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MINI-REVIEW Bacteriophage lambda display systems: developments and applications Jessica Nicastro & Katlyn Sheldon & Roderick A. Slavcev Received: 1 December 2013 /Revised: 3 January 2014 /Accepted: 4 January 2014 /Published online: 19 January 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Bacteriophage (phage) Lambda (λ) has played a key historic role in driving our understanding of molecular genetics. The lytic nature of λ and the conformation of its major capsid protein gpD in capsid assembly offer several advantages as a phage display candidate. The unique forma- tion of the λ capsid and the potential to exploit gpD in the design of controlled phage decoration will benefit future ap- plications of λ display where steric hindrance and avidity are of great concern. Here, we review the recent developments in phage display technologies with phage λ and explore some key applications of this technology including vaccine delivery, gene transfer, bio-detection, and bio-control. Keywords Bacteriophage λ . Lytic phage display . gpD translational fusion . Gene delivery . Phage vaccine Introduction to bacteriophage display Bacteriophages (phages) are bacterial parasites that, like their animal virus counterparts, exist in a variety of morphologies and with diverse genetic architectures. Most phages are limit- ed in their host range, normally infecting only a single species of bacteria, and at many times only a subset of that species, adsorbing to specific receptors on bacterial cells that define their host range. Lytic phages such as T4 and T7 enter imme- diately into a vegetative state of reproduction upon infection, lysing the cell. In contrast, temperate phages such as bacteri- ophage Lambda (λ) choosebetween vegetative growth and a quiescent state where the phage genome is stably harbored in the host cell in a state called lysogeny. Filamentous phage, such as M13 and Ff do not lyse the cell in any state but rather convert the cell into a phage-producing factory, thereby compromising the hosts growth (Gulig et al. 2008). Bacteriophage display, or phage display, was first devel- oped by George Smith in 1985 and can be defined as the process by which a heterologous protein or peptide is expressed as an exterior fusion through the genetic fusion to a coat protein gene of the bacterial virus (phage) particle (Lindqvist 2005). This revolutionary technique, which is one of the largest innovations involving phage since their discov- ery in the early 1900s (dHerelle and Smith 1926; Duckworth 1976), is based on the genetic fusion, where the outcome of this genotypic manipulation (Kaiser 1966) will produce a phenotypic outcome (Jestin 2008). The most well-known phage display methods are based on the usage of phage M13 and the related filamentous phage of Escherichia coli . Al- though filamentous phages are not the focus of this review, the principles of their early use govern phage display advance- ments. The most extensively used phages are derived from the E. coli Ff (filamentous) class, where the most commonly explored species include M13, fd, and f1. The basic structure of these phages consist of a circular single-stranded (ss) DNA genome that is encapsulated by a long tube comprised of thousands of copies of a single major coat protein and four additional minor capsid proteins at the tips. M13 phage has been especially useful for phage display as their genome is bound only by the major coat protein (as opposed to filling a phage head structure). Therefore, there is not a strict limit on the size of packaged DNA permitting for more opportunities for manipulation. Smith (1985) was the first to successfully demonstrate a phage capsid fusion by inserting an external gene into the phage genome of the filamentous phage fd. This construct demonstrated the efficient display of Eco RI endo- nuclease gene product on the filamentous phage fd minor coat protein pIII (Smith 1985; Smith and Huggins 1982). Based on this initial work, lytic phage, including phage T4 and T7, and J. Nicastro : K. Sheldon : R. A. Slavcev (*) School of Pharmacy, University of Waterloo, 200 University Avenue West Waterloo, Kitchener, ON, Canada e-mail: [email protected] Appl Microbiol Biotechnol (2014) 98:28532866 DOI 10.1007/s00253-014-5521-1

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Bacteriophage lambda display systems:developments and applications

Jessica Nicastro & Katlyn Sheldon & Roderick A. Slavcev

Received: 1 December 2013 /Revised: 3 January 2014 /Accepted: 4 January 2014 /Published online: 19 January 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Bacteriophage (phage) Lambda (λ) has played akey historic role in driving our understanding of moleculargenetics. The lytic nature of λ and the conformation of itsmajor capsid protein gpD in capsid assembly offer severaladvantages as a phage display candidate. The unique forma-tion of the λ capsid and the potential to exploit gpD in thedesign of controlled phage decoration will benefit future ap-plications of λ display where steric hindrance and avidity areof great concern. Here, we review the recent developments inphage display technologies with phage λ and explore somekey applications of this technology including vaccine delivery,gene transfer, bio-detection, and bio-control.

Keywords Bacteriophage λ . Lytic phage display . gpDtranslational fusion . Gene delivery . Phage vaccine

Introduction to bacteriophage display

Bacteriophages (phages) are bacterial parasites that, like theiranimal virus counterparts, exist in a variety of morphologiesand with diverse genetic architectures. Most phages are limit-ed in their host range, normally infecting only a single speciesof bacteria, and at many times only a subset of that species,adsorbing to specific receptors on bacterial cells that definetheir host range. Lytic phages such as T4 and T7 enter imme-diately into a vegetative state of reproduction upon infection,lysing the cell. In contrast, temperate phages such as bacteri-ophage Lambda (λ) “choose” between vegetative growth anda quiescent state where the phage genome is stably harbored inthe host cell in a state called lysogeny. Filamentous phage,

such as M13 and Ff do not lyse the cell in any state but ratherconvert the cell into a phage-producing factory, therebycompromising the host’s growth (Gulig et al. 2008).

Bacteriophage display, or phage display, was first devel-oped by George Smith in 1985 and can be defined as theprocess by which a heterologous protein or peptide isexpressed as an exterior fusion through the genetic fusion toa coat protein gene of the bacterial virus (phage) particle(Lindqvist 2005). This revolutionary technique, which is oneof the largest innovations involving phage since their discov-ery in the early 1900s (d’Herelle and Smith 1926; Duckworth1976), is based on the genetic fusion, where the outcome ofthis genotypic manipulation (Kaiser 1966) will produce aphenotypic outcome (Jestin 2008). The most well-knownphage display methods are based on the usage of phage M13and the related filamentous phage of Escherichia coli. Al-though filamentous phages are not the focus of this review,the principles of their early use govern phage display advance-ments. The most extensively used phages are derived from theE. coli Ff (filamentous) class, where the most commonlyexplored species include M13, fd, and f1. The basic structureof these phages consist of a circular single-stranded (ss) DNAgenome that is encapsulated by a long tube comprised ofthousands of copies of a single major coat protein and fouradditional minor capsid proteins at the tips. M13 phage hasbeen especially useful for phage display as their genome isbound only by the major coat protein (as opposed to filling aphage head structure). Therefore, there is not a strict limit onthe size of packaged DNA permitting for more opportunitiesfor manipulation. Smith (1985) was the first to successfullydemonstrate a phage capsid fusion by inserting an externalgene into the phage genome of the filamentous phage fd. Thisconstruct demonstrated the efficient display of EcoRI endo-nuclease gene product on the filamentous phage fd minor coatprotein pIII (Smith 1985; Smith and Huggins 1982). Based onthis initial work, lytic phage, including phage T4 and T7, and

J. Nicastro :K. Sheldon : R. A. Slavcev (*)School of Pharmacy, University of Waterloo, 200 University AvenueWest Waterloo, Kitchener, ON, Canadae-mail: [email protected]

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temperate phage λwould eventually be successfully exploitedas fusion vectors as well (Garufi et al. 2005; Kalniņa et al.2008).

