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doi:10.1093/bfgp/elt009
Bacteriophage functional genomics and
its role in bacterial pathogen detection
Jochen Klumpp, Derrick E. Fouts and Shanmuga Sozhamannan
Advance Access publication date 21 March 2013
Abstract
Emerging and reemerging bacterial infectious diseases are a major public health concern worldwide. The role of bacteriophages in the emergence of novel bacterial pathogens by horizontal gene transfer was highlighted by the May
2011 Escherichia coli O104:H4 outbreaks that originated in Germany and spread to other European countries. This
outbreak also highlighted the pivotal role played by recent advances in functional genomics in rapidly deciphering
the virulence mechanism elicited by this novel pathogen and developing rapid diagnostics and therapeutics.
However, despite a steady increase in the number of phage sequences in the public databases, boosted by the
next-generation sequencing technologies, few functional genomics studies of bacteriophages have been conducted.
Our definition of ‘functional genomics’ encompasses a range of aspects: phage genome sequencing, annotation and
ascribing functions to phage genes, prophage identification in bacterial sequences, elucidating the events in various
stages of phage life cycle using genomic, transcriptomic and proteomic approaches, defining the mechanisms of
host takeover including specific bacterial ^ phage protein interactions and identifying virulence and other adaptive
features encoded by phages and finally, using prophage genomic information for bacterial detection/diagnostics.
Given the breadth and depth of this definition and the fact that some of these aspects (especially phage-encoded
virulence/adaptive features) have been treated extensively in other reviews, we restrict our focus only on certain aspects. These include phage genome sequencing and annotation, identification of prophages in bacterial sequences
and genetic characterization of phages, functional genomics of the infection process and finally, bacterial identification using genomic information.
Keywords: next-generation DNA sequencing; prophage identification; genome annotation pathogen detection; reporter phage;
antimicrobials
INTRODUCTION
Infectious diseases are a constant threat to human
health and the second most frequent cause of
death, with an estimated more than 10 million fatalities worldwide annually. Amongst the top five foodborne diseases worldwide, four are caused by
bacterial pathogens, such as Salmonella, Clostridium,
Campylobacter and Staphylococcus, and are responsible
for more than 3 million infections and intoxications
in the USA alone in 2011 [1]. In the European
Union, foodborne infections by Salmonella,
Campylobacter and Listeria account for more than
300 000 human cases of infection annually [2]. By
their nature, bacteriophages (phages), viruses that
infect bacteria, are ideal weapons to combat bacterial
infections. They are highly species-specific, and are
Corresponding author. Jochen Klumpp, Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich,
Switzerland. Tel.: þ41 44 632 5378; Fax: þ41 44 632 1266; E-mail: [email protected]
Jochen Klumpp is a research fellow at the Institute of Food, Nutrition and Health, ETH Zurich. He is a molecular biologist and
bioinformatician, and his main research interests are DNA sequencing technologies, the bacteriophage infection process, the use of
bacteriophage particles and enzymes for food safety and transmission electron microscopy of viruses.
Derrick E. Fouts is an associate professor at the J. Craig Venter Institute, Rockville, MD. He is a molecular microbiologist,
genomicist, and bioinformaticist. His research focus is on viral and bacterial genomics, microbial pathogenesis, and community
metagenomics. He has developed a number of bioinformatics software tools, including tools for prophage identification
(Phage_Finder) and for ortholog detection (PanOCT).
Shanmuga Sozhamannan is a molecular biologist with GoldBelt Raven, LLC providing support as the technical coordinator for the
Critical Reagents Program within the Chemical Biological Medical Systems project office on Fort Detrick, MD. His research interests
focus on understanding the biology of bacterial viruses and extra chromosomal elements and their roles in horizontal gene transfer and
evolution of bacterial pathogens using genetic, molecular biological and genomic approaches.
