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Novel strategies to prevent or exploit phages in fermentations, insights from phage–host interactions
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ScienceDirect Novel strategies to prevent or exploit phages in fermentations, insights from phage–host interactions Jennifer Mahony1 and Douwe van Sinderen1,2 Phages infecting lactic acid bacteria (LAB) provide some of the most advanced model systems for (tailed) phage–host interactions. In particular the identification of receptor molecules of representative lactococcal phages combined with the elucidation of the structure of the receptor-binding protein has permitted crucial insights into the early stages of infection.Dairy and biotechnological fermentations are persistently marred by the destructive activities of phages. Here, we discuss how recent advances in our knowledge on LAB phage–host interactions have provided a basis for the next generation anti-phage strategies. Furthermore, the significant increase in genomic data has furthered our understanding of the genetics of these phages, thereby permitting the exploitation of phage-derived components for food safety and biotechnological applications.
species are the most widely used starters in dairy fermentations [3]. Consequently, this review will focus on the lessons learned from and models developed for lactococcal strains and phages, highlighting advances made over the past three years in particular, that may be broadly applied to phage–host interactions in other genera with implications for a variety of food and biotechnological fermentations.
Phage–host interactions
Introduction
Lactococcal phages are currently classified into ten groups based on their morphology and genetic relatedness [3]. Of these, three species, 936, P335 and c2, dominate in terms of persistence and frequency of isolation [4–6]. Members of the 936 and c2 observe a lytic cycle only, while P335 members may propagate lytically or may integrate their genome into that of the host and replicate in tandem with the host’s chromosome, a scenario during which they are termed lysogenic or temperate phages. The proteinaceous receptor for c2 phages is the membrane-anchored phage infection protein (PIP) [7], a functional analogue of the YueB receptor of Bacillus subtilis phage SPP1 [8,9]. The PIP-encoding gene may be altered or deleted from the host without affecting the technological traits of the host, while simultaneously providing resistance against c2 phages [10]. Since selection of c2-resistant derivatives of lactococcal strains probably harbour such alterations/ deletions, the c2 phages appear to have become less prevalent in the dairy industry. Receptor-binding proteins (RBP) of the 936 and P335 phages, which are located at the distal end of the phage tail, frequently being part of a larger protein complex termed the base plate, and host-encoded receptors that these phages recognize have been investigated thoroughly over the past decade [11–13,14,15,16,17].
Bacterio(phages) are a consistent and major threat to food fermentations, particularly dairy fermentations where infection of starter cultures may result in slow vats, lowquality and inconsistent products, and even complete fermentation failure. Since lactococcal phages were first reported as the causative agents of starter failure by Whitehead and Cox in 1935 [1], efforts have been made to improve fermentation systems. These efforts have been epitomized by the development of bacteriophageinsensitive mutants (BIMs), the formulation of blends of phage-unrelated strains and, as a result of extensive research into phage–host interactions and phage-resistance mechanisms, a knowledge-based set of tools to combat phages [2]. Phages infecting Lactococcus lactis have dominated dairy phage research since strains of this
Most significantly and after many years of speculation, structural analysis of the RBP of the 936 species phage p2 produced the direct experimental proof of the saccharidic nature of the receptor recognized by these phages [18]. This publication was followed by structural analysis of the RBPs of P335 type phages TP901-1 and Tuc2009, which were also found to possess an affinity and avidity for saccharides [13,19]. These findings were complemented with biochemical analysis of a novel surface-exposed, cell envelope macromolecule, the so-called pellicle or cell wall polysaccharide (CWPS), of a number of lactococcal strains [14,16,17], and with the identification and comparative analysis of the corresponding CWPS biosynthetic operons [20]. The ultimate outcome from these studies
Addresses 1 School of Microbiology, University College Cork, Cork, Ireland 2 Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland Corresponding author: van Sinderen, Douwe ([email protected])
Current Opinion in Biotechnology 2015, 32:8–13 This review comes from a themed issue on Food biotechnology Edited by Michiel Kleerebezem and Christophe Lacroix
http://dx.doi.org/10.1016/j.copbio.2014.09.006 0958-1669/# 2014 Elsevier Ltd. All right reserved.
