Bacteriophages and their implications on future biotechnology: a review.

0.38 MB·English

by  Haq, Irshad Ul

Haqetal.VirologyJournal2012,9:9
http://www.virologyj.com/content/9/1/9
REVIEW Open Access
Bacteriophages and their implications on future
biotechnology: a review
Irshad Ul Haq*, Waqas Nasir Chaudhry, Maha Nadeem Akhtar, Saadia Andleeb and Ishtiaq Qadri
Abstract
Recently it has been recognized that bacteriophages, the natural predators of bacteria can be used efficiently in
modern biotechnology. They have been proposed as alternatives to antibiotics for many antibiotic resistant
bacterial strains. Phages can be used as biocontrol agents in agriculture and petroleum industry. Moreover phages
are used as vehicles for vaccines both DNA and protein, for the detection of pathogenic bacterial strain, as display
system for many proteins and antibodies. Bacteriophages are diverse group of viruses which are easily manipulated
and therefore they have potential uses in biotechnology, research, and therapeutics. The aim of this review article
is to enable the wide range of researchers, scientists, and biotechnologist who are putting phages into practice, to
accelerate the progress and development in the field of biotechnology.
Keywords: Bacteriophage, Phage therapy, Antibiotics, Vaccine, Biocontrol
Introduction Bacteriophages have contributed a lot to the field of
Bacteriophages are the most abundant entities on earth. molecular biology and biotechnology and are still play-
These bacterial viruses have genetic material in the form ing its part. Many mysteries of molecular biology are
of either DNA or RNA, encapsidated by a protein coat solved by bacteriophages. Today when everything is
[1]. The capsid is attached to a tail which has fibers, much more advanced than ever before, bacteriophages
used for attachments to receptors on bacterial cell sur- are getting enormous amount of attention due to their
face. Most of the phages have polyhedral capsid except potential to be used as antibacterials, phage display sys-
filamentous phages [2]. Phages infect bacteria and can tems, and vehicles for vaccines delivery [1]. They have
propagate in two possible ways; lytic life cycle and lyso- also been used for diagnostic purposes (phage typing) as
genic life cycle. When phages multiply vegetatively they well [1]. In this review article all these applications have
kill their hosts and the life cycle is referred to as lytic been summarized.
life cycle. On the other hand some phages known as
temperate phages can grow vegetatively and can inte- Phage therapy
grate their genome into host chromosome replicating Phages as therapeutic agents in humans were first used
with the host for many generations [3]. If induction to in 1919 just when they were discovered [4]. Phage ther-
some harsh conditions like ultraviolet (UV) radiations apy started back in 1896 when Ernest Hankin first
occurs then the prophage will escape via lysis of bacteria reported the existence of antibacterial activity against
[3]. After the discovery of bacteriophages in early 20th Vibrio cholera the causative agent of cholera which was
century many researchers thought about their (phages) considered one of the deadliest peril humans had faced
potential of killing bacteria, which could undoubtedly [5]. In 1915, Frederick Twort hypothesized that antibac-
make them possible therapeutic agents. But after World terial activity could be due to the virus (phage), but he
War II when antibiotics were discovered, this natural did not pursue his discovery, therefore bacteriophages
potential therapeutic agent got little attention and was were discovered by Fe’lix d’He’relle in 1917 [5]. In 1925
only considered as a research tool for many years [1]. d’He’relle reported treatment of plague (four types) by
antiplague phages which drew attention towards phage
therapy. Later on he visited India and worked on phage
*Correspondence:[email protected]
therapy of plague at the Haffkine Institute, Bombay
NUSTCenterofVirology&Immunology(NCVI),NationalUniversityof
Sciences&Technology(NUST),H-12,Islamabad44000,Pakistan
©2012Haqetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
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(Mumbai) [6]. In west the concept of phage therapy died One way around is the use of phage lytic enzyme
out in 1940 due to the emergence of antibiotics, but in endolysin, rather than administering the whole virion
former Soviet Union it was used and is still in practice. [14-16]. Similarly genetically modified phages can be
The Eliava institute in Tbilisi Georgia is considered the used, which will only deliver the DNA essential for mak-
pioneer in this regard where phage therapy is extensively ing antibacterials that would be specific for the target
studied and applied [7]. bacteria [17].