Lytic versus filamentous phage display

The use of filamentous phage in display technology possessesmajor limitations that can be overcome with lytic phagedisplay systems. First, a tolerated fusion must have the abilityto translocate across the plasma membrane and as such, manyhydrophilic cytoplasmic proteins cannot be extruded as fusionproteins in filamentous phages. Second, filamentous phagesare severely constrained by the size of fusions possible (Garufiet al. 2005; Mikawa et al. 1996; Sternberg and Hoess 1995).Third, filamentous phages are dependent on the viability ofthe host and thus cannot effectively display peptides/proteinsthat are toxic to the cell (Garufi et al. 2005). Due to the lyticnature of double-stranded (ds) DNA bacteriophage, the fusionof toxic proteins is no longer a concern in temperate or lyticphages as they would be repressed in the phage lysogenic state(temperate phage only) and only expressed just prior to celllysis (Garufi et al. 2005).

A few lytic phage candidates have been identified forphage display, including λ, T4, and T7. The C-terminus ofthe minor T4 fibrous structural protein fibritin that comprisesthe collar/whiskers complex on the neck of the phage and theC-terminal of T4 minor capsid proteins HOC and SOC toler-ate fusions for lytic phage display in T4 (Efimov et al. 1995;Ren et al. 1996). Phage T7 has been employed as a displayplatform for various GFP-based cytoplasmic proteins thatshowed poor expression (fluorescence) with more traditionalfilamentous display vectors (Dai et al. 2008). Santini et al.(1998) developed the first chimeric phage system in which C-terminal fusions were made to the λ major capsid proteingeneral protein D or gpD. In this work, recombinant phageswere decorated with both wild-type gpD and proteins from ahepatitis C virus (HCV) cDNA expression library. Theserecombinant phages were compared to two other libraries(N-terminal fusions to the pIII and pVIII capsid proteins ofM13) using the cDNA expression library, as the majority ofthe inserts were between 100 and 300 bp in length. HCV waschosen for this study because the immune response in humanshas been heavily studied (Santini et al. 1998). In order to avoidoverpopulating the λ phage with gpD fusions, and improvethe phage stability, they expressed a second D allelepossessing an amber mutation within the gene for gpD. Here,under normal conditions gpD could not be produced by thephage unless plating was accomplished on an amber suppres-sor (SupE, SupF). This addition to the phage display processallowed for the formation of chimeric phage (Santini et al.1998). Comparing the filamentous phage proteins, pVIII’sdisplay was more efficient than pIII, and when pVIII was

compared to gpD display and selection of protein fragmentsof limited size, the two libraries showed comparable efficien-cies. However, when larger protein fragments were tested,gpD outperformed pVIII, likely because λ does not requiresecretion of the fusion protein. So unlike filamentouspIII/VIII, larger recombinant proteins can fold properly with-out interfering with phage assembly while displaying higherdensities. Non-homogeneous ligates such as human HCV-positive sera could also be used to identify positive phageafter one or a few rounds of selection when using phage λ,which can be used to identify common immunodominantantigens in developing new vaccines.

Gupta et al. (2003) later designed a λ phage display librarythat expressed fusions on the C-terminal of gpD. This librarywas compared to one created by fusing to an M13 phage (N-terminal fusions made utilizing major coat protein PVIII andminor coat protein PIII) where there was no degradation ofdisplayed products. This λ display system provided a 100-foldhigher display for all fragments compared to filamentousphage when tested with an antibody-binding assay. The λsystem generally displayed proteins of different sizes, wherethe number of fusions displayed on each phage particle was 2–3 orders of magnitude greater than that for M13. When thehigh-density display was applied to epitope mapping, the λsystem consistently outperformedM13 with a higher enzyme-linked immunosorbant assay (ELISA) reactivity shown.

Bacteriophage λ lytic phage display

The icosahedral-shaped bacteriophage λ capsid is approxi-mately 60 nm in diameter with a shell thickness of 4 nm andaccommodates a genome of 48.5 kb that contributes a mass of21×106 Da (Hohn and Katsura 1977; Witkiewiczl andSchweiger 1982). The capsid is made up of two major pro-teins, gpE and gpD, where the bottom side of gpD binds to thegpE arrangement (Fig. 1) (Wurtz et al. 1976; Yang et al. 2000).The gpD, capsid protein like gpE, is incorporated at 405–420copies on a mature phage head as strongly protruding thimble-shaped trivalent spikes. While gpE is most likely responsiblefor cutting the DNA into monomers and forming the initialphage prohead, gpD is necessary for phage head morphologyas it stabilizes the head when filled by the full-length wild-type genome (Lankes et al. 2007; Mikawa et al. 1996). In theabsence of gpD, only mutants packed with less than 82 % ofthe wild-type DNA can form mature phages (Imber et al.1980; Katsura 1988; Mikawa et al. 1996; Sternberg andHoess 1995; Sternberg and Weisberg 1977). Like eukaryoticchromosomal proteins, the gpD proteins are able to cross-reactimmunologically with eukaryotic histones, which may be thereason for gpD’s function related to the stabilization andcompaction of phage DNA. One study exploited the shp genewhich shares a 49% homology in amino acid identity with thegpDgene. This study swapped the gene ofD for shpand found

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that shp was able to stabilize the λ head shell. However, thiswas only accomplished in the presence of Mg2+ and hence,possessed high sensitivity to chelating agents (Wendt andFeiss 2004).

Early experiments of λ-fused peptides were conducted byfusing the peptide or protein of interest to the C-terminus ofthe major tail protein gpV (Dunn 1995). The initial displayvector employed the C-terminal of gpV as this end of theprotein faces outward and was considered insignificant andmore tolerant to accept fusions. Dunn (1995) later demonstrat-ed that gpV can indeed tolerate large multimeric proteins suchas β-galactosidase, but that the incorporation of fusions aswell as the yield of phages after affinity chromatography wasvery low at approximately one copy per λ tail tube and onepercent recovery, respectively (Dunn 1995; Maruyama et al.1994; Mikawa et al. 1996).