ß The Author 2013. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]
Bacteriophage functional genomics
non-toxic to animals and plants and they are
self-amplified when they infect, multiply and kill
their target bacteria [3]. Bacteriophages are the
most abundant biological entity on this planet and
it is estimated that phages kill half of all bacteria each
day and influence most of earth’s biogeochemistry
through their bacterial host [4,5]. Since their discovery in 1915 and 1917 by D’Herelle and Twort [6,7],
bacteriophages have been used primarily as research
tools in the Western world and as antibacterial agents
in the former Soviet Union countries and Eastern
Europe. The discovery of antibiotics led to the complete abandonment of phage therapy by the West
[8,9] and, hence, phages are not as widely used as
therapeutics as one would imagine. The widespread
use, and in many instances misuse, of antibiotics since
the 1940s, in a variety of healthcare settings to
combat bacterial infections in humans and livestock,
has resulted in the current crisis with antibiotic resistant bacteria. Recently, phage therapy and biocontrol approaches have received renewed research
interest. It is believed that the antibiotic potential
of phages has not yet been fully realized [3,8,10–12].
The modern day phage therapy attempts of the
Western world started around 1980. In a pioneering
study, Smith and Huggins successfully eradicated experimentally-induced Escherichia coli infection in a
mouse infection model [13]. Since then, similar studies have been conducted to cure infections of many
bacteria such as Staphylococcus aureus, VancomycinResistant Enterococci (VRE), Methicillin-Resistant
Staphylococcus aureus (MRSA) in animals,
Acinetobacter baumanii or Pseudomonas aeruginosa and animals with local and systemic disease caused by Vibrio
vulnificus, Mycobacterium avium, Mycobacterium tuberculosis
in macrophages, E. coli and fish pathogens. Phages
have also been used in treating non-systemic infections caused by enteropathogenic strains of E. coli in
calves, pigs and lambs and ileocecitis caused by
Clostridium difficile in hamsters. Phages have been
shown to be effective in preventing the destruction
of skin grafts by P. aeruginosa, against fish pathogens in
aquaculture and bacterial wilt in vegetables [14–17].
Bacteriophages have more recently been used in various other applications to counter pathogenic bacteria.
The use of bacteriophage preparations or bacteriophage-derived enzymes for detection and control of
pathogens in the food chain has recently attracted
intensive research (e.g. reviewed in [18,19]). Some
additional examples include the detection of Listeria,
Bacillus orYersinia by reporter phages carrying luciferase
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genes, which are expressed upon infection and
transcription of phage genes [20–23] and the biocontrol of pathogenic bacteria in food [24–27] (reviewed
in [28]).
Bacteriophages, especially those featuring a temperate lifestyle, are also the major driving force of
horizontal gene exchange and bacterial evolution
[29–31]. Many bacterial genomes carry either complete and fully functional prophages or defective
remnants of prophages indicative of prior infections.
In many cases, they account for a significant proportion of the bacterial genomic content. For example,
Streptococcus pyogenes features a genome with more
than 10% phage-related sequences [32], and in
E. coli O157:H7 strain Sakai, prophage elements account for 16% of the total genome [33]. In many
instances, the prophages confer adaptive features to
their host; these include genes related to bacterial
virulence, antibiotic resistance cassettes or toxin
genes in a variety of organisms [29,32]. For example,
CTX phage of Vibrio cholerae [34,35] or shiga-toxin
carrying lambdoid prophages of pathogenic E. coli,
such as the serotype O104:H4, responsible for the
recent outbreak in Germany [36].
In the classical sense, bacteriophage functional
genomics includes DNA sequencing of phage genomes, annotation and attribution of functions based
on in silico approaches and or biochemical or genetic
studies and identifying prophage regions in bacterial
sequences. With the recent expansion of ‘omics’
areas, it can also include the elucidation of various
stages of phage infection process using genomic,
transcriptomic and proteomic approaches, specific
mechanisms that phages adopt to shut down and
take over host macromolecular machinery for their
own reproduction and growth, and the study of the
interaction between specific bacterial and phage proteins. For this review, we focus on three specific
areas: (i) recent advances in bacterial and phage genomics, tools for identifying prophage regions and
genetic characterization of phages; (ii) functional
genomics of the infection process and (iii) using
phage genomic data for bacterial identification in
clinical and non-clinical samples.
