res 12(5):291-299.

22. Andersson L, (2000) Large-scale synthesis of peptides Biopolymers;55(3y.22T-50.

23. Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.

BACKGROUND OF THE INVENTION

Antibiotic resistance can result in severe adverse outcomes, such as increased mortality, morbidity and medical care costs for patients suffering from common infections, once easily treatable with antibiotics and therefore became one of the most recognized clinical problems of today's governmental, medicinal and pharmaceutical research [U.S. Congress, Office of Technology Assessment, Impacts of Antibiotic-Resistant Bacteria, OTA-H-629, Washington, D.C., U.S. Government Printing Office (1995)].

Due to the limitations associated with the use of classical antibiotics, extensive studies have been focused on finding novel, efficient and non-resistance inducing antimicrobial and antibacterial agents. Microbes (bacteria, archaea, fungi and viruses) frequently produce and secrete compounds aimed at killing other microbes which help them in their continuous struggle for survival in their ecological niche. Such compounds can be small molecule antibiotics, such as the ones produced by various Streptomyces species [1], or proteinacious antibiotics, often known as bacteriocins [2] or antimicrobial peptides (AMPs).

Proteins that target bacteria have a broad medical and biotechnological application spectrum. They can be used as direct antibiotics for human and veterinary medicine, as growth enhancers in livestock, as food preservatives, as genes engineered into probiotic bacteria, as killers of phytopathogenic bacteria for crop management, etc. In addition, they may serve as effective anti-microbial agents against antibiotic resistant organisms.

Bacteriophage T7 and its host, Escherichia coli, provide a model for systematically studying host-virus interactions. T7 is a virulent phage which, upon infection of its host E. coli, produces over 100 progeny phage per host in less than 25 min. It is an obligatory lytic phage, meaning that a successful phage growth cycle always results in lysis of the host. The genome of T7 is a 39,937-bp double- stranded DNA molecule. Despite extensive knowledge of phage T7, the mechanism by which it takes over the host molecular machinery remains obscure. Many of its gene products that create favorable conditions for phage growth in the host have not yet been assigned specific functions. One such gene product, which has neither a known function nor an obvious phenotype during phage T7 growth, is gene product (Gp) 0.4. Gene 0.4 is an early, 156- bp long gene, producing a peptide which is 51 aa in length. It is transcribed with the first genes approximately 2 min after infection [3] The polycistronic RNA encoding genes 0.3-1.3 is specifically cleaved by RNaselll upstream of gene 0.3 and downstream of gene 0.4 to yield an mPvNA fragment encoding only genes 0.3 and 0.4.

FtsZ is a bacterial protein that is essential for cell division. FtsZ is conserved in almost all known bacterial and many archaeal species, although there are some exceptions. FtsZ, a homolog of eukaryotic tubulin, is the major cytoskeletal protein in bacterial division [7]. It assembles the Z ring at the site of cytokinesis, which recruits downstream proteins that remodel the cell wall and form the septum between the daughter cells. FtsZ itself generates the constriction force on the membrane. The assembly of the Z ring is a dynamic process that requires GTP, and GTP hydrolysis produces rapid turnover of the FtsZ cytoskeleton. Several endogenous regulators and phage-derived inhibitors of FtsZ have been identified in E. coli, including MinC and SulA. The kil gene of the E. coli cryptic prophage, Rac, is another reported FtsZ phage inhibitor [9]. The kil gene of E. coli

λ phage was also implicated in inhibition of cell division, but no direct inhibition of FtsZ was shown [10]. Nevertheless, there is need for further efficient exogenous negative regulators of FtsZ for inhibiting bacterial growth.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a GpO.4 T7 bacteriophage protein, any homolog or related protein of the T7-like bacterial viruses, or any composition comprising the same for use in a method of treating an infection or infectious disease in a subject in need thereof.

In some embodiments, the homolog or related protein of the T7-like bacterial viruses may be any one of the hypothetical protein YpP-R from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06.

According to some embodiments, the GpO.4 T7 bacteriophage protein comprises an amino acid sequence at least 80 % homologous to the sequence as set forth in SEQ ID NO: 10. In yet some other embodiments, the GpO.4 T7 bacteriophage protein comprises an amino acid sequence at least 90 % homologous to the sequence as set forth in SEQ ID NO: 10.

In more specific embodiments, the GpO.4 T7 bacteriophage protein used by the method of the invention may comprise an amino acid sequence as set forth in SEQ ID NO: 10 or any derivatives or fragments thereof.

Still further alternative embodiments relate to GpO.4 T7 bacteriophage protein that may comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 11-42.

In certain embodiments, the GpO.4 T7 bacteriophage protein may be further attached to an agent that enhances penetration across the bacterial membrane. In some embodiments, the agent may be a peptide agent. In some specific embodiments such peptide may be a cell-penetrating peptide (CPP). In yet some other embodiments, the Gp0.4 T7 bacteriophage protein may be attached to a sustained-release enhancing agent.

In certain embodiments, the Gp0.4 T7 bacteriophage protein of the invention is specifically applicable for use in a method of treating infectious disease and/or infections caused by a bacterial infection.

In a second aspect, the invention provides a cytotoxic effective amount of a Gp0.4 T7 bacteriophage protein or any homolog or related protein of the T7-like bacterial viruses or any composition comprising the same for use in a method of killing a microbe. In more specific embodiments, the invention provides a Gp0.4 T7 bacteriophage protein or any homolog or related protein of the T7-like bacterial viruses or any composition comprising the same for use in a method for inhibiting microbial, specifically, bacterial growth.

In certain embodiments, the microbe or bacteria express the FtsZ protein or any homologues thereof, specifically, any FtsZ family protein.

According to certain embodiments, a homolog or related protein of the T7-like bacterial viruses may be any one of the hypothetical protein YpP-R from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06.

In yet some other embodiments the Gp0.4 T7 bacteriophage protein comprises an amino acid sequence that is at least 80 % homologous to the sequence as set forth in SEQ ID NO: 10. Still further in other embodiments the Gp0.4 T7 bacteriophage protein comprises an amino acid sequence at least 90 % homologous to the sequence as set forth in SEQ ID NO: 10. In more specific embodiments, the Gp0.4 T7 bacteriophage protein comprises an amino acid sequence as set forth in SEQ ID NO: 10 or any derivatives or fragments thereof.

In some further embodiments, the Gp0.4 T7 bacteriophage protein used for the composition of the invention may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 11- 42.

According to some embodiments, the Gp0.4 T7 bacteriophage protein of the invention is specifically suitable for use in a method of killing microbe such as bacteria. Another aspect of the invention relates to a pharmaceutical composition comprising a GpO.4 T7 bacteriophage protein or any homolog or related protein of the T7-like bacterial viruses, as the active ingredient and a pharmaceutically acceptable carrier.

In some embodiments, such homolog or related protein of the T7-like bacterial viruses may be any one of the hypothetical protein YpP-R from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06.

In some embodiments, the GpO.4 T7 bacteriophage protein comprises an amino acid sequence at least 80 % homologous to the sequence as set forth in SEQ ID NO: 10.

In other embodiments, the GpO.4 T7 bacteriophage protein comprises an amino acid sequence at least 90 % homologous to the sequence as set forth in SEQ ID NO: 10.

In more specific embodiments the GpO.4 T7 bacteriophage protein comprises an amino acid sequence as set forth in SEQ ID NO: 10 or any derivatives or fragments thereof.

In some alternative embodiments the GpO.4 T7 bacteriophage protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 11-42.

In some embodiments the GpO.4 T7 bacteriophage protein is further attached to an agent that enhances penetration across the bacterial membrane.

In some embodiments, the additional penetrating agent may be a peptide agent.

In some embodiments, the GpO.4 T7 bacteriophage protein may be attached to a sustained- release enhancing agent.

In yet further embodiments, the composition of the invention may further comprise an additional therapeutic agent.

Certain embodiments of the invention provide a composition of GpO.4 T7 bacteriophage protein or any homolog or related protein of the T7-like bacterial viruses that may be formulated for topical delivery. The invention further provides the use of a therapeutically effective amount of a Gp0.4 T7 bacteriophage polypeptide or homolog or related protein of the T7-like bacterial viruses, in the preparation of a composition for treating an infectious disease. More specifically, such infectious disease is caused by a bacterial infection. More specifically, such homolog or related protein of the T7-like bacterial viruses may be any one of the hypothetical protein YpP-R from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06.

Another aspect of the invention relates to a kit comprising: (a) a Gp0.4 T7 bacteriophage protein, any homolog or related protein of the T7-like bacterial viruses, or any fragment, peptide, analogues and derivatives thereof, optionally, in a first dosage form; and (b) at least one additional therapeutic agent, optionally, in a second dosage form. More specifically, such homolog or related protein of the T7-like bacterial viruses may be any one of the hypothetical protein YpP-R from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06.

In some embodiments, the kit of the invention may be particularly for use in a method of treatment of an infectious disease and/or infections.

A further aspect of the invention provides a solid support coated with a Gp0.4 T7 bacteriophage protein.

In some embodiments, the Gp0.4 T7 bacteriophage protein used for the solid support may comprise an amino acid sequence that is at least 80 % homologous to the sequence as set forth in SEQ ID NO: 10. In yet some other embodiments, the Gp0.4 T7 bacteriophage protein comprises an amino acid sequence that is at least 90 % homologous to the sequence as set forth in SEQ ID NO: 10. Still further, the Gp0.4 T7 bacteriophage protein may comprise an amino acid sequence as set forth in SEQ ID NO: 10 or any derivatives thereof.

In other embodiments, the Gp0.4 T7 bacteriophage protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 11-42.

In yet another aspect, the invention relates to a method of treating an infection or infectious disease in a subject in need thereof. In certain embodiments, the method comprising administering to the subject a therapeutically effective amount of a Gp0.4 T7 bacteriophage protein, any homolog or related protein of the T7-like bacterial viruses, or any composition comprising the same, thereby treating the disease.

In certain embodiments, the GpO.4 T7 bacteriophage protein may be further attached to an agent that enhances penetration across the bacterial membrane. In some embodiments, the agent may be a peptide agent. In some specific embodiments such peptide may be a cell-penetrating peptide (CPP). In yet some other embodiments, the GpO.4 T7 bacteriophage protein may be attached to a sustained-release enhancing agent.

In certain embodiments, the method of the invention is specifically applicable for treating infectious disease and/or infections caused by a bacterial infection.

In a further aspect, the invention provides a method of killing a microbe. In more specific embodiments, the invention provides a method for inhibiting microbial, specifically, bacterial growth, the method comprising contacting the microbe, specifically, bacteria with a cytotoxic effective amount of a GpO.4 T7 bacteriophage protein or any homolog or related protein of the T7-like bacterial viruses, thereby inhibiting bacterial growth and killing the bacteria.

According to certain embodiments, a homolog or related protein of the T7-like bacterial viruses may be any one of the hypothetical protein YpP-R from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06. In yet some other embodiments the Gp0.4 T7 bacteriophage protein comprises an amino acid sequence that is at least 80 % homologous to the sequence as set forth in SEQ ID NO: 10. Still further in other embodiments the Gp0.4 T7 bacteriophage protein comprises an amino acid sequence at least 90 % homologous to the sequence as set forth in SEQ ID NO: 10. In more specific embodiments, the Gp0.4 T7 bacteriophage protein comprises an amino acid sequence as set forth in SEQ ID NO: 10 or any derivatives or fragments thereof.

In some embodiments, the method of the invention involves an in vivo step of contacting the bacteria with the protein of the invention. In alternative embodiments, the contacting step may be affected ex vivo.

According to some embodiments, the method of the invention is specifically suitable for killing microbe such as bacteria.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying images. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

Figure 1. is a Western blot illustrating that FtsZ specifically pulls down T7 Gp0.4. Four proteins shown to be pulled down by Gp0.4: FtsZ (40.3 kDa), PrsA (34.2 kDa), GlpD (56.8 kDa), AceE (99.7 kDa), and a negative control— HsdM (59.3 kDa) were affinity-purified using their 6XHis tag as described in Experimental procedures. Image shows Coomasie-stained 12% polyacrylamide gel of the eluted proteins (top). A Western blot to detect pulled-down Gp0.4 was performed using an antibody against the calmodulin binding peptide tag attached to Gp0.4 (bottom). Representative images are shown from one out of three experiments showing similar results.

Figures. 2A-2B. illustrate in vivo inhibition of bacterial growth by Gp0.4.

Fig. 2A. E. coli cells encoding WT-FtsZ were transformed with plasmids encoding Gp0.4 (WT- FtsZ/Gp0.4) or an empty vector (WT-FtsZ/ctrl). Relative growth efficiency was calculated by dividing the CFU obtained in the presence of L-arabinose induction (+ara) by the number of CFU obtained in the absence of induction (-ara). Bars represent average + SD of three independent experiments.

Fig. 2B. E. coli cells encoding the WT-FtsZ or the resistant FtsZ9 were both transformed with plasmids encoding Gp0.4. Relative growth efficiency was calculated by dividing the CFU obtained in the presence of arabinose induction (+ara) by the number of CFU obtained in the absence of induction (-ara). Bars represent average + SD of three independent experiments.

Figure 3. illustrates the effect of Gp0.4 on FtsZ protofilament assembly.

Chemically synthesized Gp0.4 at 0 μΜ (circle), 1 μΜ (diamond), 3 μΜ (triangle), or 5 μΜ (square) was added to increasing concentrations of purified S 151C/Y222W-FtsZ protein. The assembly was measured via a change in the fluorescence monitored by a Shimadzu RF-5301 PC spectrofluorometer. Each sample was monitored in duplicate. Error bars represent the standard deviation of each point. The experiment was repeated twice with similar results.

Figure 4. presents photographs illustrating the change in morphology of E. coli expressing Gp0.4. E. coli encoding the WT-FtsZ or the Gp0.4-resistant FtsZ9 harboring pBAD-0.4 (Gp0.4) or a control pBAD18 (ctrl) plasmid were induced with 0.001% L-arabinose to drive expression from the plasmid promoter, as described in Experimental procedures. Images were taken 0, 2, and 4 h after induction (scale bar: 8 μιη). Images represent one out of two experiments showing similar results.

Figure 5. illustrates in vivo inhibition of bacterial growth by Gp0.4 in isogenic bacterial strains of E. coli, Amin C and Asul A, lacking endogenous inhibitors of FtsZ. E. coli lacking the indicated genes or a control gene (aceF) were transformed with the plasmid encoding Gp0.4 and grown with L-arabinose induction (+ ara) or without induction (- ara). Relative growth was calculated by dividing the number of CFUs obtained in the induced samples by the uninduced samples of each corresponding strain. Bars represent average + SD of three independent experiments.

Figures 6A-6D. illustrates PCR-quantified competition of phages.

Fig. 6A. PCR amplifying the region flanking gene 0.4 was carried out on Τ7Δ0.4 phage and WT T7FRT mixed at the indicated ratios. The upper bands are products obtained from amplifying the DNA of WT T7FRT, and the lower bands are from Τ7Δ0.4.

Fig. 6B. Graph showing the relative abundance of the indicated phages grown on an exponential- phase culture.

Fig. 6C. Graph showing the relative abundance of the indicated phages grown on a stationary- phase culture.

Fig. 6D. Graph showing the relative abundance of the indicated phages grown on an exponentialphase culture of E. coli encoding FtsZ9 or FtsZ. The PCR and relative abundance calculations were carried out as described in Experimental procedures.

Graphs show averages + SD of three independent experiments. *P = 0.01-0.05; **P = 0.001- 0.01; ***P < 0.001.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in some embodiments thereof, relates to compositions, kits and methods of inhibiting bacterial growth, thereby, killing bacteria using the Gp0.4 T7 bacteriophage protein.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Bacteriophages take over host resources primarily by expressing early proteins upon host infection. One of these early proteins, produced by the Escherichia coli phage T7, is gene product (Gp) 0.4. The present inventors have shown using a tagged affinity purification assay and mass spectrometry that Gp0.4 binds the E. coli protein FtsZ. FtsZ, a homolog of eukaryotic tubulin, is a cytoskeletal protein that is essential for bacterial cell division. Overexpression of Gp0.4 in bacterial cells resulted in a six order of magnitude drop in cell viability (Figure 2A). Further, several bacterial populations resistant to the activity of Gp0.4 were found to have mutations in the ftsZ gene. By transducing Gp0.4 sensitive bacterial populations with the mutated ftsZ gene, these populations were rendered resistant (Figure 2B) indicating that Gp0.4 specifically inhibits FtsZ, and that this inhibition can be overcome by alteration of FtsZ at a specific site. In vitro experiments with chemically synthesized Gp0.4 and purified FtsZ presented in Figure 3, demonstrate that Gp0.4 inhibits FtsZ assembly by sequestering the FtsZ subunits. Since FtsZ is conserved in almost all known bacterial and many archaeal species, the present inventors propose that Gp0.4 may be used as a broad spectrum antibiotic.

