Novel Strategies for the Delivery of Antimicrobials into Bacterial Cells
A Review
Alva Jay Smith B1009075 Supervisor: Jem Stach Word Count: 7520
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Abstract
The rise of antibiotic resistance has prompted a tremendous amount of research into the identification
and description of antimicrobial compounds. Through in depth study of the literature describing these
breakthroughs, I have identified that whilst many new, important antimicrobials (and the
development of techniques to discover them) have been outlined, these and indeed existing
antimicrobials with proven methods of inhibition lack entry into the bacterial cell cytoplasm. The
cytoplasmic targets of a plethora of antimicrobials are well known but the bacterial cell wall of Gram-
negative bacteria is proving problematic to overcome for a variety of reasons. Delivery of
antimicrobials is the next step in tackling dangerous pathogens but finding efficient ‘vehicles’ to aid in
the transport of antimicrobials across the bacterial cell membrane is disproportionate to the number
of active compounds discovered thus far which cannot access their targets in the cytoplasm. This
review hopes to explain why antibiotic drugs are difficult to deliver to the resistant Gram-negative
bacterial cytoplasm and describe major advances in the delivery of antimicrobials such as membrane
active peptides, hybrid compounds and phage and pyocin therapy, all in great detail.
Introduction
Gram-negative bacteria are the primary cause of most deadly nosocomial and (ever increasing)
community acquired (CA) bacterial infections. Media attention focused on methicillin resistant
Staphylococcus aureus in recent years has brought resistant bacteria to the forefront of public
awareness but has not yet identified the most resistant and dangerous pathogens. Gram-negative
bacteria especially those of the Enterobacteriaceae family cause urinary tract infections (UTIs), blood
poisoning, healthcare associated pneumonias and various incurable intra-abdominal infections
originating from the gut. Gram-negative pathogens which are becoming increasingly resistant to
modern antibiotics and are clinically important, include Escherichia coli, Clostridium spp, Pseudomonas
spp. and Acinetobacter spp.
Many antibiotics and antibacterial compounds have potent intercellular action; they act by inhibiting
key processes: peptidoglycan synthesis, DNA replication, cell division and protein synthesis
(translation) (Barna and Williams 1984; Chopra and Roberts 2001; Waksman and Lechevalier 1962).
However, most of these compounds have difficulty crossing the bacterial cell wall, particularly in
Gram-negative bacteria. Therefore, in this review I will explore novel methods and strategies for the
delivery of effective antimicrobial compounds into the most resistant of bacteria, outlining current
strategies and suggesting antimicrobial therapies based on emerging technologies from recent
literature published in scientific journals and older studies which I believe to be vital in discovering
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novel strategies for the delivery of antimicrobials into bacterial cells. Many of these techniques have
huge scope to be used in combination with active antimicrobials which lack entry to the bacterial
cytoplasm and should be taken into consideration when designing new anti-infective therapies.
Gram-negative bacteria have a plethora of antibiotic resistance mechanisms
In the wake of prolonged overuse of antimicrobials we are now becoming increasingly exposed to
environments which harbour lethal multi-drug resistant bacteria. In his review Resistance in Gram-
Negative Bacteria: Enterobacteriaceae (2006), Paterson describes antibiotic inhibition with emphasis
on chromosomally and plasmid mediated extended spectrum beta-lactamases (ESBLs) which inhibit
B-lactam antibiotics through hydrolysis – although beta-lactamase inhibitors can be used in
combination therapies with antibiotics, resistant varieties over produce beta-lactamases and may or
may not have mutated less porins for transport of antibiotics. AmpC beta-lactamases
(cephalosporinases) provide another important enzymatic defence mechanism (Jacoby 2009) and
coupled with organisms that reduce influx or enhance efflux (Webber and Piddock 2003), these
mechanisms present antibiotics with an armoury of defences. The top eight death causing
microorganisms, the antibiotics/antifungals which combat them and their various methods of
resistance can be found illustrated in figure 1.