Bacteriophage genome sequencing
Obtaining the complete genome sequence of a bacteriophage is an essential prerequisite for any type of
functional genomics study as well as for regulatory
approval for phage-based biocontrol and therapy applications in food industry and medicine. Recent
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advances in second- and third-generation DNA
sequencing technologies have led to an increasing
number of phage genomic sequences, either from
purified virus particles or as a result of bacterial
genome sequencing in the form of prophage sequences [37]. Conventional Sanger sequencing has
been rivaled by a number of second-generation
high throughput short-read sequencing technologies
(e.g. 454, Illumina, SOLiD and Ion Torrent) and
lately by a third-generation single-molecule longread sequencing technology (e.g. PacBio RS by
Pacific Biosciences), all of which produce more
than enough data to reliably assemble phage genomes. Although the short-read sequencing technologies produce large amounts of short, accurate reads,
they also produce context-specific errors, which
make error correction by the same method difficult.
In addition, because of the short read length, repetitive genome sequences are difficult to assemble.
Recent improvements in chemistry and device
design are targeted to address these problems. For
example, single read lengths of up to 1000 bp can
be achieved using the latest FLX plus chemistry of
the Roche 454 sequencing platform. Other
approaches include the generation of paired-end
long insert libraries, which provide scaffolding information [37]. However, single-molecule sequencing
using third-generation PacBio RS technology produces by far the longest read lengths, although the
individual reads are error prone and feature
single-pass accuracies of only around 87%. Since
the primary error model consists of truly random
single nucleotide indels, these reads can be corrected
by an increase in sequencing coverage either by circular consensus sequencing or with data from other
sequencing platforms, e.g. Illumina data for error
correction [37–39]. Error correction by deep
sequencing is an especially attractive option, as it requires only one long-insert library and a single
sequencing run. Thus, reliable bacteriophage whole
genome sequencing, regardless of the technology
used, is possible and competitively priced. Nextgeneration sequencing technologies have also
brought about the democratization of whole
genome sequencing because of the smaller foot
print needed in terms of cost, space and manpower.
Hence, generating sequence data is within any researcher’s reach. However, data handling, storage
and analysis are not trivial [37].
In addition to more than 1700 bacteriophage
genomes, the above described advances in DNA
sequencing technologies have enabled the generation
of more than 3500 complete and 11 000 incomplete
bacterial genome sequences (Gold genomes database,
November 2012). In many genome-sequencing projects, the presence of integrated genomes of temperate phages (prophage) in the host chromosome is
overlooked or not assessed. Conservative estimates
show the presence of prophages in nearly 50% of
all sequenced bacterial strains, with prophages contributing up to 20% of a bacterial genome [40]. Thus,
the phage gene pool is larger and more diverse than
the rest of the chromosome.
Bacteriophage genome annotation
More than 50% of in silico predicted phage gene
products are hypothetical and do not have an assigned function due to a lack of experimental data.
For instance, phage T4 (one of the most extensively
characterized bacteriophages) genome has about 300
probable genes (168 903 bp genome size) and almost
half of these still do not have an assigned function
[41]. Several approaches have been put forth to solve
the problem of assigning functions to hypothetical
gene products (encoded by predicted open reading
frames with no homology to any known gene) in all
phage genomes. Recently developed bioinformatic
tools, such as the structure/function prediction tool
HHPred [42] make use of Hidden Markov Models
(HMMs) and protein profiles to deduce putative
protein functions and identify functional domains
in larger proteins. Comparative genomic approaches
using closely related phages from different host organisms can fill another gap in protein function assignment. Some information can be deduced from
the genetic context or location of genes of interest,
because phage genomes are organized in a modular
fashion [31,43] and mosaicism (i.e. the exchange of
genetic modules between phages) is very common.