The invention therefore provides, in a first aspect, a Gp0.4 T7 bacteriophage protein or any homolog or related protein of the T7-like bacterial viruses for use in a method of treating an infectious disease and/or an infection in a subject in need thereof. The invention further provides a method of treating an infectious disease and/or an infection in a subject. The invention is based on the use of a therapeutically effective amount of a Gp0.4 T7 bacteriophage protein or any homolog or related protein of the T7-like bacterial viruses for treating the disease.

It should be noted that the invention further provides Gp0.4 T7 bacteriophage protein or any homolog or related protein of the T7-like bacterial viruses for use in a method for preventing and inhibiting the assembly of the FtsZ subunits in bacterial cells, thereby inhibiting cell growth and reducing viability of the cells.

Thus, according to a further aspect of the present invention there is provided Gp0.4 T7 bacteriophage protein or any homolog or related protein of the T7-like bacterial viruses for use in a method of killing microbes, specifically, inhibiting microbial, or more specifically, bacterial growth. The method of the invention is based on contacting the microbes with a cytotoxic amount of a Gp0.4 T7 bacteriophage protein or any homolog or related protein of the T7-like bacterial viruses, thereby killing the microbes.

As noted above, the present invention is base on the use of a T7 bacteriophage early protein, the Gp0.4 protein. As sued herein, "Bacteriophage T7" is a bacteriophage capable of infecting susceptible bacterial cells and infects most strains of Escherichia coli. Bacteriophage T7 is a DNA virus that has an obligatory lytic phage, unleashes more than 100 progeny phage per host in less than 25 min under optimal conditions. The 39,937-bp, double- stranded DNA genome of T7 is transcribed from left to right, with each gene of the identified 56 genes is sequentially numbered. More specifically, the essential genes are assigned integral numbers, while the nonessential genes are assigned noninteger numbers reflecting their relative positions between essential genes. Genes 2.5, 6.7, and 7.3 are essential gene exceptions to that naming practice, as is gene 7, which is now considered nonessential. Class I genes (that also comprise the Gp0.4 gene of the invention), expressed early in infection, establish favorable conditions for phage growth, class II genes are expressed later and mainly encode DNA replication proteins, and class III genes are expressed during the late stages of phage growth and mainly encode structural gene products.

In certain embodiments, the present invention further encompasses methods using any homolog or related protein of the T7-like bacterial viruses. The term "homologues" or "related" protein is used to define amino acid sequences (polypeptide) which maintain a minimal homology to the amino acid sequences defined by the invention, e.g. preferably have at least about 65%, more preferably at least about 75%, even more preferably at least about 85%, most preferably at least about 95% overall sequence homology with the amino acid sequence of any of the polypeptide as structurally defined above, e.g. of a specified sequence, more specifically, the T7 Gp0.4 protein of the invention, as defined herein after.

More specifically, "Homolog" or "related" with respect to a native polypeptide and its functional derivative is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues of a corresponding native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Neither N- nor C- terminal extensions nor insertions or deletions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art.

Still further, the homolog or related proteins applicable for the invention may be in certain embodiments, proteins of T7-like bacterial viruses. These T7-like bacterial viruses are members of the Podoviridae- phages with short tails and are characterized by a simple temporal transcriptional control system. The early genes are transcribed by the host RNA polymerase while the middle and late regions are transcribed by a single subunit phage-encoded RNA polymerase which recognizes unique 23 bp promoters sequences. These viruses are the most common types of bacteriophages with 26-29 defined or tentative species. Most of the host species are members of the γ-Proteobacteria (Erwinia, Escherichia, Klebsiella, Morganella, Pseudomonas, Salmonella, Vibrio, and Yersinia) but viral isolates also infecting a-Proteobacteria (Caulobacter, and Rhizobium) have been identified. Fifteen T7-like phages have been sequenced and deposited with GenBank. As a result of a reanalysis, at the protein level, of relationships within the "T7-like viruses" this group of bacteriophage has been classified into the subfamily Autographivirinae which currently possesses three genera: T7-like, Sp6-like and (pKMV-like viruses.

In certain embodiments, such homolog or related protein of the T7-like bacterial viruses may be for example any one of the "hypothetical protein YpP-R" from Yersinia phage YpsP-G and the "hypothetical protein Vi06_01" from Salmonella phage Vi06.

It should be noted that both T7-Llike-relatd proteins, the YpP-R protein from Yersinia phage YpsP-G and the Vi06_01 from Salmonella phage Vi06, are named or designates as "hypothetical protein". It should be noted is a protein whose existence has been predicted, but for which there is no experimental evidence that it is expressed in vivo or the activity of this protein is unknown, such protein is annotated as "hypothetical". In the present case, the biological activity of these proteins is unknown and they are therefore named "hypothetical proteins".

In more specific embodiments, the hypothetical protein YpP-R from Yersinia phage YpsP-G comprises the amino acid sequence of MANNTQYGLTAQTVLFYSDM VRCGFNWS LAM AHLKELYENNK AIALES AE , as denoted by SEQ ID NO. 55 (Protein ID. AFK13490.1) and encoded by the nucleic acid sequence of SEQ ID NO. 56 (Gene Id. JQ965703.1 base 847 to 999).

In yet another specific embodiment, the hypothetical protein Vi06_01 from Salmonella phage Vi06 comprises the amino acid sequence of MINSKHYGLT AQTVLSYSAAVRCGFDWSLAMRHLKALYENSKSLYYRDQHFED as denoted by SEQ ID NO. 57 (protein ID. CBV65199.1) and encoded by the nucleic acid sequence of SEQ ID NO. 58 (Gene Id. FR667955.1 base 491 to 652).

In certain specific embodiments, the methods of the invention use the Gp0.4 T7 bacteriophage protein that is the protein product of the T7 bacteriophage 0.4 gene. As noted herein before, the Gene 0.4 product (Gp0.4), has neither a known function nor a reported phenotype during phage T7 growth. Gene 0.4 is known therefore as a nonessential, 156-bp long gene, producing a peptide which is 51 aa in length.

More specifically, the Gp0.4 T7 protein may comprise the amino acid sequence of:

MS TTN VQ YGLT AQT VLFYS DM VRC GFNWS LAM AQLKELYENNKAIALES AE as denoted by SEQ ID NO. 10 (NCBI Reference Sequence: NP_041955.1) and encoded by the nucleic acid sequence as denoted by SEQ ID NO. 47 (NC_001604.1 base 1278 to 1433). It should be appreciated that the Gp0.4 T7 protein may be also referred to herein as "Gp0.4", "Gp0.4 protein", "T7 Gp0.4", "Gp0.4 bacteriophage T7 protein" and the like.

In certain embodiments, the term "Gp0.4 T7 polypeptide" as used herein refers to a polypeptide comprising an amino acid sequence at least 70 % homologous or identical to that set forth in SEQ ID NO: 10. According to another embodiment, the Gp0.4 T7 polypeptide comprises an amino acid sequence at least 75 % homologous or identical to that set forth in SEQ ID NO: 10. According to another embodiment, the Gp0.4 T7 polypeptide comprises an amino acid sequence at least 80 % homologous or identical to that set forth in SEQ ID NO: 10. According to another embodiment, the Gp0.4 T7 polypeptide comprises an amino acid sequence at least 85 % homologous or identical to that set forth in SEQ ID NO: 10. According to another embodiment, the Gp0.4 T7 polypeptide comprises an amino acid sequence at least 90 % homologous or identical to that set forth in SEQ ID NO: 10. According to another embodiment, the Gp0.4 T7 polypeptide comprises an amino acid sequence at least 95 % homologous or identical to that set forth in SEQ ID NO: 10. According to another embodiment, the Gp0.4 T7 polypeptide comprises an amino acid sequence at least 98 % homologous or identical to that set forth in SEQ ID NO: 10. According to another embodiment, the Gp0.4 T7 polypeptide comprises an amino acid sequence which is 100 % homologous or identical to that set forth in SEQ ID NO: 10.

It should be appreciated that the invention further encompasses the use of any derivatives of the polypeptide of SEQ ID NO. 10. Thus, the amino acids set forth in SEQ ID NO: 10 may be substituted by replacement amino acids either conservatively or non-conservatively.

It should be further appreciated that both, the hypothetical protein YpP-R protein from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06, share at least 65% homology with the Gp0.4 T7 protein of the invention. Alignment of these sequences revealed a conserved region comprising residues QYGLTAQTVLFYSDMVRCGFNWSLAMAQLKELYENNK of T7Gp0.4 (as denoted by SEQ ID NO. 64), that is almost identical in YpP-R, comprising residues, QYGLTAQTVLFYSDMVRCGFNWSLAMAHLKELYENNK, as denoted by SEQ ID NO. 65, having a substitution in position 28 from Gin to His. The hypothetical protein Vi06_01 from Salmonella phage Vi06 has a similar region comprising residues YGLT AQT VLS YS A A VRCGFD WS LAMRHLK ALYENS K, as denoted by SEQ ID NO. 66 having eight substitutions. It should be noted that the invention further encompasses specific conserved regions of these sequences that are completely identical in the three related proteins. Non limiting examples of such fragments include but are not limited to a fragment comprising amino acid sequence YGLTAQTVL, as denoted by SEQ ID NO. 67, a fragment comprising amino acid sequence VRCGF, as denoted by SEQ ID NO. 68 and a fragment comprising amino acid sequence WSLAM, as denoted by SEQ ID NO. 69.

It should be understood that the invention encompassed the use of any of the fragments disclosed herein for treating infections and inhibiting bacterial growth.

The present invention provides the use of a Gp0.4 bacteriophage protein or any peptide derived therefrom, specifically the peptides of SEQ ID NO. 10 and fragments thereof comprising the amino acid sequence of SEQ ID NO: 11-42. "Polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to naturally occurring amino acid polymers, as well as, amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid.

"Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O- phosphoserine (see Table 1). "Amino acid analogs" refers to compounds that have the same fundamental chemical structure as a naturally occurring amino acid, i.e., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. "Amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

Tables 1 and 2 below list naturally occurring amino acids (Table 1) and non-conventional or modified amino acids (Table 2) which can be used with the present invention.

Table 1: naturally occurring amino acids

Table 2: modified amino acids

Non-conventional amino acid Code Non-conventional amino acid Code

a-aminobutyric acid Abu L-N-methylalanine Nmala

a -amino- a -methylbutyrate Mgabu L-N-methylarginine Nmarg

aminocyclopropane- Cpro L-N-methylasparagine Nmasn

carboxylate L-N-methylaspartic acid Nmasp

aminoisobutyric acid Aib L-N-methylcysteine Nmcys

aminonorbornyl- Norb L-N-methylglutamine Nmgin carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D- serine Dser L-N-methyl-t-butylglycine Nmtbug

D- threonine Dthr L-norleucine Nle

D- tryptophan Dtrp L-norvaline Nva

D- tyrosine Dtyr a -methyl-aminoisobutyrate Maib

D- valine Dval a -methyl-y-aminobutyrate Mgabu

D- a -methylalanine Dmala a ethylcyclohexylalanine Mchexa

D- a -methylarginine Dmarg a -methylcyclopentylalanine Mcpen

D- a -methylasparagine Dmasn a -methyl- a -napthylalanine Manap

D- a -methylaspartate Dmasp a - methylpenicillamine Mpen

D- a -methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D- a -methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D- a -methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D- a -methylisoleucine Dmile N- amino- a -methylbutyrate Nmaabu

D- a -methylleucine Dmleu a -napthylalanine Anap

D- a -methyllysine Dmlys N-benzylglycine Nphe

D- a -methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln

D- a -methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D- a -methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D- a -methylproline Dmpro N-(carboxymethyl)glycine Nasp

D- a -methylserine Dmser N-cyclobutylglycine Ncbut

D- a -methylthreonine Dmthr N-cycloheptylglycine Nchep

D- a -methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D- a -methyltyrosine Dmty N-cyclodecylglycine Ncdec

D- a -methylvaline Dmval N-cyclododeclglycine Ncdod

D- a -methylalnine Dnmala N-cyclooctylglycine Ncoct

D- a -methylarginine Dnmarg N-cyclopropylglycine Ncpro D- a -methylasparagine Dnmasn N-cycloundecylglycine Ncund

D- a -methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D- a -methylcysteine Dnmcys N- (3 , 3 -diphenylpropyl)glycine Nbhe

D-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-yaminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-( 1 -methylpropyl) glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-( 1 -methylethyl) glycine Nva

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen

Γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr

L-r-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L- a -methylalanine Mala

L- a -methylarginine Marg L- a -methylasparagine Masn

L- a -methylaspartate Masp L- a -methyl-r-butylglycine Mtbug

L- a -methylcysteine Mcys L-methylethylglycine Metg

L- a thylglutamine Mgln L- a -methylglutamate Mglu

L- a -methyl istidine Mhis L- a -methylhomo phenylalanine Mhphe

L- a -methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-( 1 -hydroxyethyl) glycine Nthr

D-N-methyl istidine Dnmhis N-(hydroxyethyl)glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-yaminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-( 1 -methylpropyl) glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-( 1 -methylethyl) glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen

Γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr

L-r-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe L- a -methylalanine Mala

L- a -methylarginine Marg L- a -methylasparagine Masn

L- a -methylaspartate Masp L- a -methyl-r-butylglycine Mtbug

L- a -methylcysteine Mcys L-methylethylglycine Metg

L- a -methylglutamine Mgln L- a -methylglutamate Mglu

L- a ethylhistidine Mhis L- a -methylhomophenylalanine Mhphe

L- a thylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L- a -methylleucine Mleu L- a -methyllysine Mlys

L- a -methylmethionine Mmet L- a -methylnorleucine Mnle

L- a -methylnorvaline Mnva L- a -methylornithine Morn

L- a -methylphenylalanine Mphe L- a -methylproline Mpro

L- a -methylserine mser L- a -methylthreonine Mthr

L- a ethylvaline Mtrp L- a -methyltyrosine Mtyr

L- a -methylleucine Mval Nnbhm L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) N- (N- (3 , 3 -diphenylpropyl)

carbamylmethyl-glycine Nnbhm carbamylmethyl( 1 )glycine Nnbhe

1 -carboxy- 1 - (2, 2-diphenyl Nmbc

ethylamino)cyclopropane

It should be noted that the Gp0.4 T7 bacteriophage protein used by the methods, compositions and kits of the invention may comprise at least one conservative substitution. The term "conservative substitution" as used herein, refers to the replacement of an amino acid present in the native sequence in the Gp0.4 T7 polypeptide with a naturally or non-naturally occurring amino or a peptidomimetic s having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).

As naturally occurring amino acids are typically grouped according to their properties, conservative substitutions by naturally occurring amino acids can be easily determined bearing in mind the fact that in accordance with the invention replacement of charged amino acids by sterically similar non-charged amino acids are considered as conservative substitutions.

For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art. A peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled practitioner. When affecting conservative substitutions the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.

The phrase "non-conservative substitutions" as used herein refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and/or steric properties. Thus, the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and/or can have functional groups with significantly different electronic properties than the amino acid being substituted. Examples of non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or -NH-CH[(-CH2)5-COOH]-CO- for aspartic acid. Those non-conservative substitutions which fall under the scope of the present invention are those which still constitute a peptide having anti-bacterial properties.

As mentioned, the N and C termini of the Gp0.4 T7 polypeptides of the present invention may be protected by function groups. Suitable functional groups are described in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference. Preferred protecting groups are those that facilitate transport of the compound attached thereto into a cell, for example, by reducing the hydrophilicity and increasing the lipophilicity of the compounds.

These moieties can be cleaved in vivo, either by hydrolysis or enzymatically, inside the cell. Hydroxyl protecting groups include esters, carbonates and carbamate protecting groups. Amine protecting groups include alkoxy and aryloxy carbonyl groups, as described above for N-terminal protecting groups. Carboxylic acid protecting groups include aliphatic, benzylic and aryl esters, as described above for C-terminal protecting groups. In one embodiment, the carboxylic acid group in the side chain of one or more glutamic acid or aspartic acid residue in a peptide of the present invention is protected, preferably with a methyl, ethyl, benzyl or substituted benzyl ester.

