Expressing bacteriophage in mammalian cells

Expressing bacteriophage in mammalian cells

We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Is it possible to express bacteriophage in mammalian cells by codon optimization? Any relevant literature? thanks in advance.

Expressing bacteriophage in mammalian cells - Biology

Swapnil Ganesh Sanmukh and Sérgio Luis Felisbino *
Laboratory of Extracellular Matrix Biology, Department of Morphology, Institute of Biosciences Botucatu, Sao Paulo State University, Sao Paulo, Brazil

* Corresponding author: Sérgio L Felisbino, Laboratory of Extracellular Matrix Biology, Department of Morphology, Institute of Biosciences Botucatu, Sao Paulo State University, Sao Paulo, Brazil

Published: 16 May, 2017
Cite this article as: Sanmukh SG, Felisbino SL. Bacteriophages in Cancer Biology and Therapies. Clin Oncol. 2017 2: 1295.


- using the Vaccinia Virus system. Over expression of proteins in mammalian cells (hamster BHK21 cells) is achieved using an attenuated vaccinia virus vector (Modified Vaccinia Virus Ankara-MVA) that is user safe and may be handled under BSL1 conditions. Genes of interest are cloned downstream of a bacteriophage T7 promoter in a first step to check for feasibility. Protein expression is then examined after plasmid transfection into BHK21 cells and co-infection with a specialized viral vector encoding the bacteriophage T7 RNA polymerase. The IGBMC facility perform this feasibility step with any gene of interest cloned downstream of a T7 promoter and acquire results within a few days.

To obtain high level protein expression, genes that perform well in the feasibility test are cloned into specialized plasmids that enable their site directed transfer into the viral genome downstream of the bacteriophage T7 promoter. Recombinants viruses encoding genes of interest are isolated in a few weeks under non permissive conditions for recombinant protein expression. High level expression is then accomplished in mammalian cells in the presence of inducer (IPTG). Up to 10 mg of purified protein may be consistently produced from 5 litre suspension cell cultures. A general outline of methods used may be found in the following references: Anal Biochem. 2012 426(2): 106-8 Anal Biochem. 2010 Sep 1 404(1): 103-5 Protein Expr Purif. 2007 56(2): 269-78. More recent improvements improving the speed of operations and protein productivity are unpublished.

Support includes small scale expression tests with plasmids provided by users (needs the T7 promoter). Easy gene assembly using the “Biobrick” system based on compatible restriction sites (EcoRI, XbaI, SpeI, PstI) as described in (Ho-Shing et al., 2012) with a variety of N-terminal tags in a Gateway entry vector for subsequent construction of recombinants virus is available. Participation of users is encouraged for training in the isolation of viral expression vectors and protein production runs at the IGBMC. Users will be provided with specialized materials and the most recent methods available.


Bacterial strains and plasmids

The bacterial strains used were Stable 2 (Life Technologies, Breda, The Netherlands), INV㬟’ (Invitrogen, Groningen, The Netherlands), MC1061 (Stratagene Europe, Amsterdam, The Netherlands), KMBL 1164 [Δlac-proXIII, thi209, SupE + ] (16) and KMBL 1001 [SupE – ], derived from W1485 (22).

Plasmid pmMu876 was derived from pGP876 (23) by removal of the genetic code for MuA and MuB by partial EcoRI digestion and re-circularization.

The construction of plasmids pSC-NLS-MuA and pSC-NLS-MuB, which contain the transposase, MuA and transposition stimulator, MuB, respectively, was performed as described below. To isolate the Mu-A coding sequence, pGP876 was digested with the restriction enzymes EcoRV and PmeI, a fragment of 2018 bp was isolated and blunted by Klenow polymerase filling-in of the sticky-ends. Subsequently, this fragment was ligated into the eukaryotic expression vector pSuperCatch-NLS (24), a derivative of pCatch-NLS, which had been digested with BamHI and filled-in with Klenow polymerase. The construct, which contained MuA under control of the cytomegalovirus promoter (pCMV), was called pSC-NLS-MuA (Fig.  1 A).

Constructs used in the analysis of Mu transposition in mammalian cells. (A) Plasmid pSC-NLS-MuA contains the eukaryotic expression cassette encoding the Mu transposase, MuA, fused to a Flag epitope and SV40 NLS. The T7 RNA-polymerase transcription initiation site enables prokaryotic expression of this fusion protein. Plasmid pSC-NLS-MuB contains the eukaryotic expression cassette encoding the transposition stimulator, MuB, similar to the MuA construct. (B) The donor plasmids are characterized by the presence of a miniMu transposon, delimited by the L and R attachment domains (L att and R att). These att domains are depicted as arrows pointing to the location of the MuA-induced nick sites. The L att domain also includes the IAS region. The miniMu transposon of pmMuHyg contains a Hyg R marker driven by a TK promoter. Donor construct pmMuHyg-GFP contains the previously mentioned miniMu transposon with Hyg R marker and a CMV-driven GFP marker, which is located just outside this transposon. The miniMu transposon from pmMuNeo contains a Neo R marker driven by the SV40 promoter for eukaryotic expression and by the Tn5 promoter for prokaryotic expression. This element also includes a pBR322-derived origin of replication (pBR Ori). pCMV, immediate early promoter from cytomegalovirus pSV40, promoter from Simian Virus 40 PolyA, polyadenylation site ColE1, origin of replication NLS, nuclear-localization signal Amp R , Ampicilin-resistance marker T7 and Sp6, RNA-polymerase transcription-initiation sites pTK, thymidine-kinase promoter Hyg R , Hygromycin-resistance marker Cam R , Chloramphenicol-resistance marker GFP, green fluorescence protein LTR, long terminal repeat Tn5, Tn5 Neo promoter. Unless indicated otherwise, the plasmids were used in supercoiled form.

The coding sequence for MuB was obtained by digestion of pGP876 with SspI and DraIII. A fragment of 1231 bp was isolated and the DraIII-originated 3′ overhang was removed by exonuclease 3′𡤥′. This blunt-ended fragment was cloned into the BamHI-digested and Klenow polymerase-blunted pSuperCatch-NLS. The construct pSC-NLS-MuB (Fig. ​ (Fig.1A) 1 A) contained the MuB gene driven by the CMV promoter. DNA sequence analyses confirmed the integrity of the modified MuA and MuB genes in plasmids pSC-NLS-MuA and pSC-NLS-MuB.

Three donor constructs, plasmids containing a miniMu transposon, have been used in the in vivo transposition experiments (Fig. ​ (Fig.1B). 1 B). The first construct, pmMuHyg was constructed by digestion of plasmid pCep4 (Invitrogen) with NruI and partially with SalI, isolation of the 3.2-kb fragment and cloning it into SmaI- and SalI-digested pmMu876. The second donor construct, pmMuHyg-GFP, is identical to the first one, except that a GFP-marker is added outside the miniMu transposon. First, the GFP-marker was cloned into pmMu876. To that end, plasmid phGFP-S56T (Clontech Laboratories, Palo Alto, CA) was digested with MluI and BamHI, the resulting 2.3-kb fragment, which carries the complete GFP-expression cassette, was isolated and blunted with Mung Bean nuclease. This fragment was cloned into pmMu876, which had been linearized with ClaI and blunted with Mung Bean nuclease. The Hygromycin-resistance marker (Hyg R ) was subsequently cloned into the resulting construct in the same way as described for pmMuHyg. The last donor construct, pmMuNeo was made as follows. Plasmid pmMu876 was digested with AatII and SalI and a 5927-bp fragment was isolated. In this we cloned the 3707-bp AatII- and SalI-digested fragment from the retroviral vector pBAG (25).


The detection of the MuA and MuB proteins, both fused to a Flag epitope, was performed with a 400-fold dilution (3% BSA in PBS) of a mouse monoclonal antibody against the Flag epitope (m㬟lag M2 Kodak, New Haven, CT). Fluorescein-isothiocyanate-labeled goat anti-mouse antibody (GαMFitc Jackson Immunoresearch Laboratories, WestGrove, PA) was used as second antibody. Nuclear DNA was stained with 1 µg/ml 2,4-diamino-2-phenylindole (DAPI), 2% 1,4 diazabicyclo-ֲ,2,2]-octane and 0.1 M Tris–HCl pH 8.0 in glycerol.

Protein analysis by western blotting

The hybrid protein products from pSC-NLS-MuA and pSC-NLS-MuB have been produced in vitro with the TNT T7 Coupled Reticulocyte Lysate System (Promega, Leiden, The Netherlands), in accordance with the manufacturer’s protocol. Stably transfected 911 cells provided the hybrid proteins after in vivo expression. Cell lysate was obtained by scraping the cells in RIPA lysis buffer (25 mM Tris–HCl pH 7.4, 50 mM NaCl, 0.5% Doc, 2% NP-40, 0.2% SDS). After a 10 min incubation at room temperature, the lysates were cleared by centrifugation and the protein concentration of the supernatant was measured by the Bradford protein assay.

Cell-lysate and in-vitro transcription translation samples were fractionated on a 10% SDS–PAGE gel. Proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA) and incubated with m㬟lag (Kodak) as first antibody. The second antibody was a horseradish peroxidase-labeled goat anti-mouse antibody (Brunschwig Chemie, Amsterdam, The Netherlands). The resulting protein𠄺ntibody complexes were visualized by enhanced chemiluminescence.

Complementation of MuA am and MuB am phages

Cultures of the Escherichia coli strains KMBL 1001 [Sup E – ] transformed with pSC-NLS-MuA, KMBL 1001 [Sup E – ] transformed with pSC-NLS-MuB and KMBL 1164 [Sup E + ] were diluted to an ODλ650 = 0.1 (10 8 cells/ml). At this concentration, 1 ml culture samples were infected by wild-type bacteriophage Mu, MuA am or MuB am at multiplicities of infection (m.o.i.) of 2𠄳. After phage addition, the samples were incubated at 37ଌ for 20 min, without perturbation. Then, the samples were diluted 1000-fold (5 µl:5 ml) and grown at 37ଌ. After 1 h incubation, 50 µl chloroform was added, the samples were centrifuged for 10 min at 800 g and the phage-containing supernatant was collected.

The phage titre of the supernatant was determined on strains KMBL 1164 and KMBL 1001. The supernatant was diluted 10- or 1000-fold and 100 µl of these dilutions were added to a 1-ml culture of KMBL 1164 or 1001, respectively. The mixture was incubated for 20 min at 37ଌ, without perturbation. Hereafter, 3 ml top agar (1:1, agar versus LB) was added and immediately transferred to LB plates. After overnight incubation at 37ଌ the plaques were counted and the titre was calculated.

Cell culture, transfection and selection

The Ad5E1-transformed human embryonic retina cell line 911 (26) and the osteosarcoma cell line U2OS were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), antibiotics and 3 g/l glucose in a 5% CO2 atmosphere at 37ଌ. Transfection of 911 cells was performed by the calcium-phosphate technique (27). U2OS cells were transfected with Fugene (Boehringer Mannheim, Almere, The Netherlands) in accordance with the manufacturer’s protocol. Approximately 48 h post-transfection, selection of transfected cells was started by the addition of 150 µg/ml Hygromycin (Boehringer Mannheim) or 450 µg/ml G418 (Life Technologies) to the medium. One week post-transfection, all mock-transfected cells died and Hygromycin or G418-resistant colonies appeared. Selection pressure was then reduced to 50 µg/ml Hygromycin or 200 µg/ml G418. Solitary colonies were picked, cell lines established and chromosomal DNA isolated.

Analysis of chromosomal DNA for transposition events

Chromosomal DNA was isolated from G418-resistant colonies obtained after co-transfection of 911 or U2OS cells with the donor construct pmMuNeo, pSC-NLS-MuA and with or without pSC-NLS-MuB. To check for Mu-catalyzed integrations, the DNA flanking the att-sites was analyzed by the following procedure. For the analysis of the DNA flanking the L att site, 1 µg chromosomal DNA was digested overnight with 3 U of SpeI (Fig. ​ (Fig.1B). 1 B). After heat inactivation of the restriction enzyme, the samples were diluted to 4 ng/µl and the fragments circularised. After overnight incubation, DNA was precipitated and used for electroporation of E.coli strain Stable 2. Transformants containing the Neomycin-resistance marker (Neo R ) were selected by growth on Kanamycin-containing LB plates at 30ଌ. The resulting colonies were checked for Mu-related origin by contra-selection on Ampicilin (Amp). The Amp-sensitive colonies were further analyzed by SpeI and HindIII restriction analysis, PCR based on primers surrounding the Mu-induced nick site and, finally sequence analysis.

The same analyses were performed to study the DNA flanking the R att site, but in this case BamHI was used to digest the chromosomal DNA.