Current Opinion in Biotechnology 2015, 32:8–13
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Application and prevention of phages in fermentations Mahony and van Sinderen 9
was the identification of CWPS as the saccharidic receptor of members of the 936 and P335 phage species. The CWPS of L. lactis MG1363, which is host to the 936 species phages p2 and sk1, is composed of repeating units of a phospho-hexasaccharide [16], while that of L. lactis 3107, host to the P335 phages TP901-1 and phiLC3, is a phospho-pentasaccharide [17]. Most recently, the structure of the CWPS of L. lactis SMQ-388, host to the namesake of the rarely isolated 1358 lactococcal phage species was defined as repeating subunits of a phosphohexasaccharide [14] (Figure 1). While the CWPS of each of these strains contains unique elements, a core trisaccharide exists that probably represents (part of) the target for the saccharide-recognizing phages. The unique aspects of their CWPS biochemical structure explain the specificity and narrow host range of such CWPSbinding phages, which is in major contrast to the generalized PIP protein receptor of the c2 phages. These findings aid in distinguishing phages that recognize proteins from those that recognize saccharides, and such knowledge may be harnessed as a preventive measure if the target carbohydrate or protein is known (see below).
Fighting fire with fire: exploiting phages to prevent infection In order to develop next generation approaches to counter the phage problem, it is imperative to understand the molecular mechanics of the phage attachment and injection process. Historically, dairy fermentation media were adapted by employing phosphate-containing whey-based media (so-called phage inhibitory media) for bulk starter preparation to ‘mop up’ calcium and other divalent cations that were believed to be required for phage infection. Phages nevertheless remained problematic, which is now explained by the fact that not all lactococcal phages require divalent cations for infection [19]: members of the 936 species require calcium to rotate the conformation of their base plate to a cell-facing, infection-ready orientation, whereas many members of the
P335 species, including TP901-1, were observed to infect their hosts with equal efficiency in the presence or absence of calcium [19]. Consistent with this, the baseplate structure of TP901-1 was shown to be in a permanent ‘infection-ready’ conformation [19]. Interestingly, some members of the P335 species, such as Tuc2009 and Q33, have been observed to require calcium for efficient infection of their host [19]. It is clear that observations on the requirement for divalent cations by phages do not apply to all phages, therefore, limiting the effectiveness of the phage inhibitory media approach. In fact, as with the ‘hurdle approach’ applied in food production and preservation processes, it is probably that a combination of approaches should be employed to ensure limitation of phage problems. The advances made towards defining lactococcal phage– host interactions have facilitated various novel approaches to prevent or limit phage infection. For example, the distal tail region of the P335 species phages TP901-1 and Tuc2009 is observed as a so-called ‘doubledisc’ baseplate and the structure of the baseplate of both phages has been resolved [15,19]. In the case of TP9011, the baseplate is composed of multiple copies of two proteins that comprise the upper (BppU) and lower (BppL) base plate discs, while in the case of Tuc2009, the baseplate is composed of multiple copies of three proteins, namely BppU and BppL and an accessory protein, BppA. Incubation of a heterologously produced, purified phage baseplate complex with cells of the corresponding host (i.e. the base plate is derived from a phage that infects such a host), effectively coating such cells with base plate, specifically inhibits phage adsorption in a dose-dependent manner. In contrast, this complex has no effect on phage adsorption when incubated with noncorresponding (i.e. the base plate is derived from a phage that does not infect the host) host cells (Figure 2) [15]. While the base plate complex of any one phage is unlikely to exclude all phages from attaching and infecting and is, therefore, perhaps not an all-round practical solution for
Figure 1
[-6-α-G1cNAc- 3-β-Ga1f-3-β-G1cNAc- 2-β-Ga1f- 6-α-G1cNAc-P - ] 6
SMQ388 (1358 host)