Examples of N-terminal protecting groups include acyl groups (-CO-R1) and alkoxy carbonyl or aryloxy carbonyl groups (-CO-0-R1), wherein Rl is an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic or a substituted aromatic group. Specific examples of acyl groups include acetyl, (ethyl)-CO-, n-propyl-CO-, iso-propyl-CO-, n-butyl-CO-, sec-butyl-CO-, t-butyl-CO-, hexyl, lauroyl, palmitoyl, myristoyl, stearyl, oleoyl phenyl-CO-, substituted phenyl-CO-, benzyl-CO- and (substituted benzyl)-CO-. Examples of alkoxy carbonyl and aryloxy carbonyl groups include CH3-0-CO-, (ethyl)-O-CO-, n-propyl-O-CO-, iso-propyl-O-CO-, n-butyl-O-CO-, sec-butyl-O-CO-, t-butyl-O-CO-, phenyl-O- CO-, substituted phenyl-O-CO- and benzyl-O-CO-, (substituted benzyl)- 0-CO-. Adamantan, naphtalen, myristoleyl, tuluen, biphenyl, cinnamoyl, nitrobenzoy, toluoyl, furoyl, benzoyl, cyclohexane, norbornane, Z-caproic. In order to facilitate the N-acylation, one to four glycine residues can be present in the N-terminus of the molecule.

The carboxyl group at the C-terminus of the compound can be protected, for example, by an amide (i.e., the hydroxyl group at the C-terminus is replaced with -NH 2, -NHR2 and -NR2R3) or ester

(i.e. the hydroxyl group at the C-terminus is replaced with -OR2). R2 ^and R3 are independently an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aryl or a substituted aryl group. In addition, taken together with the nitrogen atom, R2 and R3 can form a C4 to C8 heterocyclic ring with from about 0-2 additional heteroatoms such as nitrogen, oxygen or sulfur. Examples of suitable heterocyclic rings include piperidinyl, pyrrolidinyl, morpholino, thiomorpholino or piperazinyl. Examples of C-terminal protecting groups include -NH2, -NHCH3, -N(CE-3)2,

-NH(ethyl), -N(ethyl)₂ , -N(methyl) (ethyl), -NH(benzyl), -N(C1-C4 alkyl)(benzyl), -NH(phenyl),

-N(C1-C4 alkyl) (phenyl), -OCH3, -O-(ethyl), -O-(n-propyl), -O-(n-butyl), -O-(iso-propyl),

-0-(sec- butyl), -O-(t-butyl), -O-benzyl and -O-phenyl.

The Gp0.4 T7 polypeptides of the present invention may also comprise non-amino acid moieties, such as for example, hydrophobic moieties (various linear, branched, cyclic, polycyclic or hetrocyclic hydrocarbons and hydrocarbon derivatives) attached to the peptides; various protecting groups, especially where the compound is linear, which are attached to the compound's terminals to decrease degradation. Chemical (non-amino acid) groups present in the compound may be included in order to improve various physiological properties such; decreased degradation or clearance; decreased repulsion by various cellular pumps, improve immunogenic activities, improve various modes of administration (such as attachment of various sequences which allow penetration through various barriers, through the gut, etc.); increased specificity, increased affinity, decreased toxicity and the like.

In yet other embodiments, the invention contemplates the use of functional fragments of the Gp0.4 T7 protein of the invention. Such functional fragments maintain the function of preventing assembly of the FstZ protein, and reducing bacterial growth. Non-limiting examples for such fragments of the Gp0.4 T7 polypeptide include polypeptides comprising at least one of the following amino acid sequences:

MS TTN VQ YGLT AQT VLFYS D - SEQ ID NO: 11;

S TTN VQ YGLT AQT VLF YS DM - SEQ ID NO: 12;

TTN VQ YGLT AQT VLFYS DM V - SEQ ID NO: 13;

TNVQYGLTAQTVLFYSDMVR - SEQ ID NO: 14;

NVQ YGLT AQT VLF YS DM VRC - SEQ ID NO: 15;

VQ YGLT AQT VLF YS DM VRC G - SEQ ID NO: 16;

QYGLTAQTVLFYSDMVRCGF - SEQ ID NO: 17;

YGLTAQTVLFYSDMVRCGFN - SEQ ID NO: 18;

GLTAQTVLFYSDMVRCGFNW - SEQ ID NO: 19;

LT AQT VLF YS DM VRC GFNWS - SEQ ID NO: 20;

TAQTVLFYSDMVRCGFNWSL - SEQ ID NO: 21;

AQT VLF YS DM VRC GFNWS LA - SEQ ID NO: 22;

QT VLF YS DM VRCGFN WS LAM - SEQ ID NO: 23;

T VLF YS DM VRCGFN WS LAMA - SEQ ID NO: 24;

VLFYS DM VRC GFNWS LAM AQ - SEQ ID NO: 25;

LF YS DM VRC GFNWS LAM AQL - SEQ ID NO: 26;

FYSDMVRCGFNWSLAMAQLK - SEQ ID NO: 27;

YS DM VRC GFNWS LAM AQLKE - SEQ ID NO: 28;

S DM VRC GFNWS LAM AQLKEL - SEQ ID NO: 29;

DMVRCGFNWSLAMAQLKELY - SEQ ID NO: 30;

M VRC GFNWS LAM AQLKELYE - SEQ ID NO: 31;

VRCGFNWS LAM AQLKELYEN - SEQ ID NO: 32;

RCGFNWS LAM AQLKELYENN - SEQ ID NO: 33;

C GFNWS LAM AQLKELYENNK - SEQ ID NO: 34;

GFNWS LAM AQLKELYENNKA - SEQ ID NO: 35;

FNWS LAM AQLKELYENNKAI - SEQ ID NO: 36;

NWSLAMAQLKELYENNKAIA - SEQ ID NO: 37;

WSLAMAQLKELYENNKAIAL - SEQ ID NO: 38;

S LAM AQLKELYENNKAIALE - SEQ ID NO: 39;

LAMAQLKELYENNKAIALES - SEQ ID NO: 40;

AM AQLKELYENNKAI ALES A - SEQ ID NO: 41; and MAQLKELYENNKAIALESAE - SEQ ID NO: 42.

In order to select for shorter Gp0.4 T7 amino acid sequences which have antimicrobial activity, assays may be performed which include the following steps:

(a) contacting a plurality of Gp0.4 T7 bacteriophage peptide agents with a population of microbial cells;

(b) analyzing a viability of the microbial cells; and

(c) identifying an agent of the plurality of agents capable of decreasing viability of the microbial cells, thereby selecting the agent comprising the antimicrobial activity.

Numerous methods of analyzing the viability of microbial cells are well known in the art.

These methods include dye exclusion which is based on the theory that live cells contain intact membranes and dead cells do not, thus dead cells absorb the dye into the cytoplasm. An exemplary dye which can be used to measure cell viability is Trypan blue dye reagent.

It should be noted that the invention encompasses the use of any of the polypeptides described above, as well as any combinations thereof and specifically, any derivatives thereof.

In some embodiments, derivatives refer to polypeptides, which differ from the polypeptides specifically defined in the present invention by insertions of amino acid residues. It should be appreciated that by the terms "insertions" or "deletions", as used herein it is meant any addition or deletion, respectively, of amino acid residues to the polypeptides used by the invention, of between 1 to 50 amino acid residues, between 20 to 1 amino acid residues, and specifically, between 1 to 10 amino acid residues. More particularly, insertions or deletions may be of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. It should be noted that the insertions or deletions encompassed by the invention may occur in any position of the modified peptide, as well as in any of the N' or C termini thereof.

The peptides of the invention may all be positively charged, negatively charged or neutral. In addition, they may be in the form of a dimer, a multimer or in a constrained conformation, which can be attained by internal bridges, short-range cyclizations, extension or other chemical modifications. The polypeptides of the invention, specifically, the peptide of SEQ ID NO. 10, or of any one of SEQ ID NO: 11-42, can be coupled (conjugated) through any of their residues to another peptide or agent. For example, the polypeptides of the invention can be coupled through their N- terminus to a lauryl-cysteine (LC) residue and/or through their C-terminus to a cysteine (C) residue.

Further, the peptides may be extended at the N-terminus and/or C-terminus thereof with various identical or different amino acid residues. As an example for such extension, the peptide may be extended at the N-terminus and/or C-terminus thereof with identical or different amino acid residue/s, which may be naturally occurring or synthetic amino acid residue/s. An additional example for such an extension may be provided by peptides extended both at the N-terminus and/or C-terminus thereof with a cysteine residue. Naturally, such an extension may lead to a constrained conformation due to Cys-Cys cyclization resulting from the formation of a disulfide bond. Another example may be the incorporation of an N-terminal lysyl-palmitoyl tail, the lysine serving as linker and the palmitic acid as a hydrophobic anchor. In addition, the peptides may be extended by aromatic amino acid residue/s, which may be naturally occurring or synthetic amino acid residue/s, for example, a specific aromatic amino acid residue may be tryptophan. The peptides may be extended at the N-terminus and/or C-terminus thereof with various identical or different organic moieties, which are not naturally occurring or synthetic amino acids. As an example for such extension, the peptide may be extended at the N-terminus and/or C- terminus thereof with an N- acetyl group.

For every single peptide sequence defined by the invention and disclosed herein, this invention includes the corresponding retro-inverse sequence wherein the direction of the peptide chain has been inverted and wherein all the amino acids belong to the D-series.

In some embodiments, the present invention also encompasses polypeptides which are variants of, or analogues to, the polypeptides specifically defined in the invention by their amino acid sequence. With respect to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to peptide, polypeptide, or protein sequence thereby altering, adding or deleting a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant", where the alteration results in the substitution of an amino acid with a chemically similar amino acid. In certain embodiments the peptide compounds of the invention may comprise one or more amino acid residue surrogate. An "amino acid residue surrogate" as herein defined is an amino acid residue or peptide employed to produce mimetics of critical function domains of peptides.

Examples of amino acid surrogate include, but are not limited to chemical modifications and derivatives of amino acids, stereoisomers and modifications of naturally occurring amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically modified amino acids, and the like. Examples also include dimers or multimers of peptides. An amino acid surrogate may also include any modification made in a side chain moiety of an amino acid. This thus includes the side chain moiety present in naturally occurring amino acids, side chain moieties in modified naturally occurring amino acids, such as glycosylated amino acids. It further includes side chain moieties in stereoisomers and modifications of naturally occurring protein amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, and the like.

It should be appreciated that the invention further encompass any of the peptides of the invention any serogates thereof, any salt, base, ester or amide thereof, any enantiomer, stereoisomer or disterioisomer thereof, or any combination or mixture thereof. Pharmaceutically acceptable salts include salts of acidic or basic groups present in compounds of the invention. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (i.e., 1,1'- methylene-bis-(2-hydroxy-3-naphthoate)) salts. Certain compounds of the invention can form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts.

It should be noted that the invention further encompasses any peptidomimetic compound mimicking the Gp0.4 bacteriophage protein of the invention and any fragment or peptide thereof. When referring to peptidomimetic s, what is meant is a compound that mimics the conformation and desirable features of a particular natural peptide but avoids the undesirable features, e.g., flexibility and bond breakdown. From chemical point of view, peptidomimetics can have a structure without any peptide bonds, nevertheless, the compound is peptidomimetic due to its chemical properties and not due to chemical structure. Peptidoinimetics (both peptide and non-peptidyl analogues) may have improved properties (e.g., decreased proteolysis, increased retention or increased bioavailability). It should be noted that peptidomimetics may or may not have similar two-dimensional chemical structures, but share common three- dimensional structural features and geometry. Each peptidomimetic may further have one or more unique additional binding elements.

It should be appreciated that all the derivatives, serogates, mimetics an analogs described herein above relate to any of the polypeptides of the invention, more specifically, any of the peptides of SEQ ID NO. 10, 11-42, 55 and 57.

In some embodiments, the polypeptides of the present invention may be attached (either covalently or non-covalently) to an agent that enhances penetration across the bacterial membrane (i.e. a penetrating agent).

As used herein the phrase "penetrating agent" or any "agent that enhances penetration" refers to an agent which enhances translocation of any of the attached Gp0.4 T7 polypeptides or any homolog or related protein of the T7-like bacterial viruses, across a cell membrane.

According to one embodiment, the penetrating agent may be a peptide and is attached to the Gp0.4 T7 polypeptides (either directly or non-directly) via a peptide bond.

In one embodiment, the penetrating agent may be a peptide. In more specific embodiments, such peptides may be Cell-penetrating peptides (CPPs). Cell-penetrating peptides (CPPs) are short peptides that facilitate cellular uptake of various molecular cargo (from nano-size particles to small chemical molecules and large fragments of DNA and polypeptides). The "cargo" is associated with the peptides either through chemical linkage via covalent bonds or through non- covalent interactions.

CPPs hold great potential as in vitro and in vivo delivery vectors for use in research and medicine. Current use is limited by a lack of cell specificity in CPP-mediated cargo delivery and insufficient understanding of the modes of their uptake. CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively.

Cell-penetrating peptides are of different sizes, amino acid sequences, and charges but all CPPs have one distinct characteristic, which is the ability to translocate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or an organelle. There has been no real consensus as to the mechanism of CPP translocation, but the theories of CPP translocation can be classified into three main entry mechanisms: direct penetration in the membrane, endocytosis-mediated entry, and translocation through the formation of a transitory structure.

Exemplary peptide penetrating agents may comprise the following consensus sequence - (RFF)₃R, such as for example, RFFRFFRFFXB - SEQ ID NO: 43, RTRTRFLRRTXB - SEQ ID NO: 44,

RXXRXXRXXB - SEQ ID NO: 45, and KFFKFFKFFKXB - SEQ ID NO: 46.

It has been found that biotinylation enhances the uptake of large peptides by Escherichia coli and Other Gram-Negative Bacteria, see for example, the contents of which are incorporated herein by reference. Accordingly, the present invention contemplates biotinylated Gp0.4 T7 polypeptides.

In yet another embodiment, the penetrating agent may be a fatty acid. Well known as efficient enhancers for transdermal delivery of drugs are fatty acids; however, their frequent dermal toxicity limits their regular use. More safe and effective biodegradable agents, which can cover a wide range of drug molecules in the transdermal permeation and other membranes absorption of physiologically active agents may include compounds having hydrophilic moiety groups such as glycolic group and N-alkyl substituted amino acidic group and lipophilic moiety groups contributing the balanced lipophilicity of the compounds. With these physical chemical features, the disclosed compound can influence the efficacy, toxicity, irritation, duration of action of the enhancers, and the reversibility of the skin, and the stability of the enhancers.

In certain embodiments, the penetration enhancing agent used by the invention may be a sustained-release enhancing agent. As used herein, the term "sustained-release enhancing agent" is an agent that in addition to its penetration enhancing properties, is also capable of gradual release of an active agent over a period of time, allowing for a sustained effect, timed-release, long-acting, prolonged- action and slow-release.

Matrices based on natural biodegradable polymers are very advantageous for controlled drug delivery. They are safe due to complete elimination and non immunologic responses in the body. Hyaluronic acid (HA) has already been exploited in a number of drug delivery applications such as long-acting HA-drug conjugate systems and sustained release formulation of protein drugs using physically and chemically crosslinked HA hydrogels. Further embodiments for sustained release hydrophilic matrixes include but are not limited to alginic acid (AA), polyhydroxyethyl methacrylate (Poly-HEMA), polyethylene glycol (PEG), glyme and polyisopropylacrylamide.

It should be noted that many cationic compounds have been found to enhance the permeability of the outer membrane of gram-negative bacteria. These agents, if nontoxic, could be applied to the therapy of bacterial infections to increase the permeability and sensitivity of gram-negative bacteria to various antibiotics. Polymyxin B nonapeptide (PMBN) is one of the cationic compounds inducing outer membrane permeabilization. PMBN binds to lipopolysaccharide (LPS) molecules in the outer membrane, and this binding is considered to trigger the disruption in the permeability barrier of the membrane.

Other polycations applicable for enhancing the penetration of the Gp0.4 protein of the invention include polymyxins and their derivatives, protamine, polymers of basic amino acids, compound 48/80, insect cecropins, reptilian magainins, various cationic leukocyte peptides (defensins, bactenecins, bactericidal/permeability-increasing protein, and others), aminoglycosides, and many more. It should be noted that the invention encompasses the use of any of the agents herein described.

It should be appreciated that the Gp0.4 T7 polypeptides of the present invention can be biochemically synthesized for example by using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. Solid phase polypeptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Polypeptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Large scale peptide synthesis is described by Andersson [22]. Synthetic peptides can be purified by preparative high performance liquid chromatography [23] and the composition of which can be confirmed via amino acid sequencing.