To overcome the low copy limitations imposed by gpV-mediated fusions (estimated at about one per phage), fusionsto the major capsid protein gpD were next investigated. Workwith cryo-electron microscopy and image processing withgpD has shown that the major capsid protein assembles intrimers that are incorporated as prominent protrusions on thesurface of the phage capsid making them more accessible forbinding to external target molecules (Dokland and Murialdo1993; Sternberg and Hoess 1995). The use of gpD to expresspeptides on the λ capsid was ideal since both its N- and C-termini protrude outward exposing its ends for the addition offused peptides (Mikawa et al. 1996; Witkiewiczl andSchweiger 1982). Mikawa et al. (1996) postulated that boththe N- and C-termini of gpD are neither at the trimer interac-tion interface, nor do they interact with the other major capsidprotein, gpE, and that the terminal tolerance and capacity assuch, depends on the fused peptide/protein (Mikawa et al.1996). This postulation was supported by the evidence for

gpD fusions of various sizes that have been successfully fusedto both the N and C termini of the protein. This would suggestthat the display of the fusions on either terminus will notjeopardize the function of the phage or prevent fusion proteinsfrom binding properly to the capsid (Dokland and Murialdo1993; Sternberg and Hoess 1995; Vilchez and Jacoby 2004).However, further studies on capsid folding and binding inter-actions have shown that the N-terminus of gpD does in factinteract with gpE and that this interaction is in fact required forthe stability of gpD (Lander et al. 2008; Yang et al. 2000).

Amino versus carboxyl fusions to gpD

As previously mentioned, the λ gpD protein is tolerant ofvarious protein and peptide fusions to the N- and C-terminiof this major capsid component, providing great flexibility tothis lytic phage display system. Pavoni et al. (2004) construct-ed a tumor cDNA display library employing the N-terminalfor fusions to gpD in order to prevent the selection of out-of-frame fusions since these fusions increased the selection ofartifact peptides. As such, the recombinant phage would bedecorated solely with wild-type gpD (Minenkova et al. 2003;Pavoni et al. 2004). This study also utilized the λ KM8 and λKM10 phage constructs, which were originally constructedbased on KM4 phage that can accept inserts of up to 3 kb inlength without disturbing λ packaging (Minenkova et al.2003). Both the use of an N-terminal fusion and engineeredphage allowed for simpler assembly of chimeric phage, but byusing enhanced green fluorescent protein, or eGFP, as a fusionpartner, an N-terminal fusion would not allow eGFP to foldproperly, and hence fluorescence would not be possible(Pavoni et al. 2004). Zucconi et al. (2001) determined thatthe number of tolerable fusion proteins displayed on thecapsid surface was based on the length and composition ofthe foreign amino acid sequence. When these fusions wereexpressed at the C-terminal, a very high incorporation rate wasachieved, which was also the case for N-terminal fusions.Neither group mentioned a reduction in phage viability or aneffect on phage assembly with the peptides they attempted tofuse, yet this work provided important initial insight into theability of gpD fusions to retain function and thus, for phage toassemble properly and form viable phage particles(Minenkova et al. 2003; Zucconi et al. 2001).

Yang et al. (2000) used cryo-electron microscopy to showthat the N-terminus of gpD is conditionally disordered whenunbound and may interact with gpE, and any fusions to itwould require the interaction of the N-terminal fusion linkerwith gpE to display the fusions on the outside of the fusionproteins. This claim was supported by a study by Lander et al.(2008) where a cryo-electron microscopy reconstruction ofthe λ capsid head was developed and showed that the first 14residues in gpD N-terminus interact with a strand of an E-loopin gpE and together form a stableβ-sheet (Lander et al. 2008).

Fig. 1 One face of the bacteriophage λ icosahedral capsid. The illustra-tion depicts 1 of the 20 faces of the bacteriophage λ capsid head. Majorcapsid protein gpD (the decoration protein labeled “D”) incorporate intothe phage head as homotrimers, while the second major capsid protein,gpE (labeled “E”) incorporate as homohexamers

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Wendt and Feiss (2004) expanded on this idea showing that achimeric protein with the N-terminus of gpD and the C-terminus of the Shp protein from phage 21 was able tostabilize the λ capsid more effectively than Shp could alone(Wendt and Feiss 2004).

Vaccaro et al. (2006) fused a functional scFv antibody(anti-CEA) to the N- and C-termini of gpD λ and found thatthe N-terminal fusions showed a relatively low recombinantprotein loading in comparison to the C-terminal fusions (50 %compared to 88 %). The C-terminal fusions also presentedunusually small plaques, which suggested that the large scFvprotein was compromising phage assembly. They also per-formed phage stability assays via immunoscreening, and ob-served that while N-terminal phage produced expected results,1 to 5 % of plaques generated by C-terminal fusions werelarger in size and negative for the assay. These plaques werelater found to be mutants possessing point mutations causingeither frameshift or premature stop signals within the scFvgene, suggesting a powerful selection against the inefficientdisplay of the antibody as a C-terminal fusion and the inhib-itory effect on the assembly of viable phages (Vaccaro et al.2006).

Controllable phage display systems

Since the development of significant applications derivedfrom the original work on lytic display, control of fusion copynumber has been generally underdeveloped. The number oftolerable fusions per phage particle is thought to depend on thesize of the fused fragment, whereby the smaller the fragmentthe greater the number of fusions expected per phage (Guptaet al. 2003). When comparing strategies for the developmentof a vaccine versus a vector for gene delivery or fusion of apeptide versus a large multimeric protein, many differentapproaches must be applied due to the differentiation in fusionnumber. In addition, various factors must be taken into con-sideration for various downstream applications, including butnot limited to the following: (1) the occurrence of over-stimulating a signal due to high affinity or high avidity inter-actions, (2) the possibility of cross-reactivity and steric hin-drance impeding protein function, and (3) ligand access. Thecontrol over fusion decoration is of paramount importanceparticularly toward the development of phage therapeutics(see Fig. 2).

Mikawa et al. (1996) were first to employ amber suppres-sion to regulate gpD, representing the only rudimentary regu-lation system to date. Although the approach was qualitativeand based on relative display, more modern approaches wereused to generate gpD fusions in vivo (Mikawa et al. 1996).These systems were able to generate libraries and purifyfunctional fusions, to increase the size and density of capsidfusions, and recently to tackle the issue of copy control

(Lankes et al. 2007; Mikawa et al. 1996; Nicastro et al.2013; Sokolenko et al. 2012). Two unique vector featuresused in these initial constructs included a peptide linker andthe conditional fusions via amber suppression strategies thatpermitted the fusion of multimeric proteins. There was only aslight difference in expressionwhen suppression was used as acrude control mechanism for capsid fusion expression byemploying various allogeneic E. coli suppressor strains SupE,SupF, SupD, Sup−, coding for glutamine, tyrosine, serine orno residue at the site of the amber stop signal, respectively(Maruyama et al. 1994; Mikawa et al. 1996). As a result of themutations in the tRNA genes of the various E. coli strains, thestrains conferred varied gpD levels of expression, generatinggpDwt, gpDQ68Y gpDQ68S, and gpD (truncated), respective-ly (Nicastro et al. 2013). However, it is important to note thatthese strains were allogeneic and that other genetic elementsor expression characteristics may have influenced variationsin plating efficiency. The genetic phenomenon of suppressionis mediated by tRNA’s mutant alleles that possess an antico-don mutation, which is complementary to a stop codon thatresults in the insertion of an amino acid (a.a.) in response toreading a termination nonsense codon as sense. This willrelieve the translational truncation imparted by the originalearly instance of a stop codon or nonsense codon. A phagewith an amber stop codon within its D gene (e.g. Dam15allele) is suppressible on a mutant bacterial strain (Sup+) thatrecognizes and decodes the termination signal with an aminoacid, resulting in a non-truncated, full-length 110 amino acidprotein (Fig. 3). In permissive strains, both the terminated andcompleted peptide chains are produced with the efficiency ofsuppression being dependent on the suppressor that is carriedby the bacterial strain (Brenner et al. 1965).