α-G1c [-6-α-G1c- 3-β-Ga1f- 3-β-G1cNAc- 2-β-Ga1f- 6-α-G1cNAc-P - ]
3107 (TP901-1 host)
[-6-α-G1cNAc-3-α-Rha- 3-β-G1cNAc- 2-β-Ga1f- 6-α-G1c-P - ] 6
MG1363 (p2 host)
α-G1c Current Opinion in Biotechnology
Schematic representing the biochemical structure of the CWPS repeating subunit of three lactococcal strains: 3107, MG1363 and SMQ-388. Each of these strains represents a host for at least one phage of three distinct species, that is, the P335, 936 and 1358, respectively. www.sciencedirect.com
Current Opinion in Biotechnology 2015, 32:8–13
10 Food biotechnology
Figure 2
(a)
Competitive exclusion
(b)
Phage infection
Current Opinion in Biotechnology
Schematic outlining the competitive exclusion of phages by supplying the base plate complex of a phage that targets a corresponding host (a). This is depicted by the corresponding colour of the outer surface of the cell and phage and its base plate (green). In this scenario, the homologous phage cannot attach to its host as it is out-competed by the base plate complex. In contrast, when the base plate complex of the same phage (green) is incubated with cells of a non-corresponding (purple) host strain (b) (i.e. a different phage–host system) in competition with a phage that typically infects that strain (purple), phage attachment infection is observed since the base plate complex has a specific cell surface target that is not found on this host. This is depicted by the noncorresponding colours of the cell surface/intact phage (purple) and base plate complex (green) and highlights the specificity of the base plate complexes for their recognized target on the cell surface (CWPS).
the dairy industry, such competitive behaviour between the base plate complex and intact phages highlights how phages and their components may be exploited in efforts to control phages and represents a new generation of molecular competitors and phage inactivators. Designed ankyrin repeat proteins (DARPins), which are proteins that specifically bind to a specified target, and neutralizing llama heavy chain antibody variable domains (VHH) directed against the head (interacting) domain of the RBP of phages have been found to be effective inhibitors of phages for both p2 (936 phage) and TP901-1 (P335 phage) [21–23]. Indeed, particular antiRBP VHHs were observed to completely prevent TP9011 infection up to at least 15 days of continuous passaging, thus highlighting the efficacy and robustness of this approach in preventing phage infection [23]. However, it is naı¨ve to presume that any phage-resistance system or Current Opinion in Biotechnology 2015, 32:8–13
physical/chemical/biological treatment is impervious as phages have proven their evolutionary elasticity in overcoming numerous treatments and systems. The only true armor is a continuous production of knowledge-based anti-phage approaches combined with constant phage monitoring to ensure that breaches by phages can be detected early enough to allow time for remedial actions. While it may be predicted that the vast majority of problematic lactococcal phages in the dairy industry belong to either the 936 or P335 species, it is essential to understand the exact phage population and population dynamics of a fermentation setting such that rational solutions may be developed to address the specific phages that dominate in a given fermentation plant. A recent study examining correlations between 936 phage RBP sequences to the lactococcal host CWPS-encoding operon genotype is a pertinent example of this [20]. The analysis identified three lactococcal CWPS-types (A, B and C) and three phage RBP phylogenetic groups (I, II and III) (while further diversity of both CWPS operons has since been defined [24] and is also anticipated for the phages) [20]. RBP groups I and II were observed to have a preference for either CWPS type C or B, respectively, while the third group was capable of infecting strains belonging to members of both CWPS-types B and C. This ground-breaking work underscores the necessity to define the CWPS/RBP nature of host strains and phages present in the fermentation in order to design a starter rotation scheme that reduces the risk of repeated phage infection problems. It also presents a rapid method of strain classification that negates labour-intensive and time-consuming techniques to assess a collection of strains and provides a ‘risk assessment’ of phage-susceptibility in any given starter producing company or fermentation facility.
