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Bacteriophage lysis in mammalian cells

Bacteriophage lysis in mammalian cells


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I have read many articles on bacteriophages (like the lambda phage1) being used for transferring genes into mammalian cells, but none of them mention any sort of lysis of the cells even though in bacteria, lysis would occur. Of course, killing the cell would beat the purpose of the gene transfer, but why doesn't the bacteriophage kill the mammalian cells? Has there been any research on lysis of the mammalian cells with phages?


Bacteriophage

A bacteriophage ( / b æ k ˈ t ɪər i oʊ f eɪ dʒ / ), also known informally as a phage ( / ˈ f eɪ dʒ / ), is a virus that infects and replicates within bacteria and archaea. The term was derived from "bacteria" and the Greek φαγεῖν (phagein), meaning "to devour". Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome, and may have structures that are either simple or elaborate. Their genomes may encode as few as four genes (e.g. MS2) and as many as hundreds of genes. Phages replicate within the bacterium following the injection of their genome into its cytoplasm.

Bacteriophages are among the most common and diverse entities in the biosphere. [1] Bacteriophages are ubiquitous viruses, found wherever bacteria exist. It is estimated there are more than 10 31 bacteriophages on the planet, more than every other organism on Earth, including bacteria, combined. [2] Viruses are the most abundant biological entity in the water column of the world's oceans, and the second largest component of biomass after prokaryotes, [3] where up to 9x10 8 virions per millilitre have been found in microbial mats at the surface, [4] and up to 70% of marine bacteria may be infected by phages. [5]

Phages have been used since the late 20th century as an alternative to antibiotics in the former Soviet Union and Central Europe, as well as in France. [6] [7] They are seen as a possible therapy against multi-drug-resistant strains of many bacteria (see phage therapy). [8] On the other hand, phages of Inoviridae have been shown to complicate biofilms involved in pneumonia and cystic fibrosis and to shelter the bacteria from drugs meant to eradicate disease, thus promoting persistent infection. [9]


Overview of Cell Lysis and Protein Extraction

All cells have a plasma membrane, a protein-lipid bilayer that forms a barrier separating cell contents from the extracellular environment. Lipids comprising the plasma membrane are amphipathic, having hydrophilic and hydrophobic moieties that associate spontaneously to form a closed bimolecular sheet. Membrane proteins are embedded in the lipid bilayer, held in place by one or more domains spanning the hydrophobic core. In addition, peripheral proteins bind the inner or outer surface of the bilayer through interactions with integral membrane proteins or with polar lipid head groups. The nature of the lipid and protein content varies with cell type and species of organism.

Cell membrane structure. Illustration of a lipid bilayer comprising outer plasma membrane of a cell.

In animal cells, the plasma membrane is the only barrier separating cell contents from the environment, but in plants and bacteria the plasma membrane is also surrounded by a rigid cell wall. Bacterial cell walls are composed of peptidoglycan. Yeast cell walls are composed of two layers of ß-glucan, the inner layer being insoluble to alkaline conditions. Both of these are surrounded by an outer glycoprotein layer rich in the carbohydrate mannan. Plant cell walls consist of multiple layers of cellulose. These types of extracellular barriers confer shape and rigidity to the cells. Plant cell walls are particularly strong, making them very difficult to disrupt mechanically or chemically. Until recently, efficient lysis of yeast cells required mechanical disruption using glass beads, whereas bacterial cell walls are the easiest to break compared to these other cell types. The lack of an extracellular wall in animal cells makes them relatively easy to lyse.

There is no universal protocol for protein sample preparation. Sample preparation protocols must take into account several factors, such as the source of the specimen or sample type, chemical and structural heterogeneity of proteins, the cellular or subcellular location of the protein of interest, the required protein yield (which is dependent on the downstream applications), and the proposed downstream applications. For instance, bodily fluids such as urine or plasma are already more or less homogeneous protein solutions with low enzymatic activity, and only minor manipulation is required to obtain proteins from these samples. Tissue samples, however, require extensive manipulation to break up tissue architecture, control enzymatic activity, and solubilize proteins.

The quality or physical form of the isolated protein is also an important consideration when extracting proteins for certain downstream applications. For instance, applications such as functional enzyme-linked immunosorbent assay (ELISA) or crystallography require not only intact proteins but also proteins that are functionally active or retain their 3D structure.