West has remained reluctant to use phage therapy due At the moment it seems a bit far that phage therapy
to the unreliable early trials of phage therapy. But still will replace antibiotics exclusively, but there is the hope
phage therapy got attention in USA. William smith and that it will be used complementary to antibiotics espe-
his fellows reported the successful use of phages against cially for antibiotic resistant strains [1]. Phages will be
E.coli in mice [8]. much more reliable when used externally and where the
One of the reasons of the avoidance of the phage ther- immune system gives it a chance by favoring it to per-
apy in most of the western countries was unreliable and sist within the body for a little while [1].
inconsistent results of many phage therapy trials. But
today it is accepted that the main reason behind the fail- Phage display
ure was poor understanding of phage biology and some The concept of phage display was first introduced in
other issues like quality control during preparation of 1985 [18] (Figure 1). Phage display is a molecular tech-
therapeutic stocks [9]. Phage therapy has been used in nique used for synthesizing polypeptides with novel
animals, plants, and humans with different degree of characteristics. The DNA that encodes the polypeptide
success. Phages have several potential advantages over is fused with phage coat protein genes, and the desired
antibiotics but at the same time it does have disadvan- protein is expressed on the surface of the phage particle
tages as well. [18,19]. For phage display filamentous phage M13 of E.
The main advantage of phages is their specificity for coli is extensively used, other phages like lambda and
target bacteria which reduces the damage to normal T7 are also used in phage display system [20,21]. Phage
flora of the host greatly. The bacteria to be targeted display libraries can be used for the screening and isola-
must be identified first or otherwise a cocktail of phages tion of peptides that are highly specific and which have
should be used. Bacteriophages are self-limiting i.e. they affinity for target proteins. These peptides can be used
require their hosts to be constantly growing; if the bac- in drug design as reagents for understanding molecular
terial pathogens they are specific for are absent they will recognition and it also minimize mimics for receptors
not persist for long enough [1]. Replication at the site of [19]. These peptides can be used as therapeutic agents
infection is another advantage of phages. They are safe by inhibiting receptor-ligand interaction or acting as
with no or less side effects [10,11]. If bacteria become agonist.
resistant to phages then phages do evolve naturally to Moreover these proteins can be used for the detection
infect the aforementioned resistant bacteria, hence mini- of pathogens and agents that are considered to be a
mizing the chances of bacterial escape, which scores potential threat to the environment [22]. Directed evolu-
another advantage of phage over antibiotics [10]. tion of proteins can be used to enhance the enzymatic
After their administration phages can dissipate very activity and binding properties [23]. The active site of
quickly throughout the body reaching almost every the enzyme is randomly altered and the activity of the
organ; but the immune system swiftly clears systemic enzyme is increased [1]. Phage display can also be varied
phages which pose yet another problem to their accep- by using phages to display the Fab antibody fragments
tance as therapeutic agent [12,13]. library mostly on filamentous phage surfaces [24]. These
One of the serious concerns about the use of phage libraries have many uses in research but one of the
therapy in vivo is a strong antibody response which most important uses of it is in the treatment against
would clear the phages more quickly and thus the use cocaine addiction where phages are administered nasally
of phages for extended period of time would not be pos- and then ultimately they make their way to central ner-
sible [1]. Other drawbacks of phages as therapeutic vous system (CNS). In central nervous system the dis-
agents are their narrow host ranges, and the fact that played antibody binds to cocaine molecule and inhibits
phages are not always lytic under certain physiological its action on brain [25]. Intensive and state of the art
conditions. During the preparation of phage stocks it work done by many researchers have made phage dis-
must be ensured that phage preparations are free of bac- play a phenomenal part of biotechnology. Amongst
teria and bacterial toxins in order to avoid secondary other applications phage antibodies have revolutionized
infections. But sterilizing phages could inactivate them. the concept of therapeutic drugs and drug design [19].