Fig. 2 Schematic of controlled phage display on the bacteriophage λcapsid. Decoration onto the capsid surface under controlled circum-stances results in a display platform where both the fused protein andthe wild-type protein are incorporated into the phage head. The schematicdepicts the scenario of a 50 % decoration rate, whereby 50 % of theinsertions are occupied by gpD/X fusions labeled “D:X” and 50 % areoccupied by wild-type gpD labeled “D”

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Combination genetic control systems

Stop codon suppression Amber suppression is a conditionalgenetic expression phenotype, where a nonsense codon is readby a complementary mutant tRNA of the E. colihost, resultingin insertion of a specific amino acid in place of the UAG(amber) stop signal (Benzer and Champe 2013; Inokuchi et al.1979). This conditional expression strategy was also appliedto bacteriophage T4D, where non-sense mutations within thehead protein were suppressed to produce a fragment of thepolypeptide chain in the presence of a suppressor host (Sup+)(Bolle 1964). This initial research led to the inference thatamber and ochre (UAA) codons were chain terminators and apermissive cell (Sup+) was capable of suppression, whilephage propagation was present, albeit at a lower frequency.Correspondingly, in a non-permissive cell (Sup−) the ambermutations were not suppressed and propagation was absent.Furthermore, a change in residue was observed from the wild-type sequence and other different types of suppressors (amber,ochre) depending on the codon present in the sequence, wherethere was a different suppressor strain for each and each wasmutually exclusive (Brenner et al. 1965). Two translationaloutcomes occur in suppressor strains: (1) non-functional pro-tein truncations and (2) full-length species conferring partial tocomplete function. To increase the efficiency of suppressors,restoration of the wild-type pristine protein sequence, or aprotein sequence that is identical to the original, is preferable(Normanly et al. 1986).

Temperature-regulated expression Lieb (1966) first charac-terized the λ CI857 temperature-labile repressor, and discov-ered that it was the dissociation of this repressor complex thatallowed downstream transcription to proceed (Lieb 1966;

Szybalski et al. 1969). It is the controllable nature of thisrepressor associated with expression from a strong promoterthat made temperature-sensitive cI[Ts] alleles attractive to usein cloning vectors to regulate expression of the gene of inter-est. In these cases, a single copy of the cI[Ts] gene producesenough repressor to effectively inhibit the strong λ PL or PRpromoter on single or multicopy replicons (Bernard et al.1979; Lukacik et al. 2012; Remaut et al. 1981). Under thisrepressor-regulated promoter’s control, a downstream gene isexpressed at increasing levels as temperature rises due to oneof the two 857 allelic mutations. The first confers thermalsensitivity that may not be as effective as chemical inductionapproaches but is less likely to be genotoxic and providescontrollable expression attributes that are very favourable,whereas the second, termed Ind− renders the repressor resis-tant to inactivation by RecA protein, the protein that catalysesrepressor autocatalysed cleavage (Mott et al. 1985; Ren et al.1996). The CI857 allele denatures at 40–42 °C, conferringmaximal expression of the gene of interest (Hayes and Hayes1986). Conversely, temperatures as low as 29 °C may berequired to fully repress transcription from PL via the CI857allele, and highest protein yields can be obtained followinginduction at 36 °C, but the latter temperature and its efficiencydepends on the protein being expressed and the host cell as theexpression of many heterologous proteins innately affect bac-terial cell growth and survival (Guzmán et al. 1994; Jechlingeret al. 1999; Lowman and Bina 1990; Villaverde et al. 1993).Strategies to avoid protein expression toxicity include: (1)suppression of basal expression from leaky inducible pro-moters, (2) suppression of read-through transcription fromcryptic promoters, (3) tight control of plasmid copy numbers,(4) protein production as inactive (but reversible) forms, and(5) the creation of special expression vectors and modified

Fig. 3 Schematic of amber suppression. The schematic illustrates theconcept of suppression as it pertains to the λ Dam15 mutation (λF7). aWhen λ F7 is grown on a suppressor strain an amino acid is inserted inplace of the translational termination signal and gpD is fully translated,resulting in viable phage. b In the nonsuppressor strain, gpD is truncated,

nonfunctional, and phages are nonviable. Sequencing of theDam15alleledefined a point mutation in the 68th codon (CAG) of gene D encodingglutamine (Q), to an amber stop codon (UAG) (Nicastro et al. 2013). Thisagrees with previous findings that show SupE (glutamine) to be thestrongest suppressor for growth of λF7

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E. coli strains (Saïda et al. 2006). CI857 repressor activity isnot limited to E. coli, but has also been effective in even gram-positive strains such as Bacillus subtilis, where downstreamexpression efficiencies were found to be similar to thosereported in E. coli with only minor codon bias modificationsrequired (Breitling et al. 1990).

Our lab has recently developed a dual combination controlsystem that employs both amber suppression and temperatureregulated expression genetic control system in tandem(Nicastro et al. 2013). This system utilizes the bacteriophageλ capsid protein gpD for the fusion of eGFP polypeptides forthe efficient display on the surface of the phage. Ambersuppression control was established through the use of gpDalleles generated during λ Dam15 infection of Sup+ isogenicderivatives. These particles carry gpD alleles of varying func-tionality depending on the amber suppressor strain of E. coli,SupD (gpDQ68S), SupE (Dwt), or SupF (gpDQ68Y) host onwhich they were grown. In combination, temperature controlwas employed through the use of the D-fusion protein,gpD::eGFP, encoded in trans from a multicopy temperature-inducible expression plasmid regulated by the CI857 repres-sor. This combinational control system generated dramaticvariation in the expression of displayed peptides through themany permutations afforded by the dual mechanisms ofcontrol. Maximal incorporation of gpD::eGFP into theλDam15 phage capsid was imparted in combinationwith the gpDQ68S allele, despite the fact that SupDproved to be the worst suppressor of the Dam15 muta-tion. Temperature regulated D::eGFP fusion gene ex-pression from the pD::eGFP plasmid that through thevarious permutations, conferring resultant phages thatdiffered in size, fluorescence and absolute protein dec-oration (Nicastro et al. 2013; Sokolenko et al. 2012).