The biggest obstacle facing the antimicrobial resistance research community is the outer membrane
(OM) of Gram-negative bacteria. This selective membrane utilises numerous beta-barrel structure
motif proteins and general diffusion porins which contain an inwardly folded extracellular loop, which
together with the opposite barrel wall, form the so called eyelet or constriction zone, determining the
size exclusion limit and other permeation properties of the barrel (Delcour 2009). These mechanisms
encompass a narrow size exclusion limit making it difficult for large peptide antimicrobials to
overcome the Gram-negative bacterial OM. It can be assumed that it is the OM rather than the
cytoplasmic membrane in Gram-negative bacteria that provides antibiotic exclusion as Lazzaroni and
Portalier’s study (1981) into E.coli cells with a leaky periplasmic (OM) membrane discovered that E.
coli mutants which leaked a large amount of periplasmic enzymes were also particularly sensitive to
peptidoglycan synthesis inhibiting antibiotics such as carbapenems and ampicillin usually reserved for
Gram-positive bacteria. Other antimicrobials like mitomycin C (DNA cross-linker), rifamycin (RNA
synthesis inhibitor) and chloramphenicol (protein synthesis inhibitor) also demonstrated action
against leaky periplasmic E. coli. This evidence provides researchers with an empyrean objective: how
to overcome the OM and access Gram-negative cell cytoplasm to enable active compounds to inhibit
cellular processes?
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Figure 1. Diagrammatic representation of the top eight death causing micro-organisms in the USA
(2012). Numbers to the left of the false colour electron microscopy (FCEM) images represent the
number of deaths caused in the year 2012. Species names can be seen above the images in either
purple (Gram-positive micro-organisms) or red (Gram-negative micro-organisms). Text to the right of
images lists antibiotics, past and present, known to combat infections caused by the related
bacterium/fungi - (*) indicates emerging or established resistance to that particular antibiotic. Text
below the illustrations describes known resistance mechanisms.
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Antimicrobial drug discovery
Modern research has given birth to efficient screening techniques, allowing large pharmaceutical
establishments and small research teams to search for new antimicrobial compounds in a streamlined
fashion. Although resistance to antibacterial compounds is becoming more common, it is believed
that most antimicrobial compounds are yet to be discovered. The potential antimicrobial compounds
derived from bacteriophage genomics alone amount to an almost incomprehensible number - if every
species of bacteria on the planet hosts one or more bacteriophage pathogens, it can be assumed that
novel antibacterial compounds lie undiscovered within the genomes of such pathogens, owing to their
mechanism of bacterial killing. In their renowned article published in Nature Biotechnology, Lui et al.
(2004) describe applying the concept of phage-mediated bacterial growth inhibition to antibiotic
discovery. The team identified 31 novel polypeptide families that inhibit bacterial growth when
expressed in S. aureus. One peptide in particular (77ORF104), of which the cellular target is DnaI (an
essential protein). Apart from utilising phage genomes to determine antibiotic compounds, one can
reduce the amount of target protein in a bacterium via RNAi (antisense RNA inhibition) making it more
sensitive to inhibition, thus aiding in the discovery of low abundance compounds from natural product
libraries. There is a large pool of literature surrounding this phenomenon including the Forsyth et al.
study (2002) and Goh et al (2009).
Natural products are an important weapon in the fight against bacteria, as they tend to have
polypharmological activities in addition to being ‘tailor made’ by evolution to overcome resistance. In
contrast, compounds that are made by design (semi-synthetic) give rise to resistant strains of bacteria
owing to the fact that they regularly only inhibit one target – this means that a point mutation in the
target protein is commonly all that is needed to render the compound obsolete. In their science article,
Haydon et al. (2008) describe an FtsZ (bacterial cell division protein) inhibitor with potent action
against S. aureus. However, due to the high occurrence of resistance, this compound must be
administered in combination therapies to avoid whole population resistance.
There are many bacterial targets available for inhibition by antimicrobial compounds which can now
be more easily discovered owing to RNAi and other antibiotic discovery techniques. However, Gram-
negative pathogens still present many conserved resistance mechanisms to antimicrobial compounds
which inhibit novel targets. For example, platensimycin – a FabF inhibitor, which shows potent, broad
spectrum Gram-positive activity in vitro and exhibits no cross-resistance to other key antibiotic
resistant bacteria including MRSA and vancomycin-intermediate Staphylococcus aureus, shows no
antibacterial activity against wild type Escherichia coli. Interestingly, platensimycin does exhibit
antibacterial activity against efflux-negative (tolC) E. coli indicating that efflux mechanisms and not
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compound specificity limit the effectiveness of platensimycin in E. coli and possibly other Gram-
negative bacteria. Other antibiotic discovery methods include genome mining (Lautru et al. 2005),
transcriptional profiling of conditional mutants (Freiberg et al. 2005) and fusion/hybrid compounds
which will be described in more detail, later in this review.
Membrane active peptides
As previously mentioned, the outer membrane of Gram-negative bacteria provides an effective,
inherent mechanism for the exclusion of large antimicrobial peptides and other antibacterial agents.
