WO2006066224A2 - Virulence targeted antibiotics - Google Patents

Abstract

The disclosure provides, in part, antimicrobial agents in the form of bacteriophage or bacteriocins that are designed so as to target one or more virulence factors of a pathogenic bacterium ('Virulence factor Targeted Bacteriophage' ['VTB'] or 'Virulence factor Targeted Bacteriocins' ['VTBC'], respectively).

Description

VIRULENCE TARGETED ANTIBIOTICS

RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 60/636238, filed December 14, 2004, entitled "Virulence Targeted Antibiotics." The provisional application is incorporated by reference herein.

BACKGROUND

Diseases caused by pathogenic bacteria contribute substantially to human mortality, especially in developing countries where people often lack access to medical care, sanitation and/or safe drinking water. Many of these diseases are caused by emerging bacterial pathogens, defined by their increasing incidence in human populations. The successful emergence of a pathogen is generally a two-step process. An interaction between humans and an emerging pathogen arises, often through an ecological change such as greater proximity of humans to an animal reservoir of potentially pathogenic bacteria, or through human exposure to bacteria that are ordinarily benign but that have acquired virulence traits through horizontal gene transfer. Subsequently, natural selection may cause the bacteria to become better adapted to humans (or to a disease vector) through mutations that improve infection and pathogenicity. Thus, through time, emerging pathogens may evolve increased virulence, allowing these pathogens to efficiently exploit humans as a host. Most bacteria that are pathogenic to humans have a set of genes that are important for virulence and pathogenicity but are not essential for commensal growth, growth outside the human body or other non-pathogenic growth conditions. The proteins encoded by these genes are referred to as virulence factors.

Antibiotics are widely relied upon to treat bacterial infections. Antibiotics have traditionally been designed to interfere with critical cellular processes, such as protein synthesis or DNA replication. Thus, a traditional antibiotic will generally kill a bacterium regardless of the environment in which the bacterium is found. Bacterial pathogens have evolved mechanisms that attenuate or compensate for the deleterious effects of exposure to antibiotics. The high frequency of drug resistance in pathogens suggests that antibiotic resistance traits can be acquired without substantial effect on the overall fitness of the pathogen. That is, traditional antibiotics do not interfere with host/pathogen interactions. The end result is that pathogenic bacteria resistant to most, if not all, available antibiotics are increasingly common. It is now crucial to develop new antimicrobial therapies.

SUMMARY

The disclosure provides, in part, antimicrobial agents in the form of bacteriophage (or "phage") or bacteriocins that are designed so as to target one or more virulence factors of a pathogenic bacterium ("Virulence factor Targeted Bacteriophage" ["VTB"] or "Virulence factor Targeted Bacteriocins" ["VTBC"], respectively). Because VTBs target virulence factors, it is expected that pathogens will develop resistance to VTBs only rarely, and with detrimental effects on the ability of the resistant strain to cause disease. The disclosure further provides methods for generating VTBs and methods for isolating targeting proteins that mediate the binding of bacteriophage or bacteriocins to the target virulence factors.

In certain aspects, the disclosure provides methods for preparing a VTB. A method may comprise altering the genome of a bacteriophage such that the genome comprises a nucleic acid encoding a virulence factor targeting protein. The process of altering the genome may include insertion, deletion or substitution of nucleotides, and may be achieved, for example, by mutagenesis, by techniques of molecular biology (e.g., splicing by restriction digest or polymerase chain reaction), by a combination thereof, or by other known techniques for introducing variability into the genome of the bacteriophage. For example, a bacteriophage that binds to a virulence factor may be selected from a library in which side tail fibers of the library phage contain representatives of a randomized peptide sequence (a variability cassette). Following selection of a variant bacteriophage that binds to the virulence factor, binding may be improved by subsequent rounds of mutagenesis of the selected bacteriophage. Mutagenesis may be achieved, for example, chemically, with a mutagen such as an alkylating agent or by production of the bacteriophage in a mutagenic bacterial host (e.g., a,MutT E. coli strain), hi certain embodiments, the virulence factor targeting protein is a variant of the a protein of the bacteriophage that normally mediates binding to a host microorganism, hi the case of the T4-like bacteriophages, the virulence factor targeting protein may be a tail fiber protein, such as p37 in the T4 phage. In certain embodiments, the nucleic acid encoding the virulence factor targeting protein has a position and orientation in the genome of the bacteriophage such that the virulence factor targeting protein is incorporated into the tail fiber of the bacteriophage. In certain embodiments, virulence factor targeting protein binds to a virulence factor of a Gram negative pathogen, particularly a member of the Enterobacteriaceae. The bacteriophage may be essentially any bacteriophage that will infect the target cell type. Lytic phages are often preferred; however, in some instances a lytic phage may be modified such that the phage kills the target bacteria without causing lysis, hi certain embodiments, the bacteriophage is a member of the family Myoviridae, including the T4-like bacteriophages, which are particularly useful as agents for killing enteropathogens. A VTB may also be constructed with a bacteriophage from the Siphoviridae family, which includes phage lambda, although it is expected that a pharmaceutical agent for administration will generally not be derived from phage lambda. In certain aspects, the disclosure provides methods for selecting a virulence factor targeting protein, hi general, such a method will involve screening a set of partially randomized polypeptides to identify those that bind to a virulence factor of interest. The partially randomized polypeptides may be based on the target binding protein of a particular phage, such as a tail fiber protein of a phage of the Siphoviridae or Myoviridae families. A tail fiber protein may be designed to include a region or a non-contiguous set of amino acid positions, which may occur at either end of the tail fiber protein, within the tail fiber protein or both, that introduces amino acid sequence variation (referred to as a "variability cassette"). A variability cassette may introduce variation through amino substitutions, additions, deletions or a combination thereof. If desired, the entire tail fiber protein may be subjected to variation, in which case the "variability cassette" should be understood as including the entire protein. For example, the nucleic acid coding for the p37 polypeptide of T4 may be randomized or otherwise modified relative to a naturally occurring form to generate a pool of variant p37 polypeptides that can be screened for binding to the virulence factor of interest. In addition to randomized polypeptide sequences, essentially any type of binding protein framework can be used to generate polypeptides that bind to the target virulence factor, including, for example, single chain antibodies and other binding proteins based on one or more antibody variable domains (e.g., V_H, V_L or V_HH)- The variants may be incorporated into a phage particle for the screening process, preferably a phage particle of a type that has been optimized for phage display screening. Variants may be expressed as a fusion protein with a proximal portion of the phage lambda Stf protein. The binding polypeptide may be selected in the context of a phage protein, or it may be selected independently (e.g., as a purified protein, optionally immobilized on a solid substrate) and later recombined with the appropriate phage protein. Ih the screening process, any of the various methods for screening protein variants may be used, including phage display techniques, PROfusion™ and other systems that allow the recovery of a nucleic acid encoding the binding polypeptide along with the polypeptide itself. In certain embodiments, a method comprises contacting a plurality of variants of a distal tail fiber protein of a bacteriophage of the family Myoviridae (e.g., T4-like phages) with a virulence factor. Preferably the virulence factor is derived from a Gram negative bacterium, particularly a pathogenic member of the Enterobacteriaceae. The term "virulence factor" includes portions of virulence factors, such as the extracellular portion, that are sufficient for the described purpose. For example, in the case of a virulence factor that is a transmembrane protein, an extracellular portion of a virulence factor may be sufficient to select for variants that bind to the virulence factor. After bringing the variants into contact with the virulence factor of interest, those variants that bind to the virulence factor may be enriched for by various techniques. The virulence factor may be immobilized in some way, as by linkage to beads or other insoluble substrate or by expression on cells, particularly a bacterial cell that is a laboratory strain of E. coli or a cell of a Gram-negative pathogen. Where the variants are expressed as part of a phage particle, the process of enrichment may comprise recovering phage that bind to purified virulence factor or to bacterial cells expressing the virulence factor. Phage particles that bind to a bacterium may also lyse or otherwise alter the bound bacterium, and phage particles that cause such alteration can likewise be recovered. In certain aspects, the disclosure provides a phage particle, particularly a phage lambda particle or other member of the Siphoviridae family comprising a variant of a distal tail fiber protein of a member of the Myoviridae family, particularly members of the T4- like group. The variant may comprise a virulence factor binding element, and particularly one that binds to a virulence factor of a Gram-negative pathogen, hi a preferred embodiment, the variant is expressed as a fusion protein with a proximal portion of the phage lambda Stf protein.