Anti-phage strategies Prevention of phage infection has been a dominant theme in dairy phage research for many decades and through such focused attention, a plethora of host and prophageencoded anti-phage strategies have been identified and engineered, including those that inhibit adsorption and block DNA injection [25–27], as well as restriction and modification (R/M) [28–30], abortive infection (Abi) [31,32] and clustered regularly interspaced short palindromic repeats (CRISPR) systems [33–36]. While significant advances have been made with respect to understanding and exploitation of each of these antiphage systems, it is the CRISPR systems and the adaptations of phages to overcome these systems that have in recent years captured scientific headlines [33,34,36–41]. While only one report exists of a (plasmid-encoded) CRISPR system in L. lactis [42], stacking of chromosomal CRISPR systems of different types has been observed for Streptococcus thermophilus and Lactobacillus spp. [37,43,44]. In technological terms, an advantage of the CRISPR system is the relative ease with which BIMs are generated. The potential to engineer L. lactis so as to add www.sciencedirect.com
Application and prevention of phages in fermentations Mahony and van Sinderen 11
CRISPR to its already diverse and impressive battery of anti-phage systems, would certainly enhance the ability of the dairy industry to rapidly respond to phage problems.
Exploiting phages in biotechnological fermentations Phage components have been exploited for many years in biotechnology fermentations and the use of phage promoters (e.g. coliphage T7 promoter in the pET range of vectors) have been employed and optimized to accommodate the expression of a plethora of proteins for the treatment of human illnesses ranging from arthritis to cancer [45,46]. Furthermore, phages and phage-derived lytic enzymes are now presenting novel and natural alternatives to more traditional chemical and thermal biocidal treatments of numerous nuisance bacterial spoilage problems in food/beverage and biotechnological fermentations. For example, some genera of the lactic acid bacteria (LAB) are spoilers and contaminants of fuel ethanol fermentations and alcoholic beverage fermentations. Lytic bacteriophages have been identified against many genera of the LAB, representing an opportunity for their exploitation as remedial agents in the food and biotech sectors. Phage SA-C12 was recently proven to specifically target hop-resistant strains of Lactobacillus brevis, a known beer spoilage organism and in small-scale food trials, extended the shelf-life of the alcoholic beverage [47]. Furthermore, streptococcal and Lactobacillus phage-derived lytic enzymes (endolysins) demonstrated lytic properties against species of Streptococcus, Lactobacillus and Staphylococcus that were isolated from fuel ethanol fermentations [48]. For focused reviews on phage therapy see [49,50]. These examples illustrate that the potential of phages and phage-derived components towards advancing antimicrobial therapies in food and in biotechnological fermentations reaches far beyond their current application range. While phage therapy is not a new concept, perhaps new ways of addressing old problems are being established based on the principles of phage therapy which is not without its challenges. For example, the Listeria monocytogenes phage P100 was applied in food trials in an effort to gauge the anti-listerial effects of the phage in soft cheeses. This resulted in a 2log to 3-log reduction in listerial numbers upon addition of phages, though viable numbers recovered during cold storage over a seven-day period [24]. This article outlined the importance of the bacterial load, phage numbers added and the storage conditions although the physical nature of the food matrix can also not be overlooked. Furthermore, phage-derived lytic enzymes may be a more promising alternative and should be assessed for their potential in such fermented food preservation trials.
Conclusions While on first inspection, phages appear to be the foe, certainly in the case of food fermentations, it is becoming www.sciencedirect.com
increasingly obvious that phages and their various encoded products may be harnessed to benefit food and biotechnological fermentations. The significant advances in phage–host interactions have permitted a first glimpse of some of the promising phage prevention methods of the future and the lessons learned in the study of lactococcal phage–host interactions serve as a springboard for other Gram-positive bacteria in particular. It seems that we are entering a golden era of knowledgebased solutions and we are enhancing the insights of our forerunners through the technological advances in genomics, molecular and structural biology. There is still much to be derived from this emerging knowledge and while regulatory bodies may currently struggle with the justification of phage therapeutics, it seems inevitable that phages and their derived components will become increasingly important as alternatives to currently employed biocidal treatments which in many cases have been subject to hyperbole or are of limited effectiveness.
Conflicts of interest The authors declare that there are no conflicts of interest associated with this manuscript.
Acknowledgement DvS is supported by a Science Foundation Ireland Principal Investigator award (Ref. No. 13/IA/1953).