Phage display applications

The small size and the enormous diversity of variants that canbe fused to the bacteriophage capsid make phages ideal can-didates for many applications across many industries. Someprimary applications include targeted therapy and detection,and conjugation with macromolecules in medicine, plant sci-ence, and nanoparticles in materials science (Petty et al. 2007;Willats 2002). Initially, phage display was developed to dis-play random and natural peptide libraries (Dulbecco 1982).Random peptide libraries consist of synthetic randomdegenerated oligonucleotide inserts that are used to identifylinear antigenic epitopes and offer a universal applicationbase. In contrast, natural peptide libraries that are composedof randomly fragmented DNA from the genomes of selectedorganisms may be used to identify vaccine components andbacterial adhesins, although most of the clones are nonfunc-tional (Mullen et al. 2006). Since initial lytic phage display

design studies, a great deal of attention has focused onprogressing towards the development of novel therapeutic orindustrial applications for the system; recent studies combinethe genomic flexibility of phages with phage display to gen-erate genomically modified phage for targeted gene delivery.The list of applications is far too large for even a comprehen-sive review so we have selected the following topics based onsignificance and extensiveness in the field.

Phage as vaccines

As vaccine delivery vehicles, bacteriophage vectors offer astrong alternative to the more classical viral and non-viralsystems since they possess inherent attributes that permit themto overcome the challenges that generally limit the utility oftraditional approaches. The dimensions of phage λ are verysimilar to many mammalian viruses and may even share someof the same ancestry (Zanghi et al. 2007). They also endowedwith many of the intrinsic mechanisms of mammalian viruses,such as cell entry and endosomal escape, and while perhapsnot as efficient their eukaryotic viral counterparts, these attri-butes provide an advantage over non-viral vectors (Min et al.2010). As phages exclusively infect bacteria and are ubiqui-tous inhabitants of our natural habitat, it is not surprising thatthey have also been proven to be completely safe in mamma-lian systems and are free of many of the serious side-effectsattributed to mammalian viral vectors (Min et al. 2010; Seowand Wood 2009).

Phage vaccine delivery follows many of the same stepsrequired in the delivery of other non-viral vectors. Research iscurrently focusing on optimizing the binding and internaliza-tion of phage by antigen-presenting cells (APCs). However,the mechanism by which phages deliver their DNA cargo tothe nucleus once the particle has been internalized is not wellunderstood. It appears that the internalization of targetedphage particles is quite efficient, whereas endosomal escapeand nuclear uptake is not (Larocca and Baird 2001). Despitethis, phage-based vaccine delivery has been successfully im-plemented suing two different strategies: (1) direct vaccinationvia the manipulation of the phage to display the protectiveantigen on the surface coat proteins, or (2) using the phageparticle as a delivery system for a DNA vaccine expressioncassette containing the sequences required for vaccine antigensynthesis packaged into the phage genome (Clark and March2006; Seow and Wood 2009). The use of phages for targetedDNA delivery offers the inherent advantage of the phagecapsid, which can act to protect the DNAwithin from degra-dation once it has been injected, thereby acting as a virus-likeparticle (Haq et al. 2012). It has also recently been shown thatphage-carrying DNA offers a more efficient vaccination strat-egy than naked DNA vaccines (Clark et al. 2011; Clark andMarch 2004; Haq et al. 2012; March et al. 2004). A potentialthird strategy combines the above two methods to develop a

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“hybrid” phage. These phages are specifically engineered todisplay both the antigen of interest as well as the gene ofinterest in the genome of the phage for a combined effort. Thedisplayed peptide may also be used as a targeting ligand forthe delivery of the phage to specific immune cells and enhancedelivery efficiency (Min et al. 2010; Clark and March 2006;Lankes et al. 2007).

Bastien et al. (1997) employed phage fd to display achimeric protein with a proven protective epitope, (G glyco-protein) against human respiratory syncytial virus (RSV), toexplore the use of filamentous phage in the development ofvaccines. This study showed that mice acquired completeresistance to a viral challenge by the absence of the virus intheir lungs and high levels of RSV-specific antibodies. Inaddition, the level of antibody was only slightly lower thanimmunizedmice with whole RSV virus. This was also the firststudy that showed recombinant phage can be selected directlyfrom random peptide libraries and then used directly forprotection assays (Bastien et al. 1997). March et al. (2004)first employed λ as an encapsulated DNAvaccine where theyimmunized mice with λ particles encoding either eGFP orhepatitis B surface antigen (HBsAG) in the commerciallyavailable λ expression vector λ -gt11 (Agilent Technologies,Mississauga ON; March et al. 2004). The expression systemprotected the DNA cargo until it was safely within the cell, butthese particular constructs did not display the protein on thephage surface (Jepson and March 2004). Both of these mod-ified DNA vaccine constructs were able to elicit an immuneresponse inmice, but results were inconsistent, suggesting thatby displaying antigens on the surface of the phage, rather thansolely a DNA format may serve as a more specific andeffective vaccine approach. Thomas et al. (2012) recentlydeveloped a λ vaccine using a combination of cis (via recom-bination) and trans (plasmid-mediated) peptide phage displayto elucidate how peptide immunization might be superior togenetic immunization. They found that phage-based peptideexpression vectors elicited a greater immune response at aphysiological pH in higher primates and humans in the ab-sence of an adjuvant than genetic vaccine (Thomas et al.2012). They also developed a divalent subunit vaccine thatwas a hybrid of a DNA and a peptide vaccine with twoseparate fusions to gpD, one with GFP and the other withthe TAT protein (protein transduction) from HIV1 that provedto be superior to the peptide vaccine. The TAT protein wasspecifically chosen for its ability for the efficient delivery intomammalian cells (Eguchi et al. 2001). Both fusions wereconstructed to the C-terminal of gpD and a short linker wasplaced between the two protein domains, which weredisplayed on the phage surface in tandem. The same phagethat lacked display for either of these two proteins was used asthe DNA vector for comparison. Similar to the system here,the plasmid expressing the fusion was inducible by IPTG andthe recombineered genome was passaged through a SupF

host. In each case, the primary immune response was humoraland λ served as an endogenous adjuvant where the phage thatdisplayed the two peptides outperformed the non-displayingDNA vaccine (Thomas et al. 2012). The preparation processimproved as the temperature-inducible promoter used in thesystem for this research study only required a change intemperature as opposed to chemical inducers.