Overcoming this barrier is essential for any compound to provide antimicrobial action and has been
the target of a plethora of recent research (Savage 2001; Schwechheimer et al 2013). Derived from
the peptides of multicellular organisms, membrane active peptides show the most promise in
overcoming the Gram-negative bacterial cell wall and beg the question, could we design anti-infective
drugs based on their properties?
The mechanism of the killing action of membrane active peptides is not understood in great detail but
three models for their activity have been proposed. (1) The Barrel-Stave model (Oren and Shai 1999)
where peptides attach to the membrane via electrostatic interaction then adopt an α-helical
conformation and self-assemble into bundles. The bundles then insert into the membrane and form
pores by placing their hydrophobic part in contact with the hydrophobic portion of the bacterial
membrane. (2) The Toroidal-Pore model which is similar to the barrel-stave model except α-helical
peptides keep their hydrophilic part in contact with the hydrophilic head groups of the lipid membrane
and bend the membrane to form pores. (3) The Carpet model (Brogden 2005). At first, the membrane
has a curvature of zero and the peptides bind preferentially to the lipid head groups. The peptides
then reorientate/realign to let their hydrophilic surface face head groups. Once a critical local
concentration is reached, transient holes are formed in the membrane and a positive curvature is
generated.
In light of these three pools of thought regarding the mechanism of membrane active peptides, all are
in agreement: these compounds act by the formation of ion channel pores that span membranes
without requiring a specific target receptor. Whilst the exact killing action exhibited on bacteria is
unknown, it is thought to be a direct result of one of two processes: damage to the bacterial cell
membrane results in the collapse of transmembrane electrochemical gradients and upon this collapse,
microorganisms lose their source of energy allowing increased water and ion flow across the
membrane resulting in cell swelling and lysis. Or, peptides act via a multi-hit mechanism that involves
more than one target (Shai 2002). Many reports including Wade et al. (1990) show that the site for
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the antibacterial action of these compounds is the cytoplasmic membrane as the bactericidal action
exhibited on these microorganisms indicates they must be initially able to cross or disintegrate the
membranes of Gram-negative bacteria.
In addition to this, membrane active peptides are safe to use in a clinical setting, bacterial membranes
are organised in such a way that the outermost bilayer is heavily populated with lipids and as a result
present negatively charged phospholipid head groups on their surface. In contrast, animal membranes
are composed mostly of lipids with no net charge. The presence of cholesterol also reduces the activity
of membrane active peptides on a cell (Zasloff 2002). So, these cationic membrane active peptides
will only exhibit action on bacterial cells as opposed to animal and plant cells – showing great potential
for use in a clinical setting.
Membrane active peptides encompass a large family of molecules with proven bactericidal action
against Gram-positive and some Gram-negative species. In particularly, cecropin P1 has been found
to exhibit antimicrobial action against all members of the Enterobacteriaceae family except the
Pseudomonas genus (Giacometti et al. 1998).
Unfortunately, as with all antimicrobial compounds, resistant bacteria can arise, however, owing to
their mode of action, only a handful of resistance mechanisms have been outlined for membrane
active peptides. One identified mechanism is most prevalent in species belonging to the Psuedomonas
genus - the production of alginate acts as an auxiliary bacterial membrane, a diffusion barrier to
cationic antibacterial agents (Chan et al. 2005). However due to their potent antimicrobial activity and
ability to disrupt the cell membrane of Gram-negative bacteria, I believe these compounds show great
promise for the delivery of antimicrobial compounds and have the potential to be used as adjuvants.
In addition to this, the very nature of these compounds means that bacteria cannot easily acquire
resistance without changing the structure of the outer membrane. The membrane active peptides
represent a conserved theme in host antimicrobial defences throughout nature and have the potential
to be used in conjunction with other compounds as fusion/hybrid antibiotics thus allowing intra
cellular access to Gram-negative bacteria.
From critical analysis of the literature surrounding membrane active peptides I have discovered
extraordinary examples of α-helical cationic antibacterial membrane active peptides which are
unaffected by common methods of bacterial resistance. A case in point is the novel membrane-active
antimicrobial peptide polybia-CP - designed, produced and discussed by Wang et al. (2012). Polybia-
CP is a compound derived from the venom of the social wasp Polybia paulista and shows antibacterial
action against both Gram-positive and Gram-negative bacteria, permeabilizing the bacterial cell
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membrane. The ability of polybia-CP to permeabilize multiple drug resistant enterohemorrhagic E. coli
(EHEC) was determined by NPN, a hydrophobic fluorescent probe that fluoresces weakly in an aqueous
environment and strongly when it enters a hydrophobic environment such as the interior of a
membrane (Loh et al. 1984). NPN is normally excluded from Gram-negative bacterial cells but upon
treatment with polybia-CP the compound shows strong fluorescence. In addition, after treatment with
polybia-CP E. coli cells demonstrated visual morphological changes and peptide induced dye leakage.