Phages may impart toxic properties to the bacteria Molecular evolution and protein-ligand interaction has
resulting in virulence [5]. been explained by phage display unambiguously [21].
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Phage particle Peptide gene fused to minor-
Phage coat proteins displaying
coat protein gene
foreign proteins
Phage particle Peptide gene fused to major-coat Foreign proteins are displayed on
protein gene some coat proteins
Phage particle Some coat proteins
Cellscontaining phagemid
displaying foreign
expressing coat protein
proteins
fusion gene are infected
Phage DNA Minor coat protein and Gene Foreign peptide and gene
Plasmid DNA Major coat protein and Gene Origin of replication
Figure1Somemethodsthatareusedtofuseforeignpeptidestothesurfaceofphage.Foreignpeptidescanbedisplayedonmorethan
onephagecoatproteins.Smallerforeignpeptidesaredisplayedinmorenumbersbutitalsodependsonthetypeofantigen,coatproteinand
thephage.(a)Thegeneforaforeignpeptideisdirectlyfusedtotheminorcoat-proteingene.Theforeignantigenisdisplayedbyallminorcoat
proteins.(b)Foreignpeptidegeneisattachedtomajorcoatproteingenewhileanothercopyofthegene(majorcoatproteins)isalsopresent.
Foreignproteinisdisplayedonsomemajorcoatproteins.(c)Cellscontainingaphagemid(plasmidthathavebothplasmidandbacteriophage
originofreplication)areinfectedwithunchangedhelperphagewhichthenexpressestheforeignpeptideorprotein.Foreignantigensare
displayedbysomecoatproteins.
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Phage typing immunodeficiency virus (HIV) tat protein and the
The specificity of phages for bacterial cells enables them simian virus 40 (SV40) T antigen nuclear localization
to be used for the typing of bacterial strains and the signal have also been used to enhance the uptake and
detection of pathogenic bacteria [1]. Phage typing is also nuclear targeting of phages like lambda that have been
known as the use of sensitivity patterns to specific modified [40]. Other displayed peptides that can facili-
phages for precisely identifying the microbial strains. tate gene delivery via phages include integrin binding
The sensitivity of the detection would be increased if peptides which enhance binding and uptake [37] and
the phages bound to bacteria are detected by specific DNA degradation reducing DNase II inhibitor [38]. To
antibodies [26]. For the detection of unknown bacterial screen the ability of phages for targeting specific cells
strain its lawn is provided with different phages, and if and tissues, phage display libraries have been used in
the plaque (clear zones) appears then it means that the mice many times and every time phages were found in
phage has grown and lysed the bacterial cell, making it specific tissues [41]. For instance isolating phages that
easy to identify the specific bacterial strain [1]. There target liver, mice were inoculated with phage display
are certain other methods which can be employed to libraries and phages were isolated after extracting the
detect pathogenic bacteria such as the use of phages livers [1]. Similar in vitro strategy is used for the isola-
that can deliver reporter genes {e.g. lux} specifically [27] tion of phage displayed peptides that enhanced cytoplas-
or using green fluorescent protein, [28] that would mic uptake into mammalian cells [42]. So again phages
express after infection of bacteria. Similarly phages hav- proved themselves to be versatile by making it possible
ing a fluorescent dye covalently attached to their coats to target specific tissues either by screening phage dis-
can be used to detect specific adsorption [29,30]. The play libraries randomly or by rational design [1].
detection of some of the released components such as
adenylate kinase [31] after the specific lysis of bacteria Phages as vehicles for vaccines delivery
and the use of antibodies and peptides that are displayed Phages have been used as vehicles for the delivery of
by phages can also be used, that will bind to toxins and vaccines (Figure 2). Phage particles can be used directly
bacterial pathogens specifically [22]. Dual phage technol- carrying the vaccine antigens expressed on their sur-
ogy is another application of phages in detection of bac- faces. But in case of DNA vaccines the sequences that
teria, in which phages are used to detect the binding of are essential for the vaccine antigen synthesis are incor-
antibodies to specific antigens [7]. Phage amplification porated into the phage genome and the phage would
assay can also be used to detect pathogenic bacteria then act as vehicle for the delivery of DNA vaccine [13].