Zanghi et al. (2005) developed a mosaic expression ap-proach to circumvent the issue of recalcitrance by decorating λwith wild-type and recombinant proteins in tandem. Thissystem utilized two separate plasmid constructs that containeddifferent origins of replication and selectable markers thatwere co-transformed into a gpD-deficient λ lysogen (Zanghiet al. 2005). When only the recombinant plasmid wasexpressed, no viable progeny were recovered, indicating thatthe fusion must either interfere with assembly or preventformation of stable phage particles. Like wild-type phage,the slight reduction in mosaic phage titers could be attributedto plasmid attributes—mainly copy number and protein ex-pression. With the presence of two plasmids that have theo-retically consistent expression and fusion capacities, the levelof control is still quite limited. In contrast, employing anindependent second dimension of control conferred by sup-pression, imparts different rates of incorporation due to func-tionality that can generate greater variability in decoration.The group expanded on this idea by using the same constructdesign to simultaneously fuse proteins to the head and tail ofλ. The tested proteins were intended to increase phage-mediated gene transfer into mammalian cells. In order toutilize phages as transgene delivery vehicles multiple intracel-lular barriers, such as cell attachment, cytoplasmic entry,endosomal escape, uncoating, and nuclear import, must beovercome. Employing this strategy, multiple peptides could bedisplayed in tandem, with each functioning to circumvent aseparate barrier. During this process, the average number offusions per phage particle was found to be about 400 copiesfor gpD and 100 copies for gpV (Zanghi et al. 2007). Inaddition, the system could potentially target intracellular path-ogens to overcome intracellular barriers, where the lowerdensity display would presumably be employed for ligandinteraction and receptor-mediated endocytosis, while a higherdensity display could be used to target the pathogen.

Hayes et al. (2010) developed a λ-based vaccine where theconstruct had multiple fusions to the C-terminal of gpD; onewhere four immunodominant regions of the porcine circoviruscapsid were fused (D-CAP), a second with GFP (D-GFP) anda third with SPA tag protein (D-SPA). Interestingly, the D-GFP and D-CAP fusions could be expressed constitutivelywithout compromising the viability of the host whereas D-SPA expression reduced phage viability by more than 50-fold,due to protein recalcitrance. Recalcitrant fusions prevent pro-tein oligomerization and the formation of trimers during phageassembly and therefore display fewer D-fusion proteins,

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thereby compromising phage stability. Overall, this group wassuccessful in creating three different D-fusions, where the D-GFP was highly fluorescent, the D-SPAwas recognized by aFLAG monoclonal antibody and the D-CAP construct wasable to elicit both a cellular and humoral response in pigs.However, once these constructs were integrated into a λdisplay particle (LDP), LDP-D-GFP and LDP-D-SPA didnot induce a meaningful antibody response, limiting theirutility as mammalian vaccines (Gamage et al. 2009; Hayeset al. 2010). By controlling the number of fusions present, thechance of encountering recalcitrant fusions was however,reduced. Despite perhaps lower incorporation of fusion intoeach capsid, phage viability would be improved.

A more recent commercial example of phage displayemployed λ phage scaffold decorated with HIV-1 envelopeprotein (Env) via in vitro complementation, where the im-mune response elicited was compared to that of solublegp140. This strategy was employed because in their nativestate, envelope spike proteins are sparsely present on thevirion and the ability to increase their density on the phagecapsid could in theory generate an increased immune re-sponse. Phages were decorated with a mixture (different molarratios) of gpD:gp140 and wild-type gpD protein. Like Zanghiet al. (2005) and Hayes et al. (2010), Mattiacio et al. (2011)discovered that phage were recalcitrant and unstable whendecorated completely with a 20-fold molar excess of fusionprotein. They also determined that some fusion proteins couldstabilize phage particles even when they only occupy a frac-tion of the gpD binding sites and that the number of fusionsincorporated into the capsid was only 30 copies of Env trimersper particle (Zanghi et al. 2005; Hayes et al. 2010; Mattiacioet al. 2011; Nicastro et al. 2013). As a result, the investigatorsdid not observe an increased immune response to the decorat-ed phage compared to the conventional protein (Mattiacioet al. 2011). This shortfall may have been circumvented bythe system described here, where a weak suppressor increasedthe number of fusions per particle, thereby eliciting a strongerimmune response than the conventional protein. Each of thesegroups also executed codon optimization in developing theirconstructs, which allowed for more efficient protein expres-sion and less homology of fusions to native λ sequences, thusdecreasing the potential for homologous recombination be-tween alleles (Zanghi et al. 2005).

Phages are considered to be natural immunostimulators,where the carrying particle can serve as a natural adjuvant(Clark et al. 2011). This adjuvant effect of phage has beenhypothesized to be related to the presence of (1) CpG motifson the foreign phage DNA and (2) the virus-like repeatingstructure of the phage coat. It has been demonstrated that theCpG motifs on the phage DNA are likely responsible for thestimulation of the Th1 immune response in mice when deliv-ered with recombinant Hepatitis B antigen (Malanchère-Brèset al. 2001) and the stimulation of B-cell responses (Clark

et al. 2011; Li et al. 2012). Clark et al. (2011) demonstratedthat phage have an increased antibody responses measuredafter vaccination in comparison to the standard recombinantprotein vaccine Engerix for the treatment of Hepatitis B inrabbits. Ling et al. (2011) also demonstrated that mice vacci-nated with a phage vaccine expressing a DNA expressioncassette for the treatment of Chlamydiphilia abortus hadhigher levels of immune cell and cytokine responses in com-parison to those treated with a naked DNA vaccine, althoughthe immune response levels were not as high as those from theattenuated commercial vaccine (Ling et al. 2011). The naturalimmunostimulatory effect of phage speaks to the potential forphage-based vaccines since the phage already possess thenatural ability to stimulate an appropriate immune response.

Although phage vectors offer promising advantages, theyare not without their limitations. Phages are rapidly removedby the reticuloendothelial system, contain potent antigens(Seow andWood 2009), and generally offer a lower efficiencyof gene delivery relative to their viral counterparts (Laroccaand Baird 2001). While these limitations will require furtherattention, the advantages imparted by phage as single admin-istration gene and vaccine are many and suggest powerfulpotential for their use in future vaccine development (Seowand Wood 2009).

Phage as gene transfer vehicles

Bacteriophages offer strong potential as gene transfer vehi-cles, particularly due to the fact that their coat proteins canprotect DNA cargo against degradation during delivery (Clarkand March 2006; Dunn 1996). Additionally, the tolerance ofcapsid fusions makes it possible for display phage to targetspecific cells of choice (Clark and March 2006); a cornerstoneof successful gene therapeutic design. Fibroblast growth fac-tors have been used as targeting sequences for the delivery ofphages to cells that have the appropriate receptors (Hart et al.1994; Sperinde et al. 2001). These sequences have beenshown to enhance the uptake and endosomal release of phagevia proteins such as penton base of adenovirus whichmediatesentry, attachment, and endosomal release (Haq et al. 2012;Piersanti et al. 2004). The transduction domain of HIV (hu-man immunodeficiency virus) TAT protein and the SimianVirus 40 (SV40) T antigen nuclear localization signal havealso been exploited to enhance cellular uptake and nuclearlocalization of lambda phages, respectively (Nakanishi et al.2003).