In certain aspects the disclosure provides libraries of variants of the distal tail fiber protein of a bacteriophage of the family Myoviridae, particularly members of the T4 group. A library will typically comprise at least 10⁴, and optionally 10⁵, 106, 10⁷, 10⁸, 10⁹ or more, species of variants of the distal tail fiber protein. Each of said variants may comprise a variability cassette having a polypeptide sequence that is distinct to each of said species of variant. The library may comprise multiple copies of each species, and may also comprise wild type distal tail fiber proteins or other proteins entirely. A library may also comprise nucleic acids that encode for such variants. Preferably, the distal tail fiber protein is the p37 protein of a T4 group bacteriophage.

In certain aspects, the disclosure provides a phage display library, such as a phage lambda library, a T4 library, an M 13 library, or any other phage display library (including phagemid libraries) that are known in the art. Such library will comprise at least 10⁴, and optionally 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ or more species of modified phage particle. Each of said species may comprise a variant of a distal tail fiber protein of a bacteriophage, such as a bacteriophage of the family Myoviridae, particularly members of the T4 group. Each variant of the distal tail fiber protein may comprise a variability cassette that is distinct to each of said species. The variants of the distal tail fiber protein may be expressed as fusion proteins with a phage lambda side tail fiber polypeptide, or other suitable display peptide with respect to other phage display libraries. In a preferred embodiment, the fusion protein comprises a proximal portion of the phage lambda Stf polypeptide, a distal portion of the T4 p37 polypeptide, and a variability cassette, which variability cassette may be positioned between the Stf and p37, within the p37, at the distal end of p37 or some combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 provides a diagram for a process of generating a T4-based VTB. Figure 2 provides a diagram of a wild type T4 bacteriophage. The numeric labels refer to the various proteins of T4. p37 is the distal protein of the T4 side tail fiber.

Figure 3 provides a genetic map of the tail fiber regions of wild type lambda and Ur- lambda* phage, showing the position of homology to the T4 g37 and g38.

Figure 4 provides an alignment of distal tail fiber proteins (gp37) from members of the T4 group of bacteriophage.

Figure 5 illustrates a genetic scheme for moving peptide display libraries into phage lambda.

Figure 6 shows a ribbon diagram of the structural organization of colicin Ia.

Figure 7 (SEQ ID NO:1) shows the sequence and map of a phage lambda construct engineered to contain the loxP cloning cassette and a tetracycline resistance marker. The lambda side tail fiber genes were also deleted from this construct. Figure 8 presents a LoxP recombination cassette constructed for moving tail fiber gene constructs into the engineered lambda phage, (SEQ ID NO:2).

Figure 9 presents the sequence for a nucleic acid encoding a chimeric tail fiber was constructed and the T4 g38 gene (required for proper assembly) was cloned between the LoxP sites in order to move the T4 tail fibers into phage lambda. (SEQ ID NO:3).

DETAILED DESCRIPTION 1. Overview

The disclosure relates, in part, to the insight that VTBs will provide a new generation of drugs that may, in addition to eliminating targeted bacterial pathogens, force such pathogens into an evolutionary tradeoff between resistance and virulence. A VTB is a bacteriophage that is designed or selected to bind to one or more surface-exposed virulence factors, including, for example, components necessary for host infection, disease progression, vector transmission, and toxin export. The term "virulence factor", as used herein, means a factor, typically a protein, that is important for the ability of the pathogen to cause disease in a host organism, but is not essential for growth in a non-pathogenic milieu. Non-pathogenic growth is often assessed in laboratory growth media or in a natural environment where such pathogens may occur in a non-pathogenic state. The contribution of a virulence factor to pathogenic versus non-pathogenic functions of a bacterium will generally be assessed by analysis of loss-of-function mutations in the gene encoding the virulence factor.

As with traditional antibiotics, pathogens are likely to develop resistance to VTBs. However, because a VTB will bind specifically to one or more virulence factors, the resistance would generally occur through loss or modification of the virulence factors themselves. Therefore, the resistant pathogen is also likely to have decreased virulence and will likely pose a decreased health risk. Thus, modification of a virulence factor under selective pressure from VTB treatment can move the pathogen away from a 'fitness peak' in a pathogenic setting, resulting in a tradeoff involving sub-optimal virulence.