Examples of protein sources for sample collection. Proteins can come from many sources, including the following: native sources such as mammalian cell cultures, tissues or bodily fluids overexpression in a model system such as bacteria, yeast, insect or mammalian cells monoclonal antibodies from hybridoma cells or plant cells used in agricultural biotechnology.


USE OF PHAGE COMPONENTS

As Prophylactic Agents

Despite reasonable success of phage therapy in diagnosis and treatment of bacterial infections by bacteriophages as discussed above, parallel research has been progressing in the use of bacteriophage components mostly as prophylaxis. Lysozymes, originally discovered by Alexander Fleming (Fleming 1922), are ubiquitous in nature, being associated with phage in mammals. Lysozymes have been used for decades as a prophylactic agent to kill Gram-positive bacteria because of their ability to degrade cell walls from outside by hydrolysis of one of the four major bonds in the peptidoglycan, which results in hypotonic bursting of the inner membrane and leaking of intracellular components and thereby causing cell death. Lysozymes from different sources including phage have been used as an adduct in the brewing industry and in yogurt and other milk products. As discussed below, phage-encoded lysozymes are of two kinds: (1) endolysin, which is made by phage during phage lytic growth after infection of bacterial host, and which lyses the cell wall from inside to help release phage progeny particles and (2) phage tail-associated murein lytic enzymes (TAME), which can hydrolyze cell wall bonds from outside after phage adsorption to the host.

Brewing industries face problems with their fermentation systems, as airborne bacteria frequently contaminate them. Bacterial contamination can make a beer turbid, aromatic, odious, or ropy. The most common contaminations are lactic-acid-producing Gram-positive bacteria (Lactobacillus and Pediococcus) and acetic-acid-producing Gram-negative bacteria (Acetobacter). The spoilage comes from the fact that acetic acid and lactic acids, among other by-products, are made by bacteria from sugar. Some measured amount of specific strains of lactic-acid-producing bacteria are allowed during fermentation to keep the pH low, thus facilitating beer production as well as lowering acidity by “malolactic acid” production. In wine making, unwanted contaminations are routinely taken care of by sanitation practices. In fact, direct lysozyme addition acts as a preservative for storage of foods like yogurt, tofu, cheese, and sake (Larson 2005).

More recently, the addition of phage endolysins has been shown to protect against a broad spectrum of Gram-positive bacteria. If microbial contamination is envisioned, lysozyme is used to kill the lactic acid bacteria. In fact, lysozyme can be added to the final product. Depending on factors like temperature, pH, etc., lysozyme starts working immediately after addition and kills bacteria at once. Lysozyme in beer remains 50% active for at least a 6-month period. Lysozyme present in beer has minimal effects on the physical and sensory properties of the beer (Feeney and Nagy 1952 Daeschel et al. 1991 Landschoot 2005 Waite and Daeschel 2007).

As Curative Agents

More recently, however, lysozymes are being developed for treatment of mammalian infections. Endolysin, used by phage to release progeny particles from bacterial hosts, acts by breaking up the cell from inside. This occurs after another phage product, called holin, creates pores in the inner membrane through which lysozymes can pass through the membrane and reach the cell wall (Wang et al. 2003). However, peptidoglycan-degrading enzymes have the ability to digest bacterial cell walls. The phage-encoded endolysins precisely do that except in a species- or genus-specific manner (Nelson et al. 2001 Fischetti 2010). Phage-encoded endolysin usually can lyse from outside only the corresponding host. Recently, cell wall lysis-mediated killing activity of phage-encoded pure lysozymes has been successfully used to its maximal level by, among others, Fischetti and colleagues, and Loessner and colleagues. Like the current surge in bacteriophage therapy, development of endolysin-mediated therapy of infection was motivated by the emergence of antibiotic-resistant bacteria in clinics. Endolysins create holes in the cell from outside by peptidoglycan digestion and expansion of the inner cytoplasmic membrane and subsequent hypotonic lysis.