Solely administered, polybia-CP demonstrates an impressive minimum inhibitory concentration (MIC)
to a range of Gram-negative and Gram-positive bacteria. However, minimum bactericidal
concentration (MBC) requires 4-8 times increase of the MIC, making it difficult to attain feasible
treatment in a clinical setting following infection by resilient bacteria namely P. aeruginosa. In light of
this information polybia-CP provides an excellent demonstration of why membrane active
antimicrobial peptides should be used as adjuvants in antimicrobial chemotherapy for the entry of
antibacterial compounds in bacterial cells. Wang et al. (2012) proved this using DNA intercalating dye
P1, which does not bind to DNA in untreated Gram-negative bacterial cells. After treatment with
polybia-CP, P1 was visualised bound to DNA through confocal laser-scanning microscopy. In theory,
quinolones - which target DNA replication and repair by binding DNA gyrase complexed with DNA,
driving double strand DNA break formation and cell death (Kohanski et al. 2007) - could be used in
conjunction with polybia-CP to access DNA in Gram-negative bacterial cells because of increased
target availability due to bacterial cell wall permeabilization by polybia-CP.
Most membrane active peptides share similar morphology. Amphiphilic secondary structures in which
cationic amino acid side chains (i.e., arginine, lysine and histidine) are orientated on one face of the
molecules while hydrophilic side chains are on the opposing face. This morphology has been dubbed
“facially amphiphilic” (Epand et al. 2010) and is an important blueprint which should always be
considered when designing Gram-negative active compounds. Ceragenins, which share facially
amphiphilic morphology (owing to their bile acid scaffolding) and exhibit antibacterial action on Gram-
negative bacteria give strong evidence for the use of this structure. The most potent ceragenin – CSA-
13 even exhibits antibacterial action on the notoriously problematic, multi-drug resistant Gram-
negative pathogen Pseudomonas aeruginosa. Figure 2 shows a diagrammatic representation of a
membrane active peptide with important morphological features highlighted – CSA-13 mimics this
morphology, providing membrane activity (Chin et al. 2007).
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Ceragenins have many advantages over membrane active peptides including their resistance to
proteolysis and amenability to large scale synthesis. Furthermore, they selectively associate with
bacterial cell membranes and disintegrate the antibiotic excluding OM of Gram-negative bacteria. This
permeabilization extenuates the activity of other antibiotics, making these compounds good
candidates for combination therapies.
Hybrid Compounds
For years, the capability of hybrid compounds to overcome and reduce the incidence of resistant
bacteria has been noted. Compounds which inhibit more than one target allow for a decline in the
number of spontaneous resistance mutants by taking the product of the amount of resistant
spontaneous mutants from both compounds so resistance rarely arises. However, I propose that
instead of joining two known antibiotics, as outlined in the expert review by Pokrovskaya and Baasov
(2010), we should investigate antibiotic compounds whose killing action can be combined with cell
wall permeabilizing compounds to overcome antibiotic resistance and gain entry to the Gram-negative
cell cytoplasm; especially that provided by compounds conferring a facially amphipathic morphology.
In theory, compounds which cause OM disruption grant at least some entry into the Gram-negative
bacterial cell cytoplasm, allowing other antibiotics access to their targets and conferring bactericidal
Figure 2. Chemical structure of the tryptophan-rich membrane-active antimicrobial peptide gp41w-
FKA. Important morphological structures are labelled: top – cationic amino acid side chain, bottom –
hydrophobic residues, and centre – helical conformation of the central backbone. Adapted from
Haney et al. (2012).
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activity. The best antimicrobials to be used as candidates for fusion with OM disrupting compounds
would be those with proven polypharmological capabilities, namely natural compounds produced by
bacteria. Although ceragenins provide potent Gram-negative OM disruption and bactericidal action
in addition to other benefits, they are synthetically produced and as a result cannot feasibly be
produced as a hybrid compound with antimicrobials (Isogai et al. 2009). Instead, I would recommend
these compounds be used only in combination therapy with other antibiotics to aid in the disruption
of the bacterial OM so providing better access to drug targets in bacteria. Further research would
require screening the efficacy of known antibiotics which lack entry into the Gram-negative cell
cytoplasm in conjunction with ceragenin therapy.