Targeting Bacteriophage to Mammalian Cell Surface Receptors for Gene Delivery

Filamentous bacteriophages represent one of nature's most elegant ways of packaging and delivering DNA. In an effort to develop novel methods for ligand discovery via phage gene delivery, we conferred mammalian cell tropism to filamentous bacteriophages by attaching basic fibroblast growth factor (FGF2), transferrin, or epidermal growth factor (EGF) to their coat proteins and measuring CMV promoter-driven reporter gene expression in target cells. In this system, FGF2 was a more effective targeting agent than transferrin or EGF. The detection of green fluorescent protein (GFP) or β-galactosidase (β-Gal) activity in cells required FGF2 targeting and was phage concentration dependent. Specificity of the targeting for high-affinity FGF receptors was demonstrated by competing the targeted phage with FGF2, by the failure of FGF2-targeted bacteriophage to transduce high-affinity FGF receptor-negative cells, and by their ability to transduce these same cells when stably transfected with FGFR1, a high-affinity FGF receptor. Long-term transgene expression was established by selecting colonies for G418 resistance, suggesting that with the appropriate targeted tropism, filamentous bacteriophage can serve as a vehicle for targeted gene delivery to mammalian cells.


Cre-Lox recombination is a special type of site-specific recombination developed by Dr. Brian Sauer and patented by DuPont that operated in both mitotic and non-mitotic cells, and was initially used in activating gene expression in mammalian cell lines. [2] [3] [4] Subsequently, researchers in the laboratory of Dr. Jamey Marth demonstrated that Cre-Lox recombination could be used to delete loxP-flanked chromosomal DNA sequences at high efficiency in specific developing T-cells of transgenic animals, with the authors proposing that this approach could be used to define endogenous gene function in specific cell types, indelibly mark progenitors in cell fate determination studies, induce specific chromosomal rearrangements for biological and disease modeling, and determine the roles of early genetic lesions in disease (and phenotype) maintenance. [5]

Shortly thereafter, researchers in the laboratory of Prof. Klaus Rajewsky reported the production of pluripotent embryonic stem cells bearing a targeted loxP-flanked (floxed) DNA polymerase gene. [6] Combining these advances in collaboration, the laboratories of Drs. Marth and Rajewsky reported in 1994 that Cre-lox recombination could be used for conditional gene targeting. [7] They observed ≈50% of the DNA polymerase beta gene was deleted in T cells based on DNA blotting. It was unclear whether only one allele in each T-cell or 50% of T cells had 100% deletion in both alleles. Researchers have since reported more efficient Cre-Lox conditional gene mutagenesis in the developing T cells by the Marth laboratory in 1995. [8] Incomplete deletion by Cre recombinase is not uncommon in cells when two copies of floxed sequences exist, and allows the formation and study of chimeric tissues. All cell types tested in mice have been shown to undergo transgenic Cre recombination.

Independently, Joe Z. Tsien has pioneered the use of Cre-loxP system for cell type- and region-specific gene manipulation in the adult brain where hundreds of distinct neuron types may exist and nearly all neurons in the adult brain are known to be post-mitotic. [9] Tsien and his colleagues demonstrated Cre-mediated recombination can occur in the post-mitotic pyramidal neurons in the adult mouse forebrain. [10]

These developments have led to a widespread use of conditional mutagenesis in biomedical research, spanning many disciplines in which it becomes a powerful platform for determining gene function in specific cell types and at specific developmental times. In particular, the clear demonstration of its usefulness in precisely defining the complex relationship between specific cells/circuits and behaviors for brain research, [11] has promoted the NIH to initiate the NIH Blueprint for Neuroscience Research Cre-driver mouse projects in early 2000. [12] [13] To date, NIH Blueprint for Neuroscience Research Cre projects have created several hundreds of Cre driver mouse lines which are currently used by the worldwide neuroscience community.

Cre-Lox recombination involves the targeting of a specific sequence of DNA and splicing it with the help of an enzyme called Cre recombinase. Cre-Lox recombination is commonly used to circumvent embryonic lethality caused by systemic inactivation of many genes. [14] [15] As of February, 2019, Cre–Lox recombination is a powerful tool and is used in transgenic animal modeling to link genotypes to phenotypes. [11] [16] [17]

The Cre-lox system is used as a genetic tool to control site specific recombination events in genomic DNA. This system has allowed researchers to manipulate a variety of genetically modified organisms to control gene expression, delete undesired DNA sequences and modify chromosome architecture.

The Cre protein is a site-specific DNA recombinase that can catalyse the recombination of DNA between specific sites in a DNA molecule. These sites, known as loxP sequences, contain specific binding sites for Cre that surround a directional core sequence where recombination can occur.

When cells that have loxP sites in their genome express Cre, a recombination event can occur between the loxP sites. Cre recombinase proteins bind to the first and last 13 bp regions of a lox site forming a dimer. This dimer then binds to a dimer on another lox site to form a tetramer. Lox sites are directional and the two sites joined by the tetramer are parallel in orientation. The double stranded DNA is cut at both loxP sites by the Cre protein. The strands are then rejoined with DNA ligase in a quick and efficient process. The result of recombination depends on the orientation of the loxP sites. For two lox sites on the same chromosome arm, inverted loxP sites will cause an inversion of the intervening DNA, while a direct repeat of loxP sites will cause a deletion event. If loxP sites are on different chromosomes it is possible for translocation events to be catalysed by Cre induced recombination. Two plasmids can be joined using the variant lox sites 71 and 66. [18]

Cre recombinase Edit

The Cre protein (encoded by the locus originally named as "Causes recombination", with "Cyclization recombinase" being found in some references) [19] [20] consists of 4 subunits and two domains: The larger carboxyl (C-terminal) domain, and smaller amino (N-terminal) domain. The total protein has 343 amino acids. The C domain is similar in structure to the domain in the Integrase family of enzymes isolated from lambda phage. This is also the catalytic site of the enzyme.

LoxP site Edit

loxP (locus of X-over P1) is a site on the bacteriophage P1 consisting of 34 bp. The site includes an asymmetric 8 bp sequence, variable except for the middle two bases, in between two sets of symmetric, 13 bp sequences. The exact sequence is given below 'N' indicates bases which may vary, and lowercase letters indicate bases that have been mutated from the wild-type. The 13 bp sequences are palindromic but the 8 bp spacer is not, thus giving the loxP sequence a certain direction. Usually loxP sites come in pairs for genetic manipulation. If the two loxP sites are in the same orientation, the floxed sequence (sequence flanked by two loxP sites) is excised however if the two loxP sites are in the opposite orientation, the floxed sequence is inverted. If there exists a floxed donor sequence, the donor sequence can be swapped with the original sequence. This technique is called recombinase-mediated cassette exchange and is a very convenient and time-saving way for genetic manipulation. The caveat, however, is that the recombination reaction can happen backwards, rendering cassette exchange inefficient. In addition, sequence excision can happen in trans instead of a in cis cassette exchange event. The loxP mutants are created to avoid these problems. [21]

13bp 8bp 13bp
Example Alternate loxP Sites [22]
Name 13bp Recognition Region 8bp Spacer Region 13bp Recognition Region

During genetic recombination, a Holliday junction is formed between the two strands of DNA and a double-stranded break in a DNA molecule leaves a 3’OH end exposed. This reaction is aided with the endonuclease activity of an enzyme. 5’ Phosphate ends are usually the substrates for this reaction, thus extended 3’ regions remain. This 3’ OH group is highly unstable, and the strand on which it is present must find its complement. Since homologous recombination occurs after DNA replication, two strands of DNA are available, and thus, the 3’ OH group must pair with its complement, and it does so, with an intact strand on the other duplex. Now, one point of crossover has occurred, which is what is called a Holliday Intermediate.

The 3’OH end is elongated (that is, bases are added) with the help of DNA Polymerase. The pairing of opposite strands is what constitutes the crossing-over or Recombination event, which is common to all living organisms, since the genetic material on one strand of one duplex has paired with one strand of another duplex, and has been elongated by DNA polymerase. Further cleavage of Holliday Intermediates results in formation of Hybrid DNA.

This further cleavage or ‘resolvation’ is done by a special group of enzymes called Resolvases. RuvC is just one of these Resolvases that have been isolated in bacteria and yeast.

For many years, it was thought that when the Holliday junction intermediate was formed, the branch point of the junction (where the strands cross over) would be located at the first cleavage site. Migration of the branch point to the second cleavage site would then somehow trigger the second half of the pathway. This model provided convenient explanation for the strict requirement for homology between recombining sites, since branch migration would stall at a mismatch and would not allow the second strand exchange to occur. In more recent years, however, this view has been challenged, and most of the current models for Int, Xer, and Flp recombination involve only limited branch migration (1–3 base pairs of the Holliday intermediate), coupled to an isomerisation event that is responsible for switching the strand cleavage specificity.

Site-specific recombination (SSR) involves specific sites for the catalyzing action of special enzymes called recombinases. Cre, or cyclic recombinase, is one such enzyme. Site-specific recombination is, thus, the enzyme-mediated cleavage and ligation of two defined deoxynucleotide sequences.

A number of conserved site-specific recombination systems have been described in both prokaryotic and eukaryotic organisms. In general, these systems use one or more proteins and act on unique asymmetric DNA sequences. The products of the recombination event depend on the relative orientation of these asymmetric sequences. Many other proteins apart from the recombinase are involved in regulating the reaction. During site-specific DNA recombination, which brings about genetic rearrangement in processes such as viral integration and excision and chromosomal segregation, these recombinase enzymes recognize specific DNA sequences and catalyse the reciprocal exchange of DNA strands between these sites.

Mechanism of action Edit

Initiation of site-specific recombination begins with the binding of recombination proteins to their respective DNA targets. A separate recombinase recognizes and binds to each of two recombination sites on two different DNA molecules or within the same DNA strand. At the given specific site on the DNA, the hydroxyl group of the tyrosine in the recombinase attacks a phosphate group in the DNA backbone using a direct transesterification mechanism. This reaction links the recombinase protein to the DNA via a phospho-tyrosine linkage. This conserves the energy of the phosphodiester bond, allowing the reaction to be reversed without the involvement of a high-energy cofactor.

Cleavage on the other strand also causes a phospho-tyrosine bond between DNA and the enzyme. At both of the DNA duplexes, the bonding of the phosphate group to tyrosine residues leave a 3’ OH group free in the DNA backbone. In fact, the enzyme-DNA complex is an intermediate stage, which is followed by the ligation of the 3’ OH group of one DNA strand to the 5’ phosphate group of the other DNA strand, which is covalently bonded to the tyrosine residue that is, the covalent linkage between 5’ end and tyrosine residue is broken. This reaction synthesizes the Holliday junction discussed earlier.

In this fashion, opposite DNA strands are joined together. Subsequent cleavage and rejoining cause DNA strands to exchange their segments. Protein-protein interactions drive and direct strand exchange. Energy is not compromised, since the protein-DNA linkage makes up for the loss of the phosphodiester bond, which occurred during cleavage.

Site-specific recombination is also an important process that viruses, such as bacteriophages, adopt to integrate their genetic material into the infected host. The virus, called a prophage in such a state, accomplishes this via integration and excision. The points where the integration and excision reactions occur are called the attachment (att) sites. An attP site on the phage exchanges segments with an attB site on the bacterial DNA. Thus, these are site-specific, occurring only at the respective att sites. The integrase class of enzymes catalyse this particular reaction.

Efficiency of action Edit

Two factors have been shown to affect the efficiency of Cre's excision on the lox pair. First, the nucleotide sequence identity in the spacer region of lox site. Engineered lox variants which differ on the spacer region tend to have varied but generally lower recombination efficiency compared to wildtype loxP, presumably through affecting the formation and resolution of recombination intermediate. [23]

Another factor is the length of DNA between the lox pair. Increasing the length of DNA leads to decreased efficiency of Cre/lox recombination possibly through regulating the dynamics of the reaction. [24] [25] [26] Genetic location of the floxed sequence affects recombination efficiency as well probably by influencing the availability of DNA by Cre recombinase. [26] The choice of Cre driver is also important as low expression of Cre recombinase tends to result in non-parallel recombination. Non-parallel recombination is especially problematic in a fate mapping scenario where one recombination event is designed to manipulate the gene under study and the other recombination event is necessary for activating a reporter gene (usually encoding a fluorescent protein) for cell lineage tracing. [26] Failure to activate both recombination events simultaneously confounds the interpretation of cell fate mapping results.