The VTBs disclosed herein are most easily developed by genetically engineering lytic phages, viruses that have evolved for millennia to specifically attack and kill bacteria. Shortly after their discovery in 1915, phages were successfully used to treat avian typhosis in chickens, shigella dysentery in rabbits, and bacillary dysentery {Shigella) in humans. In the mid-twentieth century, clinical researchers in the Soviet Union and Eastern Europe conducted hundreds of studies indicating that phage therapy is a potential alternative to antibiotics for treating bacteria-related diseases of humans. A variety of advantages of phage therapy, relative to traditional antibiotics, are set forth in the table below.

Table 1: Advantages of traditional phage therapy

One of the significant benefits of using a phage to treat human disease is that the virus is generally harmless to mammalian cells, and even to non-target bacteria. By contrast, broad-spectrum antibiotics can wipe out beneficial flora in the intestinal tract along with the targeted pathogen, and may give rise to superinfections by highly resistant bacteria such as Clostridium difficile. High specificity, however, can be a double-edged sword. The specificity of phages for infecting and destroying a narrow range of bacterial hosts may limit their effectiveness because physicians would not generally know the precise bacterial strain that is causing a particular infection, or how susceptible a pathogen would be to a particular therapeutic phage. Furthermore, if only one phage is used against a particular bacterium, the pathogen may quickly evolve resistance to phage attack, although some studies suggest that the rate of bacterial resistance to phages is roughly 10-fold lower than that for antibiotics. Many of these difficulties can be addressed by using a mixture of multiple phages (a phage cocktail) that target a variety of different hosts, or that attack the same host to delay or control the appearance of phage-resistant cells. Studies have shown that phage cocktails can be more effective than single phages in controlling pathogenic bacteria.

In most instances of phage resistance that have been studied, the resistant bacteria exhibit changes in their outer membrane components responsible for specific phage binding (phage receptors). Alteration or deletion of phage receptors from the cell surface protects bacteria from phage attack. Because these changes do not necessarily affect host fitness, the desired tradeoff between resistance and virulence may not be achieved using traditional phage therapy. However, there are instances of naturally occurring phages that have evolved to bind to host virulence factors. For example, the RNA phage phi-6 infects Pseudomonas syringae pathovar phaseolicola, a plant pathogen that causes a severe disease of beans, known as halo blight. Type-IV pili allow the bacteria to attach to leaf surfaces, and the presence of pili positively correlates with the ability of the bacteria to cause disease. Phage phi-6 uses the type-IV pilus as an initial site of attachment to host bacteria, and it appears that pilus retraction into the cell contributes to the ability of the phage to fuse with the cell membrane and initiate infection. Non-piliated mutants of P. phaseolicola are both resistant to phage phi-6 attack and show a markedly reduced ability to infect leaves. Thus, phage therapy using phi-6 should create the desired tradeoff between resistance and virulence. Unfortunately this example of naturally occurring, virulence factor targeted phage appears to be the exception, rather than the rule. Despite the resurgence of interest in phage therapy, there are significant limitations to the existing approaches. Traditional phage therapy relies on 'phage-prospecting' where naturally isolated phages are collected and screened for the ability to kill a target pathogen. The method can be efficient for identifying phages useful for therapy, but other aspects of phage biology might be ignored due to the cost of completely characterizing the new virus. Without proper evaluation, it would be unknown whether the phage is capable of lysogeny (non-lethal incorporation into the host genome) or whether the phage harbors harmful or dangerous genes (e.g., toxin-producing genes). Furthermore, there exists the possibility (however rare) that a lytic phage can mistakenly package host chromosomal genes, and horizontally transfer these genes from a donor bacterium to a recipient bacterium; a mechanism known as generalized transduction. Thus, a therapeutic phage might inadvertently move a virulence factor from a target pathogen to a non-target bacterium, possibly generating a new pathogen in the process. Most importantly, as with traditional antibiotics, phage therapy may select for resistant strains of the target pathogen. The long history of coevolution between phage and bacteria has resulted in selection pressure for bacteria to be highly variable in terms of binding sites for naturally occurring phage. Traditional phage therapy seldom investigates the binding sites for naturally isolated virus candidates and, therefore, no preference is given to using phages that target virulence factors. For this reason, resistance to these naturally occurring phages may not impose a significant cost in terms of pathogenicity of the targeted pathogen.

A rapid and efficient method of creating VTBs would not only enjoy all of the benefits of standard phage therapy (Table 1), but would avoid many of the limitations. By starting with a well-characterized lytic phage, such as T4, it is possible to minimize or eliminate the risk of introducing unknown dangerous genes from "wild" phage. The risk that a VTB would move virulence genes from pathogens to non-pathogens through generalized transduction is reduced because VTBs would be, by design, unlikely to infect non-pathogens.

Although phage resistance due to loss of the cellular receptor site is a limitation of standard phage therapy, it is a fundamental strength of VTBs. Because resistance to VTBs is not a negative outcome for the growth of the target bacteria in a non-pathogenic setting, the prophylactic use of VTBs should have little or no impact on their clinical usefulness. This would also be true for agricultural or environmental applications; for instance VTBs could be used to reduce (or attenuate the virulence of) pathogens in reservoir hosts (e.g., the human diarrheal-pathogens Escherichia coli O157:H7 in cattle, and Salmonella enterica in poultry and eggs).

VTBs can also be easily adapted into a rapid detection system that can simultaneously determine the presence of a dangerous pathogen and the specific VTB capable of treating it. For instance, a phage specific for a particular pathogen can be engineered to express a luciferase reporter gene. Environmental or clinical samples incubated with different cocktails of these "reporter VTBs" would luminesce if they contained the target pathogen. This would simultaneously indicate the presence of the targeted pathogen and which VTB cocktail could be used against it. Phage based luminescence detection methods are already in use for the detection of drug resistant Mycobacteria.

Accordingly, the present disclosure provides methods for preparing a virulence factor targeted bacteriophage, the method comprising, altering the genome of a bacteriophage such that the genome comprises a nucleic acid encoding a virulence factor targeting protein.

2. Bacteriophage:

A wide variety of bacteriophages may be used to generate the VTBs described herein. Bacteriophages are viruses that specifically infect and replicate within bacterial cells. Phage can be divided into two basic groups, lytic and temperate. In the case of lytic phages, such as T4, the host cell dies (undergoes lysis) as a result of infection, releasing hundreds of phage progeny into the environment that can initiate subsequent infections. Temperate (or lysogenic) phage, such as λ, can replicate as lytic phages, but are also capable of entering into a stable association with the host bacterium (lysogeny) in which the phage genome persists in a quiescent state (a prophage). In general, a lytic phage will be preferred for VTBs, although the lytic phage may be engineered such that host cell killing occurs through a process other than lysis. For example, one or more lysins encoded by the VTB phage may be deleted or disabled, in which case the phage may nonetheless kill host cells by production of holins or other damaging or toxic factors.