Production of hybrid peptide antibiotics is widely documented and represents and a more realistic
goal for future research. This would involve combining membrane active peptides with peptides that
have proven antibiotic activity but lack efficient transport into the Gram-negative cell cytoplasm – all
critical processes could be inhibited, with many essential proteins available for targets (highlighted in
figure 3, 1a-1d). Antibiotics which cannot overcome the OM of some resistant Gram-negative bacteria
include aminoglycosides, penicillins, cephalosporins, fatty acid synthase inhibitors like triclosan and
cerulenin, and quinolones (Mingeot-Leclercq et al. 1999; Ruiz 2003; Schweizer 2001). Creation of
hybrid antimicrobials requires the careful identification of gene clusters which code for a desired end
product and usually take the form of a polyketide synthase. After this, a synthetic gene cluster is made
and inserted into an appropriate bacterium for the in vivo synthesis and extraction of the hybrid
antibiotic. The process is complicated but end results are achievable (Ichinose et al. 2003). Other
methods are available for the creation of hybrid peptide antibiotics too. Chemical synthesis, as
demonstrated by Zhang et al. (1998) who created functionally active cell-permeable peptides via
single step ligation of two peptide molecules. Another peptide ligation technique - solid-phase peptide
synthesis is well documented (Merrifield 1969). These methods infer that non-invasive cellular import
of synthetic compounds can be achieved by incorporating a membrane permeable, and therefore
presumably a membrane disrupting, sequence. Membrane active hyrbid compounds can also be
created in vitro - Mehravar et al. (2011) isolated membrane-active metabolites produced by soil
actinomycetes.
In my opinion, future research should concentrate on the identification of membrane active
compounds which are or can be expressed in a bacterial vector. The next step is to create synthetic
gene clusters which include peptide antibiotics that cannot cross the Gram-negative OM and can also
be expressed in a bacterial vector. Cross referencing of these three variables should reveal a hybrid
compound that is able to overcome the resistant Gram-negative OM, exhibit antibiotic action and be
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produced on a large scale via heterologous expression in recombinant bacteria. I suggest that one of
the three membrane active metabolites identified by Mehravar et al. (2011) produced in 3
Streptomyces strains be combined with an innate Streptomyces peptide antibiotic such as one of the
coronamycins (Ezra et al. 2004) by one of the previously outlined techniques. This should increase the
chance of a successful hybrid peptide ligation and effective antibiotic production. If such a compound
were produced resistance mechanisms which usually inhibit either of the two components individually,
such as degradative enzymes, would be rendered ineffective due to a probable change in
conformation of the active site as demonstrated in figure 3 (4). This has huge implications for ESBL
producing resistant Gram-negative bacteria whose list of available resistance mechanisms is
substantially shortened with this theoretical hybrid cell wall permeabilizing antibiotic.
Figure 3. Diagrammatic representation of (1-5): (1) Known targets available to antibiotics; (1a) fatty
acids indicative of FAS inhibitors; (1b) DNA indicative of DNA damaging antibiotics e.g. quinolones;
(1c) ribosome indicative of protein synthesis inhibiting antibiotics; (1d) peptidoglycan structure
indicating target for cell wall synthesis inhibitors; (2) Most antibiotics cannot cross the OM of Gram-
negative bacteria; (3) Degradative enzymes (green) to certain antibiotics are produced by bacteria –
the antibiotic is complementary to the enzyme and fits into its active site; (4) Slight conformation
changes to the antibiotic when hybridized to a cell wall permeabilizing molecule (yellow) result in
lack of complementarity between it and the degradative enzyme – also able to disrupt outer
membrane of Gram-negative cell wall; (5) Disrupted cell wall allows for entry to the cytoplasm for
hybrid compound.
Gram negative bacterium cell cytoplasm
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Membrane active compounds have huge potential for use as fusion/hybrid antibiotics, but apart from
my theoretical suggestion, hybridising compounds for increased antibacterial action has been the
focus of research dating back to 1998. I believe hybrid molecules as a strategy for the delivery of
antimicrobial compounds into bacterial cells will quickly be exhausted unless biochemical research
reveals a plethora of synthetically viable compounds which can be hybridized to overcome the OM of
Gram-negative bacteria whilst exhibiting antibacterial action. Thus we must look for other strategies
for the delivery of these compounds - starting with manipulation of the bacterial pathogen
bacteriophage.
The history of phage therapy
Phage have been recognised as antimicrobial agents since early in the 20th century. But since the dawn
of penicillin, have been brushed aside and are rarely used in western medicine today. However, the
rise of antimicrobial resistance among bacteria has once again caused the world’s antimicrobial
resistance research community to study these natural predators of bacteria.