Most human infections begin at the mucosal membranes followed by their colonization, which are usually a reservoir of many pathogens. Very few anti-infective agents are known that prevent mucosal colonization. Nonetheless, if one can reduce the bacterial load of the mucous membranes in the community, in hospitals, and in nursing homes, the incidence of the diseases would very likely reduce. Precisely, endolysins are expected to be very effective in such cases. Specific endolysins have now been identified to very effectively kill Gram-positive bacteria (Nelson et al. 2001 Loeffler et al. 2003). Interestingly, the endolysins bind very tightly to their cell wall substrates and thus and do not have a turnover number requiring multiple molecules to bind and hydrolyze several cell wall bonds to make effective cell wall lysis (Loessner et al. 2002 Jervis et al. 2005). An oral colonization animal model has been developed with Streptococcus pyogenes, a nasal model with pneumococci, and a vaginal model with group B streptococci. These studies showed, for example, that nanogram quantities of endolysin kill S. pyogenes 106-fold in 2–4 h after lysozyme treatments. All current success in dealing with endolysin-mediated cell killing are only with Gram-positive bacteria simply because the enzyme when added from outside can access the cell wall peptidoglycan. Gram-negative bacteria, however, have an outer membrane that sterically interferes with lysozyme action thus making lysozyme-mediated cell killing generally ineffective. However, recently, a structurally engineered phage lysozyme containing the FyuA-binding domain of pesticin fused to the amino terminus of T4 lysozyme has given encouraging results against Gram-negative pathogens (Lukacik et al. 2012).

Immunity and Resistance

Both in bacteriophage therapy and in direct lysozyme (of phage or any other source) therapy, it has always been feared that such foreign objects or proteins when used systematically would develop neutralizing antibodies and thus hinder their antibacterial action in the future. However, it has been found that highly immune serum slows down but does not block bacteriolytic activities of pneumococcal-specific endolysins (Loeffler et al. 2001). Another concern of endolysin treatment was its short circulation time after administration (Loeffler et al. 2001). But this issue did not affect the treatment because of the very rapid action time of the lysozyme molecules. Both in antibiotic and phage therapy, another major concern is the phenomenon of the increase of resistant pathogenic strains. Interestingly, every attempt to generate resistant mutants against endolysins in the laboratory setup has so far failed (Loeffler et al. 2001). Although this gives more credence to the potential of lysozyme therapy, we note that most antibiotic-resistance elements that have infiltrated into antibiotic therapy are because of bacteria-acquiring resistance elements from natural environments by lateral gene transfer.

Use of Lysozyme for Treating Bacterial Contamination in Plant Cell Cultures

Even egg white lysozyme has been successfully used to reduce Bacillus circulans and Sphingomonas paucimobilis infection of in vitro shoot cultures quince and hybrid rootstock (Marino et al. 2003). Although lysozyme did not have a negative effect on shoot growth, under optimal conditions it was effective in eliminating B. circulans in quince shoot and hybrid rootstock cultures, and has a bacteriostatic effect on S. paucimobilis. These results suggest that lysozyme may be able to replace antibiotic treatments of in vitro shoot cultures although it may not be effective against every plant bacterial infection.

Use of Bacteriophage Tail-Associated Lytic Enzymes as Antibacterial Therapeutic Agents

It has been known for a while that peptidoglycan-degrading murein hydrolases cleave bacterial cell walls very efficiently in a species-specific manner. Thus, they have the potential of being therapeutic agents against specific pathogens. Recently, this idea has been successfully exploited. These hydrolases are different from the phage-encoded endolysins, which are made and used by phage to come out of host cells by cell wall degradation from inside as discussed above. Unlike the endolysins the murein hydrolases are present at the tip of tails of phage virions. It is believed that the tail-attached hydrolases help DNA injection after phage adsorption. If a bacterial cell is infected by phage particles at very high multiplicities, then the cell lyses before phage DNA starts replication. It has been called “lysis from without” (Delbruck 1940). These tail hydrolases are more or less ubiquitous among tailed phages. Motivated by the emergence of drug-resistant human pathogen Staphylococcus aureus, Paul et al. (2011) identified the muralytic activity of the tail of the broad host range staphylococcal-specific phage, called phage K. These investigators showed the efficacy of the K-encoded purified hydrolase as a bacteriocidal agent in cell culture. The efficacy of this protein was further enhanced by making a hybrid protein comprising the catalytic domain of the hydrolase and the staphylococcal cell wall binding domain of a bacteriocin called lysostaphin (Baba and Schneewind 1998) to generate a protein called P128. The bacteriolytic activity of P128 was more than two orders of magnitude higher than the catalytic domain of the hydrolase alone because of increased substrate specificity. The hybrid has already been tested in experimental nasal colonization of methicillin-resistant S. aureus (MRSA) in experimental rats the protein has been shown extremely effective in decolonizing the animals. Thus it is an excellent prospective therapeutic against infection.