The appropriate bacteriophage for use in a VTB will be selected depending on the pathogen to be targeted. Pathogens contemplated herein include human pathogens, as well as animal and plant pathogens.

The bacteriophage of the family Myoviridae represent a suitable group of phage, particularly for use as antimicrobial agents against bacteria of the Enterobacteriaceae. The Myoviridae are presently understood to include the following genera: Genus "T4-like phages", Genus "Pl -like phages", Genus "P2-like phages", Genus "Mu-like phages", Genus "SPOl-like Phages" and Genus "PhiH-like viruses". Virions typically contain roughly 48% nucleic acid, consisting of one molecule of linear double stranded DNA. The total genome length is typically in the range of 150 - 400 thousand nucleotides. The virions are not enveloped but consist of head and tail portions. The head is separated from the tail by a neck, a tail complex, consisting of a central tube and a contractile sheath, provided with a collar, base plate, 6 short spikes and 6 long fibers. Nucleocapsids are isometric to quasi- isometric and elongated with typical dimensions of 95-111 nm long and 65-80 nm in diameter. The symmetry is usually icosahedral. Typical dimensions for the tail are 80-455 nm long and 16 nm wide. The T4-like phages, and particularly the T4 phage are preferred phages for use in constructing VTBs. The bacteriophage of the family Siphoviridae represent a group of phage, including phage lambda, that are well-suited for use in phage display libraries. Phage of this family are double-stranded DNA viruses characterized by a long non-contractile tail and an isometric capsid (morphotype Bl) or a prolate capsid (morphotype B2). The Siphoviridae viruses have a capsid with a diameter of about 55-60 run and a long tail that can reach up to about 570 run. The double-stranded DNA is linear. This family includes the phage \ the phage x and the phage φ80. The family includes the following genera: Genus λ-like viruses, including Enterobacteria phage λ; Genus Tl -like viruses, including Enterobacteria phage Tl; Genus T5-like viruses, including Enterobacteria phage T5; Genus c2-like viruses, including Lactococcus phage c2; Genus L5-like viruses, including Mycobacterium phage L5; Genus ^Mi-like viruses, including Methanobacterium ^Ml; Genus φC31-like viruses, including Streptomyces phage φC31; and Genus N15-like viruses, including Enterobacteria phage Nl 5.

Other phage for use in phage display libraries include the Ml 3 phage.

3. Target pathogens:

The literature suggests that for every bacterium, a corresponding bacteriophage can be isolated. Moreover, it is reasonable to expect that any pathogenic bacterium will have virulence factors that facilitate the infective activities of the bacterium. Therefore, the concepts presented herein can be applied to essentially any pathogenic bacterium, regardless of whether it is a human pathogen, a pathogen for a non-human animal (such as livestock) or a pathogen for plants.

In certain embodiments, the targeted pathogen is a member of the Enterobacteriaceae, for which members of the Myoviridae family of phage provide effective killing activity. Members of the Enterobacteriaceae are typically small Gram- negative non-spore forming enteric bacilli that are oxidase negative, typically ferment glucose with acid production and have the ability, under appropriate conditions, to reduce nitrates. All members of this group are aerobic but can be facultatively anaerobic and exhibit motility via peritrichous flagella, except Shigella and Klebsiella which are non- motile. Members of this group may have a capsule, a slime layer, or neither, and will typically have fimbriae (pili) and a complex cell wall. The antigenic structure is often significant in epidemiology and classification. The family includes over 30 genera and over 120 species. The clinically important enteric bacteria are as follows: Citrobacter (C. freundii, C. diversus); Enterobacter (E. aerogenes, E. agglomerans and E. cloacae); Escherichia coli (Opportunistic E. coli: enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli and uropathogenic E. coli); Klebsiella (K pneumoniae and K. oxytoca); Morganella morganii, Proteus (P. mirabilis, P. vulgaris); Providencia (P. alcalifaciens, P. rettgeri, P. stuartii); Salmonella (S. enterica, which under present nomenclature encompasses all Salmonella); Serratia (S. marcesans, S. liquifaciens); Shigella (S. dysenteriae, S.flexneri, S. boydii, S. sonnei); and Yersinia (Y. enterocolitica, Y. pestis, Y. pseudotuberculosis). Members of the following genera are most notably pathogenic: Salmonella, Shigella, Yersinia and certain strains of Escherichia coli. Most members of the Εnterobacteriaceae are opportunistic or cause secondary infections of wounds, the urinary and respiratory tracts, and the circulatory system, occasionally resulting in a life-threating Gram-negative sepsis. Other disorders caused by members of this group include urinary tract infections (typically E. coli), pneumonia (typically K. pneumoniae), abdominal sepsis, meningitis (typically E. coli), spontaneous bacterial peritonitis (typically E. coli), endocarditis (rarely, caused by Gram- negative rods).

The following types of proteins tend to be virulence factors: toxins, adhesins, pili, lipopolysaccharide, proteases, hemolysins, secretion systems, capsules, pathogenicity islands and regulatory pathways. The type III secretion system of Yersinia species is an example of a virulence factor.

Diarrheal disease is also caused by members of the Εnterobacteriaceae. This is a rare occurrence for most people who live in the U.S. and other developed countries where sanitation is widely available, access to safe water is reliable, and personal and domestic hygiene is relatively good. World-wide around 1.1 billion people lack access to improved water sources and 2.4 billion have no basic sanitation. For these reasons, diarrheal disease is widespread throughout the developing world. Among children aged 5 years and younger, the annual burden of diarrhea is estimated to be 1.5 billion episodes, accounting for as many as 3 million deaths. Diarrheagenic Escherichia coli are the enteropathogens most frequently isolated