The recent surge of interest in phage-based antimicrobials has led to more studies being published
describing the experimental characterization of specific phage proteins, e.g. tail adhesins [44–47], host
takeover genes [48,49], core genes with therapeutic
potential [50] or lysins (see below).
Many of the hypothetical phage proteins are
likely involved in host recognition and, disruption
of host metabolism; hence, are potential candidates
for bacterial detection and antimicrobial target selection, respectively. This reasoning is best illustrated by
a pioneering study on drug discovery through phage
genomics, in which investigators at PhageTech Inc.
Bacteriophage functional genomics
used 27 S. aureus phage genome sequences to screen
potential drug targets. Screening a total of 964 ORFs
for abolition of bacterial growth produced 34 distinct
families of growth-inhibitory proteins (3.5%)
[10,51]. This screen excluded the obvious growth
inhibitory proteins such as holins, lysins or polypeptides with transmembrane domains. With an increase
in the number of genes with assigned functions,
future comparative genomic studies will likely yield
more accurate prediction of phage gene functions.
Thus, functional genomics approaches, either in
silico or experimental, present a vast potential for improving phage annotation and facilitating development of tools for food safety and medicine.
Prophage identification and using
prophages as markers for bacterial
identification
Assuming an average of three prophage regions
per genome [52], there is a potential for as many as
42 000 prophage genomes available for data mining in
the currently available bacterial genome sequences—
22 times the number of currently sequenced bacteriophage genomes. Even without prophage identification, genes encoding enzymes of interest, such as
phage lysins (see below) can easily be identified
using HMMs or BLAST searches. If, however, the
objective of a study is to generate phage tail-based
molecular diagnostic tools or tail-based bacteriocins
(see below), prophage identification is needed. The
same holds true if the study objective is to generate a
synthetic phage genome for detection or eradication
of specific bacterial taxa, as homology of tail proteins
can be too low for HMM or BLAST detection and
the nucleotide sequence of the entire prophage is
needed to construct synthetic variants.
A number of bioinformatic tools have been
developed for prophage identification in bacterial
sequences, including Phage_Finder, one of the first
automated prophage-finding open source programs
developed. Since the publication of Phage_Finder
[52], there have been at least five other prophagefinding tools described in the literature [53–56].
Phage_Finder is written in PERL and uses
BLASTP matches to a curated phage database and
HMM matches to phage-specific models to define
the rough location of a prophage region followed by
heuristic decision making to extend the region based
on annotation. Putative attachment (att) sites are
found using one of many different local alignment
options. Prophinder [54], also written in PERL also
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uses BLASTP matches to a database, but chooses
regions based on probability statistics rather than
heuristics. PHAST [55] is a web server that also
uses homology to identify prophage regions, but is
unique in that it does not require an annotated
genome as input. Prophages have also been identified based on nucleotide differences [53], training of
artificial neural networks [57] or using a combination
of AT/GC skew, protein length, gene directionality,
homology and k-mer frequencies using a random
forest classification algorithm [56]. Though each publication claims their software to be better than the
previously reported programs at finding a set of
‘known’ prophage regions, the value of these comparisons is unclear, since each group has a different
definition of what constitutes a true prophage. As
modified from a previous review on bacteriophage
bioinformatics [58], a prophage region is defined as
‘a cluster or stretch of genes, possibly encoding proteins with bacteriophage-like core functions (i.e. terminase, portal, capsid), interspersed with genes of
unknown function or functions other than phage replication, morphogenesis, packaging, immunity or lysis
of the host’. Ultimately, a true prophage is inducible,
whereas defective prophages are the non-inducible
remnants of formerly functional temperate bacteriophages. In addition, some comparisons may have been
skewed since Phage_Finder can run under two different stringency modes, producing radically different
results. PhiSpy [56] uses a training set based on known
prophages in closely related organisms to train the
random forest classification algorithm implemented
in the R statistical package, which by definition
should produce better results than other programs because the training set is highly related to the prophages
being identified.
