Introduction
Filamentous bacteriophage predominantly infect Gram-negative bacteria and make an
important contribution to host physiology, ecology, and virulence, including production
of deadly toxins, such as cholera toxin. The unique filamentous structure, small genome
size (4–12 kbp), replicative and/or integrative mode of inheritance, simple cultivation,
and easy genomic manipulation sparked considerable attention to this class of bacteriophage
for a number of applications, including cloning, sequencing, recombinant protein expression,
phage display technology, and nanotechnology. This book covers a range of topics that
can be grouped into two themes: impact of diverse filamentous phage on their host
bacteria (five chapters) and applications of Escherichia coli Ff phage (eight chapters).
Impact of diverse filamentous phage on the host bacteria
Five articles explore diverse filamentous bacteriophage, including identification,
replication, integration into the host chromosome, and effect on their bacterial host
properties, such as growth rate, biofilm dynamics, and virulence.
Nagayoshi et al. describe the first fully sequenced hyperthermophilic filamentous
phage, ΦOH3, discovered in geothermal water. This phage infects the thermophilic bacterium
Thermus thermophilus HB8. Ahmad et al. identify and describe a novel filamentous phage
isolated from soil. The phage, named XacF1, causes loss of virulence in Xanthomonas
axonopodis pv. citri, the causative agent of citrus canker, and could potentially
be used for treatment or prevention of this disease.
Both XacF1 and ΦOH3 replicate efficiently and form turbid plaques due to increase
of the host generation time, but do not kill the host. Lack of the host killing is
intrinsic to the secretion-like process of filamentous phage assembly and release,
while the superinfection is prevented due to the blocking of primary and secondary
host receptors by the production of phage-encoded receptor-binding protein pIII in
the infected cells. One exception to these universally accepted rules is prophage
Pf4 of Pseudomonas aeruginosa PAO1. This phage converts into a “superinfective” form
within the mature P. aeruginosa biofilms, infecting and killing the surrounding prophage-containing
cells. Here, Hui et al. identify a role of reactive oxygen or nitrogen species DNA-damaging
activities in the formation of superinfective phage, providing a link to the observed
high-frequency mutations in the gene encoding repressor of Pf4 phage replication in
the mature P. aeruginosa biofilms.
In contrast to the phage described above, filamentous phage YpfΦ of the plague bacillus
Yersinia pestis replicates poorly, yet allows better colonization of the mammalian
host in comparison to the phage-free strain. Derbise and Carniel review the intertwined
microevolution of Y. pestis and YpfΦ over the past 3000 years. Some peculiarities
of this phage include its broad host spectrum, elusive host receptor(s), and hard-to-reconcile
pattern of seemingly exclusively episomal or integrated states in closely related
Y. pestis strains.
Most lysogenic filamentous phage rely on a host-encoded XerCD recombinase for integration
into highly conserved dif sites of bacterial chromosomes; however the mechanisms of
integration and prophage biology vary widely. Das reviews the integration mechanisms
of three lysogenic filamentous vibriophage (CTXΦ, VGJΦ, and TLCΦ) into the Vibrio
cholerae chromosomes. Variation in DNA sequences of attP sites in the phage genomes
drives differences in the integration and excision mechanisms, which ultimately impact
on the lysogen activation, prophage replication, and efficiency of phage production.
This review therefore outlines how the attP sites in a filamentous phage can be used
to predict integration/replication modes of filamentous prophage and conversely, how
the engineered attP sites can be used to design novel types of chromosomally-integrated
bacterial expression vectors.
Applications of the Ff filamentous phage
Eight chapters in this book review or report recent applications and technological
innovations involving Ff phage of E. coli, or derived particles. Phage display is
the most prominent application of filamentous phage. It was developed on the shoulders
of versatile cloning vectors derived from the E. coli Ff (F-pilus specific) filamentous
phage (f1, fd, and M13), and knowledge about their life cycle. Combinatorial technologies
including Ff phage display are based on a physical link of coding sequence to encoded
protein displayed on the virus particle. Screening vast Ff display libraries for variants
that bind a “bait” of interest has resulted over the past 25 years in identification
of bioactive peptides or therapeutic recombinant antibodies. Two chapters, by Gagic
et al. and Henry et al., review, respectively, phage display applications for discovery
of microbial surface proteins (including vaccine targets) and non-traditional applications
of phage particles as therapeutic biologics, vaccines carriers, or bioconjugation
scaffolds.