(over 210 million diarrhea episodes), and account for approximately 380,000 annual deaths for children under the age of 5 in the developing world. These bacteria are usually associated with childhood disease, due to the substantially higher incidence in early childhood than in older age groups. However, surveillance of hospitalized cases of diarrheagenic E. coli has shown that almost half of such cases occur in persons aged >10 years. Diarrheagenic E. coli include serotype O157:H7, a strain whose severe and sometimes fatal health consequences, particularly among infants, children and the elderly, make it among the most serious of foodborne infections. The second most common is shigellosis caused by three species of Shigella (S. sonnei, S.flexneri, and S. dysenteriae type-1). The low infectious dose (10 cells) allows shigellosis to be spread effectively by contaminated food or water, as well as by person-to-person contact. Worldwide there are approximately 164.7 million cases each year, of which 163.2 million occur in developing countries leading to roughly 1.1 million deaths. Since the late 1960s pandemic waves of Shigella dysentery (bloody diarrhea) have hit Central America, South and Southeast Asia and sub-Saharan Africa, often striking areas of political upheaval and natural disaster. During the 1994 genocide in Rwanda, up to 800,000 Rwandan refugees fled into Zaire. In the first month alone, approximately 20,000 people died from dysentery caused by a strain of Shigella that was resistant to all commonly used antibiotics. The third most common is salmonellosis, most often caused by Salmonella enteriditis and S. typhimurium. In the United States alone, salmonellosis can annually account for ~30% of all deaths due to foodborne illness. In 1994 an outbreak of Salmonella enteritidis due to contaminated ice cream occurred in the US, affecting an estimated 224,000 persons. hi certain embodiments, VTBs are designed to target diarrheagenic bacteria. The causative pathogens are close relatives (family Εnterobacteriaceae), and are thus related to non-pathogenic E. coli, differing mainly by clusters of virulence-related genes known as pathogenicity islands, and chromosomally inserted prophages harboring toxin genes. This phylo genetic relatedness allows the use of laboratory strains of E. coli as a development platform. E. coli is a well-studied system of known genetic sequence that features a wealth of existing genetic tools, easing use of E. coli bacteria and its lytic phages (e.g., T4 and T7) and colicins (e.g., El and Ia) in generating VTBs. Moreover, traditional phage therapy has been successfully used against E. coli dysentery in at least a dozen studies, and colicins are also shown to be effective therapies against E. coli O157:H7. Also, diarrheal disease is treatable through oral administration of VTBs rather than injection, thereby reducing the cost of drug production by obviating the need for enterotoxin removal, hi preferred embodiments, a VTB is designed to target one or more of six clinically important isolates: an enterohemorrhagic E. coli (O157:H7), an enteropathogenic E. coli, S. sonnei, S. dysenteriae type-1, S. enteriditis and S. typhimurium. Members of the Vibrio group, particularly V. cholera, and other related causative agents of cholera, are also desirable targets.

4. Phage Display Systems and Selection of Virulence Factor Targeting Proteins

As noted in the Summary, supra, a variety of methods may be used in selecting a virulence factor targeting protein. In general, such a method will involve screening a set of partially randomized polypeptides to identify those that bind to a virulence factor of interest. Phage display technology is useful for this purpose, and allows the rapid selection of polypeptides from a large library of variants based on their ability to bind to a specific target. In addition, phage display systems offer the opportunity to isolate a virulence factor binding protein in a context that is similar to that in the ultimate VTB agent. For example, variants of a tail fiber polypeptide of a T4 phage may be expressed as a fusion protein with a structurally similar protein from a phage lambda display library. This approach reduces the risk that, upon introduction of a targeting protein into a VTB, the structural integrity of the phage is disrupted. Additionally, because each phage particle carries the genetic information for the peptide it is displaying on its surface, there is a physical link between phenotype and genotype. Since their invention in 1985, phage display systems have been refined and expanded. There are phage display systems that are based on filamentous phages such as M13, as well as those based on tailed phages such as lambda, T7 and T4. All of these systems share the same basic properties, including a method for efficiently creating the diverse library of displayed peptides, a method for selection or "panning" for phages displaying peptides capable of binding to the target and a method for amplifying the phage by infecting and multiplying in a permissive host bacterium.

There are a number of methods for panning a phage library, generally using the same principal. The phage library is exposed to the target molecule, to allow those phages that display a peptide capable of binding, to do so. The unbound phages are then washed away. Phages that bound to the target are then eluted off and amplified by growing them in a permissive host. The panning procedure is repeated one or more times to enrich the library to the point where nearly all of the phages can bind to the target.

Phage display systems have been used for a variety of applications including cloning human antibodies, displaying cDNA libraries, and epitope mapping. Although almost any standard phage display system can be used to identify polypeptides that can bind to bacterial cells, none of them are designed to allow the phage to infect a target cell based on the binding ability of the displayed peptide. This would require that the peptide library be displayed at the end of the phage tail fibers. Several phage display systems based on the filamentous phage M13 do display their peptide libraries at the end of the phage's tail fibers, however, preliminary results suggest that Ml 3 phage may be unsuitable for this purpose, or may require additional work. A preferred phage display system is one that permits infection of target cells based on the peptide library (tail fiber variants). Panning in such a system can be done in vivo, and there would be no need to purify the target molecule and immobilize it for in vitro panning. The panning procedure itself would be significantly simplified; for instance there would be no elution step. Because the phage can directly infect the cell expressing the target molecule, the panning procedure goes directly from binding to the amplification step.

5. Virulence Targeted Bacteriocins ("VTBCs")

As an alternative embodiment, the disclosure provides antimicrobial agents based on bacteriocins that are engineered to target bacteria. The same general methods and techniques can be used to engineer VTBCs. Bacteriocins are protein toxins produced by bacteria that kill other closely related bacteria. A well studied subclass of bacteriocins, the colicins (so named because they are produced by E. coli) have attracted interest as a possible new class of therapeutic antibiotics. Purified colicin administered orally has been shown to clear colicin sensitive strains from mice in a single dose. Much of the research into the antibiotic application of bacteriocins has been focused on their possible use as probiotics. Probiotics are live microbes, which when ingested improve health by modulating the intestinal microbial flora. Recent work has demonstrated the ability of a colicin producing strain of E. coli to displace, and prevent the recolonization by, a colicin sensitive strain in a mouse model.

Colicins possess some interesting properties that make them good candidates for virulence targeted antimicrobial agents. Colicins are modular proteins composed of three functional domains; an amino-terminal translocation domain that is involved in transporting the toxin into the cell, a central binding domain that recognizes and binds to specific receptors on the surface of target cells, and a carboxy-terminal killing domain that kills the cell. Evolutionary studies of colicins have shown the extraordinary modularity of these molecules with numerous examples of domain swapping between different colicins. The binding domain of colicins shows the most variation and it is this domain that primarily dictates the specificity and range of killing of a particular colicin. The disclosure provides, therefore, VTBCs comprising (a) an amino-terminal translocation domain of a colicin, (b) a central virulence factor targeting polypeptide, and (c) a carboxy-terminal killing domain of a colicin. This is illustrated in Figure 6 with respect to colicin Ia. The virulence factor targeting domain may be generated according to any of the methods disclosed herein. Other examples of suitable colicins and bacteriocins are set forth in Riley and Wertz, Annu. Rev. Microbiol., 2002, 56:117-37/

EXAMPLES

The following examples provide a detailed protocol and research plan by which a system for generating VTBs may be implemented. Example 1 : Preparing a phage display system containing a T4 distal tail fiber domain expressed by phage λ.