Temporal control Edit

Inducible Cre activation is achieved using CreER (estrogen receptor) variant, which is only activated after delivery of tamoxifen. [27] This is done through the fusion of a mutated ligand binding domain of the estrogen receptor to the Cre recombinase, resulting in Cre becoming specifically activated by tamoxifen. In the absence of tamoxifen, CreER will result in the shuttling of the mutated recombinase into the cytoplasm. The protein will stay in this location in its inactivated state until tamoxifen is given. Once tamoxifen is introduced, it is metabolized into 4-hydroxytamoxifen, which then binds to the ER and results in the translocation of the CreER into the nucleus, where it is then able to cleave the lox sites. [28] Importantly, sometimes fluorescent reporters can be activated in the absence of tamoxifen, due to leakage of a few Cre recombinase molecules into the nucleus which, in combination with very sensitive reporters, results in unintended cell labelling. [29] CreER(T2) was developed to minimize tamoxifen-independent recombination and maximize tamoxifen-sensitivity.

Conditional cell lineage tracing Edit

Cells alter their phenotype in response to numerous environmental stimuli and can loose the expression of genes typically used to mark their identity, making it difficult to research the contribution of certain cell types to disease. Therefore, researchers often use transgenic mice expressing CreER t2 recombinase induced by tamoxifen administration, under the control of a promoter of a gene that marks the specific cell type of interest, with a Cre-dependent fluorescent protein reporter. The Cre recombinase is fused to a mutant form of the oestrogen receptor, which binds the synthetic oestrogen 4-hydroxytamoxifen instead of its natural ligand 17β-estradiol. CreER(T2) resides within the cytoplasm and can only translocate to the nucleus following tamoxifen administration, allowing tight temporal control of recombination. The fluorescent reporter cassette will contain a promoter to permit high expression of the fluorescent transgene reporter (e.g. a CAG promoter) and a loxP flanked stop cassette, ensuring the expression of the transgene is Cre-recombinase dependent and the reporter sequence. Upon Cre driven recombination, the stop cassette is excised, allowing reporter genes to express specifically in cells in which the Cre expression is being driven by the cell-specific marker promoter. Since removal of the stop cassette is permanent, the reporter genes are expressed in all the progeny produced by the initial cells where the Cre was once activated. Such conditional lineage tracing has proved to be extremely useful to efficiently and specifically identify vascular smooth muscle cells (VSMCs) and VSMC-derived cells and has been used to test effects on VSMC and VSMC-derived cells in vivo. [30] [31] [32] [33] [34] [35]

The P1 phage is a temperate phage that causes either a lysogenic or lytic cycle when it infects a bacterium. In its lytic state, once its viral genome is injected into the host cell, viral proteins are produced, virions are assembled, and the host cell is lysed to release the phages, continuing the cycle. In the lysogenic cycle the phage genome replicates with the rest of the bacterial genome and is transmitted to daughter cells at each subsequent cell division. It can transition to the lytic cycle by a later event such as UV radiation or starvation.

Phages like the lambda phage use their site specific recombinases to integrate their DNA into the host genome during lysogeny. P1 phage DNA on the other hand, exists as a plasmid in the host. The Cre-lox system serves several functions in the phage: it circularizes the phage DNA into a plasmid, separates interlinked plasmid rings so they are passed to both daughter bacteria equally and may help maintain copy numbers through an alternative means of replication. [36]

The P1 phage DNA when released into the host from the virion is in the form of a linear double stranded DNA molecule. The Cre enzyme targets loxP sites at the ends of this molecule and cyclises the genome. This can also take place in the absence of the Cre lox system [37] with the help of other bacterial and viral proteins. The P1 plasmid is relatively large (≈90Kbp) and hence exists in a low copy number - usually one per cell. If the two daughter plasmids get interlinked one of the daughter cells of the host will lose the plasmid. The Cre-lox recombination system prevents these situations by unlinking the rings of DNA by carrying out two recombination events (linked rings -> single fused ring -> two unlinked rings). It is also proposed that rolling circle replication followed by recombination will allow the plasmid to increase its copy number when certain regulators (repA) are limiting. [36]

Multiple variants of loxP, [38] in particular lox2272 and loxN, have been used by researchers with the combination of different Cre actions (transient or constitutive) to create a "Brainbow" system that allows multi-colouring of mice's brain with four fluorescent proteins.

Another report using two lox variants pair but through regulating the length of DNA in one pair results in stochastic gene activation with regulated level of sparseness. [24]

Interactions between Bacteriophages and Eukaryotic Cells

As the name implies, bacteriophage is a bacterium-specific virus. It infects and kills the bacterial host. Bacteriophages have gained attention as alternative antimicrobial entities in the science community in the western world since the alarming rise of antibiotic resistance among microbes. Although generally considered as prokaryote-specific viruses, recent studies indicate that bacteriophages can interact with eukaryotic organisms, including humans. In the current review, these interactions are divided into two categories, i.e., indirect and direct interactions, with the involvement of bacteriophages, bacteria, and eukaryotes. We discuss bacteriophage-related diseases, transcytosis of bacteriophages, bacteriophage interactions with cancer cells, collaboration of bacteriophages and eukaryotes against bacterial infections, and horizontal gene transfer between bacteriophages and eukaryotes. Such interactions are crucial for understanding and developing bacteriophages as the therapeutic agents and pharmaceutical delivery systems. With the advancement and combination of in silico, in vitro, and in vivo approaches and clinical trials, bacteriophages definitely serve as useful repertoire for biologic target-based drug development to manage many complex diseases in the future.

1. Introduction

Bacteriophages (phage, for short) were independently discovered as bacteria-specific viruses by the British microbiologist Frederick Twort in 1915 and French-Canadian scientist Felix d’Herelle in 1917 [1]. In the following years, bacteriophages were regarded as promising candidates for antimicrobial therapy, until the idea was abandoned in the western world because of the introduction of antibiotics [2]. Antibiotics were chosen over bacteriophages because they are fixed chemical compounds and are easy to manufacture. Nevertheless, bacteriophage research has been continued in some regions of the world, e.g., Georgia, Poland, and the Soviet Union, during the Second World War until today [3, 4]. With the emerging cases of antibiotic-resistant superbugs around the world, bacteriophages have regained scientific attention and are currently extensively studied as an alternative antimicrobial therapy [5]. Bacteriophage research has greatly expanded and encompasses many aspects of bacteriophage biology, from in vitro-level experiments to clinical trials.

The most recent clinical trial regarding the utilization of bacteriophage therapy in burn wounds infected by Pseudomonas aeruginosa had been conducted in the years 2015–2017. Despite the insufficient efficacy of the therapy compared with the control antibiotic sulfadiazine, because of a low amount of administered bacteriophages, the results sparked interest in the science community [6]. In February 2019, the US Food and Drug Administration has approved a clinical trial of bacteriophage therapy, initiated by the University of California, San Diego, School of Medicine and AmpliPhi Biosciences Corporation, in patients with ventricular assist devices infected by Staphylococcus aureus [7]. The bacteriophage therapy is administered via an intravenous route and is the first clinical trial of bacteriophage treatment in North America [7]. These two clinical trials and a few other unmentioned here mark new stages of the development of bacteriophages from the experimental stage to a regular commercially available therapy to eradicate highly resistant superbugs.

Other than the typical interactions with bacteria and its application in infectious disease, bacteriophages can also interact and affect eukaryotes in many unexpected ways [8]. In the current review, these interactions are divided into two categories: indirect and direct interactions. During the former, a bacteriophage affects a eukaryote by affecting eukaryotic-related bacteria, whereas, during the latter, the bacteriophage and eukaryote are in immediate contact. We present these interactions below and emphasize their impact on the future application of bacteriophages in the clinical setting.

2. Indirect Interactions between Bacteriophages and Eukaryotes

2.1. Bacteriophages Assist to Kill Bacterial Pathogens of Eukaryotes

The ability of bacteriophages to kill pathogenic bacteria of eukaryotes underpins the revived interest in bacteriophages as an alternative antimicrobial therapy. The detailed mechanisms used by bacteriophages to assist eukaryotes vary.

Barr et al. showed that bacteriophages, particularly T4 bacteriophages, act as non-host-derived immunity against bacterial invaders at human mucosal surfaces [9]. T4 phage binds weakly to a mucin glycoprotein, one of the essential building blocks of the mucus (secreted by the epithelial mucosa), using an immunoglobulin-like domain of Hoc protein. The weak binding provides additional protection to the epithelial cells by facilitating the T4 phage killing of and preventing colonization by bacterial pathogens [9]. The weak binding also maximizes the ability of T4 phage to kill bacteria by enabling it to move across mucosal surfaces in a subdiffusive fashion. Subdiffusive motion increases the probability of bacteriophage-bacterium encounters, as it allows the phage a thorough exploration of certain regions of the mucus [10].

Kaur et al. point out the ability of some bacteriophages to kill intracellular pathogens [11]. Intracellular pathogens are more dangerous and harder to eradicate than extracellular pathogens because they can evade the immune system by hiding inside the host cell. An early study showed that a broad-host-range lytic bacteriophage MR-5 is a promising candidate for phage therapy [11]. The bacteriophage kills methicillin-resistant S. aureus (MRSA) that had been phagocytosed by murine BALB/c macrophages, reducing the bacterial numbers so that the bacteria are handled more easily by macrophages. Furthermore, it also reduces the cytotoxic effect of S. aureus against macrophages by killing the bacteria. Therefore, MR-5 phage indirectly assists the macrophages in eliminating MRSA, providing major assistance, especially in the case of high bacterial loads. Instead of directly entering the macrophages, MR-5 phage uses S. aureus as a ride.

In a similar study, Zhang et al. found that another broad-host-range lytic bacteriophage, vB_SauM_JS25, can kill MRSA inside bovine epithelial cells (MAC-T bovine mammary epithelial cells) [12]. The authors reported that vB_SauM_JS25 phage can penetrate into the nucleus of MAC-T cell, although the underlying mechanism is not known. Subsequent investigations confirmed that several bacteriophages are indeed able to penetrate the eukaryotic cells, but not quite reach the nuclei [13, 14]. Bacteriophage penetration of the eukaryotic cells is discussed in detail as follows.

The occurrence of interactions between phages, bacteria, and eukaryotic cells raises the question about the effectiveness of phage therapy. A further study investigated the safety issues surrounding the effect of experimental bacteriophage therapy in vitro on the intracellular killing ability of granulocytes and monocytes [15]. The authors found that therapy involving T2, T4, and A3 bacteriophages does not affect the intracellular killing capability of the mentioned cells. A later investigation in humans [16] reached similar conclusions. Namely, that bacteriophage therapy, administered as a cocktail of several lytic bacteriophages, does not affect the killing ability of polymorphonuclear neutrophils and peripheral blood mononuclear cells during chronic infection caused by pathogenic Escherichia coli, Enterococcus faecalis, P. aeruginosa, and S. aureus, regardless of the route of administration and infection type. Furthermore, the bacteriophage therapy improved the killing ability of peripheral blood mononuclear cells during nonpathogenic E. coli B infections in patients who responded positively to the therapy. Based on these observations, the authors suggested that bacteriophage therapy is sufficiently safe to be employed in humans, especially in cases of chronic infection.

The studies confirm that in some systems bacteriophages can support the antibacterial activity of eukaryotic cells. Bacteriophages, however, may also interact indirectly with bacteria to harm eukaryotes, either by (1) disrupting a mutualistic relationship between eukaryotes and bacteria or by (2) supporting eukaryotic bacterial pathogen, as discussed in detail hereinafter.

2.2. Bacteriophages Disrupt the Mutualistic Microbial Equilibrium within the Human Body

The mutualistic relationship between microbes and humans is considered to be vital for maintaining physiological functions and homeostasis in the human body. The human host provides nutrition and habitat necessary to support bacterial growth. The microbiota produce metabolites which serve as signaling molecules to the gut, brain, immune and hormone system, and other functions of the host [17]. The relationship is important as the human body is a sanctuary of nearly 100 trillion microbes representing a wide range of species, especially within the digestive tract. The presence of bacteriophages that can kill bacteria, including beneficial bacteria, may shift the balance towards dysbiosis (maladaptation of the microbial composition), thus triggering diseases [18–21]. In other words, bacteriophages can also become human pathogens [22].

According to this hypothesis, bacteriophages may be the possible initiators of Parkinson’s disease [23]. Parkinson’s disease is marked by the accumulation of misfolded α-synuclein in dopaminergic neurons of the substantia nigra because of a depletion of the neurotransmitter dopamine. According to the proposed pathophysiologic pathway of Parkinson’s disease, the misfolding of α-synuclein begins in the enteric nervous system and spreads gradually to the substantia nigra. The misfolding is thought to be the result of the absence of certain lactic acid bacteria, Lactococcus spp., in the gut, which are responsible for maintaining the proper levels of dopamine in the enteric nervous system [24]. L-DOPA, as part of Parkinson’s disease drug regiment, affects the microbial population in the gut [25]. The analysis of the fecal samples from L-DOPA-naive Parkinson’s disease patients showed alteration of microbiota [26] and phageota [23]. The gut population of Lactococcus spp. is 10 times lower than that in control individuals. The depletion of Lactococcus spp. is associated with the overabundance of specific lytic bacteriophages, Lactococcus bacteriophage 936 and Lactococcus virus c2. Lactococcus bacteriophages are thought to kill Lactococcus spp., thereby promoting the development of Parkinson’s disease [23]. Since Lactococcus bacteriophages are frequently found in milk, cheese, and yogurt, the consumption of dairy products may lead to their high abundance in the human gut [27]. Additional data are required to validate such assumptions, perhaps by determining the average concentration of Lactococcus bacteriophage 936 and Lactococcus virus c2 in dairy products and investigating the correlation between consumption of dairy products containing these bacteriophages and the incidence of Parkinson’s disease symptoms.