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Current Opinion in Biotechnology 2015, 32:8–13
ScienceDirect Novel strategies to prevent or exploit phages in fermentations, insights from phage–host interactions Jennifer Mahony1 and Douwe van Sinderen1,2 Phages infecting lactic acid bacteria (LAB) provide some of the most advanced model systems for (tailed) phage–host interactions. In particular the identification of receptor molecules of representative lactococcal phages combined with the elucidation of the structure of the receptor-binding protein has permitted crucial insights into the early stages of infection.Dairy and biotechnological fermentations are persistently marred by the destructive activities of phages. Here, we discuss how recent advances in our knowledge on LAB phage–host interactions have provided a basis for the next generation anti-phage strategies. Furthermore, the significant increase in genomic data has furthered our understanding of the genetics of these phages, thereby permitting the exploitation of phage-derived components for food safety and biotechnological applications.
species are the most widely used starters in dairy fermentations [3]. Consequently, this review will focus on the lessons learned from and models developed for lactococcal strains and phages, highlighting advances made over the past three years in particular, that may be broadly applied to phage–host interactions in other genera with implications for a variety of food and biotechnological fermentations.
Phage–host interactions
Introduction
Lactococcal phages are currently classified into ten groups based on their morphology and genetic relatedness [3]. Of these, three species, 936, P335 and c2, dominate in terms of persistence and frequency of isolation [4–6]. Members of the 936 and c2 observe a lytic cycle only, while P335 members may propagate lytically or may integrate their genome into that of the host and replicate in tandem with the host’s chromosome, a scenario during which they are termed lysogenic or temperate phages. The proteinaceous receptor for c2 phages is the membrane-anchored phage infection protein (PIP) [7], a functional analogue of the YueB receptor of Bacillus subtilis phage SPP1 [8,9]. The PIP-encoding gene may be altered or deleted from the host without affecting the technological traits of the host, while simultaneously providing resistance against c2 phages [10]. Since selection of c2-resistant derivatives of lactococcal strains probably harbour such alterations/ deletions, the c2 phages appear to have become less prevalent in the dairy industry. Receptor-binding proteins (RBP) of the 936 and P335 phages, which are located at the distal end of the phage tail, frequently being part of a larger protein complex termed the base plate, and host-encoded receptors that these phages recognize have been investigated thoroughly over the past decade [11–13,14,15,16,17].
Bacterio(phages) are a consistent and major threat to food fermentations, particularly dairy fermentations where infection of starter cultures may result in slow vats, lowquality and inconsistent products, and even complete fermentation failure. Since lactococcal phages were first reported as the causative agents of starter failure by Whitehead and Cox in 1935 [1], efforts have been made to improve fermentation systems. These efforts have been epitomized by the development of bacteriophageinsensitive mutants (BIMs), the formulation of blends of phage-unrelated strains and, as a result of extensive research into phage–host interactions and phage-resistance mechanisms, a knowledge-based set of tools to combat phages [2]. Phages infecting Lactococcus lactis have dominated dairy phage research since strains of this
Most significantly and after many years of speculation, structural analysis of the RBP of the 936 species phage p2 produced the direct experimental proof of the saccharidic nature of the receptor recognized by these phages [18]. This publication was followed by structural analysis of the RBPs of P335 type phages TP901-1 and Tuc2009, which were also found to possess an affinity and avidity for saccharides [13,19]. These findings were complemented with biochemical analysis of a novel surface-exposed, cell envelope macromolecule, the so-called pellicle or cell wall polysaccharide (CWPS), of a number of lactococcal strains [14,16,17], and with the identification and comparative analysis of the corresponding CWPS biosynthetic operons [20]. The ultimate outcome from these studies
Addresses 1 School of Microbiology, University College Cork, Cork, Ireland 2 Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland Corresponding author: van Sinderen, Douwe ([email protected])
Current Opinion in Biotechnology 2015, 32:8–13 This review comes from a themed issue on Food biotechnology Edited by Michiel Kleerebezem and Christophe Lacroix
http://dx.doi.org/10.1016/j.copbio.2014.09.006 0958-1669/# 2014 Elsevier Ltd. All right reserved.