Step 3: Replication

Enzymes coded by the bacteriophage genome shut down the bacterium's macromolecular (protein, RNA, DNA) synthesis. The bacteriophage replicates its genome and uses the bacterium's metabolic machinery to synthesize bacteriophage enzymes and bacteriophage structural components (Figure (PageIndex<3>) and Figure (PageIndex<4>)).

Figure (PageIndex<4>): Late Replication during the Lytic Life Cycle of a Lytic Bacteriophage. The production of bacteriophage components and enzymes progresses.


4. Discussion

Since development of phage therapy against E. faecalis appears to be important to establish effective treatments of infections by antibiotic-resistant strains of this bacterium [1,2,3,4,5,6,7,8,9,10,11,12], and biofilm formation on catheters is one of the most difficult medical challenges caused by this species [13], we aimed to test efficiency of recently isolated and described bacteriophage vB_EfaS-271 [26] in killing E. faecalis cells included in biofilms formed on Foley silicone catheters. We found that vB_EfaS-271 is able to efficiently reduce number of viable bacterial cells under such conditions ( Figure 1 ), providing evidence for its possible use in medical practice to either prevent colonization of catheters by E. faecalis or eradication of this bacterium from already formed biofilms in these medical devices. Nevertheless, after 24-h incubation under experimental conditions, appearance of phage-resistant bacteria was found, particularly when high m.o.i. (10) was used.

When testing effects of bacteriophage vB_EfaS-271 on mammalian cells (BALB/c3T3 mouse fibroblasts), neither toxicity nor changes in cell morphology could be observed ( Figure 2 and Figure 3 ). This indicated that the investigated phage is safe for mammalian cells. However, efficiency of protection of mouse fibroblasts against E. faecalis by phages was higher at lower m.o.i., while appearance of phage-resistant bacteria was more rapid at higher m.o.i ( Figure 2 ). These results again suggested that selection of phage-resistant bacteria was more effective under conditions when one bacterial cell was infected by several virions rather than under conditions of rare infection events in cell population. Such a scenario was confirmed in experiments where appearance of phage-resistant E. faecalis was investigated in 24-h long experiments ( Figure 4 ).

We suggest that more efficient selection of vB_EfaS-271-resistant bacteria at high m.o.i. may result from population dynamics. Namely, according to the theory of evolution, mutations appear randomly in populations of any organisms under certain environmental conditions. Therefore, one should assume that vB_EfaS-271-resistant E. faecalis cells appear at certain frequency. However, it is likely that a phage-resistant mutant, although viable, is at least slightly deficient in one physiological processes relative to wild-type cell. Therefore, in the absence of bacteriophages, such mutants are outgrown by wild-type bacteria. However, in the presence of viruses that can efficiently infect and kill bacterial cells, phage-resistant mutant are selected and can propagate efficiently, contrary to bacteria susceptible to infection. If so, when E. faecalis culture is infected with vB_EfaS-271 at high m.o.i. (i.e., under conditions where each cell is supposed to be infected by phage), only previously formed phage-resistant mutants can survive, and then they propagate effectively as multiplicated phages cannot infect them. On the other hand, under low m.o.i. conditions, only a small fraction of bacterial cell population is infected by the phage. Therefore, pre-existing phage-resistant mutants, which are at the same time slightly defective in at least one other feature relative to wild-type cells, at outgrown by phage-sensitive, non-infected bacteria, at the beginning of the experiment. Only when bacteriophages multiplicate efficiently in more and more susceptible cells, and number of such cells became drastically limited, the phage-resistant mutant can efficiently compete with their wild-type counterparts and appear as predominant bacteria in the population. Such a hypothesis may be corroborated by results presented in Figure 4 C, where fraction of phage-resistant cells dropped after one passage without the presence of phage vB_EfaS-271, indicating that phage resistance is accompanied by weakness of at least one other physiological feature of the cell, making these mutants less competitive when the phage is absent in the culture. Therefore, any revertant mutants, with restored wild-type phenotype, would again effectively compete with phage-resistant mutants.