2.3. Bacteriophages Can Assist Bacterial Pathogens of Eukaryotes

Bacteriophages may assist in pathogenic bacterial infection in several different ways. Addy et al. investigated the involvement of filamentous phage φRSS1 during Ralstonia solanacearum infection of tomato plants [28]. First, φRSS1 enhances the R. solanacearum virulence via the attachment and accumulation of phage particles on the surface of the bacterial cell membrane. This subsequently increases the density of bacterial colonies and induces early expression of the gene for transcriptional regulator PhcA, responsible for the activation of many other virulence factors. These included the production of copious amounts of extracellular polysaccharide, which plays a vital role in expanding stem colonization in tomato. Moreover, φRSS1 enhances the expression of PilA protein (type IV pilus), which further increases the twitch motility and attachment of bacterial colonies to plant cells. R. solanacearum strain infected by φRSS1 causes complete wilt more rapidly in tomato plant (5 d after inoculation) than an uninfected strain (8 d after inoculation). Interestingly, this phenomenon could be more common in nature as similar observations were made for filamentous phage Xf2 which enhances the virulence of Xanthomonas campestris pv. oryzae [29].

Bacteriophages may also assist bacterial pathogens in biofilm formation [30]. Biofilm is an aggregation of bacteria in well-organized polymers. The extracellular matrix protects bacteria and allows them to attach to multiple surfaces within the host or nonliving objects. It is one of the key features of bacterial pathogens, enabling their survival in a harsh environment, evasion of the host’s immune system, and promoting chronic infections [31, 32]. Accordingly, the existence of prophage SV1 in the Streptococcus (St.) pneumoniae genome is correlated with the bacterial ability to form biofilm [33]. SV1 prophage can spontaneously alter its life mode from static lysogenic to active lytic. Such alteration is sufficient to lyse small numbers of St. pneumoniae cells in a colony, providing extracellular DNA which serves as adhesion required for biofilm formation [34]. In other words, some St. pneumoniae cells are sacrificed via SV1-mediated lysis for biofilm construction to protect other cells. Although St. pneumoniae is already equipped with an autolytic mechanism, in the form of the lytic enzyme LytA [35], the presence of another lytic pathway (SV1-mediated) increases its capacity to form a biofilm. St. pneumoniae strain with SV1 prophage constructs thicker and denser biofilms in a shorter amount of time than St. pneumoniae without SV1 prophage [33].

Bacteriophages could also assist biofilm formation of other bacterial species like P. aeruginosa and E. coli. In cystic fibrosis, filamentous bacteriophages Pf and Fd assist P. aeruginosa and E. coli in constructing highly ordered biofilms containing stable liquid crystal structures [36]. The filamentous bacteriophages are continuously extruded from, but not lyse and kill, the bacterial host. The shape and negative charge of these filamentous bacteriophages appear to correlate with their ability to associate with polymers inside the biofilms. The resulting biofilms protect the bacteria from aminoglycoside antibiotics and dehydration and promote tighter attachment to surfaces than that of biofilms formed in the absence of bacteriophages [37]. Moreover, the association is maintained by a depletion attractive force, which depends on the ionic strength, polymer size, and polymer concentration. In a murine model of pneumonia, biofilms formed by bacteriophage Pf-aided P. aeruginosa allow the bacterium to remain in the lung by evading phagocytosis by macrophages and inhibiting inflammatory responses [38]. This interaction is quite remarkable because P. aeruginosa and E. coli act as hosts for bacteriophage Pf and Fd, respectively. Many laboratory and clinical strains of P. aeruginosa and E. coli harbor prophages Pf and Fd, which are activated when the bacteria form biofilms [38].

In studies of Bille et al., it was reported that prophage of a filamentous bacteriophage MDAφ (Meningococcal Disease-Associated) enhances the physical contact between Neisseria meningitidis cells during the attachment and colonization of epithelial cells in the human nasopharynx, before infection and penetration of the blood-brain barrier [39]. The initial attachment and colonization stage is mediated by a type IV pilus, creating the first layer of N. meningitidis cells that bind tightly to the apical surface of epithelial cells. Soon after the attachment, the cells multiply and create another layer of bacteria to form colonies. However, type IV pilus does not mediate the creation of this next layer because the expression of the pilus is repressed after the first layer is formed [40]. The authors found that N. meningitidis expresses and utilizes the MDAφ prophage as a replacement of type IV pilus. In other words, the bacteriophage particles are used like the pilus to create the next layer of bacteria attached to the first layer. Interestingly, the bacteriophage particles are secreted and directly embedded in the outer membrane of the bacteria without lysing or killing them. Many bacteriophage particles form huge bundles that aggregate the bacteria and protect the colonies from shear stress and the flow movement of epithelial cell cilia. The deletion of prophage MDAφ from the N. meningitidis genome results in aggregation and colonization failure [39].

The integral involvement of filamentous bacteriophages in promoting bacterial infection against eukaryotes reveals mutualistic behaviors of bacteria and bacteriophages. Consequently, these types of bacteriophages are becoming new potential targets for drug development against pathogenic bacteria. As a vital link between filamentous bacteriophages and resistant microbes is identified, perhaps there is a chance to combat these superbugs, not by directly attacking them but by destroying their allies.

3. Direct Interactions between Bacteriophages and Eukaryotes

Viruses (including bacteriophages) are obligate intracellular pathogens and are generally classified based on the respective host domain (bacteria, archaea, or eukaryotes) [41]. This classification is based on the general assumption that no virus can infect and interact directly with representatives of more than a single domain of life [42]. However, even though bacteriophages cannot infect domains of life other than bacteria, they can nonetheless interact directly and affect representatives of such other domains, especially eukaryotes. We describe some findings on direct interaction between bacteriophages and eukaryotes below.

3.1. Bacteriophages Can Penetrate and Disperse within a Eukaryotic Host

One important factor that enables bacteriophages to interact directly and affect eukaryotes is the ability to penetrate the cell membrane and spread freely within a eukaryotic host [43–45]. Studies involving monolayer epithelial cells from the gut (T84 and CaCo2), lung (A549), liver (Huh7), brain (hBMec), and kidney (MDCK) derived from human demonstrated that the bacteriophage penetration of cell relies on transcytosis, which involves the endomembrane systems of the eukaryotic cells, particularly the Golgi apparatus [14]. Transcytosis starts when a bacteriophage particle is engulfed by the cell membrane and transferred to the cytoplasm inside a small vesicle. The vesicle then transits inside the Golgi apparatus before being discharged on the other side of the same cell. The process repeated by the neighboring cells, thus enabling the bacteriophage particle crossing cell layers. This phenomenon has been observed for several bacteriophages, such as T4, T5, T7, SP01, SPP1, and P22 phages [14]. Detailed observations revealed that transcytosis of bacteriophages mainly proceeds in the apical to basolateral direction, with an estimated rate of 0.325 × 10 −12 mL/(μm 2 h), and is considered a dose-dependent process [14].

Similar observations were made in another study using E. coli bacteriophage PK1A2 and human neuroblastoma cells kSK-N-SH that express a copious amount of polysialic acid on the membrane surface [13]. PK1A2 bacteriophage can bind to polysialic acid and enters kSK-N-SH cells by endocytosis. The bacteriophages accumulate in the late endosome compartment close to the perinuclear region, residing therein until gradual degradation by the cell, approximately 48 h after endocytosis. The binding between PK1A2 and polysialic acid of kSK-N-SH cell is specific, probably because of the structural similarity with the polysialic acid lipopolysaccharide of E. coli K1, the main host of PK1A2 bacteriophage. The binding is needed for the initiation of endocytosis and is temperature-dependent [13].

Bacteriophage penetration of the eukaryotic cell could explain the observation that bacteriophages are found in many multicellular eukaryotes, even in isolated regions, e.g., the human brain, which had been long considered sterile [46]. This observation provides valuable information on the pharmacokinetic aspects of bacteriophage treatment, which is vital in the context of using bacteriophages as an alternative antimicrobial therapy [47]. It also emphasizes the possibility of using bacteriophages as vectors in drug and gene delivery systems, including therapies targeted towards the brain tissue, gastrointestinal tract, and lung via systemic or local delivery [48–51]. Furthermore, it also provides a reasonable explanation of the mechanism of direct horizontal gene transfer (HGT) between phages and eukaryotes, discussed in a later section.

3.2. Bacteriophages Can Bind to and Hamper Metastasis of Cancer Cells

Bacteriophage can interact with cancer cells, inhibiting metastasis by using specific protein-protein configuration involving GP24 of the bacteriophage and integrin β3, HSP90 receptor, or other proteins of the cancer cells. This unusual interaction was investigated in melanoma and lung cancer cells in a mouse model (B16 and LLC cells, respectively) and in humans (HS294T and A549 cells, respectively) [52, 53]. The authors suggested that in vitro and in vivo metastasis of both cancer types is inhibited by the binding of bacteriophages T4 and HAP1 (a substrain of T4 bacteriophage). The hypothesis is that this inhibition is mediated by the specific interactions between GP24 to integrin β3 (αIIβ3/αvβ3) on cancer cells. GP24 is a capsid protein with a specific Lys-Gly-Asp motif (the KGD motif). It forms a pentamer on every corner of the T4 and HAP1 head. Integrin β3 is a surface protein that is involved in various aspects of cell biology, such as tissue integrity, cell migration, cell survival, and angiogenesis [54]. In cancer cells, integrin β3 is highly abundant and regarded as one of the possible factors promoting metastasis [55, 56]. The KGD motif in GP24 is thought to participate in the binding between GP24 and integrin β3. Both T4 and HAP1 bacteriophages can bind integrin β3 via GP24, but HAP1 phage binding is significantly stronger than that of T4 phage [53]. The exact mechanism that triggers the inhibition of cancer metastasis after binding of GP24 and integrin β3 is unknown. Probably, the binding downregulates integrin β3 expression or prevents integrin β3 interaction with other proteins involved in some pathways related to cancer metastasis.

Detailed investigation revealed that the stronger binding by HAP1 phage is associated with a missense mutation in hoc gene, which encodes Hoc protein [57]. Hoc protein is a highly immunogenic outer capsid protein of the head of T4 and HAP1 phages. It has a dumbbell-like shape, which protrudes approximately 6 nm away from the capsid surface, including GP24 [58, 59]. Instead of a full-length Hoc protein like T4 phage, HAP1 phage produces a truncated Hoc protein because of a change of C496 residue to T, which subsequently changes the Gln166 codon into a stop codon (UAA). Hoc truncation in HAP1 phage possibly increases the probability of binding between GP24 and integrin β3, as it does not protrude away from the head and interferes with a direct contact between these two proteins. This notion is supported by a similar binding capacity of HAP1 phage and T2 bacteriophage, which does not possess the Hoc protein but does possess GP24 and KGD motif, to integrin β3 in cancer cells [57].

Investigation of T4 and HAP1 phage behavior in mouse harboring B16 melanoma cells yielded another interesting observation. It revealed that despite having a much greater affinity for integrin β3 in the cancer cells, HAP1 phage is removed more rapidly by Kupffer cells in the liver by phagocytosis than T4 phage [53]. It was proposed that HAP1 is more prone to phagocytosis because the short version of Hoc protein does not conceal the head capsid proteins, which are thus easily detected by Kupffer cells. Wild-type Hoc protein has four domains, three of which are similar to eukaryotic immunoglobulin. Domain 1 is similar to Fc receptors, domain 2 to that of the third immunoglobulin-like domain of Perlecan, and domain 3 to the V-set family of immunoglobulin superfamily [60]. Therefore, the Hoc protein probably evolved as a form of adaptation of T4 bacteriophages to avoid immune system recognition, thereby allowing them to survive inside eukaryotes.

Another study demonstrated that bacteriophages T4 and M13 can suppress the expression of HSP90 gene in human prostate cancer cells (PC3), responsible for the promotion of mitosis, DNA repair, and prevention of apoptosis and autophagy [61]. The suppression is mediated by the interaction of T4 and M13 phages with HSP90 receptors and results in apoptosis and autophagy of cancer cells. Similar to GP24-integrin β3 binding, the exact mechanism that promotes the downregulation of the expression of the HSP90 gene after T4 and M13 phage binding to HSP90 receptors is unknown. However, these findings highlight the unusual interactions between bacteriophages and eukaryotes and also present a new approach to explore the possible use of bacteriophages as anticancer agents.