Phage-Finder does more than just finding prophage regions—it takes advantage of the power of
HMMs to identify, with high confidence, specific
phage genes of interest. Lysins, toxins and tail components can be mined for applications in bacterial
detection or destruction (see below). In addition,
HMM matches to family-specific proteins can be
used to characterize the prophage region (e.g.
Mu-like or P2-like prophage).
These in silico approaches to prophage identification are also successful at identifying cryptic, defective prophages or remnants of prophages. One such
class of defective prophages is the phage tail-like bacteriocins (PTLBs), for which the P. aeruginosa R-type
or F-type pyocins are the prototypes [59]. PTLB
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regions are defective prophages which are different
from prophage regions in that they primarily contain
genes for tail fibers and tail appendages, regulators
and lysis genes, but lack genes for packaging and
head morphogenesis. PTLB particles can kill bacteria
of similar species by disrupting the membrane of the
target bacteria [60]. The bacteriocins are referred to
by many different names based on the bacterium that
produces it (monocin for Listeria monocytogenes, pestisin for Yersinia pestis, cerecin for Bacillus cereus, colicins
for E. coli, staphylococcin for Staphylococcus and diffocin for C. difficile) [61]. Like bacteriophage, the bacteriocins have proven useful for the typing/
identification of pathogenic bacterial strains [62,63].
The presence of prophages can be used to detect
and type bacterial pathogens and define their roles in
gene regulation and pathogenicity of their host bacteria [64,65]. Newly emerging V. cholerae variants can
be discriminated by PCR targeted to mobile genetic
elements and phages [66]. Prophage polymorphism
has been employed to detect and differentiate exfoliative toxin A-producing S. aureus strains [67]. Likewise,
the chromosomal insertion sites of Shiga
toxin-producing E. coli define the three major
genotypes (clusters 1–3) among clinical isolates of E.
coli O157:H7 [68]. In addition, it has been found
that prophage regions are critical for competitiveness
of P. aeruginosa strains [69]. Prophages also provide
unique signatures for bacterial identification. For example, Bacillus anthracis genome encodes four prophages and the collective presence of all four is a
unique feature of B. anthracis and can be exploited
for developing a multiplex PCR assay for B. anthracis
[70]. Similarly, the recent German outbreak
strains can be distinguished from their near neighbor
E. coli O104:H4 strains by prophage content [71].
Experimental approaches to identify prophages in
bacterial chromosomes rely on the induction of prophages by UV irradiation or chemical DNA damaging agents [72]. However, this approach is only
applicable to intact prophages that can undergo excision from the chromosome and undergo the lytic
life cycle to be detected as plaques on a bacterial
lawn. In some pathogens, such as V. cholerae, only
subpopulations of bacteria undergo lysis and produce
toxin. Studies of these subpopulations require a
method to isolate cells that are normally killed by
the prophage after induction [73]. Methods such as
recombination-based in vivo expression technology
and selective in vivo expression technology have
been developed for this purpose [73–75].
Functional genomics of the phage
infection process and the use of
phage and phage proteins for
pathogen control and detection
Phages are found in nearly every environment where
they have been searched for and they can transduce
genetic markers between related host bacteria. As
alluded to in an earlier section, phage-mediated horizontal gene transfer between intestinal bacteria and
autochthonous bacteria is of interest with respect to
bacterial pathogenicity [76]. For example, the presence of prophages enables Clostridium botulinum and
C. difficile toxin production [77,78], increases
Salmonella virulence [79] and influences gene expression within the Staphylococcus pathogenicity islands
[80]. Phages with transduction or lysogenic potential
are generally not desired in any biotechnological application and identification of such phages or of
genes contributing to lysogeny in a phage is critical
before embarking on any whole phage based therapeutic application. [81]. Also, temperate phages often
possess a narrow host range which limits their utility
as biocontrol agents but may be useful as diagnostic
tools [82].