Although appearance of vB_EfaS-271-resistant mutants might suggest a limitation in the use of this virus in phage therapy, the impaired growth and lower competitiveness of such mutants implicates that combination of phage therapy with other anti-bacterial treatment(s) can still be effective. Moreover, such impaired competitiveness might suggest that vB_EfaS-271-resistant E. faecalis mutants could be eliminated by natural microbiome of patients.


Structure of Bacteriophage

Schematic representation of main types of phages
(Image source: Brock Biology of Microorganisms)

Bacteriophage structures are diverse, but most of them share some common characteristics. For example, bacteriophage T4 of Escherichia coli has an icosahedral head structure made of repeat protein sub-units known as the capsid. This head structure contains a linear double-stranded viral genome.

Structure of a Phage λ (lambda)

Phage genome varies in size from approximately 2 to 200 kilobases per strand of nucleic acid. Considerable variability is found in the nucleic acid of phages, and it may be ds DNA, ds RNA, ss DNA, ss RNA. Most known bacteriophages contain dsDNA genomes.

The head of bacteriophage T4 is attached to a helical tail through a collar (neck). Tails contain a series of tail fibers and tail pins at the end. These specialized syringe-like structures bind to receptors on the cell surface. All bacteriophages do not contain a ‘tail’ structure.


Discussion

The issue of bacteriophage interactions with the mammalian immune system and its components is still not precisely defined. The significance of such interactions may be crucial for the development of bacteriophage therapy, which is undisputed. The recruitment of patients who may undergo such therapy might become less restricted when all aspects of human–phage interactions are strictly known. However, the clinical applications of phages are not limited to phage therapy. Bacteriophages may also be beneficial in transplantation. They may inhibit the activation of allograft-induced T cells and the nuclear transcription factor NF-κB, which is believed to be strongly related to transplant tolerance [40]. Diminution of NF-κB activity by bacteriophages is an effect opposite to that caused by HSV-1 virus, which was proved to promote activation of this factor [1]. The effect of phage presence on graft infiltration (mononuclear cells and neutrophils) was also studied. Skin transplants removed and examined on days 1–3 post-transplant did not reveal any differences between the phage and control groups. However, on subsequent days the intensity of graft infiltration differed in both the analyzed groups. On days 7–8 post-transplant, the phages appeared to diminish the infiltration of mostly mononuclear cells, but also partially that of neutrophils. Reducing inflammatory infiltration may be extremely beneficial in preventing graft injury or its loss and may also result in allograft-induced T-cell activation [1].

The constant development of molecular biological techniques may lead to more significant application of bacteriophages than now. Bacteriophages, as objects of intense study, may have a great influence on human life in the future. The phage-display technique, which is based on genetic modifications of phages, allows the display of foreign proteins on their capsids. This method has been successfully used for the development of vaccines with phage capsids as platforms for antigens. Significantly, the creation of a vaccine is not limited by the protein’s size. It is possible to obtain phage particles displaying several kinds of antigens, as was shown by, among others, Sathaliyawala et al. [41] in 2006 for HIV p24, Nef and g41 proteins. These proteins were displayed simultaneously on T4 phages’ capsids deprived of Hoc protein. In vivo studies showed that such vaccines may elicit a strong humoral and cellular immune response [41–44]. Because the treatment of animals with these vaccines brought good results and considering the abundant advantages of this technique, it seems an interesting direction for further study. Another application of phage vaccines is anti-cancer immunotherapy. Identified tumour antigens are subsequently displayed on the phage capsid to induce an immunological response. Such a strategy was used, for example, with antigens of 4T1 breast adenocarcinoma. Peptides were displayed on T7 virions and orally applied to mice, inducing a specific immune response. As a result of this response, tumor growth and metastasis were inhibited [45]. The development of anticancer phage-based vaccines seems to be one of the most meaningful applications of phages. The good results of studies conducted so far suggest the necessity of their continuation.

Bacteriophages are also one of the best known genetic vectors. Increased interest in genetic methods may also result in augmented bacteriophage application in biotechnological branches connected with genetic modifications.

The developing techniques of molecular engineering may also soon bring the possibility of designing phages with particular properties and their usage in a directed way. In 2001, Di Giovine et al. [46] introduced a gene encoding a protein responsible for the adhesion and internalization of adenovirus into the bacteriophage M13 genome. Modified phages could bind integrin receptors on mammalian cells and penetrate and transduce them however, they could neither propagate nor induce cell lysis. It is possible that the inevitable future development of molecular biology will allow free manipulation of interactions between phages and organisms other than bacterial ones.