The common lab strain of λ expresses a single central tail fiber (J), whose receptor is the maltose binding protein. It has been discovered, however that wild type λ carries genes for side tail fibers that have been inactivated due to a single base pair deletion (Figure 2). The distal portion of the side tail fiber (stf) structural gene and the adjacent tfa gene show homology to the distal tail fiber genes of T4 (g37 and g38). A version of λ (Ur-λ) that expresses its side tail fibers is able to bind to E. coli cells that do not express the maltose binding protein (λ's normal receptor). It has also been shown that replacement of the distal portion of T4's tail fiber genes with the homologous sequence from λ results in a viable phage with an altered host range. Therefore, it is expected that the distal portion of the g37 gene of T4 can be spliced in frame with the proximal portion of the lambda stf gene to provide a functionally expressed and packaged side tail fiber comprising a portion of the T4 p37.

While it is expected that virulence factor targeting polypeptides can be generated by any of the various methods disclosed herein, it would be desirable to introduce variability into the T4 p37 protein at the positions that naturally participate in receptor binding (OmpC and OmpF in E. coli K12, and LPS in E. coli B). The distal portion of gene 37 contains six conserved His boxes that have the motif GXHXH (labeled A-F in Fig. 3). These His boxes are thought to act as recombinational hotspots between related phages for the exchange of cell-binding domains. Between the His boxes are thought to exist the cell-binding domains responsible for binding specific cellular receptors (labeled 1-5 in Fig. 4). By analyzing sets of nested deletions from each cell-binding domain of gene 37 and monitoring which amino acid changes result in a modified binding phenotype for each cellular receptor it is expected that receptor binding portions of T4 p37 can be defined with greater precision.

Nucleic acid encoding one or more of the existing receptor binding domains of T4 p37 may be replaced with unique restriction sites to aid in library construction. The final construct may be reinserted into the native T4 binding domain to ensure that any amino acid substitutions caused by the insertion of the restriction sites do not interfere with proper tail fiber assembly and function.

Appropriate plasmid vectors for displaying random peptide libraries as binding domains in g37 of phage T4 may be generated according to any of the methods disclosed herein. In a preferred approach, the tail fiber phage display system is designed such that peptide moieties that bind to the surface of bacteria are rapidly identified. A /ox-Cre based system may be used. The first step in constructing the lambda-T4 hybrid display system is to replace the portion of the lambda genome from the distal portion of the J gene to the EaSl ORF with the lacZ gene and a truncated chloramphenicol resistance gene (cat) (Figure 5A). The stf-g37 protein fusion can then be moved onto λ using a suicide vector p- stf-g37 (Figure 5B). Homologous recombination at the J and cat genes would result in an easily selected phage that has functional J tail fibers, a functional cat gene, and no longer encodes beta-galactosidase (Figure 5C). In addition to the stf-g37 fusion, the new sequence will contain two different 34bp loxP sites, loxP_wt and loxPsπ. The loxP sites are part of the high-efficiency /ox-Cre recombination system from phage Pl. The Cre (named for cyclization of recombination) enzyme catalyzes recombination between identical loxP (named for locus of X-over Pl) sites. The loxP_wt and loxPsn sites vary by a single nucleotide, but are incompatible with each other.

A custom peptide display cloning vector (p-stf-g37L, Figure 5D) for the efficient cloning of peptide library sequences will be generated. The appropriate placement and size of library cassettes in the stf-g37 hybrid tail fiber gene will be determined, for example, based on the results from the preceding examples. Cre mediated recombination at the loxP sites may be used to efficiently move stf-g37 genes containing the library sequence from the plasmid to λ, creating the actual phage display library λ- stf-g37L (Figure 5E). This scheme employs a double crossover (one at each loxP site) to successfully generate the desired construct. If only a single crossover occurs at either loxP site, the entire p-stf-g37L plasmid would be integrated into the λ prophage. Subsequent recombination at the other loxP site would then result in the desired construct, whereas a second recombination at the same loxP site would just regenerate the starting τp-stf-g37L plasmid. The single crossover intermediate is predicted to be unstable because it would result in the plasmid high copy number origin of replication being on the chromosome. It would also result in a λ genome too large to be efficiently packaged (>54Kb) by the phage. The inclusion of an ampicilin resistance {bid) gene in the tail fiber library cassette will allow us to easily determine the efficiency of this phase of library construction. We will be able to infect cells using the wild type J tail fibers and determine what fraction of the phages carry the library (resistant to both chloramphenicol and ampicilin) versus those without the library cassette (resistant to just chloramphenicol). By starting with a strain of λ that has a temperature sensitive repressor (cI857) we can do all of the engineering while λ is a lysogen and then efficiently induce the phage by increasing the incubation temperature. Maintaining the phage library as lysogens will also allow us to amplify a library by just growing the cells, without worrying about introducing bias based on the structure of the recombinant tail fibers. Example 2: Developing T4 phages targeted to a virulence factor of E. coli O157:H7.

This example provides three different strategies for developing a T4-based VTB targeted to E. coli O157:H7.

Panning will use a strain of E. coli O157:H7 that is incapable of binding the phage display vector prior to library construction (lamlT), and that has been cultured in the laboratory under conditions that induce virulence. Initial libraries will be screened for the ability to infect the strain of E. coli O157:H7 by both plaque assay, and the ability to form lysogens (indicated by a phage marker). If the library is able to infect E. coli O157:H7 we will then subject it to more stringent screening. More stringent panning regimens consist of exposing the phage libraries to non-pathogenic laboratory strains of E. coli such as K12 and B, and then filtering out all of the cells and bound phages. This step is useful for removing from the library any phages capable of binding to non-virulent E. coli. A similar process can be used to cull the library to contain only candidates that target a specific virulence factor of ΕHΕC. To do so, the library may be pre-exposed to wild type K12, and then the remaining library screened with a Kl 2 strain expressing a cloned virulence factor. The cloned virulence factors used in these panning procedures may be adhesins, bacterial surface proteins (e.g., filamentous projections such as pili or fimbriae) that aid in pathogenicity by binding to specific receptors on eukaryotic cell membranes. Adhesins are an example of likely targets for VTBs, because there are many of them, they are surface- exposed and they have been shown to be important for virulence in EHEC (Table 2). At the end of the screening process, the novel cell-binding domains may be moved into phage T4 for further testing.