Due to a general lack of functional annotation of
many phage gene products and the enormous diversity seen in the strategies adopted by phages, our
understanding of the mechanism of the phage infection process is rather sketchy at best. In general, functional data, which could bolster in silico prediction of
protein function based on sequence homology, are
lacking. Only a few model phages have been studied
in detail with respect to their life cycle (mostly
infecting Gram-negative organisms) and structure/
functions of only a few phage proteins have been
determined. The infection of a bacterial cell by a
bacteriophage is initiated by a highly specific and
irreversible interaction of the phage particle associated protein to cell surface receptors, such as
outer membrane proteins or LPS structures in
Gram-negative bacteria or teichoic acids or the peptidoglycan backbone in Gram-positive bacteria. This
binding is mediated by highly specific receptor binding protein (RBP) of the phage that is very difficult
to predict simply based on sequence homology.
Besides a trial-and-error cloning strategy of candidate
receptor genes, genomics approaches such as structure prediction and identification of conserved domains play an important role in the discovery of
RBP proteins. The three-dimensional (3D) atomic
RBP structures of three Lactococcus siphoviruses
Bacteriophage functional genomics
(p2, bIL170 and TP901-1) have been solved by
X-ray crystallography and Cryo-EM techniques
[44–47]. General saccharide and phosphoglycerol
binding has been demonstrated for some RBPs
[44,83,84]. The baseplate of Lactococcal phage
Tuc2009 has been shown to possess a 6-fold symmetry with six RBP trimers attached around the
baseplate and a tail-associated lysin located in the
middle of the base plate [83].
Bacteriophages of two other organisms have been
subject to intensive research recently. Streptococcus
thermophilus is an important organism in industrial
fermentation. The RBP of the small Siphovirus
DT1 of S. thermophilus is encoded by gp18 [85].
The receptors for S. thermophilus phages are most
likely glucosamine, N-acetylglucosamine or rhamnose [86,87]. Other phages of Streptococcus have
been investigated for identifying the host receptor
[87,88]. Bacillus subtilis Siphovirus SPP1 has been the
focus of intensive structural studies. Besides 3D structures of the virion and details of DNA packaging, the
baseplate proteins (gp 22–24.1) have been investigated [89–91]. The baseplate proteins have been
shown to be essential for binding of the host cell
and hence may have utility in bacterial detection
assays. Cell wall teichoic acids have been proposed
as the major target of SPP1 reversible initial binding,
followed by binding to the membrane receptor
YueB. Adsorption to cell wall teichoic acids has
been shown to accelerate irreversible binding to
YueB [92]. Thus, these functional genomic
approaches lay the foundation for modeling based
structural prediction of phage encoded RBPs.
Following adsorption, the phage must penetrate
the rigid cell wall and inject its DNA into the host
cytosol. Specialized tail-located lytic proteins mediate localized cell wall degradation and allow DNA
translocation. The potential application of lytic structural proteins (LSPs) in biocontrol is exemplified by
the identification of improved adsorption of the
SPO1-related phage EF34C infecting Enterococcus
faecalis [93,94] due to a spontaneous point mutation
in a putative long tail fiber protein encoded by
ORF31 [95], using a functional genomics approach.
With the exception of well-studied enteric phages
such as bacteriophage P22 [96], little is known
about the molecular details of DNA transport
across cell membranes, especially in phages infecting
Gram-positive bacteria [97]. Localized cell wall hydrolysis is mediated by tail tip-associated lytic proteins (LPs) [98–100]. It is believed that LSPs are
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commonly found among all phages [101–103]. In
Lactococcus phage Tuc2009, a tail-tip associated protein (Tal2009) has been found to feature cell wall
degrading activity [101,104]. The presence of LSP
proteins in phages infecting other Firmicutes has
been demonstrated [103,105,106]. The LSP of
Staphylococcus phage MR11 could be located at the
tail tip [107], whereas in mycobacteriophage TM4,
the lytic activity lies in the tape measure protein
[108]. Recently, a tail-associated muralytic enzyme
was described for Staphylococcus phage K and its
activity demonstrated [103].