The studies involving phage–mammal interactions also include the influence of individual proteins on the biological activity of normal and cancer cells. Especially interesting from the immunological point of view seems to be the recently described Hoc protein of bacteriophage T4, which possesses an immunoglobulin-like domain. Since the immunoglobulin superfamily (IgSF) includes members of fundamental importance in the mammalian immune system, the similarity of its domains and Hoc protein domains may be significant [47]. Moreover, analyses show that many phage peptides more frequently show closer sequence similarity with eukaryotic than with prokaryotic homologs [48], which may be of great importance when considering phage–mammal interactions.

Although phage influence on the migration of mammalian cells seems to be extremely important, there are no reliable and abundant data related to this problem. The inhibition of melanoma cell migration and the resulting inhibition of tumor spread by phages discussed earlier are evidence that this phenomenon may be of great importance. The study of the influence of phages on the metastatic migration of melanoma cells has been continued in our laboratory. Recently, we have also undertaken studies on the influence of phages on the migration of human immune cells, i.e., granulocytes, mononuclear cells and also human leukemia tissue. The studies include the T-family phage preparations investigated previously and also preparations used in the treatment of staphylococcal and pseudomonal infections of patients of our therapy center. We have also studied the biological properties of T-family phage proteins, which in our opinion is extremely important and to our knowledge a pioneering direction of studies. It is important to mention that, because of virion propagation, phage preparations contain some amounts of bacterial endotoxins occurring after bacterial lysis. Complete endotoxin removal from a protein solution was previously described [49]. However, such procedures lead to marked phage loss and may therefore be of limited practical value. LPS is highly immunogenic and its effects on the immune system have to be considered along with the actions of phages. We have observed that bacteriophage T4 preincubated with granulocytes (1 h) caused a slight stimulation of cell migration compared with the PBS group. This effect was, however, stronger than that of LPS and the phages seemed to suppress the inhibiting effect of lipopolysaccharide. Bacteriophage HAP1 caused inhibition of granulocyte migration only slightly more weakly than LPS (data not shown). Considering the great importance of this issue and the sparse (if any) data related to the described interactions, we believe that this direction of study is indispensable.

Bacteriophages introduced into the mammalian organism may penetrate it quite freely. They are able to enter the bloodstream almost irrespective of the way of administration [50–55]. Recent studies showed that bacteriophages may pass the intestinal wall by exploiting gut immune cells (enterocytes, M cells and, particularly, dendritic cells) [56]. Interactions with dendritic cells and downregulation of their actions may be significant in preventing inflammation leading to gut injuries. The easy and direct contact with human/animal tissues implies the necessity of a narrow circumscription of all the possible interactions between introduced virions and mammalian cells. One of the most important areas of interest seems to be the immunological system, which is the first target of phage contact. The small amount of data on the interactions of bacteriophages with cells of the mammalian immune system, especially that of humans, in connection with increasing the significance of these viruses in contemporary medicine and biotechnology seems to be a problem. We believe that explaining all the aspects of such interactions and the influence of bacteriophages on the animal/human immune system is indispensable and that this direction of research is pivotal.


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This work was supported by the Basic Research Program of the Ministry of Science and Technology of China (2012CB721102), the Chinese Academy of Sciences (KJZD-EW-L02), the National Natural Science Foundation of China (31400126), and the Key Laboratory on Emerging Infectious Diseases and Biosafety, CAS.

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Keywords: lysin, chimeolysin, artilysin, bacteriophage, lysin engineering, enzybiotics

Citation: Yang H, Yu J and Wei H (2014) Engineered bacteriophage lysins as novel anti-infectives. Front. Microbiol. 5:542. doi: 10.3389/fmicb.2014.00542

Received: 23 August 2014 Accepted: 29 September 2014
Published online: 16 October 2014.

Marta Martins, University College Dublin, Ireland

Melinda J. Mayer, Institute of Food Research, UK
Taoufik Ghrairi, Faculty of Medicine Ibn El Jazzar of Sousse, Tunisia
Pedro Fernandes, Instituto Superior Tecnico - Universidade de Lisboa, Portugal

Copyright © 2014 Yang, Yu and Wei. 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.


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