[32]. The technique has most extensively been used for Phage display can be used to construct phages that
the detection of Mycobacterium tuberculosis, E.coli, would display the specific antigenic peptide on their sur-
Pseudomonas, Salmonella, Listeria, andCampylobacter faces [1]. Phage display libraries can be screened with
species [33]. specific antiserum to detect novel antigens and mime-
topes. Mimetopes are the peptides that mimic the anti-
Targeted gene delivery through Phages genic properties and secondary structures of protective
Phages are the potential therapeutic gene delivery vehi- protein, lipid or carbohydrate, although having different
cles [33,34]. The rationale of using phages for targeted primary structure [43,44]. Phage display libraries can
gene delivery is similar to that of using phages for DNA also be screened against the serum of convalescents for
vaccines delivery in which the phage coat protects the the identification of potential vaccines against specific
DNA inside from degradation after it has been injected. diseases [45]. There are some cases in which whole
But conceptually both are different. Phages ability to phage particles that displayed antigenic peptides have
display foreign proteins on their surfaces enable them to been used as vaccines in animal models [46,47].
target specific cell types which is a prerequisite for suc- Rather than transcriptional fusion to a coat protein,
cessful gene therapy [1]. Phage display and artificial some substances can be artificially conjugated to the
covalent conjugation are used to display targeting and phage surface after growth, which will increase the
processing molecules on the surfaces of phages [35,36]. range of displayed antigens [48]. Phages are considered
For the delivery of phages, targeting sequences such as to be natural immunostimulators [13,49] therefore an
fibroblast growth factor have been used to the cells hav- antigen that is presented on the phage coat protein
ing the appropriate receptors [37,38]. Enhancing the would come ‘ready conjugated’ with a natural adjuvant
uptake and endosomal release of phages, proteins activity, needing no separate protein purification and
sequences such as penton base of adenovirus which subsequent conjugation to a carrier protein before
mediates entry, attachment and endosomal release are immunization. Recently it has been shown that
used [39]. The protein transduction domain of human unchanged phages can be used to deliver DNA vaccines
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(cid:87)(cid:396)(cid:381)(cid:410)(cid:286)(cid:272)(cid:410)(cid:349)(cid:448)(cid:286)(cid:3)(cid:258)(cid:374)(cid:410)(cid:349)(cid:336)(cid:286)(cid:374)(cid:3)(cid:336)(cid:286)(cid:374)(cid:286)(cid:3)(cid:296)(cid:437)(cid:400)(cid:286)(cid:282)(cid:3)(cid:410)(cid:381)(cid:3)(cid:393)(cid:346)(cid:258)(cid:336)(cid:286)(cid:3)(cid:272)(cid:381)(cid:258)(cid:410)(cid:3) (cid:381)(cid:374)(cid:3)(cid:400)(cid:437)(cid:396)(cid:296)(cid:258)(cid:272)(cid:286)(cid:3)(cid:258)(cid:400)(cid:3)(cid:393)(cid:396)(cid:381)(cid:364)(cid:258)(cid:396)(cid:455)(cid:381)(cid:410)(cid:349)(cid:272)(cid:3)
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Figure2Someexamplesofmethodsforvaccinesdeliveryviaphages.(a)Hostcouldbeinoculatedforphage-deliveredproteinvaccine.(b)
Asin(a)hostisinoculatedforphage-deliveredproteinvaccinebuttheprotectiveantigenisexpressedasprokaryoticcoatproteinfusion.(c)
InoculationofhostforphagemediatedDNAvaccination.(d)Hostcanbeinoculatedforhybridphagevaccination,whereinoneconstruct
proteinandDNAvaccinesaredeliveredthroughphage.(e)HostinoculationforstandardDNAvaccination.