Infection of a bacterial host by a bacteriophage is
usually accompanied by dramatic rearrangements in
bacterial gene expression and changes in cell physiology and metabolism. Understanding these steps
using functional genomics approaches may lead to
development of rapid diagnostics and therapeutics
against bacteria. After an initial takeover phase, the
virus down-regulates cellular functions and reprograms the bacterial cell for virus progeny production.
Later in the life cycle, virion proteins and progeny
DNAs are produced, packaged and virions are
assembled, finally resulting in cell lysis and release of
progeny phage particles. In case of temperate bacteriophages, the infection process can be stalled at an
early stage and the phage genome is integrated in the
host chromosome, resulting in some cases, host gene
disruption and possible effects on gene expression
(e.g. as recently shown for comK disruption in
Listeria by phage A118 [65]). Once induced by stress
signals, temperate phages enter into a lytic cycle and
take over host cell functions. Many studies have
focused on only certain stages of the infection process,
although in some phages, the entire life cycle has been
investigated. Escherichia coli phage T4 is one such example that has been extensively studied in using
microarrays [109] and proteomics (described in
[41,98] and many more). Host modification and takeover functions have been described for some phages
[110,111]. Many studies have focused on the changes
in gene expression and cell physiology during virus
infection [112,113]. Other authors have demonstrated the complete shutoff of macromolecular synthesis in the infected host [114,115]. Such functional
genomic approaches reveal specific phage–bacterial
interactions in shutting down and take over of host
machinery and also possibly provide new drug targets.
The identification of DnaI (the helicase loader) as a
target for ORF 104 of a S. aureus phage 77, led to the
development of novel small molecule antimicrobial
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Klumpp et al.
compounds that mimic the phage protein (ORF 104
product) activity (69). More such studies are needed
to unravel the various strategies that phages adopt to
tame their host.
All stages in the bacteriophage life cycle can be
harnessed for pathogen detection and for the development of novel antimicrobials. Bacterial detection
can be achieved either by manipulation of the genomes of virulent phages to give a detectable signal
during the infection process (reporter phages) or by
the use of the highly specific phage binding proteins,
either RBPs or the anchoring domain of endolysins
[84,116–118] for specific tagging and/or immobilization of bacteria. Phage tail lysins [102] and endolysins
[119,120] represent a novel class of lytic protein
(enzybiotics) for pathogen control.
Another pathogen detection approach, which follows the genomics to protein function path, is the
use or reporter bacteriophages. Reporter phages are
usually virulent phages containing a genetically modified genome in which a reporter gene is expressed
under the transcriptional control of phage promoters.
Expression of the reporter gene upon infection of
the susceptible host generates a detectable response.
The advantage of such an approach is that only viable
target cells are detected, as the expression of the reporter gene relies on successful infection and initiation of phage gene expression. Construction of such
reporter phages requires functional genomic knowledge of the gene expression profiles and location of
promoters and appropriate insertion sites for the reporter genes and the effect of reporter gene insertion
on the overall phage gene expression and a productive life cycle. One of the early approaches involved
cloning of the luxAB genes from Vibrio harveyi or
Vibrio fischeri behind a strong late phase phage promoter of Listeria phage A511 [20]. This type of reporter assay has subsequently been developed for a
number of other bacterium–phage pairs (i.e. Bacillus
[23], Salmonella [121] or Yersinia [22]). In principle,
nearly every available reporter gene can be used for
diagnostic purposes (e.g. luc, ina, beta-galactosidase or
fluorescent molecules [122–124]). Recently, a
hyperthermostable glycosidase reporter gene cloned
into phage A511 was used for the detection of
Listeria. The assay offers flexibility in the use of fluorescent, chemilumnescent or chromogenic substrates
for pathogen detection [125]. The reporter-phage
assay is highly specific, yields no false-positive results
and is easy to conduct in a standard laboratory setting
by lab personnel with little specialized experience or
expertise. The assay detects only viable, phage-sensitive bacteria and is also somewhat dependent on
correct folding of the reporter protein (which
should be chosen accordingly) and incubation temperature [125]. Recently, phage genomes have been
manipulated to express biotin-binding peptide on
the capsid surface, which were subsequently captured
upon adsorption to cognate bacteria using streptavidin coated quantum dots for high sensitive detection
of bacteria [126,127].