A technology report (Tjhung et al.) addresses an issue that has plagued phage display
libraries of proteins and peptides fused to the N-terminus of virion protein pIII,
in that some peptide variants are more likely to be degraded than others. Recombinant
phage encoding these degradable variants have advantage at amplification step over
other library clones, due to more efficient pIII-mediated infection of the host, and
may outcompete the true binders in the library screens. The authors demonstrate that
this can be prevented by displaying peptides between the pIII N1 and N2 domains instead
of display at the N-terminus. Given that N1 domain is essential for infection, amplification
of recombinant phage clones depends on preservation of displayed peptide (and thereby
retention of the N1 domain in the phage). This strategy eliminates those recombinant
clones in the library whose displayed peptides are degraded. It is very likely that
phage display between N1 and N2 domains of pIII will be taken up by many researchers
in the future.
Two research reports describe novel applications of Ff-phage-derived particles in
tumor targeting. Gillespie et al. describe a new approach for assembly of tumor-targeting
drug-loaded liposomes, by enabling spontaneous insertion of cancer-cell-binding peptide-pVIII
fusion protein. The insertion via pVIII hydrophobic core without damaging the liposome
was achieved by applying a novel method for direct purification from the phage particles,
using 2-propanol. This protocol greatly simplifies the assembly of cancer-targeting
drug-loaded liposomes, allowing screening of multiple peptides for targeting efficiency
and drug delivery. Dor-On and Solomon report brain tumor targeting by naked Ff phage
(not displaying any brain-targeting peptides) in a mouse model of glioblastoma after
intranasal application. Interestingly, particle-associated lipopolysaccharides may
be the key to brain targeting and anti-tumor activity of Ff in this model.
Three reports describe applications of Ff phage as nanoparticles. Sattar et al. report
development of a method to functionalize and efficiently produce extremely short Ff-derived
particles (50 nm in length) that contain no genes or antibiotic markers. The authors
show that the short particles perform better than the full-length phage of the same
composition as diagnostic particles in lateral-flow diagnostic assays. In a short
review, Bernard and Francis discuss modifications that are essential for applications
of Ff phage as functionalized nanoparticles. These include chemical conjugation to
organic molecules such as fluorophores, pigments, carbohydrates, or inorganic molecules.
One fascinating property of filamentous phage is that they are liquid crystals at
high concentrations. Review by Dogic gives a clear, biologist-friendly, and up-to-date
account of the liquid crystalline properties filamentous phage and their applications
in the soft matter physics.
Perspective
Future holds discovery of many novel filamentous phage. Some of these will likely
be used as genetic tools for bacterial engineering, utilizing knowledge about their
attP sites, integration, and replication. Many filamentous phage modulate bacterial
pathogenicity, hence therapeutic interventions against pathogenic bacteria, based
on known and novel filamentous bacteriophage, are eagerly anticipated. Filamentous
phage of innocuous bacteria other than currently used Ff (f1, fd, and M13) will find
applications in biotechnology, biomedicine, and nanotechnology, allowing exploration
of novel properties, with the aim of decreasing the production cost and environmental
footprint. Upscaling and eliminating safety concerns (removal of antibiotic-resistance
genes and ability to replicate) will allow transition of filamentous-phage-particle-based
technology from the laboratory containment to the consumer. In parallel, filamentous-phage-derived
particles of ever more imaginative functions or physical properties will be designed
and assembled into advanced nanostructures and nanomachines.
Author contributions
Manuscript was written by JR and BD; it was edited by all three authors.
Funding
Funding to JR laboratory by Palmerston North Medical Research Foundation, Massey University,
Institute of Fundamental Sciences, Anonymous Donor and the Maurice Wilkins Centre
for Molecular Biodiscovery is gratefully acknowledged. Work in BD laboratory was funded
by Department of Biotechnology, Govt. of India (Grant No. BT/MB/THSTI/HMC-SFC/2011).
RD acknowledges funding from the Alberta Glycomics Centre.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial
or financial relationships that could be construed as a potential conflict of interest.