Current Opinion in Biotechnology 2015, 32:8–13
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Application and prevention of phages in fermentations Mahony and van Sinderen 9
was the identification of CWPS as the saccharidic receptor of members of the 936 and P335 phage species. The CWPS of L. lactis MG1363, which is host to the 936 species phages p2 and sk1, is composed of repeating units of a phospho-hexasaccharide [16], while that of L. lactis 3107, host to the P335 phages TP901-1 and phiLC3, is a phospho-pentasaccharide [17]. Most recently, the structure of the CWPS of L. lactis SMQ-388, host to the namesake of the rarely isolated 1358 lactococcal phage species was defined as repeating subunits of a phosphohexasaccharide [14] (Figure 1). While the CWPS of each of these strains contains unique elements, a core trisaccharide exists that probably represents (part of) the target for the saccharide-recognizing phages. The unique aspects of their CWPS biochemical structure explain the specificity and narrow host range of such CWPSbinding phages, which is in major contrast to the generalized PIP protein receptor of the c2 phages. These findings aid in distinguishing phages that recognize proteins from those that recognize saccharides, and such knowledge may be harnessed as a preventive measure if the target carbohydrate or protein is known (see below).
Fighting fire with fire: exploiting phages to prevent infection In order to develop next generation approaches to counter the phage problem, it is imperative to understand the molecular mechanics of the phage attachment and injection process. Historically, dairy fermentation media were adapted by employing phosphate-containing whey-based media (so-called phage inhibitory media) for bulk starter preparation to ‘mop up’ calcium and other divalent cations that were believed to be required for phage infection. Phages nevertheless remained problematic, which is now explained by the fact that not all lactococcal phages require divalent cations for infection [19]: members of the 936 species require calcium to rotate the conformation of their base plate to a cell-facing, infection-ready orientation, whereas many members of the
P335 species, including TP901-1, were observed to infect their hosts with equal efficiency in the presence or absence of calcium [19]. Consistent with this, the baseplate structure of TP901-1 was shown to be in a permanent ‘infection-ready’ conformation [19]. Interestingly, some members of the P335 species, such as Tuc2009 and Q33, have been observed to require calcium for efficient infection of their host [19]. It is clear that observations on the requirement for divalent cations by phages do not apply to all phages, therefore, limiting the effectiveness of the phage inhibitory media approach. In fact, as with the ‘hurdle approach’ applied in food production and preservation processes, it is probably that a combination of approaches should be employed to ensure limitation of phage problems. The advances made towards defining lactococcal phage– host interactions have facilitated various novel approaches to prevent or limit phage infection. For example, the distal tail region of the P335 species phages TP901-1 and Tuc2009 is observed as a so-called ‘doubledisc’ baseplate and the structure of the baseplate of both phages has been resolved [15,19]. In the case of TP9011, the baseplate is composed of multiple copies of two proteins that comprise the upper (BppU) and lower (BppL) base plate discs, while in the case of Tuc2009, the baseplate is composed of multiple copies of three proteins, namely BppU and BppL and an accessory protein, BppA. Incubation of a heterologously produced, purified phage baseplate complex with cells of the corresponding host (i.e. the base plate is derived from a phage that infects such a host), effectively coating such cells with base plate, specifically inhibits phage adsorption in a dose-dependent manner. In contrast, this complex has no effect on phage adsorption when incubated with noncorresponding (i.e. the base plate is derived from a phage that does not infect the host) host cells (Figure 2) [15]. While the base plate complex of any one phage is unlikely to exclude all phages from attaching and infecting and is, therefore, perhaps not an all-round practical solution for
Figure 1
[-6-α-G1cNAc- 3-β-Ga1f-3-β-G1cNAc- 2-β-Ga1f- 6-α-G1cNAc-P - ] 6
SMQ388 (1358 host)