The first acknowledgement of phage was in 1897 when British chemist E.H. Hankin reported that
water straight from the sewage ridden Ganges could kill the cholera pathogen. It was not until twenty
years later the cause of the bactericidal activity was suggested when British bacteriologist Frederick
W. Twort described an “ultramicroscopic virus” that by some means killed bacteria. French scientist
d’Herre gave the bacteria killing virus fame after he and his wife isolated “anti-shiga” microbes from
the faeces of patients with dysentery and grew them using bacterial hosts. D’Herre dubbed these
microbes ‘bacteriophage’ and was also first to appreciate their potential in antimicrobial therapy.
After characterization and improved bacteriophage culture methods it was possible to use
bacteriophage as an antimicrobial therapy, dubbed ‘phage therapy’. Since one bacteriophage will only
target one type of bacterium, therapies can be tailored once an infective bacterium is identified.
During World War Two and the ensuing Stalin era of the Soviet Union, this technique was employed
to cure patients of various antimicrobial resistant bacteria with great success (Stone 2002).
Following the early success of bacteriophage, which can be grown easily and in great numbers, a huge
decline in clinical use by the western world has occurred – small molecule antibiotics became cheaper
and easier to manufacture in large homogenous quantities. In my opinion, the profit driven business
of pharmaceutical corporations (particularly in the USA) has encouraged the use of antimicrobial drugs,
which are made in large quantities following massive investment. As a result there has been a lack of
research and production of cheaper effective therapies such as bacteriophage. Another complication
that should be considered whilst assessing the administration of bacteriophage to patients are
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society’s potential ethical issues with the technology. Lack of understanding could lead to fear of the
virus particles and therefore proper education should be given to the public about this potentially
lifesaving therapy.
Whilst a lack of proper understanding of phage therapy continues in the western world, research
conducted in studies from the former Soviet Union is abundant, harbouring excellent success rates in
the treatment of resistant bacteria residing in mainly external infections. Phage study successes
include: Markoishvilli et al. (2002) reporting a 70% success rate in curing ulcers and wounds infected
with medically relevant resistant bacteria including the Gram-negative organisms E. coli and
Pseudomonas spp, Lazareva et al. (2001) reporting reduced septic complications, better temperature
normalization and two fold reduction in bacteria when treating resistant infection with phage in tablet
form, Perepanova et al. (1995) curing 92% of patients who had acute and chronic urogenital
inflammation caused by resistant bacteria and Miliutina and Vorotyntseva (1993) who studied the
efficacy of phage in combination with antibiotic therapy, discovering that combination therapy was
only effective against resistant infections vs. antibiotics alone. The success of phage therapy in the
former Soviet Union can be additionally determined by noting the production of phage related medical
products including PhageBioDerm, a novel wound-healing preparation consisting of a biodegradable
polymer impregnated with and antibiotic and lytic bacteriophages, recently licenced in the Republic
of Georgia (former soviet union) (Markoishvili et al. 2002).
Exploiting phage
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Lytic phage kill bacteria during their normal life cycle and as a result require no modification to be
used as an antibacterial therapy - the mechanism is well understood. After location of their specific
bacteria, phage inject pathogenic DNA to shut down bacterial processes and commence with the
replication of themselves simultaneously. Phage proteins, known as lysins, initiate bacteriolysis during
which a number of clones of the original bacteriophage are released into the environment. See figure
4 for a detailed representation of the T4 phage life cycle - how tailed-phage infect, replicate and
escape infected cells upon bacteriolysis.
Firstly phage need to locate and identify their specific targets, this is known as adsorption. As
previously mentioned, one type of phage can only usually infect one species of bacteria. Specificity
allows phage to be used on the body without fear of affecting an individual’s natural flora. Adsorption
usually occurs between a receptor protein on the bacteriophage tail fibre and a particular recognisable
target on the bacterial surface, usually a protein or lipopolysaccharide. These are encoded by a largely
Figure 4. Life cycle of T4 lytic phage as described in Josslin (1970). (1) Attachment and injection of
DNA; (2) transcription of early genes; (3) replication and the beginning of capsid head formation; (4)
transcription of late genes; (5) head assembly; (6) tail assembly; (7) attachment of the head to tail;
(8) attachment of tail fibres – complete phage; (9) cell lysis and release of the mature phage.
4
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conserved section of the genome within species and genera (Mahony et al. 2013). Until recently, these
tail fibres were little understood. The Bartual et al. study (2010) gave insight into the structure of tail
fibres when they determined the blueprint of the bacteriophage T4 long tail fibre receptor-binding tip.