Hart et al. (1994) reported the first use of filamentous phagefor gene transfer and delivery. In this study, phage fd wasemployed to display a cyclic-binding peptide sequence viatranslational fusion to the major coat protein pVIII. This N-terminal fusion, at approximately 300 copies of fusion proteinper phage, was bound to cells and was efficiently internalized,as determined by competition and cell binding assays,

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staining, and microscopy. The same peptide sequence wasthen fused to the major tail protein where λ was hypothesizedto be a more suitable candidate for transfecting mammaliancells due to its dsDNA genome. These modified phages wereshown to transfect mammalian cells at a remarkable frequencycompared to the control counterparts that showed no signifi-cant interaction with cells (Dunn 1996; Hart et al. 1994).Lankes et al. (2007) expanded the application of λ as a genedelivery vector by executing phage-mediated gene transferin vivo. Here, they constructed recombinant λ particlesencoding firefly luciferase (luc) in order to visualize genedelivery in real-time via the use of bioluminescence imaging.They also determined that pre-immunization of mice withwild-type λ a few weeks before immunization with recombi-nant phage conferred increased luciferase expression com-pared to unprimed control mice (Lankes et al. 2007). Theythen fused a αvβ3 (CD51/CD61) receptor integrin-bindingpeptide to gpD to increase its uptake by receptor-mediatedpinocytosis. This integrin was chosen because it is known toplay a role in the binding/internalization in a number ofmammalian viruses and it had been used to enhance targetingmodified virus vectors to dendritic cells, a particularly usefulaccessory cell target for a vaccine vector. Through the use ofthis recombinant phage, the study showed an increased inter-nalization versus a non-fused phage, where internalizationwas decreased in a dose-dependent fashion. Vaccaro et al.(2006) noted a similar decrease in internalization upon addi-tion of competitor protein indicating a receptor-mediated pro-cess. The recombinant phage outperformed the control cellsboth in vitro and in vivo. In addition, they noted a 100-foldincrease in the efficiency of phage internalization intointegrin-positive versus control cells, but only a three-foldincrease in the level of phage-mediated gene expression,indicating that an increase in internalization does not neces-sarily correspond to a comparable increase in the delivery ofgenetic material (Vaccaro et al. 2006). Overall, this studyprovided a proof-of-concept for the use of recombinant phageto increase the gene transfer in vivo and a compelling argu-ment for the use of phage in transgene delivery (Lankes et al.2007; Vaccaro et al. 2006).

Sapinoro et al. (2008) explored pre-immunization as anapproach to increase the uptake of recombinant phage, termedantibody-dependent enhancement (ADE). In concept, ADErelates to the observation that opsonization, or binding ofantibodies to an antigen, permits the more efficient infectionof susceptible host cells such as monocytes and macrophagesthat possess receptors for the antibody isotype. The objectiveof the group was to develop an in vitro model for this phe-nomenon in mammalian cells by bacteriophage λ vectors anddetermine their mechanisms. Since it is known that this effectcan be mediated through cellular receptors specific for the Fcportion of IgG, the group used a cell line expressing human Fcgamma receptor (FcγR) and found that antibody-dependent

enhancement of phage-mediated gene transfer required a re-ceptor on the surface of target cells (Sapinoro et al. 2008). Inaddition, prior immunization with wild-type phage resulted inmore efficient phage-mediated gene expression in live miceand this increased transduction was undertaken through areceptor-mediated endocytic mechanism rather than throughphagocytosis (Sapinoro et al. 2008). Overall, this researchstudy showed that the system was advantageous toward thedevelopment of downstream vaccines and targeted gene ther-apy approaches.

Although numerous methods have been developed forgene delivery, an efficient platform for protein delivery andmore specifically protein delivery in tandem with gene deliv-ery does not currently exist. Recently, Tao et al. (2013) devel-oped a T4 DNA packaging machine using T4-based“progene” nanoparticles that were targeted to antigen-presenting cells and were expresses both in vitro andin vivo. The group fused DNA molecules on the capsid headproteins Soc and Hoc that would later be displayed on thephage heads. Foreign cell penetration peptides (CPPs) andproteins (β-galactosidase, dendritic cell specific receptor 205monoclonal antibody, and CD40 ligand) were chosen fordisplay onto Hoc. The encapsidated DNA included GFP andluc (luciferase) genes to enable quantifiable expression withinmammalian cells. Overall, the group showed evidence forefficient in vitro and in vivo progene delivery and expressionof self-replicating genes into mammalian cells. Although theresults were promising, there is some room for further inves-tigation with the in vivo studies in particular where, unexpect-edly, the strongest luciferase signal came from mice infectedwith the nanoparticles that did not display a targeting ligand.The authors have attributed this finding to migration of thetargeted cells to other parts of the body; an inference that willrequire further investigation (Tao et al. 2013).

When undertaking a vaccination approach, high-densitydisplay may be preferred as functions of the fused proteinmay not be critical. In contrast, an application such as targetedgene therapy relies upon ligand interaction and proper folding/function of the fusion to improve cell specificity or enhancedendocytosis. The ability to control the number of fusionsprovides power and flexibility to cater mosaic phage to differ-ent scenarios and needs. Moreover, the ability to control thenumber of fusions and gpD molecules could reduce thechance of recalcitrance and improve viability. Finally, thissystem can be expanded to generate multiple fusions andpolyvalent therapeutics.

Phage as bacterial sensors

There is a strong and growing demand for new technologiesthat can detect pathogens in food, water, environmental, andpatient samples provided that they can address a real need: (1)pathogen detection in food, water, or air is required to identify

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pathogens or agents of bioterrorism before they can causeharm; (2) diagnose disease so that the appropriate therapycan be administered; or (3) identify disease-causing organ-ism(s) to allow preventative measures and to detect ongoingoutbreaks (Gulig et al. 2008). Classical methods of bacterialdetection employed biochemical tests that can be time con-suming and require microbes to grow on differential media.More recently, significant efforts have been applied to nucleicacid detection (i.e., real-time PCR) and the use of ELISA,antibody arrays, fiber optics, and surface plasmon resonanceto detect pathogens (Gulig et al. 2008). Herein, we will inves-tigate methods that have been developed for bacterial detec-tion using phage reporters, and specifically those that utilizephage display technologies.

Bacteriophage-based detection strategies are generallyquicker than many antisera/antibody methods, offering resultstypically in days as compared to months by conventionalmethods. Phage typing has been most extensively used forthe detection of Mycobacterium, Escherichia, Pseudomonas,Salmonella, and Campylobacter species (Barry et al. 1996).Developing phage reagents is also quite simple, rapid, eco-nomical, and easy to manipulate genetically. There is a con-siderable amount of literature from investigations that usephage display methods for the detection of a variety of anti-gens for a variety of purposes. We will highlight those thatcontribute a novel aspect to the use of phage display forbacterial detection purposes. The specificity of bacteriophagefor bacterial cells enables them to be easily used for the typingof bacterial strains and pathogens. This natural specificity canbe improved when phage display targeting peptides on thesurface of the phage coat proteins. The most commonly usedantibody for this is the single chain F variable (scFv) portionthat contains the antigen binding regions of the heavy andlight chains; an antibody portion can normally be obtainedfrom animals that are immunized with the target antigen/microbe. Phage particles that display the fusion peptide ofinterest are then selected by a process called panning whichinvolves the binding of phage to the antigen and the washingaway of unbound phage and then eluting of bound phagefollowed by amplification in the appropriate host cell (Smith1985; Gulig et al. 2008). Unbound scFv is normally unsuc-cessful for the purposes of bacterial detection. Sorokulovaet al. (2005) developed a random 8-mer landscape phagelibrary expressing fusions to coat protein (pVIII) of fd phage.The phage was panned against S. typhimurium whole cellsresulting in the isolation of a phage that was highly specifictowards Salmonellawhole cells (Sorokulova et al. 2005). In afollow-up study, Olsen et al. (2006) were able to detectSalmonella with this phage at a concentration as low as 100cells/mL (Olsen et al. 2006). This technique was also used topan against Bacillus anthracis, an organism of particularimportance in thwarting bioterrorist threats, leading to theidentification of multiple phages with the ability to capture

anthrax spores with reasonable specificity (Brigati et al.2004).