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more efficiently than standard plasmid DNA vaccination medical science. The applications of phages range from
[13,50-52]. The gene for vaccine is cloned under the the diagnosis of the disease, through phage typing, and
control of eukaryotic expression cassette in a lambda its prevention (phage vaccine), to the treatment (phage
bacteriophage and purified phage particles are injected therapy). There is the hope that phages could be useful
into the host. The coat protects DNA from degradation to humans in many ways. By making a cock tail of
and as it acts as a virus-like particle it would target the phages it would become easy to treat a wide variety of
vaccine to the antigen presenting cells [1]. When it was bacterial infections that are otherwise resistant to the
compared with the standard DNA vaccination, the anti- latest generations of antibiotics. A phage can be used
body response was very much superior in mice [52] and individually to treat a bacterial infection by lysing the
rabbits [50]. Recently the possibility of producing a bacterial cell as it is having the lytic potential. At the
hybrid phage has been proposed, a DNA vaccine con- same time the versatility of phages would allow us to
tained in phage particle under the eukaryotic promoter use the antibodies against the bacteria that have been
and a phage display variant of the same antigen is pre- displayed on the phage surface. Similarly a protective
sent on the phage surface [1]. Such a vaccine would effi- antigen could be delivered as a DNA or phage display
ciently target both humoral and cellular immune vaccine. So a mixture of phages that are modified
systems [13]. It can also be extended to the modification genetically would be more helpful in addressing all these
of surface of the phage vaccine by incorporating specific problems. Phages have also been good to cope with the
protein sequences to target particular immune cells food spoilage problem, and to treat the bacterial infec-
types like galactose residues that will target galactose tion of plants and fruits.
recognizing hepatic receptors in the liver [48]. Similarly There are some concerns about the use of phages. It
dendritic [53] and langerhans cells could be targeted by includes the safety and efficacy issues, as well as
isolating peptide from the phage display libraries [54]. immune response to the administered phages. Growth
optimization and purification strategies of phages are
Phages as biocontrol and bacteriophage also some issues needed to be addressed. Due to the
bioprocessing rapid progress in the fields of biotechnology and mole-
Phages could be used as predators of pests (bacteria) cular biology it is hoped that these entities (phages)
found in association withplants, fungi or their products which are present abundantly in the biosphere could
[55,56]. Phage mediated biocontrol of plant pathogens answer many questions human beings are having.
has successfully been attempted against Xanthomonas
pruni associated bacterial spot of peaches to control
Abbreviations
infectionsofpeaches,cabbage and peppers.Phageshave
HIV:Humanimmunodeficiencyvirus;CNS:Centralnervoussystem;SV40:
also been used to control Ralstonia solanacearum of Simianvirus40.
tobacco. They have been successfully employed against
Acknowledgements
Xanthomonascampestriswhichcausespotsontomatoes.
WeacknowledgeNationalUniversityofSciences&Technology(NUST)
SimilarlybacterialblotchofmushroomscausedbyPseu- Islamabadforprovidingfinancialassistance.
domonastolaasiicanbetreatedwithphages[57].Phages
Authors’contributions
have also been considered as a means of controlling the
IUHsurveyedtheliterature,collectedthereferencesconcernedwiththis
biofoulingofthermalpowerplantscondensertubes[58]. reviewanddraftedthemanuscript.MNAdesignedthefigures.WNC,SAand
Bacteriophages in bioprocessing are used to reduce the IQcontributedinrevisingandeditingthemanuscript.Alltheauthorsread
andapprovedthefinalmanuscript.
bacterialloadinfoodsusuallyintheminimallyprocessed
foods to avoid cooking associated flavor or texture [59]. Competinginterests
Controllingpathogensoffruitsandvegetablesisofmuch Theauthorsdeclarethattheyhavenocompetinginterests.
concernasthese foods cannot be further processedthat
Received:11June2011 Accepted:10January2012
would kill any pathogen present. Control of pathogens Published:10January2012
viaphagesisanon-thermalinterventionbywhichgrowth
of Salmonella and Campylobacter on chicken skin [60] References
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doi:10.1186/1743-422X-9-9
Citethisarticleas:Haqetal.:Bacteriophagesandtheirimplicationson
futurebiotechnology:areview.VirologyJournal20129:9.
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