It is described as “an unusual elongated six-stranded antiparallel beta strand needle domain
containing seven iron ions coordinated by histidine residues arranged collinearly along the core of the
biological unit. At the end of the tip, the three chains intertwine forming a broader head domain,
which contains the putative receptor interaction site.” This structure suggests a framework for
mutations to expand or modulate receptor binding-activity. If targets change on the bacterial outer
membrane then phage may be able to ‘adjust’ it’s recognition site through the process of natural
selection.
Interestingly, experiments involving hybrid phage have revealed the receptor-binding region of phage
tail fibres to be around 89 amino acids long (Montag et al. 1990). Future research should be aimed at
the identification of particular targets on the bacterial cell surface and genomic isolation of these 89
amino acids and their place in the phage genome so that they can be replicated by a genetically
modified phage. Outer membrane proteins/antigenic proteins which are recognised by bacteriophage
could be prepared from cell envelope suspension by differential Sarkosyl solubility. Outer membrane
proteins can then be identified from spots resolved by two-dimensional electrophoresis and LC-
MS/MS (Dumetz et al. 2008). This technology has recently been employed for developing vaccines
against flavobacteriosis infection in aquaculture and if achieved for bacteriophage targets, we could
tailor lytic phage therapy to target any bacterial species, including multi-drug resistant bacteria.
Identification of the part of the phage genome which codes for the 89 amino acids would be the result
of determining the amino acid sequence and cross referencing the thousands of combinations of
nucleic acid order with the bacteriophage genome.
Entry into the Gram-negative cell cytoplasm is inferred by bacteriophage who have evolved a ‘hole
punch’ mechanism for the transport of their own genome into bacteria. After receptor binding, a
recognition signal is sent to the baseplate causing the short tail fibres to extend and bind irreversibly
to the outer core region of the lipopolysaccharides. This is then followed by contraction of the outer
tail sheath, penetration of the bacterial membrane by the hollow inner tail tube and injection of the
viral DNA from the bacteriophage. See figure 5, adapted from Bartual et al. (2010) for a diagrammatic
demonstration of this mechanism. Viral DNA either inserts itself into the genome – known as the
lysogenic cycle, or, if conditions permit, viral DNA will commence the lytic cycle creating many virus
particles to be released into the environment upon bacteriolysis.
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Figure 5. Bacteriophage T4 and its long tail fibres, annotations of the capsid head and protein tail are
provided - adapted from Bartual et al. (2010). (A) Bacteriophage before initiation of the ‘hole punching’
mechanism and delivery of viral DNA; (B) after this event has taken place. Receptor region of long tail
fibre is highlighted by grey box. This mechanism could provide an efficient vehicle for the delivery of
compounds other than DNA in the future.
In addition to the ‘hole punching’ mechanism and phage ability to adhere to specific bacteria phage
genomes can be exploited to aid in antibiotic discovery. In a previously mentioned example published
in Nature Biotechnology (2004), Lui et al. demonstrated identification of 31 novel peptide families
from the Stapholococcus spp. phage. This technique could be applied to genomes of phage which
infect Gram-negative bacteria, thus identifying Gram-negative active compounds. However, these
compounds would probably lack entry to the Gram-negative cell cytoplasm without phage, their
normal transport being from the phage body. In addition, a plethora of research has been conducted
concerning the bacteriophage lysins. These are phage-encoded peptidoglycan cell wall hydrolases that
accumulate in the bacterial cytoplasm during a lytic infection cycle. Compounds have been developed
using phage lysins to clear in vivo populations of Gram-positive bacteria. Nonetheless, a major
limitation of this approach is that lysins cannot normally pass the OM of Gram-negative bacteria
without the aid of accessory proteins or cell wall disrupting agents. The OM shields periplasmic
peptidoglycan so that lysins cannot exhibit their killing action. Genetic screens are available to search
for these compounds in the phage genome (Schuch et al. 2008) and can be applied to any phage.
More recently, research has turned towards the synthetic structural engineering of phage lysins that
can target Gram-negative pathogens and overcome the OM. Pesticin, a type B bacteriocin which
requires the gene products of tonB, exbB, exbD and fyuA is produced in times of stress by Yersinia
pesticis. It is harboured on a 10kb plasmid called pPCPI (Schuenemann et al. 2011) with the pesticin
immunity protein and plasminogenic activator. Interaction between FyuA (which is an outer
membrane, TonB-dependant iron transporter protein (Noinaj et al. 2010) expressed on some
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pathogenic Gram-negative bacteria) and pesticin, is required for entry of the compound into Gram-
negative cell periplasm. Pesticin is a soluble protein with two domains, one that binds to FyuA and the
other which shares homology with the T4 phage lysozyme. Upon determining the crystal structure of
pesticin, Lukacik et al. (2012) went on to design a hybrid compound which shared the FyuA domain of
pesticin fused with the N-terminus of the T4 lysozyme. This hybrid phage lysin showed bactericidal
activity against pathogenic E. coli and was not inhibited by the pesticin immunity protein due to a
change in conformation. Figure 6 shows a cartoon representation of the mode of action of pesticin
versus this hybrid compound.