α-G1c [-6-α-G1c- 3-β-Ga1f- 3-β-G1cNAc- 2-β-Ga1f- 6-α-G1cNAc-P - ]
3107 (TP901-1 host)
[-6-α-G1cNAc-3-α-Rha- 3-β-G1cNAc- 2-β-Ga1f- 6-α-G1c-P - ] 6
MG1363 (p2 host)
α-G1c Current Opinion in Biotechnology
Schematic representing the biochemical structure of the CWPS repeating subunit of three lactococcal strains: 3107, MG1363 and SMQ-388. Each of these strains represents a host for at least one phage of three distinct species, that is, the P335, 936 and 1358, respectively. www.sciencedirect.com
Current Opinion in Biotechnology 2015, 32:8–13
10 Food biotechnology
Figure 2
(a)
Competitive exclusion
(b)
Phage infection
Current Opinion in Biotechnology
Schematic outlining the competitive exclusion of phages by supplying the base plate complex of a phage that targets a corresponding host (a). This is depicted by the corresponding colour of the outer surface of the cell and phage and its base plate (green). In this scenario, the homologous phage cannot attach to its host as it is out-competed by the base plate complex. In contrast, when the base plate complex of the same phage (green) is incubated with cells of a non-corresponding (purple) host strain (b) (i.e. a different phage–host system) in competition with a phage that typically infects that strain (purple), phage attachment infection is observed since the base plate complex has a specific cell surface target that is not found on this host. This is depicted by the noncorresponding colours of the cell surface/intact phage (purple) and base plate complex (green) and highlights the specificity of the base plate complexes for their recognized target on the cell surface (CWPS).
the dairy industry, such competitive behaviour between the base plate complex and intact phages highlights how phages and their components may be exploited in efforts to control phages and represents a new generation of molecular competitors and phage inactivators. Designed ankyrin repeat proteins (DARPins), which are proteins that specifically bind to a specified target, and neutralizing llama heavy chain antibody variable domains (VHH) directed against the head (interacting) domain of the RBP of phages have been found to be effective inhibitors of phages for both p2 (936 phage) and TP901-1 (P335 phage) [21–23]. Indeed, particular antiRBP VHHs were observed to completely prevent TP9011 infection up to at least 15 days of continuous passaging, thus highlighting the efficacy and robustness of this approach in preventing phage infection [23]. However, it is naı¨ve to presume that any phage-resistance system or Current Opinion in Biotechnology 2015, 32:8–13
physical/chemical/biological treatment is impervious as phages have proven their evolutionary elasticity in overcoming numerous treatments and systems. The only true armor is a continuous production of knowledge-based anti-phage approaches combined with constant phage monitoring to ensure that breaches by phages can be detected early enough to allow time for remedial actions. While it may be predicted that the vast majority of problematic lactococcal phages in the dairy industry belong to either the 936 or P335 species, it is essential to understand the exact phage population and population dynamics of a fermentation setting such that rational solutions may be developed to address the specific phages that dominate in a given fermentation plant. A recent study examining correlations between 936 phage RBP sequences to the lactococcal host CWPS-encoding operon genotype is a pertinent example of this [20]. The analysis identified three lactococcal CWPS-types (A, B and C) and three phage RBP phylogenetic groups (I, II and III) (while further diversity of both CWPS operons has since been defined [24] and is also anticipated for the phages) [20]. RBP groups I and II were observed to have a preference for either CWPS type C or B, respectively, while the third group was capable of infecting strains belonging to members of both CWPS-types B and C. This ground-breaking work underscores the necessity to define the CWPS/RBP nature of host strains and phages present in the fermentation in order to design a starter rotation scheme that reduces the risk of repeated phage infection problems. It also presents a rapid method of strain classification that negates labour-intensive and time-consuming techniques to assess a collection of strains and provides a ‘risk assessment’ of phage-susceptibility in any given starter producing company or fermentation facility.