Table 2: Colonization virulence factors of EHEC

One strength of phage based VTBs over traditional antibiotics is their ability to evolve. Experimental evolution in the laboratory is an excellent means to select for phage traits that improve infection on a new host. Thus, techniques similar to laboratory evolution (e.g., prolonged serial-passage experiments) maybe used to enhance traits of the "first-pass" VTBs, such as improved binding affinity and host specificity. Applicants have previously examined the importance of co-infection of the same host cell by multiple phages, where genetic exchange (e.g., recombination) can lead to formation of hybrid progeny that feature a mixture of traits found in the co-infecting parents. Similarly, two or more VTBs that bind to different virulence factors can undergo co-infection, followed by screening for recombinant phages that bind to multiple virulence factors of a single target cell.

Example 3. Evaluating the efficacy of constructed VTBs

A desirable VTB should efficiently kill the targeted organism. Most likely, intrinsic to the design of VTBs is their ability to force an evolutionary tradeoff between resistance and virulence. The incidence of resistance to VTBs, and whether resistance leads to a concomitant reduction in virulence, will be assessed.

Different VTBs will vary in the rate at which they eliminate the same target cell. The most promising of the VTBs obtained may be tested to determine the rate at which the VTB reduces a population of E. coli O157:H7 cells. Because killing efficiency may vary under different conditions, Applicants will examine the efficacy of VTBs under a variety of conditions; for example, in E. co/z-contaminated raw hamburger, water, food-preparation surfaces, and laboratory tissue culture.

E. coli O157:H7 will likely vary in its ability to evolve resistance to different VTBs. Bacterial resistance to a phage or colicin is typically achieved by a mutation that knocks out a cellular receptor. Less commonly, the resistance arises through a mutation that only alters the specific portion of the cellular receptor that interacts with the binding domain of the phage or colicin. Applicants will measure the frequency of both types of resistance to the engineered VTBs. These data relate to the therapeutic value of a given VTB, because the desired tradeoff between resistance and virulence should be maximized if the pathogen gains resistance by completely eliminating the virulence factor.

Because the effects of virulence factor knockouts on E. coli O157:H7 pathogenicity are well studied, testing may focus on those rare mutations that confer resistance to VTBs by modifying but not eliminating virulence factors. Virulence will be measured using standard tissue culture assay methods such as the Adhesion assay and the Fluorescent Actin Staining assay. The precise virulence assay methods will depend on which virulence factors our VTBs successfully target. m furtherance of the experimental protocols set forth in these examples, Applicants have prepared a phage lambda construct engineered to contain the loxP cloning cassette and a tetracycline resistance marker. The lambda side tail fiber genes were also deleted from this construct. The sequence and map of this construct are presented in Figure 7 (SΕQ ID NO:1). Additionally, a LoxP recombination cassette was constructed for moving tail fiber gene constructs into the engineered lambda phage, as presented in Figure 8 (SΕQ ID NO:2). Finally, a chimeric tail fiber was constructed and the T4 g38 gene (required for proper assembly) was cloned between the LoxP sites, see Figure 9 (SΕQ ID NO:3), in order to move the T4 tail fibers into phage lambda.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. 1. Andersson, D. L, and B. R. Levin. 1999. The biological cost of antibiotic resistance. Curr Opin Microbiol 2:489-93.

2. Antia, R., R. Regoes, J. Koella, and C. Bergstrom. 2003. The role of evolution in the emergence of infectious diseases. NATURE 426:658-661. 3. Cravioto, A., R. Gross, S. Scotland, and B. Rowe. 1979. An adhesive factor found in strains of Escherichia coli belonging to the traditional infantile enterophato genie serotypes. Curr Microbiol 3:95-99.

4. Deng, W., J. Puente, S. Gruenheid, Y. Li, B. Vallance, A. Vazquez, J. Barba, J. Ibarra, P. O'Donnell, P. Metalnikov, K. Ashman, S. Lee, D. Goode, T. Pawson, and B. Finlay. 2004. Dissecting virulence: systematic and functional analyses of a pathogenicity island. Proc Natl Acad Sci U S A 101:3597-602.

5. Desplats, C, and H. Krisch. 2003. The diversity and evolution of the T4- type bacteriophages. Res Microbiol 154:259-67.

6. Froissart, R., C. O. Wilke, R. Montville, S. K. Remold, L. Chao, and P. E. Turner. 2004. Co-infection Weakens Selection Against Epistatic Mutations in RNA

Viruses. Genetics 168:9-19.

7. Gupta, A., M. Onda, I. Pastan, S. Adhya, and V. Chaudhary. 2003. High- density functional display of proteins on bacteriophage lambda. J MoI Biol 334:241-54.

8. Hendrix, R., and R. Duda. 1992. Bacteriophage lambda PaPa: not the mother of all lambda phages. Science 258: 1145-8.

9. Henning, U., and S. Hashemolhosseini. 1994. Receptor recognition by T- even coliphages, p. 291-298. hi J. Karani (ed.), Molecular Biology of Bacteriophage T4. American Society for Microbiology, Washington, DC.

10. Hoess, R., A. Wierzbicki, and K. Abremski. 1986. The role of the loxP spacer region in Pl site-specific recombination. Nucleic Acids Res 14:2287-300.

11. Knutton, S., A. Phillips, H. Smith, R. Gross, R. Shaw, P. Watson, and E. Price. 1991. Screening for enteropathogenic Escherichia coli in infants with diarrhea by the fluorescent-actin staining test. Infect hnmun 59:365-71.

12. LeBlanc, J. 2003. Implication of virulence factors in Escherichia coli O157-.H7 pathogenesis. Crit Rev Microbiol 29:277-96. 13. Lenski, R. E. 2002. Experimental evolution, p. 339-344. In M. Pagel (ed.), Encyclopedia of Evolution. Oxford University Press, Oxford.

14. Lenski, R. E., and R. M. May. 1994. The Evolution of Virulence in Parasites and Pathogens: Reconciliation Between Two Competing Hypotheses. Journal of Theoretical Biology 169:253-265.

15. Levin, B., and J. Bull. 2004. Population and evolutionary dynamics of phage therapy. Nat Rev Microbiol 2:166-73.

16. Lipsitch, M. 2001. The rise and fall of antimicrobial resistance. Trends Microbiol 9:438-44. 17. Merril, C, D. Scholl, and S. Adhya. 2003. The prospect for bacteriophage therapy in Western medicine. Nat Rev Drug Discov 2:489-97.