Although the knowledge regarding interactions between β3 integrin and HSP90 receptor with bacteriophages is scarce, we speculate that the two proteins probably allow the bacteriophage to interact with eukaryotes in general, not only in cancer cells. This speculation is based on the information that β3 integrin and HSP90 receptors are found in many species, even though they may be not as abundant as in cancer cells. In other words, β3 integrin and HSP90 receptors are used by bacteriophages to interact with eukaryotes and vice versa. Further, this interaction probably initiates transcytosis of bacteriophages to eukaryotic cells via receptor-mediated endocytosis, similar to polysialic acid-mediated endocytosis of PK1A2 bacteriophage, as discussed earlier. If that is indeed the case, then a direct interaction between bacteriophages and eukaryotes is more common than currently assumed. Furthermore, β3 integrin, HSP90 receptor, and polysialic acid most probably are not the only types of mediators that mediate such direct interactions.

3.3. Human Cells Assist Bacteriophages in Infecting the Bacterial Host

The interaction between bacteriophages and human cells may be crucial for bacteriophage infection of bacteria. phiCDHS1 bacteriophage kills the pathogenic bacterium Clostridium difficile more rapidly when both are placed in a culture of a human colon cancer line HT-29 cells [62]. In other words, HT-29 cells seemingly help to maximize the killing ability of phiCDHS1 bacteriophage to eradicate C. difficile. This phenomenon is associated with the attachment of phiCDHS1 phage and C. difficile to HT-29 cells, which places phiCDHS1 phage and C. difficile cells in close contact, providing more opportunities for the phage to infect the bacterium. The propensity for this attachment is high as approximately 70% of phiCDHS1 phages were found attached to HT-29 cells in the study [62]. Further, the attachment is specific, as replacing HT-29 cells with HeLa cells (human cervical cancer line) led to no attachment and no increment of the phiCDHS1 killing capability or that of other C. difficile bacteriophages (phiCDHM3 and phiCDHM6). Since the attachment would enhance the eradication of C. difficile in the human gut and increase the population of phiCDHS1 phages, it can be classified as a direct mutualistic relationship between bacteriophages and humans. Unfortunately, the mediator of the attachment and the underlying mechanism are not clearly understood.

The attachment of phiCDHS1 phage to HT-29 cells supports our earlier speculation that direct interactions between bacteriophages and eukaryotes are more common in nature than currently proposed. Although the mechanism of the attachment remains unknown, it is possible that it is mediated by molecules with properties similar to those of β3 integrin, HSP90 receptor, and polysialic acid. In silico studies employing homology/comparative modeling, molecular docking, quantitative structure-activity relationship (QSAR) methods, and conformational analysis of bacteriophage, bacteria, and human proteome may identify the candidate protein(s) involved in such interactions.

3.4. Bacteriophages Engage in Direct HGT with Eukaryotes

HGT is defined as a transfer of genes from one organism to another unrelated organism [63, 64]. The transfer can occur between organisms from the same species, different species, and even organisms representing different domains. HGT occurs primarily in bacteria, by three pathways: transduction, transformation, and conjugation. The knowledge regarding HGT within the eukaryote domains is limited, but many lines of evidence indicate that it might also take place frequently in plants, animals, and humans [65]. HGT is common between bacteriophage and the bacterial host and also between bacteria and eukaryotes, particularly among obligate intracellular bacteria and their respective eukaryote host. At first glance, there is no direct HGT between bacteriophages and eukaryotes. However, such interactions occur, as it was shown for bacteriophage WO carrying several arthropod genes [42, 66]. Bacteriophage WO naturally infects Wolbachia. Wolbachia, in turn, is an intracellular bacterium of arthropods. By infecting Wolbachia, bacteriophage WO can be in contact with arthropod cells, which would allow HGT between them. Genes that are harbored by bacteriophage WO are collectively called eukaryotic association module. The module includes such genes as ones encoding latrotoxin-C-terminal domain, eukaryotic furin cleavage, ankyrin C-terminus, ankyrin and tetratricopeptide, and NACHT (Neuronal Apoptosis inhibitory protein, MHC Class II transcription activator, incompatibility locus protein from Podospora anserina HET-E, telomerase-associated protein TP1). Bacteriophage WO harbors all these genes, presumably to adapt to and survive within arthropod bodies and to efficiently infect Wolbachia. The mechanism that allows HGT between bacteriophage WO and arthropods is not fully understood. It is assumed that HGT might involve three different pathways: (1) direct gene transfer when bacteriophage WO enters the arthropod cells, (2) transfer mediated by Wolbachia, and (3) transfer mediated by other types of viruses that also infect the arthropods.

By contrast, several eukaryotes carry genes that relate to specific bacteriophages. Nematode and woodland strawberry carry an orthologous gene called VP1, which encodes a major capsid protein of φChp4, a Chlamydia bacteriophage from the Microviridae family [67]. It is possible that a fragment of bacteriophage gene(s) managed to integrate into the eukaryotes genomes, either by nonhomologues recombination or inserted during DNA replication.

The existence of a nuclear localization signal was demonstrated within Terminal Protein (TP) of several bacteriophages, e.g., Φ29, Nf, PRD1, Bam35, and Cp-1 [68]. The nuclear localization signal is a specific sequence that allows an uptake and delivery of proteins into the eukaryotic nucleus thus, these TP-DNA molecules are a useful tool to amplify and subsequently deliver genes efficiently into the eukaryotic nucleus [69]. Meanwhile, TP is a protein used by bacteriophages to prime the DNA for replication. In that process, TP is covalently bound to the 5′-end of the DNA product. Therefore, following TP-mediated DNA replication, TP-bound bacteriophage DNA product has a nuclear localization signal and can enter the nucleus. This is another possible mechanism enabling direct HGT between bacteriophages and eukaryotes and also enabling eukaryotes to obtain bacteriophage genes. However, for such HGT to occur, the bacteriophage has to penetrate eukaryotic cells before releasing the TP-bound DNA. Therefore, bacteriophage penetration via transcytosis plays a vital role in allowing direct HGT to occur. This finding supports a new theory about the bacteriophage role in shaping the diversity of eukaryotic genomes.

4. Conclusion

Since their discovery in the early 20th century, bacteriophage characteristics and roles have triggered many basic and applied researches. Over time, the studies regarding bacteriophages extended from phage-bacteria interactions to interactions with nonbacterial cells, including cells of eukaryotes. These interactions include i.a. binding of phages with specific receptors on eukaryotic cells, transcytosis, and horizontal gene transfer. Furthermore, the potential roles of bacterial viruses in neurodegenerative disease and cancer have also been explored. Considering the remarkable diversity of phages and eukaryotes, more studies are required to identify new mechanisms of interactions and also to explain which interaction modes are common and which are unique. Not only are bacteriophages are exceptional microbes but they may also be used as research tools and valuable repertoire for biologic target-based drug development and drug delivery systems. Thus, interactions of bacterial viruses with eukaryotic cells are current and relevant research topic.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this paper.


This work was supported by the Division of Clinical Pharmacy, Faculty of Medicine, Brawijaya University.