Recombinant techniques may also be used to generate the Gp0.4 T7 polypeptides of the present invention as well as any homolog or related protein of the T7-like bacterial viruses. To produce a peptide of the present invention using recombinant technology, a polynucleotide encoding the peptide of the present invention is ligated into a nucleic acid expression vector, which comprises the polynucleotide sequence under the transcriptional control of a cis-regulatory sequence (e.g., promoter sequence) suitable for directing constitutive, tissue specific or inducible transcription of the polypeptides of the present invention in the host cells.

A variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptides of some embodiments of the invention. These include, but are not limited to; yeast transformed with recombinant yeast expression vectors containing the coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the coding sequence. Mammalian expression systems can also be used to express the polypeptides of some embodiments of the invention.

Examples of bacterial constructs include the pUC series of E. coli vectors disclosed in Table 3.

Other than containing the necessary elements for the transcription and translation of the inserted coding sequence, the expression construct of some embodiments of the invention can also include sequences engineered to enhance stability, production, purification, yield or toxicity of the expressed antimicrobial peptide. For example, the expression of a fusion protein or a cleavable fusion protein comprising the antimicrobial peptides of some embodiments of the invention and a heterologous protein can be engineered. Such a fusion protein can be designed so that the fusion protein can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the heterologous protein. Where a cleavage site is engineered between the antimicrobial and the heterologous protein, the antimicrobial can be released from the chromatographic column by treatment with an appropriate enzyme or agent that disrupts the cleavage site. Recovery of the recombinant polypeptide is affected following an appropriate time in culture. The phrase "recovering the recombinant polypeptide" refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification. Notwithstanding the above, polypeptides of some embodiments of the invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

In addition to being synthesizable in host cells, the polypeptides of the present invention can also be synthesized using in vitro expression systems. These methods are well known in the art and the components of the system are commercially available.

As indicated above, in certain embodiments, the invention provides isolated and purified Gp0.4 bacteriophage protein of the invention or any peptides or fragments thereof. As used herein, "isolated" or "substantially purified", in the context of an amino acid molecule, means the protein or amino acid sequence has been removed from its natural milieu or has been altered from its natural state. As such "isolated" does not necessarily reflect the extent to which the amino acid molecule has been purified. However, it will be understood that a protein or any amino acid molecule derived therefrom, that has been purified to some degree is "isolated". If the protein or amino acid molecule does not exist in a natural milieu, i.e. it does not exist in nature, the molecule is "isolated" regardless of where it is present.

Furthermore, the term "isolated" or "substantially purified", when applied to a protein, denotes that the amino acid sequence or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state, although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified.

As mentioned, the Gp0.4 T7 bacteriophage proteins or any homolog or related protein of the TV- like bacterial viruses, described herein comprises antimicrobial activity. The phrase "antimicrobial activity" as used herein, refers to an ability to suppress, control, inhibit or kill microorganisms, such as bacteria, archaea and fungi. Thus for example the antimicrobial activity may comprise bactericidal or bacteriostatic activity, or both.

According to a particular embodiment, the antimicrobial activity comprises bactericidal activity.

A bactericide (also bactericide, bacteriocidal) agent refers to a substance that kills bacteria as opposed to the bacteriostatic antibiotics that slow their growth or reproduction. Bacteriocides include for example agents that inhibit cell wall synthesis. Examples for known bacteriocides include the Beta-lactam antibiotics (penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems) and vancomycin and also daptomycin, fluoroquinolones, metronidazole, nitrofurantoin, co-trimoxazole, telithromycin. Non- antibiotic bacteriocide agents may include disinfectants, e.g. active chlorine, active oxygen, iodine, concentrated alcohols, phenolic substances, strong oxydizers and heavy metals and their salts. It should be noted that in certain embodiments, the Gp0.4 bacteriophage protein of the invention that exhibits a bactericidic action, may be also combined with other bacteriocides, for example, those indicated above.

A bacteriostatic agent (also bacteriostat) is a biological or chemical substance that stops bacteria from reproducing, while not necessarily harming them otherwise. Depending on their application, bacteriostatic antibiotics, disinfectants, antiseptics and preservatives can be distinguished from bacteriocides, as upon removal of the bacteriostat, the bacteria usually start to grow again. Bacteriostatic agents limit the growth of bacteria by interfering with bacterial protein production, DNA replication, or other aspects of bacterial cellular metabolism.

However, it must be understood that there is not always a precise distinction between them and bactericidal agents; high concentrations of some bacteriostatic agents are also bactericidal, whereas low concentrations of some bacteriocidal agents are bacteriostatic. The group of bacteriostatic antibiotics may include tetracyclines, sulfonamides, spectinomycin, trimethoprim, chloramphenicol, macrolides, lincosamides.

In this connection, in some embodiments, the present invention provides a bacteriostatic agent based on the Gp0.4 peptide or the above specified fragments or derivatives thereof. In yet some other embodiments, the Gp0.4 peptide or the above specified fragments or derivatives thereof according to the invention may be used as a bacteriocidal agent.

In certain embodiments, the bacteriostatic or bacteriocidal agent of the invention may be administered in conjunction with one or a number of known bacteriocidic or bacteriostatic antibiotics described above.

One of the aspects of the invention provides a method of inhibiting microbial growth, specifically, bacterial growth, by contacting the bacteria or any other microbe with a cytotoxic effective amount of a Gp0.4 T7 bacteriophage protein or any homolog or related protein of the T7-like bacterial viruses, thereby inhibiting bacterial growth and killing the bacteria.

The terms "inhibition", "moderation" or "attenuation" as referred to herein, relate to the retardation, restraining or reduction of microbial or specifically, bacterial growth or to an inhibition, elimination or reduction of bacterial viability. Such inhibition may be of about 1% to 99.9%, specifically, about 1% to about 95%, about 5% to 90%, about 10% to 85%, about 15% to 80%, about 20% to 75%, about 25% to 70%, about 30% to 65%, about 35% to 60%, about 40% to 55%, about 45% to 50%. More specifically, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9%.

In further embodiments, the Gp0.4 bacteriophage protein of the invention or any homolog or related protein of the T7-like bacterial viruses may lead to an increase, enhancement, induction or elevation in bacterial death, said increase, induction or elevation of bacterial death may be an increase by about 1% to 99.9%, specifically, about 1% to about 95%, about 5% to 90%, about 10% to 85%, about 15% to 80%, about 20% to 75%, about 25% to 70%, about 30% to 65%, about 35% to 60%, about 40% to 55%, about 45% to 50%. More specifically, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9%. More specifically, an increase of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% as compared to untreated control.

With regards to the above, it is to be understood that, where provided, percentage values such as, for example, 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 etc., respectively. Typically, the microbes which are killed according to embodiments of the present invention express the FtsZ protein.

The term FtsZ (named after "Filamenting temperature-sensitive mutant Z"), as meant herein, refers to the prokaryotic cell division protein and tubulin-homologue protein responsible for the septum formation at the midpoint of a pre-divisional bacterial cell. At least nine bacterial proteins have been implicated in proper septation during bacterial cell division (products of genes ftsA, ftsl, ftsK, ftsL, ftsN, fisQ, ftsW, ftsZ and zipA), among which FtsZ appears to act at the earliest step in septation and is required through the final step of cytokinesis. Most interestingly, the N-terminal of FtsZ contains the tubulin signature GTP-binding sequence motif, owing to which it too, like tubulin, has a GTPase activity. In this respect, tubulin and FtsZ form a distinct family of GTP-hydrolyzing proteins which clearly differ from other GTPases. In certain embodiment, as used herein the term refers to the bacterial FtsZ, specifically, the E. coli FtsZ protein of 383 amino acid (a.a.) disclosed by GeneBank accession No. NP_414637.1, as denoted by SEQ ID NO. 59 encoded by the ftsZ gene of accession number NC_000913 bases 104,305 to 106,456, as denoted by SEQ ID NO. 60.

As FtsZ is vital to bacterial reproduction and growth, FtsZ proteins encoded by the bacterial chromosome or by extra-chromosomal plasmids are found in many bacterial strains. Thus in the present context, the term FtsZ encompasses a family of proteins sharing a sequence and functional homology, which may occur in Gram-positive and Gram-positive bacteria, and sphere-shaped (coccus) or rod-shaped (bacillus) bacteria. To name but a few examples, FtsZ of another rod-shape bacteria, Bacillus anthracis (Gram-positive) the etiologic agent of anthrax, may be represented by the 386 a.a. protein with NCBI Acc. Num. GI: 229604092, as denoted by SEQ ID NO. 61. FtsZ of Salmonella (rod-shaped, Gram- negative), may be represented by the 383 a.a. protein with NCBI Acc. Num. GI: 205337491, as denoted by SEQ ID NO. 62, FtsZ of Mycobacterium tuberculosis (Gram-positive), may be represented by the 379 a.a. protein with NCBI Acc. Num. GI: 378545347, as denoted by SEQ ID NO. 63. In Pseudomonas aeruginosa (Gram- negative), a common bacterium found in soil, water, skin flora and most man-made environments throughout the world that can cause generalized inflammation and sepsis, FtsZ may be represented by the 394 a.a. protein with NCBI Acc. Num. GI: 6715615, as denoted by SEQ ID NO. 53. In Streptococcus pneumonia or pneumococcus (Gram-positive), the major cause of community acquired pneumonia and meningitis in children and the elderly, FtsZ may be represented by the 419 a.a. protein with NCBI Acc. Num. GI: 556561406, as denoted by SEQ ID NO. 54.

According to one embodiment, the microbes comprise bacteria.

The bacteria may be gram positive or gram negative.

The term "Gram-positive bacteria" as used herein refers to bacteria characterized by having as part of their cell wall structure peptidoglycan as well as polysaccharides and/or teichoic acids and are characterized by their blue-violet color reaction in the Gram-staining procedure. Representative Gram-positive bacteria include: Actinomyces spp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum, Clostridium perfringens, Clostridium spp., Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcus faecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae, Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum, Leuconostoc spp., Mycobacterium abcessus, Mycobacterium avium complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium haemophilium, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium smegmatis, Mycobacterium terrae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Nocardia spp., Peptococcus niger, Peptostreptococcus spp., Proprionibacterium spp., Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdanensis, Staphylococcus saccharolyticus, Staphylococcus saprophyticus, Staphylococcus schleiferi, Staphylococcus similans, Staphylococcus warneri, Staphylococcus xylosus, Streptococcus agalactiae (group B streptococcus), Streptococcus anginosus, Streptococcus bovis, Streptococcus canis, Streptococcus equi, Streptococcus milleri, Streptococcus mitior, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes (group A streptococcus), Streptococcus salivarius, Streptococcus sanguis.

The term "Gram-negative bacteria" as used herein refer to bacteria characterized by the presence of a double membrane surrounding each bacterial cell. Representative Gram-negative bacteria include Acinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans, Aeromonas hydrophila, Alcaligenes xylosoxidans, Bacteroides, Bacteroides fragilis, Bartonella baciUiformis, Bordetella spp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chromobacterium violaceum, Citrobacter spp., Eikenella corrodens, Enterobacter aerogenes, Escherichia coli, Flavobacterium meningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilus spp., Helicobacter pylori, Klebsiella spp., Legionella spp., Leptospira spp., Moraxella catarrhalis, Morganella morganii, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Plesiomonas shigelloides, Prevotella spp., Proteus spp., Providencia rettgeri, Pseudomonas aeruginosa, Pseudomonas spp., Rickettsia prowazekii, Rickettsia rickettsii, Rochalimaea spp., Salmonella spp., Salmonella typhi, Serratia marcescens, Shigella spp., Treponema carateum, Treponema pallidum, Treponema pallidum endemicum, Treponema pertenue, VeiUonella spp., Vibrio cholerae, Vibrio vulnificus, Yersinia enterocolitica, Yersinia pestis.

As mentioned above, the method for inhibiting bacterial growth and killing bacteria, according to certain embodiments of the invention, involves contacting the bacteria with an inhibitory effective amount of the protein of the invention, Gp0.4 or of any homolog or related protein of the T7-like bacterial viruses,. As used herein the term "contacting" refers to the positioning of the Gp0.4 T7 polypeptide of the present invention such that it is in direct or indirect contact with the microbial cells. Thus, the present invention contemplates both applying the Gp0.4 T7 polypeptide to a desirable surface and/or directly to the microbial cells.

Contacting the Gp0.4 T7 polypeptide or any homolog or related protein of the T7-like bacterial viruses, with a surface can be effected using any method known in the art including spraying, spreading, wetting, immersing, dipping, painting, ultrasonic welding, welding, bonding or adhering. The Gp0.4 T7 polypeptide of the present invention may be attached as monolayers or multiple layers.

In yet a further aspect, the invention provides an in vitro application of the Gp0.4 T7 polypeptide of the present invention. The present invention coating a wide variety of surfaces with the Gp0.4 T7 polypeptide of the present invention including fabrics, fibers, foams, films, concretes, masonries, glass, metals, plastics, polymers, and like.

An exemplary solid surface that may be coated with the peptides of the present invention is an intracorporial or extra-corporial medical device or implant.

An "implant" as used herein refers to any object intended for placement in a human body that is not a living tissue. The implant may be temporary or permanent. Implants include naturally derived objects that have been processed so that their living tissues have been devitalized. As an example, bone grafts can be processed so that their living cells are removed (acellularized), but so that their shape is retained to serve as a template for ingrowth of bone from a host. As another example, naturally occurring coral can be processed to yield hydroxy apatite preparations that can be applied to the body for certain orthopedic and dental therapies. An implant can also be an article comprising artificial components.

Thus, for example, the present invention therefore envisions coating vascular stents with the peptides of the present invention. Another possible application of the peptides of the present invention is the coating of surfaces found in the medical and dental environment.

Surfaces found in medical environments include the inner and outer aspects of various instruments and devices, whether disposable or intended for repeated uses. Examples include the entire spectrum of articles adapted for medical use, including scalpels, needles, scissors and other devices used in invasive surgical, therapeutic or diagnostic procedures; blood filters, implantable medical devices, including artificial blood vessels, catheters and other devices for the removal or delivery of fluids to patients, artificial hearts, artificial kidneys, orthopedic pins, plates and implants; catheters and other tubes (including urological and biliary tubes, endotracheal tubes, peripherably insertable central venous catheters, dialysis catheters, long term tunneled central venous catheters peripheral venous catheters, short term central venous catheters, arterial catheters, pulmonary catheters, Swan-Ganz catheters, urinary catheters, peritoneal catheters), urinary devices (including long term urinary devices, tissue bonding urinary devices, artificial urinary sphincters, urinary dilators), shunts (including ventricular or arterio-venous shunts); prostheses (including breast implants, penile prostheses, vascular grafting prostheses, aneurysm repair devices, heart valves, artificial joints, artificial larynxes, otological implants), anastomotic devices, vascular catheter ports, clamps, embolic devices, wound drain tubes, hydrocephalus shunts, pacemakers and implantable defibrillators, and the like. Other examples will be readily apparent to practitioners in these arts.

Surfaces found in the medical environment include also the inner and outer aspects of pieces of medical equipment, medical gear worn or carried by personnel in the health care setting. Such surfaces can include counter tops and fixtures in areas used for medical procedures or for preparing medical apparatus, tubes and canisters used in respiratory treatments, including the administration of oxygen, of solubilized drugs in nebulizers and of anesthetic agents. Also included are those surfaces intended as biological barriers to infectious organisms in medical settings, such as gloves, aprons and face shields. Commonly used materials for biological barriers may be latex -based or non-latex based. Vinyl is commonly used as a material for non- latex surgical gloves. Other such surfaces can include handles and cables for medical or dental equipment not intended to be sterile. Additionally, such surfaces can include those non-sterile external surfaces of tubes and other apparatus found in areas where blood or body fluids or other hazardous biomaterials are commonly encountered.

Other surfaces related to health include the inner and outer aspects of those articles involved in water purification, water storage and water delivery, and those articles involved in food processing. Thus the present invention envisions coating a solid surface of a food or beverage container to extend the shelf life of its contents.

Surfaces related to health can also include the inner and outer aspects of those household articles involved in providing for nutrition, sanitation or disease prevention. Examples can include food processing equipment for home use, materials for infant care, tampons and toilet bowls.

According to another embodiment the surface is comprised in a biological tissue, such as for example, mammalian tissues e.g. the skin.