Other approaches to pathogen detection harness
the function of bacteriophage encoded enzymes.
Phage genomes usually encode at least two types of
highly specific cellular binding proteins. Tail proteins
feature binding domains, which mediate the initial
contact with the host cell surface (RBP proteins, see
above). These are highly strain- or even serovarspecific and can be used for pathogen detection.
They feature a single binding-site and are in this respect superior to antibodies, because no agglutination
is triggered.
The final stage of a successful infection is marked
by the release of the progeny virus particles. Phageencoded cell wall hydrolytic enzymes lyse the bacterial cell from within [119]. These enzymes are also
very useful for pathogen detection and control. Large
libraries of lytic enzyme-encoding genes identified
by either genome sequencing, prophage identification or functional (meta-) genomics approaches [128]
are available for different bacterial species, e.g.
Streptococcus and Staphylococcus [120]. Phage lysins
are composed of an enzymatic active domain(s) and
a cell wall-binding domain(s) (CBDs), directing the
enzyme to its target structure [119]. CBDs have
efficiently been used to target and selectively decorate bacteria in several matrices and media [117,129–
131]. CBD domains can be fused to reporter molecules, such as Green Fluorescent Protein (GFP), for
easy readout of the signal upon binding to the target
bacteria. Different CBD molecules, tagged with different fluorophores can be used to simultaneously
identify several different bacterial species or serovariants in a one-step multiplex assay [117]. CBDs feature equilibrium constants in the picomolar range
and are highly species specific and more sensitive
than conventional antibodies [117,131]. Recently,
CBD domains have been used in combination with
gold screen-printed electrodes to develop a biosensor
for Listeria [132]. Because of the usefulness of these
small phage proteins in combination with powerful
detection methods, we expect many more such
Bacteriophage functional genomics
biosensor platforms targeting many different bacteria
to be developed in the near future.
In general, CBD proteins are not as effective for
detection of Gram-negative cells, as the outer membrane precludes binding to the cell wall receptor
structure. For Gram-negative bacteria, phage-tail
based affinity molecules are much better suited for
immobilization and detection. Moreover, these
proteins exhibit similar desirable features as CBD
molecules, such as high affinity, high specificity and
a single binding site. Besides specific cell decoration,
both CBDs and tail adhesins can also be used to
immobilize bacterial cells, for example to paramagnetic beads. Enrichment of a specific pathogen from
a complex matrix is possible with this technique in a
matter of minutes, compared to hours or even days
when using conventional enrichment methods [131].
Similar to methods employing whole phage particles,
coupling of these phage proteins to specific surfaces
for detection applications, paves the way for the development of a new class of biosensor based on
phage proteins [133,134].
In conclusion, bacteriophage functional genomics
offers exciting new possibilities for detection and
control of bacterial pathogens. The high specificity
of phages and some phage proteins to their bacterial
hosts, combined with the absence of undesired side
effects and the relatively straightforward discovery
and large-scale production of phages make them
ideal tools for food industry and medical applications.
However, our knowledge of phage-encoded gene
functions, and their influence on the bacterial host
gene expression and our general understanding of
the molecular diversity of phage life cycle are
rather limited. Recent advances in genome sequencing, comparative genomics combined with functional genomic studies will undoubtedly play a
major role in filling this knowledge gap and increase
our understanding of phage biology for better utilization of these organisms for bacterial detection and
therapeutics.
Functional genomic studies addressing all stages of the bacteriophage infection cycle can be manipulated in developing methods
for pathogen detection.
Acknowledgements
J.K. is grateful to Martin J. Loessner for providing the research
environment which enabled the bacteriophage functional genomics studies mentioned in this manuscript. The views expressed
in this article are those of the authors and do not necessarily
reflect the official policy or position of the JPEO-CBDCBMS-BSV-CRP, Department of Defense, nor the US
Government.
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