  1. X. Wittebole, S. De Roock, and S. M. Opal, “A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens,” Virulence, vol. 5, no. 1, pp. 226–235, 2014. View at: Publisher Site | Google Scholar
  2. D. R. Roach and L. Debarbieux, “Phage therapy: awakening a sleeping giant,” Emerging Topics in Life Sciences, vol. 1, no. 1, pp. 93–103, 2017. View at: Publisher Site | Google Scholar
  3. A. El-Shibiny and S. El-Sahhar, “Bacteriophages: the possible solution to treat infections caused by pathogenic bacteria,” Canadian Journal of Microbiology, vol. 63, no. 11, pp. 865–879, 2017. View at: Publisher Site | Google Scholar
  4. W. C. Summers, “The strange history of phage therapy,” Bacteriophage, vol. 2, no. 2, pp. 130–133, 2012. View at: Publisher Site | Google Scholar
  5. L. L. Furfaro, M. S. Payne, and B. J. Chang, “Bacteriophage therapy: clinical trials and regulatory hurdles,” Frontiers in Cellular and Infection Microbiology, vol. 8, p. 376, 2018. View at: Publisher Site | Google Scholar
  6. P. Jault, T. Leclerc, S. Jennes et al., “Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): a randomised, controlled, double-blind phase 1/2 trial,” The Lancet Infectious Diseases, vol. 19, no. 1, pp. 35–45, 2019. View at: Publisher Site | Google Scholar
  7. R. Voelker, “Eye-tracking test approved to help diagnose concussion,” JAMA, vol. 321, no. 7, p. 638, 2019. View at: Publisher Site | Google Scholar
  8. A. Chatterjee and B. A. Duerkop, “Beyond bacteria: bacteriophage-eukaryotic host interactions reveal emerging paradigms of health and disease,” Frontiers in Microbiology, vol. 9, p. 1394, 2018. View at: Publisher Site | Google Scholar
  9. J. J. Barr, R. Auro, M. Furlan et al., “Bacteriophage adhering to mucus provide a non-host-derived immunity,” Proceedings of the National Academy of Sciences, vol. 110, no. 26, pp. 10771–10776, 2013. View at: Publisher Site | Google Scholar
  10. J. J. Barr, R. Auro, N. Sam-Soon et al., “Subdiffusive motion of bacteriophage in mucosal surfaces increases the frequency of bacterial encounters,” Proceedings of the National Academy of Sciences, vol. 112, no. 44, pp. 13675–13680, 2015. View at: Publisher Site | Google Scholar
  11. S. Kaur, K. Harjai, and S. Chhibber, “Bacteriophage-aided intracellular killing of engulfed methicillin-resistant Staphylococcus aureus (MRSA) by murine macrophages,” Applied Microbiology and Biotechnology, vol. 98, no. 10, pp. 4653–4661, 2014. View at: Publisher Site | Google Scholar
  12. L. Zhang, L. Sun, R. Wei et al., “Intracellular Staphylococcus aureus control by virulent bacteriophages within MAC-T bovine mammary epithelial cells,” Antimicrobial Agents and Chemotherapy, vol. 61, no. 2, 2017. View at: Publisher Site | Google Scholar
  13. T. A. Lehti, M. I. Pajunen, M. S. Skog, and J. Finne, “Internalization of a polysialic acid-binding Escherichia coli bacteriophage into eukaryotic neuroblastoma cells,” Nature Communications, vol. 8, no. 1, Article ID 1915, 2017. View at: Publisher Site | Google Scholar
  14. S. Nguyen, K. Baker, B. S. Padman et al., “Bacteriophage transcytosis provides a mechanism to cross epithelial cell layers,” mBio, vol. 8, no. 6, 2017. View at: Publisher Site | Google Scholar
  15. A. Kurzepa-Skaradzinska, M. Lusiak-Szelachowska, G. Skaradzinski et al., “Influence of bacteriophage preparations on intracellular killing of bacteria by human phagocytesin vitro,” Viral Immunology, vol. 26, no. 2, pp. 150–162, 2013. View at: Publisher Site | Google Scholar
  16. E. Jończyk-Matysiak, M. Łusiak-Szelachowska, M. Kłak et al., “The effect of bacteriophage preparations on intracellular killing of bacteria by phagocytes,” Journal of Immunology Research, vol. 2015, Article ID 482863, 13 pages, 2015. View at: Publisher Site | Google Scholar
  17. B. O. Schroeder and F. Bäckhed, “Signals from the gut microbiota to distant organs in physiology and disease,” Nature Medicine, vol. 22, no. 10, pp. 1079–1089, 2016. View at: Publisher Site | Google Scholar
  18. L. De Sordi, V. Khanna, and L. Debarbieux, “The gut microbiota facilitates drifts in the genetic diversity and infectivity of bacterial viruses,” Cell Host & Microbe, vol. 22, no. 6, pp. 801.e3–808.e3, 2017. View at: Publisher Site | Google Scholar
  19. S. R. Carding, N. Davis, and L. Hoyles, “Review article: the human intestinal virome in health and disease,” Alimentary Pharmacology & Therapeutics, vol. 46, no. 9, pp. 800–815, 2017. View at: Publisher Site | Google Scholar
  20. V. Aggarwala, G. Liang, and F. D. Bushman, “Viral communities of the human gut: metagenomic analysis of composition and dynamics,” Mobile DNA, vol. 8, no. 1, p. 12, 2017. View at: Publisher Site | Google Scholar
  21. A. N. Shkoporov and C. Hill, “Bacteriophages of the human gut: the “known unknown” of the microbiome,” Cell Host & Microbe, vol. 25, no. 2, pp. 195–209, 2019. View at: Publisher Site | Google Scholar
  22. G. Tetz and V. Tetz, “Bacteriophages as new human viral pathogens,” Microorganisms, vol. 6, no. 2, p. 54, 2018. View at: Publisher Site | Google Scholar
  23. G. Tetz, S. M. Brown, Y. Hao, and V. Tetz, “Parkinson’s disease and bacteriophages as its overlooked contributors,” Scientific Reports, vol. 8, no. 1, Article ID 10812, 2018. View at: Publisher Site | Google Scholar
  24. L. Klingelhoefer and H. Reichmann, “Pathogenesis of Parkinson disease-the gut-brain axis and environmental factors,” Nature Reviews Neurology, vol. 11, no. 11, pp. 625–636, 2015. View at: Publisher Site | Google Scholar
  25. A. Fasano, N. P. Visanji, L. W. C. Liu, A. E. Lang, and R. F. Pfeiffer, “Gastrointestinal dysfunction in Parkinson’s disease,” The Lancet Neurology, vol. 14, no. 6, pp. 625–639, 2015. View at: Publisher Site | Google Scholar
  26. J. R. Bedarf, F. Hildebrand, L. P. Coelho et al., “Erratum to: functional implications of microbial and viral gut metagenome changes in early stage L-DOPA-naive Parkinson’s disease patients,” Genome Medicine, vol. 9, no. 1, p. 39, 2017. View at: Publisher Site | Google Scholar
  27. J. Mahony, J. Murphy, and D. van Sinderen, “Lactococcal 936-type phages and dairy fermentation problems: from detection to evolution and prevention,” Frontiers in Microbiology, vol. 3, p. 335, 2012. View at: Publisher Site | Google Scholar
  28. H. S. Addy, A. Askora, T. Kawasaki, M. Fujie, and T. Yamada, “The filamentous phage ϕRSS1 enhances virulence of phytopathogenic Ralstonia solanacearum on tomato,” Phytopathology, vol. 102, no. 3, pp. 244–251, 2012. View at: Publisher Site | Google Scholar
  29. H. Kamiunten and S. Wakimoto, “Cleavage of replicative form DNAs of filamentous phages Xf and Xf2 by restriction endonucleases,” Japanese Journal of Phytopathology, vol. 49, no. 5, pp. 633–638, 1983. View at: Publisher Site | Google Scholar
  30. I. W. Sutherland, K. A. Hughes, L. C. Skillman, and K. Tait, “The interaction of phage and biofilms,” FEMS Microbiology Letters, vol. 232, no. 1, pp. 1–6, 2004. View at: Publisher Site | Google Scholar
  31. L. Chen and Y. M. Wen, “The role of bacterial biofilm in persistent infections and control strategies,” International Journal of Oral Science, vol. 3, no. 2, pp. 66–73, 2011. View at: Publisher Site | Google Scholar
  32. H. K. DeBardeleben, E. S. Lysenko, A. B. Dalia, and J. N. Weiser, “Tolerance of a phage element by Streptococcus pneumoniae leads to a fitness defect during colonization,” Journal of Bacteriology, vol. 196, no. 14, pp. 2670–2680, 2014. View at: Publisher Site | Google Scholar
  33. M. Carrolo, M. J. Frias, F. R. Pinto, J. Melo-Cristino, and M. Ramirez, “Prophage spontaneous activation promotes DNA release enhancing biofilm formation in Streptococcus pneumoniae,” PLoS One, vol. 5, no. 12, Article ID e15678, 2010. View at: Publisher Site | Google Scholar
  34. S. Vilain, J. M. Pretorius, J. Theron, and V. S. Brözel, “DNA as an adhesin: Bacillus cereus requires extracellular DNA to form biofilms,” Applied and Environmental Microbiology, vol. 75, no. 9, pp. 2861–2868, 2009. View at: Publisher Site | Google Scholar
  35. M. J. Frias, J. Melo-Cristino, and M. Ramirez, “The autolysin LytA contributes to efficient bacteriophage progeny release in Streptococcus pneumoniae,” Journal of Bacteriology, vol. 191, no. 17, pp. 5428–5440, 2009. View at: Publisher Site | Google Scholar
  36. P. R. Secor, J. M. Sweere, L. A. Michaels et al., “Filamentous bacteriophage promote biofilm assembly and function,” Cell Host & Microbe, vol. 18, no. 5, pp. 549–559, 2015. View at: Publisher Site | Google Scholar
  37. P. R. Secor, L. Jennings, L. Michaels et al., “Biofilm assembly becomes crystal clear-filamentous bacteriophage organize the Pseudomonas aeruginosa biofilm matrix into a liquid crystal,” Microbial Cell, vol. 3, no. 1, pp. 49–52, 2016. View at: Publisher Site | Google Scholar
  38. P. R. Secor, L. A. Michaels, K. S. Smigiel et al., “Filamentous bacteriophage produced by Pseudomonas aeruginosa alters the inflammatory response and promotes noninvasive infection in vivo,” Infection and Immunity, vol. 85, no. 1, 2017. View at: Publisher Site | Google Scholar
  39. E. Bille, J. Meyer, A. Jamet et al., “A virulence-associated filamentous bacteriophage of Neisseria meningitidis increases host-cell colonisation,” PLoS Pathogens, vol. 13, no. 7, Article ID e1006495, 2017. View at: Publisher Site | Google Scholar
  40. A.-E. Deghmane, D. Giorgini, M. Larribe, J.-M. Alonso, and M.-K. Taha, “Down-regulation of pili and capsule of Neisseria meningitidis upon contact with epithelial cells is mediated by CrgA regulatory protein,” Molecular Microbiology, vol. 43, no. 6, pp. 1555–1564, 2002. View at: Publisher Site | Google Scholar
  41. A. Nasir, P. Forterre, K. M. Kim, and G. Caetano-Anollés, “The distribution and impact of viral lineages in domains of life,” Frontiers in Microbiology, vol. 5, p. 194, 2014. View at: Publisher Site | Google Scholar
  42. S. R. Bordenstein and S. R. Bordenstein, “Eukaryotic association module in phage WO genomes from Wolbachia,” Nature Communications, vol. 7, no. 1, Article ID 13155, 2016. View at: Publisher Site | Google Scholar
  43. K. Dabrowska, K. Switala-Jelen, A. Opolski, B. Weber-Dabrowska, and A. Gorski, “Bacteriophage penetration in vertebrates,” Journal of Applied Microbiology, vol. 98, no. 1, pp. 7–13, 2005. View at: Publisher Site | Google Scholar
  44. J. Van Belleghem, K. Dąbrowska, M. Vaneechoutte, J. Barr, and P. Bollyky, “Interactions between bacteriophage, bacteria, and the mammalian immune system,” Viruses, vol. 11, no. 1, p. 10, 2018. View at: Publisher Site | Google Scholar
  45. J. Xu and Y. Xiang, “Membrane penetration by bacterial viruses,” Journal of Virology, vol. 91, no. 13, Article ID e00162, 2017. View at: Publisher Site | Google Scholar
  46. J. J. Barr, “A bacteriophages journey through the human body,” Immunological Reviews, vol. 279, no. 1, pp. 106–122, 2017. View at: Publisher Site | Google Scholar
  47. D. J. Malik, I. J. Sokolov, G. K. Vinner et al., “Formulation, stabilisation and encapsulation of bacteriophage for phage therapy,” Advances in Colloid and Interface Science, vol. 249, pp. 100–133, 2017. View at: Publisher Site | Google Scholar
  48. A. Ksendzovsky, S. Walbridge, R. C. Saunders, A. R. Asthagiri, J. D. Heiss, and R. R. Lonser, “Convection-enhanced delivery of M13 bacteriophage to the brain,” Journal of Neurosurgery, vol. 117, no. 2, pp. 197–203, 2012. View at: Publisher Site | Google Scholar
  49. K. Namdee, M. Khongkow, S. Boonrungsiman et al., “Thermoresponsive bacteriophage nanocarrier as a gene delivery vector targeted to the gastrointestinal tract,” Molecular Therapy-Nucleic Acids, vol. 12, pp. 33–44, 2018. View at: Publisher Site | Google Scholar
  50. H. Xu, X. Bao, Y. Wang et al., “Engineering T7 bacteriophage as a potential DNA vaccine targeting delivery vector,” Virology Journal, vol. 15, no. 1, p. 49, 2018. View at: Publisher Site | Google Scholar
  51. H. Huh, S. Wong, J. S. Jean, and R. Slavcev, “Bacteriophage interactions with mammalian tissue: therapeutic applications,” Advanced Drug Delivery Reviews, vol. 145, pp. 4–17, 2019. View at: Publisher Site | Google Scholar
  52. K. D[aogon]browska, A. Opolski, J. Wietrzyk et al., “Activity of bacteriophages in murine tumor models depends on the route of phage administration,” Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics, vol. 15, no. 4, pp. 183–187, 2005. View at: Publisher Site | Google Scholar
  53. K. Dabrowska, A. Opolski, J. Wietrzyk et al., “Antitumor activity of bacteriophages in murine experimental cancer models caused possibly by inhibition of beta3 integrin signaling pathway,” Acta virologica, vol. 48, no. 4, pp. 241–248, 2004. View at: Google Scholar
  54. C. J. Avraamides, B. Garmy-Susini, and J. A. Varner, “Integrins in angiogenesis and lymphangiogenesis,” Nature Reviews Cancer, vol. 8, no. 8, pp. 604–617, 2008. View at: Publisher Site | Google Scholar
  55. F. Danhier, A. Le Breton, and V. Préat, “RGD-based strategies to target alpha(v) beta(3) integrin in cancer therapy and diagnosis,” Molecular Pharmaceutics, vol. 9, no. 11, pp. 2961–2973, 2012. View at: Publisher Site | Google Scholar
  56. B. Pan, J. Guo, Q. Liao, and Y. Zhao, “β1 and β3 integrins in breast, prostate and pancreatic cancer: a novel implication,” Oncology Letters, vol. 15, no. 4, pp. 5412–5416, 2018. View at: Publisher Site | Google Scholar
  57. K. Dabrowska, M. Zembala, J. Boratynski et al., “Hoc protein regulates the biological effects of T4 phage in mammals,” Archives of Microbiology, vol. 187, no. 6, pp. 489–498, 2007. View at: Publisher Site | Google Scholar
  58. A. Fokine, P. R. Chipman, P. G. Leiman, V. V. Mesyanzhinov, V. B. Rao, and M. G. Rossmann, “Molecular architecture of the prolate head of bacteriophage T4,” Proceedings of the National Academy of Sciences, vol. 101, no. 16, pp. 6003–6008, 2004. View at: Publisher Site | Google Scholar
  59. P. G. Leiman, S. Kanamaru, V. V. Mesyanzhinov, F. Arisaka, and M. G. Rossmann, “Structure and morphogenesis of bacteriophage T4,” Cellular and Molecular Life Sciences (CMLS), vol. 60, no. 11, pp. 2356–2370, 2003. View at: Publisher Site | Google Scholar
  60. A. Fokine, M. Z. Islam, Z. Zhang, V. D. Bowman, V. B. Rao, and M. G. Rossmann, “Structure of the three N-terminal immunoglobulin domains of the highly immunogenic outer capsid protein from a T4-like bacteriophage,” Journal of Virology, vol. 85, no. 16, pp. 8141–8148, 2011. View at: Publisher Site | Google Scholar
  61. S. G. Sanmukh, S. A. A. Dos Santos, and S. L. Felisbino, “Natural bacteriophages T4 and M13 down-regulates Hsp90 gene expression in human prostate cancer cells (PC-3) representing a potential nanoparticle against cancer,” Virology Research Journal, vol. 1, no. 1, pp. 21–23, 2017. View at: Google Scholar
  62. J. Shan, A. Ramachandran, A. M. Thanki, F. B. I. Vukusic, J. Barylski, and M. R. J. Clokie, “Bacteriophages are more virulent to bacteria with human cells than they are in bacterial culture insights from HT-29 cells,” Scientific Reports, vol. 8, no. 1, Article ID 5091, 2018. View at: Publisher Site | Google Scholar
  63. H. Jeong, B. Arif, G. Caetano-Anollés, K. Mo Kim, and A. Nasir, “Horizontal gene transfer in human-associated microorganisms inferred by phylogenetic reconstruction and reconciliation,” Scientific Reports, vol. 9, no. 1, Article ID 5953, 2019. View at: Publisher Site | Google Scholar
  64. C. L. Schneider, “Bacteriophage-mediated horizontal gene transfer: transduction,” in Bacteriophages: Biology, Technology, Therapy, D. Harper, Ed., pp. 1–42, Springer, Berlin, Germany, 2017. View at: Publisher Site | Google Scholar
  65. B. Koskella and T. B. Taylor, “Multifaceted impacts of bacteriophages in the plant microbiome,” Annual Review of Phytopathology, vol. 56, no. 1, pp. 361–380, 2018. View at: Publisher Site | Google Scholar
  66. S. R. Bordenstein, M. L. Marshall, A. J. Fry, U. Kim, and J. J. Wernegreen, “The tripartite associations between bacteriophage, Wolbachia, and arthropods,” PLoS Pathogens, vol. 2, no. 5, p. e43, 2006. View at: Publisher Site | Google Scholar
  67. A. G. Rosenwald, B. Murray, T. Toth, R. Madupu, A. Kyrillos, and G. Arora, “Evidence for horizontal gene transfer between Chlamydophila pneumoniae and Chlamydia phage,” Bacteriophage, vol. 4, no. 4, Article ID e965076, 2014. View at: Publisher Site | Google Scholar
  68. M. Redrejo-Rodríguez and M. Salas, “Multiple roles of genome-attached bacteriophage terminal proteins,” Virology, vol. 468470, pp. 322–329, 2014. View at: Publisher Site | Google Scholar
  69. M. Redrejo-Rodriguez, D. Muñoz-Espín, I. Holguera, M. Mencía, and M. Salas, “Nuclear localization signals in phage terminal proteins provide a novel gene delivery tool in mammalian cells,” Communicative & Integrative Biology, vol. 6, no. 2, Article ID e22829, 2013. View at: Publisher Site | Google Scholar