Anti-phage strategies Prevention of phage infection has been a dominant theme in dairy phage research for many decades and through such focused attention, a plethora of host and prophageencoded anti-phage strategies have been identified and engineered, including those that inhibit adsorption and block DNA injection [25–27], as well as restriction and modification (R/M) [28–30], abortive infection (Abi) [31,32] and clustered regularly interspaced short palindromic repeats (CRISPR) systems [33–36]. While significant advances have been made with respect to understanding and exploitation of each of these antiphage systems, it is the CRISPR systems and the adaptations of phages to overcome these systems that have in recent years captured scientific headlines [33,34,36–41]. While only one report exists of a (plasmid-encoded) CRISPR system in L. lactis [42], stacking of chromosomal CRISPR systems of different types has been observed for Streptococcus thermophilus and Lactobacillus spp. [37,43,44]. In technological terms, an advantage of the CRISPR system is the relative ease with which BIMs are generated. The potential to engineer L. lactis so as to add www.sciencedirect.com
Application and prevention of phages in fermentations Mahony and van Sinderen 11
CRISPR to its already diverse and impressive battery of anti-phage systems, would certainly enhance the ability of the dairy industry to rapidly respond to phage problems.
Exploiting phages in biotechnological fermentations Phage components have been exploited for many years in biotechnology fermentations and the use of phage promoters (e.g. coliphage T7 promoter in the pET range of vectors) have been employed and optimized to accommodate the expression of a plethora of proteins for the treatment of human illnesses ranging from arthritis to cancer [45,46]. Furthermore, phages and phage-derived lytic enzymes are now presenting novel and natural alternatives to more traditional chemical and thermal biocidal treatments of numerous nuisance bacterial spoilage problems in food/beverage and biotechnological fermentations. For example, some genera of the lactic acid bacteria (LAB) are spoilers and contaminants of fuel ethanol fermentations and alcoholic beverage fermentations. Lytic bacteriophages have been identified against many genera of the LAB, representing an opportunity for their exploitation as remedial agents in the food and biotech sectors. Phage SA-C12 was recently proven to specifically target hop-resistant strains of Lactobacillus brevis, a known beer spoilage organism and in small-scale food trials, extended the shelf-life of the alcoholic beverage [47]. Furthermore, streptococcal and Lactobacillus phage-derived lytic enzymes (endolysins) demonstrated lytic properties against species of Streptococcus, Lactobacillus and Staphylococcus that were isolated from fuel ethanol fermentations [48]. For focused reviews on phage therapy see [49,50]. These examples illustrate that the potential of phages and phage-derived components towards advancing antimicrobial therapies in food and in biotechnological fermentations reaches far beyond their current application range. While phage therapy is not a new concept, perhaps new ways of addressing old problems are being established based on the principles of phage therapy which is not without its challenges. For example, the Listeria monocytogenes phage P100 was applied in food trials in an effort to gauge the anti-listerial effects of the phage in soft cheeses. This resulted in a 2log to 3-log reduction in listerial numbers upon addition of phages, though viable numbers recovered during cold storage over a seven-day period [24]. This article outlined the importance of the bacterial load, phage numbers added and the storage conditions although the physical nature of the food matrix can also not be overlooked. Furthermore, phage-derived lytic enzymes may be a more promising alternative and should be assessed for their potential in such fermented food preservation trials.
Conclusions While on first inspection, phages appear to be the foe, certainly in the case of food fermentations, it is becoming www.sciencedirect.com
increasingly obvious that phages and their various encoded products may be harnessed to benefit food and biotechnological fermentations. The significant advances in phage–host interactions have permitted a first glimpse of some of the promising phage prevention methods of the future and the lessons learned in the study of lactococcal phage–host interactions serve as a springboard for other Gram-positive bacteria in particular. It seems that we are entering a golden era of knowledgebased solutions and we are enhancing the insights of our forerunners through the technological advances in genomics, molecular and structural biology. There is still much to be derived from this emerging knowledge and while regulatory bodies may currently struggle with the justification of phage therapeutics, it seems inevitable that phages and their derived components will become increasingly important as alternatives to currently employed biocidal treatments which in many cases have been subject to hyperbole or are of limited effectiveness.
Conflicts of interest The authors declare that there are no conflicts of interest associated with this manuscript.
Acknowledgement DvS is supported by a Science Foundation Ireland Principal Investigator award (Ref. No. 13/IA/1953).
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