It will be appreciated that the contacting may also be effected in vivo (i.e. within a mammalian body) or ex vivo (i.e. in cells removed from the body).

Accordingly, the present invention contemplates administering of the Gp0.4 T7 polypeptide or any homolog or related protein of the T7-like bacterial viruses, per se (or pharmaceutical compositions comprising same) to subjects in need thereof in order to prevent or treat infections in the body.

Thus, as mentioned above, the invention provides as one of its aspects, a method of treating an infectious disease in a subject in need thereof. The method of the invention may comprise the administration of a therapeutically effective amount of a Gp0.4 T7 bacteriophage protein or any composition comprising the same, thereby treating the disease.

Infectious diseases, or infection as used herein, is the invasion of a host organism's bodily tissues by disease-causing organisms, their multiplication, and the reaction of host tissues to these organisms and the toxins they produce. Infectious diseases, also known as transmissible diseases or communicable diseases, comprise clinically evident illness (i.e., characteristic medical signs and/or symptoms of disease) resulting from the infection, presence and growth of pathogenic biological agents in an individual host organism.

Infections are caused by infectious agents such as viruses, viroids, and prions, microorganisms such as bacteria, nematodes such as roundworms and pinworms, arthropods such as ticks, mites, fleas, and lice, fungi such as ringworm, and other macroparasites such as tapeworms.

The appearance and severity of disease resulting from any pathogen, depends upon the ability of that pathogen to damage the host as well as the ability of the host to resist the pathogen. Clinicians therefore classify infectious microorganisms or microbes according to the status of host defenses - either as primary pathogens or as opportunistic pathogens. Primary pathogens cause disease as a result of their presence or activity within the normal, healthy host, and their intrinsic virulence (the severity of the disease they cause) is, in part, a necessary consequence of their need to reproduce and spread. Many of the most common primary pathogens of humans only infect humans, however many serious diseases are caused by organisms acquired from the environment or which infect non-human hosts. An opportunistic infection is an infection caused by pathogens, particularly opportunistic pathogens, those that take advantage of certain situations, such as bacterial, viral, fungal or protozoan infections that usually do not cause disease in a healthy host, one with a healthy immune system. Such infections may be caused by microbes that are ordinarily in contact with the host, such as pathogenic bacteria or fungi in the gastrointestinal or the upper respiratory tract, and they may also result from (otherwise innocuous) microbes acquired from other hosts (as in Clostridium difficile colitis) or from the environment as a result of traumatic introduction (as in surgical wound infections or compound fractures). An opportunistic disease requires impairment of host defenses, which may occur as a result of genetic defects (such as Chronic granulomatous disease), exposure to antimicrobial drugs or immunosuppressive chemicals (as might occur following poisoning or cancer chemotherapy), exposure to ionizing radiation, or as a result of an infectious disease with immunosuppressive activity (such as with measles, malaria or HIV disease). Primary pathogens may also cause more severe disease in a host with depressed resistance than would normally occur in an immuno sufficient host. Infections or infectious diseases can be classified by the anatomic location or organ system that is infected, including: Urinary tract infection, Skin infection, Respiratory tract infection, Odontogenic infection (an infection that originates within a tooth or in the closely surrounding tissues), Vaginal infections and Intra- amniotic infection. In addition, locations of inflammation where infection is the most common cause include pneumonia, meningitis and salpingitis. It should be understood therefore, that the method of the invention may be applicable for treating any infectious disease as described herein, as well as any related disorders.

As used herein, the term "treating" refers to curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a pathogen infection. The term "treatment or prevention" refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, infectious disease symptoms or undesired side effects of such infectious disease related conditions. More specifically, treatment or prevention includes the prevention or postponement of development of the disease, prevention or postponement of development of symptoms and/or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing- additional symptoms and ameliorating or preventing the underlying metabolic causes of symptoms.

As used herein, "disease", "disorder", "condition" and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms.

It is understood that the interchangeably used terms "associated", "linked" and "related", when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder condition or pathology causes the second disease, disorder, condition or pathology.

The present invention relates to the treatment of subjects, or patients, in need thereof. By "patient" or "subject in need" it is meant any organism who may be affected by the above- mentioned conditions, and to whom the treatment methods herein described are desired, including humans, domestic and non-domestic mammals such as canine and feline subjects, bovine, simian, equine and murine subjects, rodents, domestic birds, aquaculture, fish and exotic aquarium fish. It should be appreciated that the treated subject may be also any reptile or zoo animal. More specifically, the methods and compositions of the invention are intended for mammals. By "mammalian subject" is meant any mammal for which the proposed therapy is desired, including human, equine, canine, and feline subjects, most specifically humans. It should be noted that specifically in cases of non-human subjects, the method of the invention may be performed using administration via injection, drinking water, feed, spraying, oral gavage and directly into the digestive tract of subjects in need thereof. It should be further noted that particularly in case of human subject, administering of the compositions of the invention to the patient includes both self-administration and administration to the patient by another person.

In yet another aspect, the invention provides a pharmaceutical composition comprising a GpO.4 T7 bacteriophage protein or any homolog or related protein of the T7-like bacterial viruses, as the active ingredient and a pharmaceutically acceptable carrier.

In certain embodiments, such homolog or related protein of the T7-like bacterial viruses may be any one of the hypothetical protein YpP-R from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06.

In some specific embodiments, the invention provides a pharmaceutical composition comprising the GpO.4 T7 bacteriophage protein as the active ingredient in an effective amount sufficient for treating infectious disease in a subject in need thereof. The composition may further comprise a pharmaceutically acceptable carrier.

In yet another embodiment, the pharmaceutical composition of the invention comprises an effective amount of the GpO.4 T7 bacteriophage protein sufficient for inhibition of bacterial growth in a subject in need thereof.

The phrase "pharmaceutical composition", as used herein refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

As used herein the term "active ingredient" refers to the GpO.4 T7 polypeptide of the present invention accountable for the intended biological effect. It will be appreciated that a polynucleotide encoding the GpO.4 T7 polypeptide of the present invention may be administered directly into a subject (as is, or part of a pharmaceutical composition) where it is translated in the target cells i.e. by gene therapy. Accordingly, the phrase "active ingredient" also includes such polynucleotides. An exemplary polynucleotide which encodes the GpO.4 T7 polypeptide is provided in SEQ ID NO: 47. Still further, in some embodiments, the polynucleotide sequence may by a sequence encoding the homolog or related protein of the T7-like bacterial viruses. More specifically, the nucleic acid sequence of SEQ ID NO. 56 encoding the hypothetical protein YpP-R from Yersinia phage YpsP-G, or alternatively, the nucleic acid sequence of SEQ ID NO. 58, encoding the hypothetical protein Vi06_01 from Salmonella phage Vi06.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier," which may be used interchangeably, refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, which is herein fully incorporated by reference and are further described herein below.

Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

It will be appreciated that for oral administration the polypeptides of the present invention may be attached to sustained-release enhancing agent. Exemplary sustained-release enhancing agents include matrices based on natural biodegradable polymers, specifically, hydrophilic matrixes such as, but are not limited to hyaluronic acid (HA), alginic acid (AA), polyhydroxyethyl methacrylate (Poly-HEMA), polyethylene glycol (PEG), glyme and polyisopropylacrylamide.

Attaching the amino acid sequence component of the polypeptides of the invention to other non- amino acid agents may be by covalent linking, by non-covalent complexion, for example, by complexion to a hydrophobic polymer, which can be degraded or cleaved producing a compound capable of sustained release; by entrapping the amino acid part of the peptide in liposomes or micelles to produce the final peptide of the invention. The association may be by the entrapment of the amino acid sequence within the other component (liposome, micelle) or the impregnation of the amino acid sequence within a polymer to produce the final peptide of the invention. It should be further appreciated that attachment of the Gp0.4 any homolog or related protein of the T7-like bacterial viruses, to an enhancing agent may be either directly or via a linker. In certain embodiments, said linker may comprise an amino acid sequence of about 10 to 100 amino acid residues. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions, which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.

Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

The preparation of the present invention may also be formulated as topical compositions, such as a spray, a cream, a mouthwash, a wipe, a foam, a soap, an oil, a solution, a lotion, an ointment, a paste and a gel.

More specifically, topical application or administration, as meant in the preset context, refers to a medication that is applied to body surfaces such as the skin or mucous membranes to treat ailments via a large range of classes including but not limited to creams, foams, gels, lotions and ointments. Many topical medications are epicutaneous, meaning that they are applied directly to the skin. Topical medications may also be inhalational, such as asthma medications, or applied to the surface of tissues other than the skin, such as eye drops applied to the conjunctiva, or ear drops placed in the ear, or medications applied to the surface of a tooth. As a route of administration, topical medications are contrasted with enteral (in the digestive tract) and intravascular/intravenous (injected into the circulatory system). Recently, transdermal patches have become a popular means of administering some topical drugs. A topical effect, in the pharmacodynamic sense, may refer to a local, rather than systemic, target for a medication. However, some topically administered drugs have systemic effects, such as some hydrophobic chemicals, such as steroid hormones, can be absorbed into the body.

Specific embodiments of the invention may contemplate skin infectious conditions. As such, treatment by topical administration of the affected skin areas of an ointment, cream, suspensions, paste, lotions, powders, solutions, oils, encapsulated gel, liposomes containing the Gp0.4 bacteriophage protein of the invention, any nano-particles containing the Gp0.4 protein of the invention, or sprayable aerosol or vapors containing a the same. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. The term "topically applied" or "topically administered" means that the ointment, cream, emollient, balm, lotion, solution, salve, unguent, or any other pharmaceutical form is applied to some or all of that portion of the skin of the patient skin that is, or has been, affected by, or shows, or has shown, one or more symptoms of the infectious disease.

It should be noted that the term "skin" as used herein means the air-contacting part of the human body, to a depth of about 7 mm from the air interface; as such, it also includes the nails.

In certain embodiments, topical administration of the Gp0.4 bacteriophage protein of the invention or any composition comprising the same may include topical dressing. The term "dressing" means a covering for a wound or surgical site, typically composed of a cloth, fabric, synthetic membrane, gauze, or the like. It is usually a polymer-containing matrix covering an area of the skin. The dressing may or may not be in intimate contact with the skin. It can be, for example, a cloth or gauze, or it can be a polymer solution painted or sprayed on the skin, the polymer solidifying on the skin when the solvent dries off and/or when the polymer crosslinks. Dressings also include gels, typically cross-linked hydrogels, which are intended principally to cover and protect wounds, surgical sites, and the like.

Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

It will be appreciated that the Gp0.4 T7 polypeptides of the present invention or any homolog or related protein of the T7-like bacterial viruses can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself.

Exemplary agents that may be formulated in the compositions of the present invention (or administered together with the composition of the present invention) include antibiotics (e.g. rifampicin, chloramphenicol and spectinomycin); DNA damaging agents (e.g. mitomycin, nalidixic acid and trimethoprim), serine analogues (e.g. serine hydroxamate), anti-inflammatory agents or anti-mycotic (or anti-fungal) drugs. As noted above, the present invention involves the use of different active ingredients, for example, the Gp0.4 T7 bacteriophage protein of the invention, specifically the Gp0.4 protein of SEQ ID NO. 10 or any fragments or peptides thereof, specifically the peptides of any one of SEQID NO: 11-42 or any o the polypeptides of SEQ ID NO. 55 and 57, and at least one additional therapeutic agent that may be administered through different routes, dosages and combinations. More specifically, the treatment of an infectious diseases and conditions with a combination of active ingredients may involve separate administration of each active ingredient. Therefore, a kit providing a convenient modular format of the Gp0.4 T7 bacteriophage protein of the invention, would allow the required flexibility in the above parameters.

Thus, in another aspect, the invention provides a kit. In some embodiments, the kit of the invention may include at least two separate pharmaceutical compositions that are required for treating an infectious disease. Thus, in certain embodiments, the present invention provides a kit comprising: (a) a Gp0.4 T7 bacteriophage protein, any homolog or related protein of the T7- like bacterial viruses, or any fragment, peptide, analogues and derivatives thereof, optionally, in a first dosage form; and (b) at least one additional therapeutic agent, optionally, in a second dosage form. In other embodiments the kit of the invention may optionally further comprise container means for containing said first and second dosage forms.

In one particular embodiment, the additional therapeutic agent may be another known antibiotic agent such as bacteriostatic or a bactericidal antibiotic adapted for a particular application. For example, several antibiotics are currently used for topical applications, i.e. treatment and prevention of bacterial infections in the skin (e.g. acne) or in minor skin wounds and burns, specifically clindamycin, erythromycin, etronidazole and chloramphenicol. The purpose of the additional antibiotic agent may be to widen the spectrum of the combination therapy.

An "antibiotic agent" as used herein, may be any agent that inhibits bacterial growth or kills bacteria. The term is often used synonymously with the term antibacterial agent. Most of today's antibacterials chemically are semisynthetic modifications of various natural compounds. These include, for example, the beta-lactam antibacterials, which include the penicillins (produced by fungi in the genus Penicillium), the cephalosporins, and the carbapenems. Compounds that are still isolated from living organisms are the aminoglycosides, whereas other antibacterials, for example, the sulfonamides, the quinolones, and the oxazolidinones, are produced solely by chemical synthesis. In accordance with this, many antibacterial compounds are classified on the basis of chemical/biosynthetic origin into natural, semisynthetic, and synthetic. Another classification system is based on biological activity; in this classification, antibacterials are divided into two broad groups according to their biological effect on microorganisms: bactericidal agents kill bacteria, and bacteriostatic agents slow down or stall bacterial growth.

Antibacterial antibiotics are commonly classified based on their mechanism of action, chemical structure, or spectrum of activity. Most target bacterial functions or growth processes. Those that target the bacterial cell wall (penicillins and cephalosporins) or the cell membrane (polymyxins), or interfere with essential bacterial enzymes (rifamycins, lipiarmycins, quinolones, and sulfonamides) have bactericidal activities. Those that target protein synthesis (macrolides, lincosamides and tetracyclines) are usually bacteriostatic (with the exception of bactericidal aminoglycosides). Further categorization is based on their target specificity. "Narrow-spectrum" antibacterial antibiotics target specific types of bacteria, such as Gram-negative or Gram-positive bacteria, whereas broad- spectrum antibiotics affect a wide range of bacteria. New classes of antibacterial antibiotics have been brought into clinical use: cyclic lipopeptides (such as daptomycin), glycylcyclines (such as tigecycline), oxazolidinones (such as linezolid) and lipiarmycins (such as fidaxomicin). It should be noted that the present invention encompasses the combined use of the Gp0.4 protein of the invention with any antibiotic agent as classified herein.

In more specific embodiments, the Gp0.4 bacteriophage protein of the invention may be administered with an additional therapeutic agent that may be at least one β-lactam antibiotics. Generally, β-lactams are classified and grouped according to their core ring structures, where each group may be divided to different categories. The term "penam" is used to describe the core skeleton of a member of a penicillin antibiotic, i.e. a β-lactam containing a thiazolidine rings. Penicillins contain a β-lactam ring fused to a 5-membered ring, where one of the atoms in the ring is a sulfur and the ring is fully saturated. Penicillins may include narrow spectrum penicillins, such as benzathine penicillin, benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), procaine penicillin and oxacillin. Narrow spectrum penicillinase-resistant penicillins include methiciUin, dicloxaciUin and flucloxaciUin. The narrow spectrum β-lactamase-resistant penicillins may include temocillin. The moderate spectrum penicillins include for example, amoxicillin and ampicillin. The broad spectrum penicillins include the co-amoxiclav (amoxicillin+clavulanic acid). Finally, the penicillin group also includes the extended spectrum penicillins, for example, azlocillin, carbeniciUin, ticarcillin, mezlocillin and piperacillin.

Penicillins are sometimes combined with other ingredients called β -lactamase inhibitors, which protect the penicillin from bacterial enzymes that may destroy it before it can do its work. Members of this class include pivampicillin, hetacillin, bacampicillin, metampicillin, talampiciUin, epicillin, carbeniciUin, carindacillin, ticarcillin, azlocillin, piperacillin, mezlocillin, mecillinam, pivmecillinam, sulbenicillin, clometocillin, procaine benzylpenicillin, azidocillin, penamecillin, propicillin, pheneticillin, cloxacillin and nafcillin. β-lactams containing pyrrolidine rings are named carbapenams. A carbapenam is a β-lactam compound that is a saturated carbapenem. They exist primarily as biosynthetic intermediates on the way to the carbapenem antibiotics.