Copyright © 2020 Ramendra Dirgantara Putra and Diana Lyrawati. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Barr, J. J. (2017). A bacteriophages journey through the human body. Immunol. Rev. 279, 106�. doi: 10.1111/imr.12565

Barr, J. J., Auro, R., Furlan, M., Whiteson, K. L., Erb, M. L., Pogliano, J., et al. (2013). Bacteriophage adhering to mucus provide a non-host-derived immunity. Proc. Natl. Acad. Sci. U.S.A. 110, 10771�. doi: 10.1073/pnas.1305923110

Bichet, M. C., Chin, W. H., Richards, W., Lin, Y. W., Avellaneda-Franco, L., Hernandez, C. A., et al. (2020). Bacteriophage uptake by Eukaryotic cell layers represents a major sink for phages during therapy. bioRxiv. Available online at: (accessed December 10, 2020).

Borysowski, J., Miedzybrodzki, R., Przybylski, M., Owczarek, B., Weber-Dabrowska, B., and Gorski, A. (2020). The effects of T4 and A5/80 phages on the expression of immunologically important genes in differentiated Caco-2 cells. Adv. Hyg. Exp. Med. 74, 371�, doi: 10.5604/01.3001.0014.3919

Borysowski, J., Przybylski, M., Miedzybrodzki, R., Owczarek, B., and Górski, A. (2019). The effects of bacteriophages on the expression of genes involved in antimicrobial immunity. Adv. Hyg. Exp. Med. 73, 414�. doi: 10.5604/01.3001.0013.4081

Cafora, M., Brix, A., Forti, F., Loberto, N., Aureli, M., Briani, F., et al. (2020). Phages as immunomodulators and their promising use as anti-inflammatory agents in a cftr loss-of-function zebrafish model. J. Cyst. Fibros. doi: 10.1016/j.jcf.2020.11.017. [Epub ahead of print].

Chuquimia, O. D., Petursdottir, D. H., Periolo, N., and Fernández, C. (2013). Alveolar epithelial cells are critical in protection of the respiratory tract by secretion of factors able to modulate the activity of pulmonary macrophages and directly control bacterial growth. Infect. Immun. 81, 381�. doi: 10.1128/IAI.00950-12

Dabrowska, K., Opolski, A., Wietrzyk, J., Switala-Jelen, K., Boratynski, J., Nasulewicz, A., et al. (2004). Antitumor activity of bacteriophages in murine experimental cancer models caused possibly by inhibition of beta3 integrin signaling pathway. Acta Virol. 48, 241�.

Dabrowska, K., Skaradziński, G., Joᑌzyk, P., Kurzepa, A., Wietrzyk, J., Owczarek, B., et al. (2009). The effect of bacteriophages T4 and HAP1 on in vitro melanoma migration. BMC Microbiol. 9:13. doi: 10.1186/1471-2180-9-13

Dufour, N., Delattre, R., Chevallereau, A., Ricard, J. D., and Debarbieux, L. (2019). Phage therapy of pneumonia is not associated with an overstimulation of the inflammatory response compared to antibiotic treatment in mice. Antimicrob. Agents Chemother. 63, e00379�. doi: 10.1128/AAC.00379-19

Górski, A., Dabrowska, K., Miedzybrodzki, R., Weber-Dabrowska, B., Lusiak-Szelachowska, M., Jonczyk-Matysiak, E., and Borysowski, J. (2017). Phages and immunomodulation. Future Microbiol. 12, 905�. doi: 10.2217/fmb-2017-0049

Górski, A., Miedzybrodzki, R., Jonczyk-Matysiak, E., Zaczek, M., and Borysowski, J. (2019). Phage-specific diverse effects of bacterial viruses on the immune system. Future Microbiol. 14, 1171�. doi: 10.2217/fmb-2019-0222

Górski, A., and Weber-Dabrowska, B. (2005). The potential role of endogenous bacteriophages in controlling invading pathogens. Cell. Mol. Life Sci. 62, 511�. doi: 10.1007/s00018-004-4403-6

Khan Mirzaei, M., Haileselassie, Y., Navis, M., Cooper, C., Sverremark-Ekström, E., and Nilsson, A. S. (2016). Morphologically distinct Escherichia coli bacteriophages differ in their efficacy and ability to stimulate cytokine release in vitro. Front. Microbiol. 7:437. doi: 10.3389/fmicb.2016.01974

Koeninger, L., Armbruster, N. S., Brinch, K. S., Kjaerulf, S., Andersen, B., Langnau, C., et al. (2020). Human β-Defensin 2 mediated immune modulation as treatment for experimental colitis. Front. Immunol. 11:93. doi: 10.3389/fimmu.2020.00093

Mason, R. J. (2020). Pathogenesis of COVID-19 from a cell biology perspective. Eur. Respir. J. 55:2000607. doi: 10.1183/13993003.00607-2020

Merril, C. R. (1973). Effects of bacteriophage on eukaryotes. Johns Hopkins Med. J. Suppl. 2, 73�.

Nguyen, S., Baker, K., Padman, B. S., Patwa, R., Dunstan, R. A., Weston, T. A., et al. (2017). Bacteriophage transcytosis provides a mechanism to cross epithelial cell layers. mBio. 8, e01874�. doi: 10.1128/mBio.01874-17

Oie, C. I., Wolfson, D. L., Yasunori, T., Dumitriu, G., Sorensen, K. K., McCourt, P. A., et al. (2020). Liver sinusoidal endothelial cells contribute to the uptake and degradation of entero bacterial viruses. Sci. Rep. 10:898. doi: 10.1038/s41598-020-57652-0

Parducho, K. R., Beadell, B., Ybarra, T. K., Bush, M., Escalera, E., Trejos, A. T., et al. (2020). The antimicrobial peptide human Beta-Defensin 2 inhibits biofilm production of Pseudomonas aeruginosa without compromising metabolic activity. Front. Immunol. 11:805. doi: 10.3389/fimmu.2020.00805

Pincus, N. B., Reckhow, J. D., Saleem, D., Jammeh, M. L., Datta, S. K., and Myles, I. A. (2015). Strain specific phage treatment for Staphylococcus aureus infection is influenced by host immunity and site of infection. PLoS ONE 10:e0124280. doi: 10.1371/journal.pone.0124280

Pinkerton, J. W., Kim, R. Y., Koeninger, L., Armbruster, N. S., Hansbro, N. G., Brown, A. C., et al. (2020). Human β-defensin-2 suppresses key features of asthma in murine models of allergic airways disease. Clin. Exp. Allergy. doi: 10.1111/cea.13766. [Epub ahead of print].

Shan, J., Ramachandran, A., Thanki, A. M., Vukusic, F., Barylski, J., and Clokie, M. (2018). Bacteriophages are more virulent to bacteria with human cells than they are in bacterial culture insights from HT-29 cells. Sci. Rep. 8:5091. doi: 10.1038/s41598-018-23418-y

Shelley, J. R., Davidson, D. J., and Dorin, J. R. (2020). The dichotomous responses driven by β-Defensins. Front. Immunol. 11:1176. doi: 10.3389/fimmu.2020.01176

Shiozawa, A., Kajiwara, C., Ishii, Y., and Tateda, K. (2020). N-acetyl-cysteine mediates protection against Mycobacterium avium through induction of human β-defensin-2 in a mouse lung infection model. Microbes Infect. 22, 567�. doi: 10.1016/j.micinf.2020.08.003

Trend, S., Fonceca, A. M., Ditcham, W. G., Kicic, A., and Cf, A. (2017). The potential of phage therapy in cystic fibrosis: Essential human-bacterial-phage interactions and delivery considerations for use in Pseudomonas aeruginosa-infected airways. J. Cyst. Fibros. 16, 663�. doi: 10.1016/j.jcf.2017.06.012

Uluçkan, Ö., and Wagner, E. F. (2017). Chronic systemic inflammation originating from epithelial tissues. FEBS J. 284, 505�. doi: 10.1111/febs.13904

Van Belleghem, J. D., Clement, F., Merabishvili, M., Lavigne, R., and Vaneechoutte, M. (2017). Pro- and anti-inflammatory responses of peripheral blood mononuclear cells induced by Staphylococcus aureus and Pseudomonas aeruginosa phages. Sci. Rep. 7:8004. doi: 10.1038/s41598-017-08336-9

Van Belleghem, J. D., Dabrowska, K., Vaneechoutte, M., Barr, J. J., and Bollyky, P. L. (2018). Interactions between bacteriophage, bacteria, and the mammalian immune system. Viruses 11:10. doi: 10.3390/v11010010

Zaczek, M., Górski, A., Szkaradzinska, A., Lusiak-Szelachowska, M., and Weber-Dabrowska, B. (2020). Phage penetration of eukaryotic cells: practical implications. Future Virol. 11, 745� 6. doi: 10.2217/fvl-2019-0110

Zhang, L., Hou, X., Sun, L., He, T., Wei, R., Pang, M., et al. (2018). Staphylococcus aureus bacteriophage suppresses LPS-induced inflammation in MAC-T bovine mammary epithelial cells. Front. Microbiol. 9:1614. doi: 10.3389/fmicb.2018.01614

Keywords: phage therapy, epithelial cells, inflammation, eukaryotic cells, cytokines

Citation: Górski A, Borysowski J and Mi⊝zybrodzki R (2021) Bacteriophage Interactions With Epithelial Cells: Therapeutic Implications. Front. Microbiol. 11:631161. doi: 10.3389/fmicb.2020.631161

Received: 19 November 2020 Accepted: 22 December 2020
Published: 18 January 2021.

Luís D. R. Melo, University of Minho, Portugal

Gabriel Almeida, University of Jyväskylä, Finland
Anders S. Nilsson, Stockholm University, Sweden

Copyright © 2021 Górski, Borysowski and Mi⊝zybrodzki. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Senior Mammalian Cell Expression Scientist – Cell and Molecular Biology

Today, Lonza is a global leader in life sciences operating across three continents. While we work in science, there’s no magic formula to how we do it. Our greatest scientific solution is talented people working together, devising ideas that help businesses to help people. In exchange, we let our people own their careers. Their ideas, big and small, genuinely improve the world. And that’s the kind of work we want to be part of.

Every day, Lonza’s products and services have a positive impact on millions of people. For us, this is not only a great privilege, but also a great responsibility. How we achieve our business results is just as important as the achievements themselves. At Lonza, we respect and protect our people and our environment. Any success we achieve is no success at all if not achieved ethically.

The Lonza Pharma and Biotech segment (LPB) focuses on the contract development and manufacturing of recombinant protein biotherapeutics that dramatically improve patients’ lives. With a renewed commitment to innovation in the space, and the ambition to build the mammalian expression system of the future, we are seeking highly competent, experienced Senior Scientists (bio-processing) to join us on our journey and complement our team based at the research center of excellence in Cambridge.

Leading projects from the bench, you will need a strong background in the delivery of technical innovation projects in the bioprocessing space. Working with a multi-disciplinary team of scientists dedicated to achieving a step change improvement in mammalian cell expression platforms, you will bring a proven set of skills in mammalian cell culture, recombinant protein expression, and cell and molecular biology. LPB R&D is a fast-paced working environment where innovative thinking, creativity and collaboration are all actively encouraged, and to succeed you will need to be flexible, open to new ideas, and excited by the chance to work with colleagues across the R&D network, including external collaborators such as leading academics.