18. Montag, D., H. Schwarz, and U. Henning. 1989. A component of the side tail fiber of Escherichia coli bacteriophage lambda can functionally replace the receptor- recognizing part of a long tail fiber protein of the unrelated bacteriophage T4. J Bacteriol 171:4378-84.

19. Nagaev, I., J. Bjorkman, D. I. Andersson, and D. Hughes. 2001. Biological cost and compensatory evolution in fusidic acid-resistant Staphylococcus aureus. MoI Microbiol 40:433-439.

20. Nataro, J. P., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin Microbiol Rev 11 : 142-201.

21. Palumbi, S. 2001. Humans as the world's greatest evolutionary force. Science 293:1786-90.

22. Rogers, D. J., and M. J. Packer. 1993. Vector-borne diseases, models, and global change. The Lancet 342:1282-1284. 23. Sternberg, N., and D. Hamilton. 1981. Bacteriophage Pl site-specific recombination. I. Recombination between loxP sites. J MoI Biol 150:467-86.

24. Sulakvelidze, A., Z. Alavidze, and J. G. Morris, Jr. 2001. Bacteriophage therapy. Antimicrob Agents Chemother 45:649-659.

25. Summers, W. 2001. Felix d'Herelle and the Origins of Molecular Biology. Yale University Press, New Haven, CT. 26. Tetart, F., F. Repoila, C. Monod, and H. Krisch. 1996. Bacteriophage T4 host range is expanded by duplications of a small domain of the tail fiber adhesin. J MoI Biol 258:726-31.

27. Thapar, N., and I. Sanderson. 2004. Diarrhoea in children: an interface between developing and developed countries. Lancet 363:641-53.

28. Thiel, K. 2004. Old dogma, new tricks~21st Century phage therapy. Nat Biotechnol 22:31-6.

29. Tjaniadi, P., M. Lesraana, D. Subekti, N. Machpud, S. Komalarini, W. Santoso, C. Simanjuntak, N. Punjabi, J. Campbell, W. Alexander, H. r. Beecham, A. Corwin, and B. Oyofo. 2003. Antimicrobial resistance of bacterial pathogens associated with diarrheal patients in Indonesia. Am J Trop Med Hyg 68:666-670.

30. Turner, P. 2004. Phenotypic plasticity in bacterial plasmids. Genetics 167:9- 20.

31. Turner, P., V. Cooper, and R. Lenski. 1998. Tradeoff between horizontal and vertical modes of transmission in bacterial plasmids. 52:315-329. Evolution 52:315-329.

32. Turner, P. E., and L. Chao. 1998. Sex and the evolution of intrahost competition in RNA virus Phi-6. Genetics 150:523-532.

33. Whittam, T., M. Wolfe, I. Wachsmuth, F. Orskov, I. Orskov, and R. Wilson. 1993. Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea. Infect Immun 61:1619-1629.

EQUIVALENTS

While specific embodiments of the subject inventions are explicitly disclosed herein, the above specification is illustrative and not restrictive. Many variations of the inventions will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the inventions should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

WE CLAM:

I . A method for preparing a virulence factor targeted bacteriophage (VTB), the method comprising, altering the genome of a bacteriophage such that the genome comprises a nucleic acid encoding a virulence factor targeting protein.

2. The method of claim 1 , wherein the virulence factor targeting protein is a variant of a tail fiber protein of said bacteriophage.

3. The method of claim 2, wherein the nucleic acid encoding the virulence factor targeting protein is inserted into the genome of the bacteriophage in a position and orientation such that the virulence factor targeting protein is incorporated into the tail fiber of the bacteriophage.

4. The method of claim 1, wherein said virulence factor targeting protein binds to a virulence factor of a Gram negative pathogen.

5. The method of claim 1 , wherein said bacteriophage is a member of the Myoviridae family.

6. A method for selecting a virulence factor targeting protein, the method comprising:

(a) contacting a plurality of variants of a distal tail fiber protein of a bacteriophage of the Myoviridae family with a virulence factor of a Gram negative bacterium; and (b) enriching for those variants that bind to the virulence factor.

7. The method of claim 6, wherein the variants are incorporated into a phage particle.

8. The method of claim 7, wherein the variants are expressed as a fusion protein with a proximal portion of the phage lambda Stf protein.

9. The method of claim 7, wherein each of said variants comprises a variability cassette.

10. The method of any of claims 6-9, wherein said virulence factor is expressed on a bacterial cell.

I 1. The method of claim 10, wherein the bacterial cell is a cell of a laboratory strain of E. coli or a cell of a Gram negative pathogen.

12. The method of claim 10, wherein (b) comprises recovering phage that bind to bacterial cells expressing the virulence factor.

13. The method of claim 10, wherein (b) comprises recovering phage that kill bacterial cells expressing the virulence factor.

14. The method of claim 6, wherein the bacteriophage is a member of the T4 group.

15. The method of claim 6, wherein the variant is a variant of the g37 protein of the T4 bacteriophage.

16. A modified phage particle of the Siphoviridae family, comprising a variant of a distal tail fiber protein of a bacteriophage of the Myoviridae family.

17. The modified phage particle of claim 16, wherein the variant comprises a virulence factor binding element.

18. The modified phage particle of claim 17, wherein the variant binds to a virulence factor of a Gram negative pathogen.

19. The modified phage particle of claim 16, wherein the phage particle is phage lambda phage particle and the variant is expressed as a fusion protein with a proximal portion of the phage lambda Stf protein.

20. A library of Myoviridae bacteriophage distal tail fiber protein variants, the library comprising at least 10⁵ species of a variant of a distal tail fiber protein of a Myoviridae bacteriophage, and wherein each of said variants comprises a variability cassette having a polypeptide sequence that is distinct to each of said species of variant.

21. The library of claim 20, wherein the distal tail fiber protein is the p37 protein of a T4 group bacteriophage.

22. A phage library, wherein the library comprises at least 10⁵ species of modified phage particle, wherein each of said species comprises a variant of a distal tail fiber protein of a T-even bacteriophage, and wherein said variant of a distal tail fiber protein comprises a variability cassette that is distinct to each of said species.

23. The phage lambda library of claim 22, wherein the phage library is a phage lambda library and the variant of a distal tail fiber protein is expressed as a fusion protein with a phage lambda side tail fiber polypeptide.

24. The phage library of claim 23, wherein the fusion protein comprises a proximal portion of the phage lambda Stf polypeptide, a distal portion of the T4 g37 polypeptide, and a variability cassette.