Carbapenems have a structure that renders them highly resistant to β-lactamases and therefore are considered as the broadest spectrum of β-lactam antibiotics. The carbapenems are structurally very similar to the penicillins, but the sulfur atom in position 1 of the structure has been replaced with a carbon atom, and hence the name of the group, the carbapenems. Carbapenem antibiotics were originally developed from thienamycin, a naturally-derived product of Streptomyces cattleya. The carbapenems group includes: biapenem, doripenem, ertapenem, imipenem, meropenem, panipenem and PZ-601. β-lactams containing 2, 3-dihydrothiazole rings are named penems. Penems are similar in structure to carbapenems. However, where penems have a sulfur, carbapenems have another carbon. There are no naturally occurring penems; all of them are synthetically made. An example for penems is faropenem. β-lactams containing 3, 6-dihydro-2H-l, 3-thiazine rings are named cephems. Cephems are a sub-group of β-lactam antibiotics and include cephalosporins and cephamycins. The cephalosporins are broad- spectrum, semisynthetic antibiotics, which share a nucleus of 7- aminocephalosporanic acid. First generation cephalosporins, also considered as the moderate spectrum includes cephalexin, cephalothin and cefazolin. Second generation cephalosporins that are considered as having moderate spectrum with anti-Haemophilus activity may include cefaclor, cefuroxime and cefamandole. Second generation cephamycins that exhibit moderate spectrum with anti- anaerobic activity include cefotetan and cefoxitin. Third generation cephalosporins considered as having broad spectrum of activity includes cefotaxime and cefpodoxime.

Finally, the fourth generation cephalosporins considered as broad spectrum with enhanced activity against Gram positive bacteria and β-lactamase stability include the cefepime and cefpirome. The cephalosporin class may further include: cefadroxil, cefixime, cefprozil, cephalexin, cephalothin, cefuroxime, cefamandole, cefepime and cefpirome.

Cephamycins are very similar to cephalosporins and are sometimes classified as cephalosporins. Like cephalosporins, cephamycins are based upon the cephem nucleus. Cephamycins were originally produced by Streptomyces, but synthetic ones have been produced as well. Cephamycins possess a methoxy group at the 7-alpha position and include: cefoxitin, cefotetan, cefmetazole and flomoxef. β-lactams containing 1, 2, 3, 4-tetrahydropyridine rings are named carbacephems. Carbacephems are synthetically made antibiotics, based on the structure of cephalosporin, a cephem. Carbacephems are similar to cephems but with a carbon substituted for the sulfur. An example of carbacephems is loracarbef.

Monobactams are β-lactam compounds wherein the β-lactam ring is alone and not fused to another ring (in contrast to most other β-lactams, which have two rings). They work only against Gram-negative bacteria. Other examples of monobactams are tigemonam, nocardicin A and tab toxin. β-lactams containing 3, 6-dihydro-2H-l, 3-oxazine rings are named oxacephems or clavams. Oxacephems are molecules similar to cephems, but with oxygen substituting for the sulfur. Thus, they are also known as oxapenams. An example for oxapenams is clavulanic acid. They are synthetically made compounds and have not been discovered in nature. Other examples of oxacephems include moxalactam and flomoxef. Another group of β-lactam antibiotics is the β-lactamase inhibitors, for example, clavulanic acid. Although they exhibit negligible antimicrobial activity, they contain the β-lactam ring. Their sole purpose is to prevent the inactivation of β-lactam antibiotics by binding the β- lactamases, and, as such, they are co-administered with β-lactam antibiotics, β-lactamase inhibitors in clinical use include clavulanic acid and its potassium salt (usually combined with amoxicillin or ticarcillin), sulbactam and tazobactam.

It should be appreciated that any other class of antibiotic agents mentioned in the application may be used as an additional therapeutic agent in the compositions, kits and methods of the invention.

In yet in another specific embodiment, the additional therapeutic agent may be an antiinflammatory drug. Anti-inflammatory drugs refer to the property of a substance or treatment that reduces inflammation. Many steroids, to be specific glucocorticoids, reduce inflammation or swelling by binding to glucocorticoid receptors. These drugs are often referred to as corticosteroids.

Corticosteroids have been classified into groups based on potency, for example, the corticosteroid clobetasol proprionate, is ranked as a very potent steroid, while betametasone diproprionate and fluocinolone acetonide can range from potent to moderately potent. Antiinflammatory drugs containing hydrocortisone are the least potent. It should be further noted that the invention also contemplates the use of Nonsteroidal anti-inflammatory drugs, usually abbreviated to NSAIDs. NSAIDs inhibit the activity of both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), and thereby, the synthesis of prostaglandins and thromboxanes. It is thought that inhibiting COX-2 leads to the anti-inflammatory, analgesic and antipyretic effects. The most prominent members of this group of drugs, aspirin, ibuprofen and naproxen that may be used for the kits and compositions of the invention.

In certain embodiments, combining the Gp0.4 peptide of the invention or any homolog or related protein of the T7-like bacterial viruses, with an anti-inflammatory drug may be specifically applicable to topical applications as well as to eye and ear inflammations. Yet in other specific embodiments, the additional therapeutic agent may be an anti-mycotic (or anti-fungal) drug. Such kit may be in certain embodiments specifically applicable for vaginal inflammation, wherein the Gp0.4 peptide of the invention may be combined with an anti- mycotic drug.

Anti mycotic or an antifungal medication as used herein, is a pharmaceutical fungicide used to treat mycoses and include different classes. For example, polyene antifungals that are amphiphilic molecules with multiple conjugated double bonds (include Amphotericin B, Candicidin, Hamycin, Natamycin, Nystatin, Rimocidin and the like), Imidazole, triazole, and thiazole antifungals (Azole antifungal drugs inhibit the enzyme lanosterol 14 a-demethylase), Allylamines, and Echinocandins.

The kit of the invention may further include container means for containing separate compositions; such as a divided bottle or a divided foil packet however, the separate compositions may also be contained within a single, undivided container. Typically the kit includes directions for the administration of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.

According to one embodiment, the kit of the invention is intended for achieving a therapeutic effect, specifically, inhibiting bacterial growth, or more specifically, eliminating or killing bacteria.

It should be appreciated that each of the multiple components of the kit may be administered simultaneously.

Alternatively, each of said multiple dosage forms may be administered sequentially in either order.

More specifically, the kits described herein can include a composition as described, or in separate multiple dosage unit forms, as an already prepared liquid topical, nasal or oral dosage form ready for administration or, alternatively, can include the composition as described as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid oral dosage form. When the kit includes a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid dosage form (e.g., for oral administration), the kit may optionally include a reconstituting solvent. In this case, the constituting or reconstituting solvent is combined with the active ingredient to provide liquid oral dosage forms of each of the active ingredients or of a combination thereof. Typically, the active ingredients are soluble in so the solvent and forms a solution. The solvent can be, e.g., water, a non-aqueous liquid, or a combination of a non-aqueous component and an aqueous component. Suitable non-aqueous components include, but are not limited to oils, alcohols, such as ethanol, glycerin, and glycols, such as polyethylene glycol and propylene glycol. In some embodiments, the solvent is phosphate buffered saline (PBS).

As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1- 317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

Experimental procedures

Reagents, Strains, and Plasmids

Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract and 5 g/L NaCl) was from Acumedia, and agar was from Difco. Antibiotics, isopropyl- -D-thiogalactopyranoside (IPTG) and L-arabinose were from Sigma-Aldrich. Restriction enzymes were from New England Biolabs. Rapid ligation kit was from Roche. The bacterial strains, phages, plasmids and oligonucleotides used in this study are listed in Table 3, herein below.

Table 3 Bacterial strains, phages, plasmids, and oligonucleotides used

Bacterial strains Description

[Source]

NEB 5 a [New F , §WlacZAM\5A(lacZYA-argF), U169, deoR, recAl, endAl, hsdR17 (r_k ,

England Biolabs] m_k ⁺), gal , phoA, supE44, X, thi 1, gyrA96, relAl

BW25113AirxA V,A(araD-araB)567, AlacZ4787( nnB-3), , rph-1, AtrxA732::kan,

[16] A(rhaD-rhaB)568, hsdR514

E. coli K-12 Wild-type

[Laboratory

collection]

BW25113 [17] V,A(araD-araB)567, AlacZ4787(::nnB-3),□^", rph-1, A{rhaD- rhaB)568, hsdR514

BW25113Aace ¥,A(araD-araB)567, AaceF733::kan, AlacZ4787(::rrnB-3), X, rph-

[16] 1, A(rhaD-rhaB)568, hsdR514

RK6497 [This ¥,A(araD-araB)567, AaceF733::kan, AlacZ4787(::rrnB-3), X, rph- study] 1, A(rhaD-rhaB)568, hsdR514 tsZ9

BW25113AmmC V,A(araD-araB)567, AlacZ4787{: :rrnB-3), λ^', rph-1, AminCr. kan,

[16] A(rhaD-rhaB)568, hsdR514

BW25113As«/A V,A(araD-araB)567, AlacZ4787{: :rrnB-3), , rph-1, AsulA: :kan,

[16] A(rhaD-rhaB)568, hsdR514 BW25113A;yi//zQ V,A(araD-araB)567, AlacZ4787{: :rrnB-3), , rph-1, AydhQ::kan,

[16] A(rhaD-rhaB)568, hsdR514

Phage

WT T7 Wild-type T7

[Laboratory

collection]

0.4-CTAP T7 T7 encoding Gp0.4 with CTAP tag

[This study]

1.6-CTAP T7 T7 encoding Gpl.6 with CTAP tag

[Laboratory

collection]

Τ7Δ0.4 [This T7 with an FRT scar instead of gene 0.4

study]

WT T7_FRT [This T7 with an FRT scar downstream gene 0.4

study]

Plasmids

pUC19 [18] Cloning vector, ampicillin^r

p0.4-CTAP [This pUC19 cloned with CTAP and trxA gene, ampicillin^r

study]

pBAD18 [19] L-arabinose-inducible expression vector, ampicillin^r

pBAD-0.4 [This pBAD18 cloned with Gp0.4 under arabinose promoter, ampicillin^r study]

pUC-0.4 [This pUC19 cloned with trxA gene, flanked by FRT sites and by 50 bp study] upstream and downstream of the DNA sequence encoding gene 0.4, ampicillin^r

pUC-ctrl [This pUC19 cloned with trxA gene, flanked by FRT sites and by 50 bp study] immediately upstream and downstream the stop codon ofgene 0.4, ampicillin^r

Oligonucleotides 5'→3'

RK1F CTAGCATATGTCAAAGAACTGTACGAAAACAACAAGGCAA

TAGCTTTAGAATCTGCTGAGACGCGGCCGCCAGCTGAAGC

- SEQ ID NO: 1

RK1R CTAGTCTAGAAGTAAAGTGATAATCATAAAGGCCACTCGC

T AGG AGC G ACCTTG AGTCT ATT AC GCC AGGTT AGC GTC G A -

SEQ ID NO: 2

RKl lFa GACTCCCGGGGAGGAGGATGAAGAGTAATG - SEQ ID NO: 3

RKl lRa CTAGTCTAGATCACTCAGCAGATTCTAAAG - SEQ ID NO: 4

RK28F CTGTGCTCAGCGCGTGTTTC - SEQ ID NO: 5 RK28Ra CGCATCCAGCAGGGAGATAC - SEQ ID NO: 6

RK28Rb CCAACCGAAGTGTACTATAC - SEQ ID NO: 7

RK53F GTACCAGCTGTCAATGAATACTTGGAGGAAGTCGAGGAGT

ACGAGGAGGATGAAGAGTAAGATCCGTCAGCCTGCAGTTC

SEQ ID NO:48

RK53R TAAGCAGCTGAGTAAAGTGATAATCATAAAGGCCACTCGCTAG

GAGCGACCTTGAGTCTATGTAGGCTGGAGCTGCTTCG SEQ ID

NO:49

RK60F GTACCAGCTGAAGAACTGTACGAAAACAACAAGGCAATAGCTT

TAGAATCTGCTGAGTGAGAT CCGTCAGCCTGCAGTTC SEQ ID

NO:50

85F TGGTCTGATGCCTGACACCA SEQ ID NO:51

SM24R1 ATTACTGCAGTCATTTGCGTAGTGCCCCTT SEQ ID NO:52

Plasmid Construction

Plasmid p0.4-CTAP encodes the last 50 nt of gene 0.4 fused, in frame, to a tandem- affinity tag (CTAP), which encodes a C-terminal calmodulin-binding peptide, a specific TEV protease recognition sequence, and an IgG-binding unit of protein A of Staphylococcus aureus. This tag was followed by the stop codon of gene 0.4 50 nt downstream and Q:2 then by trxA, a positive selection marker for T7 grown on hosts lacking trxA. Plasmid p0.4-CTAP was constructed by PCR amplification of phage T7 encoding the CTAP-trxA fragment by using primers RK1F and RK1R (Table 3) containing Ndel and Xbal restriction sites, respectively. The PCR fragment was digested with Ndel and Xbal and ligated to a compatibly digested pUC19 plasmid. Plasmid pBAD-0.4 was constructed by PCR amplification of WT phage T7 using primers RKl lFa and RKl lRa (Table 3), containing restriction sites Xmal and Xbal, respectively. The PCR fragment was digested with Xmal and Xbal and consequently ligated to compatibly digested plasmid pBAD18. Plasmid pUC-0.4 was constructed to delete gene 0.4 Q:3 in the T7 genome, resulting in a DNA "scar" in the form of FRT sites. Plasmid pUC-ctrl (Table 3) was constructed to insert an identical DNA scar immediately downstream of gene 0.4. Both plasmids encode the trxA gene, a positive selection marker for T7 grown on hosts lacking trxA, flanked by FRT sites. The trxA in the pUC-0.4 and pUC-ctrl plasmids is flanked by 50 bp upstream and downstream of the DNA sequence encoding gene 0.4 or the 50 bp immediately downstream and upstream the stop codon of gene 0.4, respectively. The plasmids were constructed by PCR amplification of T7 phage encoding the trxA gene flanked by FRT sites by using primers RK53F and RK53R for the pUC-0.4 plasmid and primers RK53R and RK60F (Table 3) for the pUC-ctrl plasmid, which contained the PvuII restriction sites. The PCR fragment was digested with PvuII and was ligated to a compatibly digested pUC19 plasmid.

Homologous Recombination of Bacteriophage T7

T7 containing gene 0.4 with a CTAP tag was constructed by homologous recombination. Plasmid p0.4-CTAP was transformed into E. coli strain NEB5a (Table 3). E. coli NEB5a/p0.4- CTAP were aerated overnight in LB supplemented with 100 μg/mL ampicillin at 37 °C. The overnight culture was diluted 1:20 in fresh LB supplemented with 100 μg/mL ampicillin at 42 °C and aerated to an OD₆oo of 0.5. The cells were then infected with WT phage T7 at a multiplicity of infection (MOI) of 0.1. The infected bacteria were aerated at 42 °C until complete clearing of the culture. The obtained lysate was used to infect E. coli lacking trxA [strain JW5856 (Table 3)]. This procedure selected for phages which had recombined the fragment containing trxA into their genome. Strain JW5856 was aerated overnight in LB supplemented with 25 μg/mL kanamycin at 37 °C. The overnight culture was diluted 1: 1 in 3 mL of fresh LB supplemented with 25 μg/mL kanamycin at 37 °C, and aerated for 1 h. The culture was then centrifuged at ~4500g, 4 °C, for 10 min. The pellet was resuspended in 3 mL warm LB medium supplemented with 0.7% agar and 0.5 mL of the T7 lysate was added. The suspension was poured onto an LB plate and was incubated at 37 °C for 3 h. A single plaque from several emerging on the plate was purified and the correct insertion was verified by sequencing.

T7 having trxA instead of the 0.4 gene and T7 having trxA at the end of the 0.4 gene were constructed by homologous recombination as described above, using pUC-0.4 and pUC-ctrl plasmids. The correct insertions, one in a T7 phage encoding trxA in place of the DNA encoding gene product (Gp) 0.4 (T7A0.4trxA)Q:4 and the second in a T7 phage encoding trxA at the end of the 0.4 gene (T7FRTtrxA), were verified by DNA sequencing.