The key responsibilities include:

  • Lead lab-based R&D activities to develop next-generation expression technology using platforms based on Chinese hamster ovary (CHO) cells
  • Employ your knowledge and skills in mammalian cell culture and molecular biology to improve all aspects of recombinant protein expression, focusing on the DNA expression vectors and the host cell line
  • Generate, collate and present high quality experimental data in written reports and for oral presentations
  • Initiate and lead scientific discussions on the latest bioprocessing R&D
  • Act as a mentor for junior members of the team
  • Work closely with other groups within R&D to deliver solutions or improvements in recombinant protein expression
  • Actively keep abreast of relevant academic literature
  • PhD Degree in Life Sciences / Molecular Biology/ Biotechnology, together with practical experience in an industrial setting
  • Experience in mammalian (CHO) cell line development for recombinant protein expression
  • R&D technical background in relevant mammalian cell culture techniques e.g. generation of recombinant CHO cell lines, stable and transient transfection processes, fed-batch culture, etc.
  • Experience with flow cytometry FACS experience is desirable but not essential
  • Extensive molecular biology experience in methods such as gene cloning, expression vector design and manipulation, PCR/qPCR, agarose gel electrophoresis, SDS-PAGE and Western blotting.
  • Experience in the use of transposon systems (e.g. piggyBac or similar) is desirable
  • Must be able to work with minimal direction and guidance, with a high level of independence and flexibility
  • Experience in the lab-based supervision of junior scientists
  • Basic protein chemistry/analytical techniques (HPLC, electrophoresis, immunoassays etc.)

This is an exciting opportunity to join an R&D team of scientists with a rich heritage of delivering innovative solutions in the bioprocessing space. People come to Lonza for the challenge and creativity of solving complex problems and developing new ideas in life sciences. In return, we offer the satisfaction that comes with improving lives all around the world. The satisfaction that comes with making a meaningful difference.


Anderson, C., Shen, M., Eisenstein, R., and Leibold, E. (2012). Mammalian iron metabolism and its control by iron regulatory proteins. Biochem. Biophys. Acta 1823, 1468�. doi: 10.1016/j.bbamcr.2012.05.010

Anderson, G., and Vulpe, C. (2009). Mammalian iron transport. Cell. Mol. Life Sci. 66, 3241�. doi: 10.1007/s00018-009-0051-1

Andrews, S., Andrea, K., and Rodriquez-Quinones, F. (2003). Bacterial iron homeostasis. FEMS Microbiol. Rev. 27, 215�. doi: 10.1016/S0168-6445(03)00055-X

Belton, M., Commerford, K., Hall, J., Prato, F., and Carson, J. (2008). Real-time measurement of cytosolic free calcium concentration in Hl-60 cells during static magnetic field exposure and activation by ATP. Bioelectromagnetics 29, 439�. doi: 10.1002/bem.20409

Bin Na, H., Song, I., and Hyeon, T. (2009). Inorganic nanoparticles for MRI contrast agents. Adv. Mater. 21, 2133�. doi: 10.1002/adma.200802366

Burtea, C., Laurent, S., Vander Elst, L. and Muller, R. (2008). 𠇌ontrast agents: magnetic resonance,” in Handbook of Experimental Pharmacology. Molecular Imaging I. eds W. Semmler and M. Schwaiger (Heidelberg: Springer-Verlag Berlin).

Chung, M., Hogstrand, C., and Lee, S.-J. (2006). Cytotoxicity of nitric oxide is alleviated by zinc-mediated expression of antioxidant genes. Exp. Biol. Med. 231, 1555�.

Cohen, B., Ziv, K., Plaks, V., Harmelin, A., and Neeman, M. (2009). Ferritin nanoparticles as magnetic resonance reporter gene. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 1, 181�. doi: 10.1002/wnan.11

Daniels, T., Delgado, T., Rodriguez, J., Helguera, G., and Penichet, M. (2006). The transferrin receptor part I: biology and targeting with cytotoxic antibodies for the treatment of cancer. Clin. Immunol. 121, 144�. doi: 10.1016/j.clim.2006.06.010

Deans, A. E., Wadghiri, Y. Z., Bernas, L. M., Yu, X., Rutt, B. K., and Turnbull, D. H. (2006). Cellular MRI contrast via coexpression of transferrin receptor and ferritin. Magn. Reson. Med. 56, 51�. doi: 10.1002/mrm.20914

Ganz, T. (2005). Cellular iron: ferroportin is the only way out. Cell Metabol. 1, 155�. doi: 10.1016/j.cmet.2005.02.005

Gelman, N., Gorell, J., Barker, P., Savage, R., Spickler, E., Windham, J., et al. (1999). MR imaging of human brain at 3.0 T: preliminary report on transverse relaxation rates and relation to estimated iron content. Radiology 210, 759�. doi: 10.1148/radiology.210.3.r99fe41759

Genove, G., Demarco, U., Xu, H., Goins, W. F., and Ahrens, E. T. (2005). A new transgene reporter for in vivo magnetic resonance imaging. Nat. Med. 11, 450�. doi: 10.1038/nm1208

Goldhawk, D., Lemaire, C., Mccreary, C., Mcgirr, R., Dhanvantari, S., Thompson, R., et al. (2009). Magnetic resonance imaging of cells overexpressing MagA, an endogenous contrast agent for live cell imaging. Mol. Imaging 8, 129�.

Goldhawk, D., Rohani, R., Sengupta, A., Gelman, N., and Prato, F. (2012). Using the magnetosome to model effective gene-based contrast for magnetic resonance imaging. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 4, 378�. doi: 10.1002/wnan.1165

Greene, S., and Komeili, A. (2012). Biogenesis and subcellular organization of the magnetosome organelles of magnetotactic bacteria. Curr. Opin. Cell Biol 24, 490�. doi: 10.1016/

Huang, J., Zhong, X., Wang, L., Yang, L., and Mao, H. (2012). Improving the magnetic resonance imaging contrast and detection methods with engineered magnetic nanoparticles. Theranostics 2, 86�. doi: 10.7150/thno.4006

Jogler, C., and Schuler, D. (2009). Genomics, genetics, and cell biology of magnetosome formation. Annu. Rev. Microbiol. 63, 501�. doi: 10.1146/annurev.micro.62.081307.162908

Kim, T., Momin, E., Choi, J., Yuan, K., Zaidi, H., Kim, J., et al. (2011). Mesoporous silica-coated hollow manganese oxide nanoparticles as positive T1 contrast agents for labeling and MRI tracking of adipose-derived mesenchymal stem cells. J. Am. Chem. Soc. 133, 2955�. doi: 10.1021/ja1084095

Komeili, A. (2012). Molecular mechanisms of compartmentalization and biomineralization in magnetotactic bacteria. FEMS Microbiol. Rev. 36, 232�. doi: 10.1111/j.1574-6976.2011.00315.x

Kuhlpeter, R., Dahnke, H., Matuszewski, L., Persigehl, T., Von Wallbrunn, A., Allkemper, T., et al. (2007). R2 and R2* mapping for sensing cell-bound superparamagnetic nanoparticles: in vitro and murine in vivo testing. Radiology 245, 449�. doi: 10.1148/radiol.2451061345

Laemmli, U. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680�. doi: 10.1038/227680a0

Linklater, H., Galsworthy, P., Stewart-Dehaan, P., D𠆚more, T., Lo, T., and Trevithick, J. (1985). The use of guanidinium chloride in the preparation of stable cellular homogenates containing ATP. Anal. Biochem. 148, 44�. doi: 10.1016/0003-2697(85)90625-6

Nakamura, C., Burgess, J. G., Sode, K., and Matsunaga, T. (1995a). An iron-regulated gene, magA, encoding an iron transport protein of Magnetospirillum sp. Strain AMB-1. J. Biol. Chem. 270, 28392�. doi: 10.1074/jbc.270.47.28392

Nakamura, C., Kikuchi, T., Burgess, J. G., and Matsunaga, T. (1995b). Iron-regulated expression and membrane localization of the MagA protein in Magnetospirillum sp. Strain AMB-1. J. Biochem. 118, 23�.

Nyholm, S., Mann, G., Johansson, A., Bergeron, R., Graslund, A., and Thelander, L. (1993). Role of ribonucleotide reductase in inhibition of mammalian cell growth by potent iron chelators. J. Biol. Chem. 268, 26200�.

Pantopoulos, K., Porwal, S., Tartakoff, A., and Devireddy, L. (2012). Mechanisms of mammalian iron homeostasis. Biochemistry 51, 5705�. doi: 10.1021/bi300752r

Ponka, P., and Lok, C. (1999). The transferrin receptor: role in health and disease. Int. J. Biochem. Cell Biol. 31, 1111�. doi: 10.1016/S1357-2725(99)00070-9

Recalcati, S., Locati, M., Marini, A., Santambrogio, P., Zaninotto, F., De Pizzol, M., et al. (2010). Differential regulation of iron homeostasis during human macrophage polarized activation. Eur. J. Immunol. 40, 824�. doi: 10.1002/eji.200939889

Renier, C., Vogel, H., Offor, O., Yao, C., and Wapnir, I. (2010). Breast cancer brain metastases express the sodium iodide symporter. J. Neurooncol. 96, 331�. doi: 10.1007/s11060-009-9971-8

Richter, M., Kube, M., Bazylinski, D. A., Lombardot, T., Glockner, F. O., Reinhardt, R., et al. (2007). Comparative genome analysis of four magnetotactic bacteria reveals a complex set of group-specific genes implicated in magnetosome biomineralization and function. J. Bacteriol. 189, 4899�. doi: 10.1128/JB.00119-07

Rohani, R., Figueredo, R., Bureau, Y., Koropatnick, J., Foster, P., Thompson, R., et al. (2013). Imaging tumor growth non-invasively using expression of MagA or modified ferritin subunits to augment intracellular contrast for repetitive MRI. Mol. Imaging Biol. 16, 63�. doi: 10.1007/s11307-013-0661-8

Shpyleva, S., Tryndyak, V., Kovalchuk, O., Starlard-Davenport, A., Chekhun, V., Beland, F., et al. (2011). Role of ferritin alterations in human breast cancer cells. Breast Cancer Res. Treat. 126, 63�. doi: 10.1007/s10549-010-0849-4

Smith, P., Krohn, R., Hermanson, G., Mallia, A., Gartner, F., Provenzano, M., et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76�. doi: 10.1016/0003-2697(85)90442-7

Takahashi, M., Yoshino, T., Takeyama, H., and Matsunaga, T. (2009). Direct magnetic separation of immune cells from whole blood using bacterial magnetic particles displaying protein G. Biotechnol. Prog. 25, 219�. doi: 10.1002/btpr.101

Tobin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350�. doi: 10.1073/pnas.76.9.4350

Uebe, R., Henn, V., and Schuler, D. (2012). The MagA protein of Magnetospirilla is not involved in bacterial magnetite biomineralization. J. Bacteriol. 194, 1018�. doi: 10.1128/JB.06356-11

Weinberg, E. (1992). Roles of iron in neoplasia. promotion, prevention, and therapy. Biol. Trace Element Res. 34, 123�. doi: 10.1007/BF02785242

Wood, J., Enriquez, C., Ghugre, N., Tyzka, J., Carson, S., Nelson, M., et al. (2005). MRI R2 and R2* mapping accurately estimates hepatic iron concentration in transfusion-dependent thalassemia and sickle cell disease patients. Blood 106, 1460�. doi: 10.1182/blood-2004-10-3982

Yan, L., Zhang, S., Chen, P., Liu, H., Yin, H., and Li, H. (2012). Magnetotactic bacteria, magnetosomes and their application. Microbiol. Res. 167, 507�. doi: 10.1016/j.micres.2012.04.002

Zurkiya, O., Chan, A. W., and Hu, X. (2008). MagA is sufficient for producing magnetic nanoparticles in mammalian cells, making it an MRI reporter. Magn. Reson. Med. 59, 1225�. doi: 10.1002/mrm.21606

Keywords : magnetic resonance imaging, MagA, modified ferritin subunits, relaxation rates, iron, cancer cells

Citation: Sengupta A, Quiaoit K, Thompson RT, Prato FS, Gelman N and Goldhawk DE (2014) Biophysical features of MagA expression in mammalian cells: implications for MRI contrast. Front. Microbiol. 5:29. doi: 10.3389/fmicb.2014.00029

Received: 11 November 2013 Accepted: 17 January 2014
Published online: 05 February 2014.

Kevin C. Chan, University of Pittsburgh, USA
Laimonas Kelbauskas, Arizona State University, USA

Copyright © 2014 Sengupta, Quiaoit, Thompson, Prato, Gelman and Goldhawk. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.