Ide et al. (2003) used the New England Biolabs 12-merlibrary of random peptides in an effort to isolate peptides forthe identification of the H7 flagella of E. coli O157:H7(Enterohemorrhagic E. coli, or EHEC). This is a pathogenicstrain of bacteria that is responsible for the onset of hemor-rhagic colitis and fatal kidney damage, due to the release ofphage-encoded Shiga toxin (H7 antigen) that is sharedamongst most strains. They were able to develop specificclones to H7, one of which could bind to intact E. coli cellsexpressing flagella (Ide et al. 2003). Similarly, Turnbough(2003) developed phage libraries using New England Biolabs7- and 12-mer libraries to isolate phage clones that couldrecognize a variety of Bacillus spores, including those formedby B. anthracis species. They were also able to differentiatebetween the spore types, an important aspect in the develop-ment of anti-spore reagents (Turnbough 2003).

Phage can also be used to deliver reporter genes such asgreen fluorescent protein that are expressed upon infection ofa bacterium (Loessner et al. 1997; Funatsu et al. 2002).Funatsu et al. (2002) manipulated λ by direct cloning toexpress a GFP reporter gene. The researchers were able todetect an E. coli infection 4 to 6 h post-infection, usingfluorescent microscopy to detect fluorescing bacteria(Funatsu et al. 2002). In a similar study by Oda et al. (2004),virulent phage PP01 specific to E. coli O157:H7 was used.Here, The GFP gene was displayed on the surface of the SOCcapsid protein without compromising the phage host range.Visual detection of infected target cells was again accom-plished by fluorescent microscopy after an incubation timeof only 10 min. The test worked on viable cells, viable butnon-culturable cells, and most notably dead cells, althoughwith a far lower fluorescence level (Oda et al. 2004). The samegroup constructed a T4-based reporter phage possessing aGFP fusion to the SOC protein of the capsid. This phagemutant was deficient in the ability to produce T4 lysozymeat the end of its replication cycle, thereby trapping the reporterphage within the target cells. Detection and visualization ofthe targeted cells was thus, relatively easy and efficient as GFPcontinued to accumulate within (Tanji et al. 2004).

Future applications of phage display to bacterial detectionwill likely look into the development and utilization of newnon-immunoglobulin target-binding tools that can beengineered to increase the randomness of binding. As exam-ples: (1) affibodies that are small proteins isolated from Staph-ylococcus aureus protein A can be manipulated by randomiz-ing the 13 amino acid binding domain and highly specificaffibodies to prophages in targets have already been obtained;and (2) anticalins are proteins based on lipocalin proteins thattransport or store molecules that are soluble and can be ma-nipulated to recognize a variety of targets with high affinitybinding (Gulig et al. 2008).

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Phage as bio-control agents

Expanding on the concept of bacterial detection, phage prod-ucts can also be manipulated to recognize and clear bacterialinfections. Typical phage-based bio-control procedures in-clude the use of purified lytic enzymes from phage (Fischetti2005), while other approaches employ genetically modifiedphages to not lyse cells, but rather to serve as targeted deliveryvehicles for DNA encoding antibacterials, to targeted patho-gens (Westwater et al. 2003). Particulate phages are relativelyeasy and inexpensive to purify, and phage display can alsoserve as an economical means to purify a particular protein/antibody (Clark and March 2006). Phage display libraries caneasily be screened against a set of target proteins with affinitiessimilar to antibodies, which can then be used as therapeuticagonists or conversely, inhibitors of receptor-ligand interac-tions (Clark and March 2006). As an added advantage, thegeneration of phage display antibodies does not require theuse of animals when working with existing libraries, eliminat-ing the need to inoculate animals with often toxic substancesor highly lethal infectious agents that increases cost and pro-duction time (Gulig et al. 2008). Phage display methods canalso be performed in vitro from randomly generated aminoacid sequences or randomly assorted antibody fragments,where the agents can be used directly to determine the detec-tion efficiency (Gulig et al. 2008).

Phage as therapeutic binding agents

Finally, phage can be used to bind to released componentswithin circulation, including toxins, and bacterial pathogensspecifically to prevent them from harming the host (Petrenkoand Vodyanoy 2003). An especially novel use of phage dis-play was the nasal administration of phage displaying anti-bodies against cocaine as a treatment against cocaine addic-tion (Dickerson et al. (2005). The whole filamentous phageparticles were able to effectively cross the blood–brain barrierand penetrate the central nervous system where the specificphage displayed antibodies could bind cocaine molecules,thereby preventing the behavioral effects from the drug(Dickerson et al. 2005).

Phage-based techniques for the control of bacterial patho-gens have undergone a great deal of research to date wherethese methods offer a set of advantages over conventionalmethods of detection, particularly when considering timeand specificity. Furthermore, many of the outlined methodsfeature high sensitivity and can be offered at relatively lowcost. Following administration, phages dissipate rapidlythroughout the mammal and reach the organs at varying ratesand to varying degrees (Dabrowska et al. 2005). While this isan advantage for detection and treatment purposes it alsomeans that phage are cleared quickly by the immune systemand as strong immunogens, are targets of antibody

neutralization. While phage offer promising alternatives fortherapeutic uses, the physicochemical characteristics thatmake them highly immunogenic, limits their utility as repet-itive therapeutics (reviewed in Kaur et al. 2012).


With an estimated 1031 phage particles worldwide (Gulig et al.2008), bacteriophages offer a practically unlimited reservoirof exploitable genetic information that can be applied to aplethora of purposes. The applications of phage display aremany and the rich and growing research in this area suggests apromising future for their exploitation in medicine, food sci-ence, biotechnology, and nanotechnology, just to name a few.

Acknowledgments We would like to thank Elisabeth Huang for hergreat help in editing this manuscript. This work was funded by theCanadian Institute for Health Research (DSECT) trainee program to JNand, Human Resources and Skill Development Canada (HRSDC) andNatural Sciences and Engineering Research Council of Canada (NSERC)funding to RAS.


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