Cleaving Out the trxA in Phages T7A0.4trxA and T7FRTtrxA

FLP recombinase was used to flip the trxA gene out from the engineered T7 phages. T7A0.4trxA and T7FRTtrxA phages were diluted 1: 1,000 in fresh LB. These stocks were further diluted 1: 1,000 in 1 Ml of a culture of E. coli NEB5a/pCP20, encoding the FLP recombinase [20], and aerated at 32 °C until the culture lysed. The infection

cycles were repeated four times to enrich for phage with a flipped-out trxA. These lysates contained phages with flippedout trxA that were screened as follows. Dilutions of the obtained ly sates (100 μί) and diluted NEB5a/pCP20 bacteria (100 μί) were plated in LB medium supplemented with 0.7% (wt/vol) agar onto LB plates and incubated at 32 °C for 3 h. Single plaques were transferred into wells of a 96- well microtiter plate containing 50 μΐ_^ LB medium. Each plaque was replicated to two fresh LB plates, with bacterial lawns of BW25113AtrxA (200 HL) or BW25113AydhQ (Table 3) (200 \LL) bacteria in LB medium supplemented with 0.7% (wt/vol) agar. The plates were incubated at 37 °C for 3 h. Phages growing on the BW25113AydhQ lawn but not on the BW25113AtrxA lawn were DNA-sequenced. Phages T7A0.4trxA and WT T7FRTtrxA that lost the trxA gene were named Τ7Δ0.4 and WT T7FRT, respectively (Table 3).

Interaction Validation Using His-Tag Purification Followed by Western Blot:

Overnight culture of E. coli strains harboring plasmid pCA24N encoding genes ftsZ, prsA, glpD, aceE, or hsdM (from the ASKA library (A Complete Set of E. coli K- 12 ORF Archive) [21]) wereQ:5 diluted 1 :50 in LB and aerated at 30 °C to an OD600 of 0.2. IPTG was added to a final concentration of 1 mM, and growth was continued for an additional 2 h. At this point, each culture was infected at a MOI of about 4 with T7 encoding either CTAP-tagged Gp0.4 or CTAP- tagged Gpl .6 as a control. After 14 min of infection, cultures were transferred directly into slurry ice. The cultures were then centrifuged at 4 °C for 20 min at 4,000 x g. The pellet was resuspended in lysis buffer [20 mM sodium phosphate buffer (pH 7.4), 300 mM NaCl, 10 mM imidazole, 200 μg/mL hen-egg lysozyme, 2.5 U/mL benzonase and Complete Mini EDTA-free tablet] and frozen at -80 °C overnight. The samples were thawed in a room temperature (RT) water bath and incubated for 30 min on ice. They were then frozen for 2 min in liquid nitrogen and thawed for 2.5 min in an RT water bath. This freeze-thaw cycle was repeated twice more. Cell debris was removed by centrifugation at 4 °C for 10 min at 20,000 x g. Supernatants were transferred into new tubes prewashed with lysis buffer, into a 50% (vol/vol) slurry of Ni-NTA beads (Thermo/Q:6 Pierce), and incubated on a rotator shaker at 4 °C for 1 h. The bead-protein complex was washed three times with wash buffer [20 mM sodium phosphate buffer (pH 7.4), 300 mM NaCl, 25 mM imidazole] and eluted with elution buffer [20 mM sodium phosphate buffer (pH 7.4), 300 mM NaCl, 250 mM imidazole] . Protein concentration was measured with a Nanodrop 2000 spectrophotometer (Thermo), and equal amounts of total protein were loaded on a 12% polyacrylamide gel. The gel was electrophoresed and subsequently stained with Imperial protein stain (Thermo/Pierce). Western blotting was performed to detect the CTAP-tagged proteins using Anti-Calmodulin-Binding Protein Epitope Tag (Merck, Upstate) according to the manufacturer' s instructions. Transduction of FtsZ

Transduction was used to replace the ftsZ Q:7 gene with the mutant ftsZ gene. PI lysate was prepared as follows: the donor strain, E. coli encoding the ftsZ9 allele and a kanamycin- resistance cassette in a gene located about 20 kbp from FtsZ (RK6497 strain; Table 3), was aerated overnight in LB supplemented with 25 μg/mL kanamycin at 37 °C. The probability of the kanamycin cassette to cotransduce with the ftsZ9 allele is about 50%. This overnight culture was diluted 1: 100 in LB supplemented with 25 μg/mL kanamycin, 5 mM CaC12, and 0.2% (wt/vol) glucose (Merck). After 1 h aeration at 37 °C, 0 or 100

of phage PI was added. Cultures were aerated for 1 to 3 h, until lysis occurred. Phage PI lysate (0 or 30 mixed with 100

of overnight culture of the recipient strain BW25113 (Table 3) and of 1 M CaC12. After incubation at 30 °C for 30 min, 100 of 1 M Na-citrate (Merck) and 500 μΐ, of LB medium were added to each tube. Infected cultures were incubated at 37 °C for 1 h, and then 3 mL of warm LB medium supplemented with 0.7% (wt/vol) agar was added to each tube. The suspension was poured onto a plate containing 25 μg/mL kanamycin. Transductants were streaked several times on LB plates containing 25 μg/mL kanamycin and their ftsZ gene was then DNA sequenced to differentiate between ftsZ and ftsZ9 transductants.

In Vitro FtsZ Inhibition Assays

FtsZ assembly was measured by the increase in fluorescence of a tryptophan mutant (L189W) [14, 15] FtsZ was pre-incubated with Gp0.4 (0, 3, 5 μΜ) in HMK100 (50 mM HEPES pH 7.7, 5 mM MgAc, 100 mM KAc) for 5-10 min. Assembly was initiated by the addition of 200 μΜ GTP. To measure the CcApp, the fluorescence before addition of GTP was subtracted from that at steady state, for a range of starting FtsZ concentrations. Measurements were performed with a Shimadzu RF-5301 PC spectrofluorometer with a thermostatically controlled cell at 25 °C. The fluorescence was monitored at 330 nm with excitation at 295 nm. FtsZ assembly was also assayed by quenching of BODIPY fluorescence, as described previously [14, 15].

Negative-Stain EM

FtsZ mutant L189W (2 μΜ) was preincubated with Gp0.4 (0, 5, or 10 μΜ) for 5-10 min in HMK100. Assembly was induced by the addition of 100 μΜ GTP. After 2 min, 10 μΐ, of the reaction was applied to a UV-treated carbon-coated grid [4] and stained with 2% (w/v) uranyl acetate. Images were collected on a Phillips EM420 equipped with a CCD camera. GTPase Activity Assay

GTPase activity was measured using a continuous, regenerative coupled GTPase assay [5, 6] . Various concentrations of FtsZ were preincubated with 10 μΜ Gp0.4 in HMK100, followed by the addition of assay buffer (1.4 mM phosphoenol pyruvate, 1.2 mM NADH, and 20 units/mL pyruvate kinase and lacatate dehydrogenase) and 0.5 mM GTP. Measurements were performed with a Shimadzu UV2401PC spectrophotometer with a thermostatically controlled cell at 25 °C.

TAP Assay

E. coli strain K-12 was aerated overnight in LB at 37 °C. The overnight culture was diluted 1 : 100 in 750 mL of LB medium at 30 °C and aerated to an OD600 of 0.5. The cells were then infected with phage T7 having a tagged gene 0.4 with at a multiplicity of infection (MOI) of 4. The culture was aerated for 14 min at 30 °C and then cooled immediately to 0 °C. The bacteria were centrifuged for 10 min at 9,000 x g and 4 °C. The pellet was resuspended in 5 mL of lysis buffer [20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 150 mM NaCl, 0.1% wt/vol Nonidet P-40 (Sigma), 200 μg/mL hen-egg Q:31 lysozyme (Calbiochem), 2.5 U/mL benzonase (Novagen), and Complete Mini EDTA-free tablet (Roche)] and was frozen at -80 °C overnight. The sample was thawed in a room temperature (RT) water bath and then incubated for 30 min. It was then frozen again in liquid nitrogen and thawed in an RT water bath twice more. The lysate was injected through a 23-gauge needle five times and then centrifuged in a Sorvall SS34 rotor for 10 min at 9,000 x g and 4 °C. The samples were filtered through a 45-μί filter. IgG Sepharose beads (400 μί; Pharmacia) were added, and the suspension was incubated on a rotating platform at 4 °C for 1 h. The lysate and beads were poured onto a Bio-Rad Poly-Prep Chromatography Column (0.8 x 4 cm). The beads were washed three times with 10 mL of IPP150 buffer [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% Nonidet P-40] and then with 10 mL TEV cleavage buffer [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% Nonidet P-40, 0.5 mM EDTA, 1.0 mM DTT (Calbiochem)] . The bottom of the column was sealed, and 500 μΐ_^ of TEV cleavage buffer and 50 μΐ_^ of TEV enzyme were added. The samples were incubated on a rotating platform at 16 °C for 2 h. TEV cleavage buffer (1 mL) was added to the columns. Three volumes of calmodulin binding buffer [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM Mg2+ acetate (Merck), 1 mM imidazole (Sigma), 2 mM CaC12 (Merck), 10 mM β-mercaptoethanol (Sigma), and 0.1% Nonidet P-40], 3 μΐ_^ 1 M CaC12 per mL IgG eluate, and 300 μΐ_^ calmodulin affinity resin (Stratagene) were added to the TEV supernatant. The sample was incubated on a rotating platform at 4 °C for 1 h. The lysate and beads were poured onto a new Bio-Rad Poly-Prep Chromatography Column. The beads were washed twice with 8 mL calmodulin binding buffer and then with 8 mL calmodulin binding buffer with a lower detergent concentration (0.02% Nonidet P-40 instead of 0.1% Nonidet P-40). The samples were eluted with 1 mL calmodulin elution buffer [10 mM Tris HCl (pH 8.0), 150 mM NaCl, 0.02% Nonidet P-40, 1 mM Mg2+ acetate, 1 mM imidazole, 20 mM EGTA (Calbiochem) and 10 mM β-mercaptoethanol]. The elution was precipitated in TCA (Calbiochem) by adjusting it to 25% TCA, vortexing and placing it on ice overnight. The overnight elu-Q:32 tion was centrifuged at maximum speed at 4 °C for 5 min. The TCA pellet was first washed with 1 mL cold (-20 °C) acetone containing 0.05 N HC1 (Bio-Lab) and centrifuged for 5 min at 13,000 x g and 4 °C. The second wash was carried out with 1 mL cold (-20 °C) acetone and centrifugation for 5min at 13,000 x g and 4 °C. The supernatant was carefully removed. The pellet was dried for 5min in a vacuumconcentrator (Concentrator 5301; Eppendorf) with heatingQ: 33 (45 °C). The pellet was used for mass spectrometry (Technion). Interactions of proteins identified by mass spectrometry with Gp0.4 were validated as described herein.

Interactions of proteins identified by mass spectrometry with Gp0.4 were validated as described herein.

Gp0.4 Toxicity Assay

E. coli cells harboring pBAD-0.4 or pBAD18 as a control were aerated overnight in LB supplemented with 100 μg/mL ampicillin at 37 °C. Overnight cultures were diluted 1: 100 in fresh LB supplemented with 100 μg/mL ampicillin at 37 °C and aerated to an OD₆oo of 0.1. The cultures were then centrifuged for 10 min at ~4500g, 4 °C and resuspended to an OD₆oo of 1. Cultures were serially diluted and plated overnight at 37 °C on LB agar with 100 μg/mL ampicillin, with or without 0.2% (w/v) arabinose. The toxicity was calculated by dividing the number of CFU obtained in the presence of arabinose by that in the absence of arabinose. E. coli NEB5a/pBAD-0.4 and E. coli NEB5a/pBAD18 colonies surviving on plates supplemented with arabinose were used as a template for PCR amplification using primers RK28F, RK28Ra and RK28Rb (Table 3). The PCR product amplifying the ftsZ gene was DNA sequenced. PI transduction for transferring the obtained ftsZ mutations were carried out as described herein above.

Microscopy Indicated E. coli strains harboring pBAD-0.4 or pBAD18 plasmids were aerated overnight in LB medium supplemented with 100 μg/mL ampicillin at 32 °C and 0.2% glucose. These overnight cultures were diluted 1:50 in fresh LB supplemented with 100 μg/mL ampicillin at 32 °C, and then induced with 0.001% L-arabinose for 0, 2, and 4 h at 32 °C with shaking. Each sample was centrifuged for 10 min, at ~4500g, 4 °C. The cells were resuspended to an OD₆oo of 10. Cells (5 μL· per slide) were viewed under an Olympus Provis AX-70 microscope. Images were taken by an Olympus DP72 camera.

Phage Competition Assay

BW25113A<2ce (Table 3) bacteria were aerated overnight in LB supplemented with 25 μg/mL kanamycin at 37 °C. This culture was then kept on ice and used as the host culture in the stationary phase representing the non-dividing bacteria. A sample of this overnight culture was diluted 1: 100 in LB supplemented with 25 μg/mL kanamycin at 37 °C and aerated to an OD₆oo of -0.15. This culture was kept on ice and used as the culture in the exponential phase representing dividing bacteria. A mixture of Τ7Δ0.4 and WT T7_FRT was used to infect the exponential- and stationary-phase cultures at a MOI of -0.01, at 37 °C. Each infection cycle was defined as co- incubation of the phage mixture and the bacteria for 1 h at 37 °C with shaking. A diluted lysate was transferred from one cycle to the next, resulting in cumulative effect of the competition over time. The relative abundance of Τ7Δ0.4 compared to WT T7_FRT in a phage mixture was determined for each cycle by PCR amplification of the region flanking gene 0.4 using primers 85F and SM24R1. The amplified DNA was 592 bp and 437 bp for WT T7_FRT and Τ7Δ0.4, respectively. Band intensities were quantified by their digital densities using ImageJ software. Intensity values for the band representing WT T7_FRT were multiplied by 0.74 to correct for size differences compared to Τ7Δ0.4. The relative abundance of each phage was calculated by dividing the density of the representative band by the total intensity of both bands. To determine the effect of gene 0.4 on E. coli encoding WT-ftsZ compared to E. coli encoding ftsZ9, BW25l l3AaceF and RK6497 [BW25l l3AaceF, ftsZ9] (Table 3) bacteria were used as described above for the logarithmic-phase culture but grown at 32 °C due to growth deficiency of E. coli encoding ftsZ9 at higher temperature. An unpaired t-test was used to analyze competition in each infection cycle. Statistical significance was determined using the GraphPad Prism software. EXAMPLE 1

Gp0.4 of Bacteriophage T7 Interacts Specifically with FtsZ in Vivo

To identify the interactions of GpO.4 with host proteins, a tandem affinity purification (TAP) assay was used [11]. A DNA fragment encoding an IgG-binding protein followed by a calmodulin-binding peptide (CBP) was cloned downstream of gene 0.4 in the T7 genome. E. coli hosts were infected with the genetically engineered phage and the infected cells, harboring the tagged GpO.4, were collected. The cells were then concentrated and ruptured. The soluble content was affinity-purified on IgG beads and then on calmodulin beads. This procedure yielded the purified GpO.4 with its interacting proteins along with some non-specific contaminants. These proteins were identified by mass spectrometry as: FtsZ, the essential division protein described above; PrsA, an essential enzyme that synthesizes the essential co-factor phosphoribosylpyrophosphate; GlpD, a non-essential enzyme catalyzing the synthesis of dihydroxyacetone phosphate, and AceE, a non-essential subunit of the pyruvate dehydrogenase enzyme that catalyzes the synthesis of acetyl-CoA [12]. Additional proteins that were also pulled down with the control tagged proteins were not further analyzed due to their putative nonspecific binding mode.

Since TAP occasionally generates false positives, the binding of GpO.4 was reciprocally validated to each of the four pulled-down proteins, and to a negative control, HsdM. Cells were infected with T7 encoding the tagged GpO.4, but this time, rather than pulling down GpO.4, each of the identified proteins was identified through its N-terminal 6XHis tag using Ni-coated beads. The beads were washed and the tagged proteins eluted using imidazole. The total pulled-down proteins were adjusted to similar levels (Figure 1), and a Western blot was performed to detect GpO.4 using an antibody against the CBP tag attached to it. As shown in Figure 1, the only protein found to pull down GpO.4 was FtsZ. None of the other proteins, including the negative control, pulled down GpO.4 at detectible levels. These results indicate that GpO.4 binds to FtsZ either directly or indirectly through another protein.

EXAMPLE 2

Toxicity of Gp0.4 Is Overcome by Host Mutations inftsZ

The effect of GpO.4 on E. coli cells was unknown, nor its interaction with FtsZ. Since FtsZ is an essential gene product, its inhibition may possibly affect viability of cells. The present inventors therefore tested the viability of bacterial cells following overexpression of GpO.4. As shown in Figure 2A, a six orders of magnitude drop in cell viability was found in cells over expressing Gp0.4, indicating that the interaction of Gp0.4 with FtsZ inhibits its action leading to bacterial cell death. Notably, similar toxicity was observed in isogenic strains lacking minC or sulA (Figure 5), the two known endogenous inhibitors of FtsZ, indicating that Gp0.4 does not exert its effect through enhancement of the activity of these known inhibitors, as is the case for DicB [8].

If toxicity is indeed related to inhibition of FtsZ, then mutants resistant to Gp0.4 overexpression should arise with mutations in ftsZ. To test this, the present inventors isolated nine mutants surviving overexpression of Gp0.4 and sequenced their entire ftsZ gene. In three of these mutants, a 6-nt insertion (TCGGCG) was identified adjacent to a segment that encodes a duplication of this same insertion (TCGGCG/TCGGCG as denoted by SEQ ID NO. 9), thus resulting in a triplicate sequence of the 6 nt (TCGGCG/TCGGCG/TCGGCG as denoted by SEQ ID NO. 8). It seems likely that the replicating strand briefly denatures from the template, and slips backward to anneal to the upstream 6 nt, thus resulting in another copy of these 6 nt. The resulting FtsZ contains a duplication of glycine-valine at position 18. This same insertion mutant, named FtsZ9, was identified previously and shown to overcome inhibition by the FtsZ inhibitors SulA and MinC [13]. The ftsZ gene of the other Gp0.4 resistant mutants was also completely sequenced, but no mutations were identified. It is likely that the other resistant mutants, lacking ftsZ mutations, have other genetic changes such as loss of Gp0.4 from plasmid or loss of sensitivity to the expression-inducer reagent arabinose (e.g. arabinose transporter mutation), or another supporession mutation. This indicates that the insertion mutation is a dominant mutation, under the tested conditions, that renders the bacteria resistance to Gp0.4 expression. To demonstrate that the mutation ftsZ renders the cells resistant to inhibition by Gp0.4, the present inventors used PI phage to transduce the mutation back into the parental E. coli cells that were sensitive to Gp0.4 toxicity. These cells were then transformed with a plasmid encoding Gp0.4 and were tested for their susceptibility to Gp0.4 overexpression. All bacterial cells that were transduced with the altered ftsZ became resistant to Gp0.4 overexpression, whereas control cells that were transduced with the wild-type (WT) ftsZ remained sensitive to Gp0.4 overexpression (Figure 2B). These results indicated that Gp0.4 specifically inhibits FtsZ, and that this inhibition can be overcome by alteration of FtsZ at a specific site.

EXAMPLE 3

Purified Gp0.4 Inhibits Purified FtsZ To rule out indirect interaction of Gp0.4 with FtsZ, FtsZ was purified to homogeneity and a chemically synthesized Gp0.4 peptide was purchased. Inhibition of FtsZ assembly by Gp0.4 was examined by fluorescence-based assay. This assay monitors filament assembly of an altered FtsZ (S 151C/Y222W), via changes in the quenching of the fluorescent dye BODIPY coupled to the cysteine at position 151, as described previously [14]. In this assay, there is no assembly until the concentration of FtsZ reaches a critical concentration (Cc), above which there is a linear increase in fluorescence upon assembly. Addition of increasing concentrations of Gp0.4 inhibited assembly of the altered FtsZ filaments, as demonstrated by an upward shift in the apparent Cc (Figure 3). The mutant protein FtsZ9 was also purified to homogeneity, however, its assembly activity as well as its GTPase activity could not be detected, consistent with previous reports on the activity of FtsZ9 protein [13]. Therefore, its sensitivity to Gp0.4 could not be assessed. Nevertheless, the results indicate that Gp0.4 directly blocks the assembly of WT FtsZ in vitro.

EXAMPLE 4

Expression of Gp0.4 Results in Elongation of Bacteria Encoding FtsZ If Gp0.4 inhibition of FtsZ indeed directly inhibits FtsZ in vivo, then its expression should result in elongation of the cells. In contrast, bacterial cells having the resistant form FtsZ9, should show a normal morphology upon Gp0.4 expression. To test this, the inventors used light microscopy to monitor bacterial morphology of WT E. coli and an E. coli encoding the ftsZ9 mutant, both induced to express Gp0.4 at low levels (0.001% arabinose). Indeed, E. coli cells harboring the plasmid encoding Gp0.4, but not those harboring a control empty vector, demonstrated cumulative elongation at all time points of the experiment (Figure 4). E. coli encoding ftsZ9 were significantly less affected by Gp0.4 expression at the expression levels used, however, at higher induction of expression levels (0.2.% arabinosse), these bacteria were also inhibited. These results demonstrated that Gp0.4 inhibits division of cells encoding WT FtsZ. EXAMPLE 5

Gp0.4 Is Toxic in Bacterial Strains Lacking Endogenous FtsZ Inhibitors

To demonstrate that GpO.4 directly interacts with FtsZ, rather than via enhancement of endogenous FtsZ inhibitors, the inventors tested GpO.4 toxicity in isogenic strains lacking the endogenous FtsZ inhibitors as is the case for DicB [8]. E. coli lacking the indicated genes (AminC and AsulA) or a control gene (aceF) were transformed with the plasmid encoding GpO.4 and grown with L-arabinose induction (+ ara) or without induction (- ara). Relative growth was calculated by dividing the number of CFUs obtained in the induced samples by the uninduced samples of each corresponding strain. Bars represent average + SD of three independent experiments. Notably, similar toxicity was observed in isogenic strains lacking minC and sulA (Figure 5), indicating that GpO.4 does not exert its effect through enhancement of the activity of these two known endogenous inhibitors of FtsZ.

EXAMPLE 6

Gp0.4 Increases Competitiveness of Bacteriophage T7 by Inhibiting Cell Division

Finally, the inventors tested the hypothesis that GpO.4 may confer competitive advantage to the phage infecting a dividing cell. If a cell divides early in infection, while there is only a single phage genome in the cell, one daughter cell will escape and phage propagation will be confined to only half of the cell resources. However, if the phage inhibits this daughter-cell escape using GpO.4, then the entire cell resources are available for its progeny. To test if this is indeed the case, the inventors generated T7 phages lacking gene 0.4 (Τ7Δ0.4) and compared its competitive ability against the WT T7_FRT (a phage having a similar "genetic scar" as does Τ7Δ0.4, but encodes gene 0.4 in dividing or non-dividing hosts. It would be expected that the WT T7_FRT phage would have a significant advantage over Τ7Δ0.4 in dividing hosts, whereas this advantage would vanish in non-dividing hosts. To determine the relative abundance of Τ7Δ0.4 compared to WT T7_FRT in a phage mixture, a PCR that amplifies the region flanking gene 0.4 was carried out. This PCR discriminates between the two phages because amplification of the WT T7_FRT results in a longer product than amplification of the deletion mutant. As can be seen in Figure 6A, this assay is quantitative, enabling detection of different ratios of phage mixtures. To measure competition ability, the inventors used a mixture containing an equal ratio of WT T7_FRT to Τ7Δ0.4 to infect dividing E. coli hosts in the exponential growth phase and non-dividing E. coli hosts in the stationary growth phase. Phage lysates were collected following each infection cycle and PCR was carried out on these samples to measure the ratio of each phage. A phage that is more competitive will produce more progeny in each infection cycle, and therefore will eventually outcompete the other phage. As shown in Figure 6B, WT T7_FRT had a significant growth advantage on exponentially growing bacteria, indicating that Gp0.4 significantly increases the competitiveness of WT T7_FRT phage on dividing bacteria. Remarkably, on non- dividing hosts, the trend reversed showing that WT T7_FRT was significantly less competitive than Τ7Δ0.4 (Figure 6C). These results suggest that inhibition of cell division by Gp0.4 confers an advantage to the phage. However, since stationary-phase bacteria also differ from exponential- phase bacteria in aspects other than cell division, the inventors further specifically tested the effect on division inhibition using E. coli encoding FtsZ9, the variant identified as resistant to Gp0.4 inhibition. It would be expected that in this host, growing exponentially, the competitive advantage of WT T7_FRT would be reduced because FtsZ9 is not inhibited. Indeed, on FtsZ9- encoding hosts WT T7_FRT had a significantly lower competitive advantage compared to its advantage on FtsZ-encoding hosts (Figure 6D). The fact that WT T7_FRT still retains some competitive advantage is most likely because FtsZ9 is not completely refractory to Gp0.4 (Figure 4). To verify that the measured DNA quantity in the above experiments corresponds to functional assembled phages, dilution of the ly sates from the last cycle of each experiment were plated. Individual plaque forming units (PFU) were then picked and analyzed by PCR to determine whether they are WT T7_FRT or Τ7Δ0.4. As expected, the percentage of the PFU in each lysate corroborated the results obtained by the quantitative PCR assay. The percentage of functional assembled phages of WT T7_FRT recovered from the exponential phase was 78%, from the stationary phase - 20%, from FtsZ-encoding cells - 80% and from FtsZ9-encoding cells - 60%. These results indicate that the measured DNA levels correspond to functional phage particles. It should be noted that single-step burst size experiments showed that Τ7Δ0.4 produces less progeny per infected bacterium compared with WT T7_FRT Taken together, these results indicated that Gp0.4 has a physiological role in conferring a competitive advantage to the T7 phage through inhibition of FtsZ and consequently, cell division.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

CLAIMS:

1. A Gp0.4 T7 bacteriophage protein, any homolog or related protein of the T7-like bacterial viruses, or any composition comprising the same for use in a method of treating an infectious disease in a subject in need thereof.

2. The Gp0.4 bacteriophage protein of claim 1, wherein said homolog or related protein of the T7-like bacterial viruses is any one of the hypothetical protein YpP-R from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06.

3. The Gp0.4 bacteriophage protein of claim 1, wherein said protein comprises an amino acid sequence at least 80 % homologous to the sequence as set forth in SEQ ID NO: 10.

4. The Gp0.4 bacteriophage protein of claim 1, wherein said protein comprises an amino acid sequence at least 90 % homologous to the sequence as set forth in SEQ ID NO: 10.

5. The Gp0.4 bacteriophage protein of claim 1, wherein said protein comprises an amino acid sequence as set forth in SEQ ID NO: 10 or any derivatives or fragments thereof.

6. The Gp0.4 bacteriophage protein of claim 1, wherein said protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 11-42.

7. The Gp0.4 bacteriophage protein of claim 1, wherein said protein is further attached to an agent that enhances penetration across the bacterial membrane.

8. The Gp0.4 bacteriophage protein of claim 5, wherein said agent is a peptide agent.

9. The Gp0.4 bacteriophage protein of claim 1, wherein said Gp0.4 T7 bacteriophage protein is attached to a sustained-release enhancing agent.

10. The Gp0.4 bacteriophage protein of claim 1, wherein said infectious disease is caused by a bacterial infection.

11. A Gp0.4 T7 bacteriophage protein, any homolog or related protein of the T7-like bacterial viruses, or any composition comprising the same for use in a method of inhibiting bacterial growth.

12. The Gp0.4 bacteriophage protein of claim 11, wherein said bacteria express the FtsZ protein.

13. The Gp0.4 bacteriophage protein of claim 11, wherein said homolog or related protein of the T7-like bacterial viruses is any one of the hypothetical protein YpP-R from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06.

14. The Gp0.4 bacteriophage protein of claim 11, wherein said Gp0.4 T7 bacteriophage protein comprises an amino acid sequence at least 80 % homologous to the sequence as set forth in SEQ ID NO: 10.

15. The Gp0.4 bacteriophage protein of claim 11, wherein said Gp0.4 T7 bacteriophage protein comprises an amino acid sequence at least 90 % homologous to the sequence as set forth in SEQ ID NO: 10.

16. The Gp0.4 bacteriophage protein of claim 11, wherein said Gp0.4 T7 bacteriophage protein comprises an amino acid sequence as set forth in SEQ ID NO: 10 or any derivatives or fragments thereof.

17. The Gp0.4 bacteriophage protein of claim 11, wherein said Gp0.4 T7 bacteriophage protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 11-42.

18. A pharmaceutical composition comprising a Gp0.4 T7 bacteriophage protein or any homolog or related protein of the T7-like bacterial viruses, as the active ingredient and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition of claim 18, wherein said homolog or related protein of the T7-like bacterial viruses is any one of the hypothetical protein YpP-R from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06.

20. The pharmaceutical composition of claim 18, wherein said Gp0.4 T7 bacteriophage protein comprises an amino acid sequence at least 80 % homologous to the sequence as set forth in SEQ ID NO: 10.

21. The pharmaceutical composition of claim 18, wherein said Gp0.4 T7 bacteriophage protein comprises an amino acid sequence at least 90 % homologous to the sequence as set forth in SEQ ID NO: 10.

22. The pharmaceutical composition of claim 18, wherein said Gp0.4 T7 bacteriophage protein comprises an amino acid sequence as set forth in SEQ ID NO: 10 or any derivatives or fragments thereof.

23. The pharmaceutical composition of claim 18, wherein said Gp0.4 T7 bacteriophage protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 11-42.

24. The pharmaceutical composition of claim 18, wherein said Gp0.4 T7 bacteriophage protein is further attached to an agent that enhances penetration across the bacterial membrane.

25. The pharmaceutical composition of claim 24, wherein said agent is a peptide agent.

26. The pharmaceutical composition of claim 18, wherein said Gp0.4 T7 bacteriophage protein is attached to a sustained-release enhancing agent.

27. The pharmaceutical composition of claim 18, wherein said composition further comprises an additional therapeutic agent.

28. The pharmaceutical composition of claim 18, wherein said Gp0.4 T7 bacteriophage protein or any homolog or related protein of the T7-like bacterial viruses, is formulated for topical delivery.

29. Use of a therapeutically effective amount of Gp0.4 T7 bacteriophage polypeptide or any homolog or related protein of the T7-like bacterial viruses, in the preparation of a composition for treating an infectious disease.

30. The use of claim 29, wherein said homolog or related protein of the T7-like bacterial viruses is any one of the hypothetical protein YpP-R from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06.

31. The use of claim 29, wherein said disease is caused by a bacterial infection.

32. A kit comprising:

a. a Gp0.4 T7 bacteriophage protein, any homolog or related protein of the T7-like bacterial viruses or any fragment, peptide, analogues and derivatives thereof, optionally, in a first dosage form; and

b. at least one additional therapeutic agent, optionally, in a second dosage form.

33. The kit of claim 32, wherein said homolog or related protein of the T7-like bacterial viruses is any one of the hypothetical protein YpP-R from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06.

34. The kit of claim 32 for use in a method of treatment of an infectious disease.

35. A solid support coated with a Gp0.4 T7 bacteriophage protein or with any homolog or related protein of the T7-like bacterial viruses.

36. The solid support of claim 35, wherein said homolog or related protein of the T7-like bacterial viruses is any one of the hypothetical protein YpP-R from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06.

37. The solid support of claim 35, wherein said Gp0.4 T7 bacteriophage protein comprises an amino acid sequence at least 80 % homologous to the sequence as set forth in SEQ ID NO: 10.

38. The solid support of claim 35, wherein said Gp0.4 T7 bacteriophage protein comprises an amino acid sequence at least 90 % homologous to the sequence as set forth in SEQ ID NO: 10.

39. The solid support of claim 35, wherein said Gp0.4 T7 bacteriophage protein comprises an amino acid sequence as set forth in SEQ ID NO: 10 or any derivatives or fragments thereof.

40. The solid support of claim 35, wherein said Gp0.4 T7 bacteriophage protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 11-42.

41. A method of treating an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a Gp0.4 T7 bacteriophage protein, any homolog or related protein of the T7-like bacterial viruses, or any composition comprising the same, thereby treating said disease.

42. The method of claim 41, wherein said homolog or related protein of the T7-like bacterial viruses is any one of the hypothetical protein YpP-R from Yersinia phage YpsP-G and the hypothetical protein Vi06_01 from Salmonella phage Vi06.

43. The method of claim 41, wherein said Gp0.4 T7 bacteriophage protein comprises an amino acid sequence as set forth in SEQ ID NO: 10 or any derivatives or fragments thereof.

44. The method of claim 41, wherein said Gp0.4 T7 bacteriophage protein is further attached to an agent that enhances penetration across the bacterial membrane.

45. The method of claim 41, wherein said infectious disease is caused by a bacterial infection.

46. A method of inhibiting bacterial growth, the method comprising contacting said bacteria with a cytotoxic effective amount of a Gp0.4 T7 bacteriophage protein, thereby inhibiting bacterial growth and killing the bacteria.

47. The method of claim 46, wherein said bacteria express the FtsZ protein.

48. The method of claim 41, wherein said contacting is affected in vivo or ex vivo.