Figure 6. Mode of action of pesticin and the hybrid phage lysin as described by Lukacik et al. (2012).
(1) Pesticin – a 2 component molecule consisting of a FyuA binding domain and a lysozyme like domain)
enters the periplasm through binding to FyuA protein in Gram-negative outer membrane (OM). After
traversing the outer membrane, pesticin degrades the bacterial peptidoglycan layer to exhibit cell
death. This traversing depends on the presence of an outer membrane virulence protein FyuA. Pesticin
can be inhibited by bacteria expressing the pesticin resistance protein in the periplasm. (2) The
lysozyme like domain was substituted for a similar T4 lysozyme during creation of the hybrid
compound and can traverse the Gram-negative bacterial OM in the presence of FyuA. Translocation
of this hybrid compound also results in the killing of cells via peptidoglycan degradation, activity is not
affected by pesticin resistance protein. (3) Without additional means of disrupting the OM, externally
added T4 lysozyme cannot effect killing.
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In addition, FyuA outer membrane protein is only expressed when pathogenicity occurs and therefore
this hybrid compound would not exhibit killing action on the normal gut flora, for example, due to a
lack of transport into the bacterial cell cytoplasm. Some modern antibiotics currently offer an ‘all or
nothing’ approach to eliminating infection, during which healthy bacteria can also be compromised
leading to increased production of resistant populations. With the FyuA targeting system only
pathogenic bacteria would be killed, allowing normal function of the body’s healthy microbial
populations. Figure 7, taken from Lukacik et al. (2012) demonstrates this hybrid phage lysins
bacteriolytic action against E.coli cells.
Figure 7. Cyro-electron micrograph taken directly from Lukacik et al. (2012). Cells were exposed to (A)
no treatment; (B and C) treatment with hybrid T4 lysozyme/FyuA binding compound. Extensive
membrane breakdown of the treated samples is apparent. As a result the cell is weakened and,
consequently spreads and flattens on the thin film of buffer while largely preserving its 3D surface
area so it projects a larger 2D surface area. As the cell is much thinner, membrane vesiculation occurs
all over the cell, not just at the edges. Inset (B and C), this vesiculation is shown with increased
magnification. Inset (A) – increased magnification highlights the outer membrane (OM), periplasm (PG)
and inner membrane (IM).
Phage lysins seem to yield considerable potential, yet they will always need hybridization with
accessory compounds to aid with transport to the Gram-negative periplasm. The above example is
impressive but identification of specific binding/transport proteins is difficult – they are usually part
of antibiotic compounds produced by bacteria. In contrast, targeting FyuA for antimicrobial therapy
offers new hope. Recent findings indicate that fyuA appears to be associated with the persistence of
urinary tract infections and multi-drug resistance in animal and human infections (Platell et al. 2012).
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Since bacteria expressing FyuA are pathogenic and not a part of the normal microbial flora, we can, in
theory, selectively kill the detrimental bacteria and leave the rest unharmed.
The future of phage therapy
Phage can be exploited in a variety of ways, with huge scope for overcoming antibiotic resistance and
the Gram-negative OM. Future therapies must base their technology on recent innovations in phage
therapy. The rise of the genomic era has given us the ability to genetically engineer phage and holds
much promise for the future. An excellent demonstration of phage potential is the Westwater et al.
(2003) study where the team used genetically modified (GM) phage to deliver lethal DNA encoding
bactericidal proteins. These proteins were ‘addiction molecules’ consisting of two components (one
with lethal action, the other usually transcribed as an antidote) which mediate programmed cell death
under particular conditions. A non-lytic phage delivery system (phage M13) transformed with helper
phage R408 (to introduce phagemids into the delivery system) transferred the lethal genes into the
bacterial cell cytoplasm. A high copy number plasmid containing the lethal genes was constructed and
placed under the control of a LacI/IPTG regulated promoter for screening purposes. When induced,
viable counts of bacterial populations dropped dramatically and did not recover. One drawback to this
research is that E. coli was used because of the wealth of molecular tools available. To utilise this
technology for other, more problematic Gram-negative bacteria, specific phage delivery systems must
be created to target conserved proteins in pathogenic bacteria, FyuA for example.
Phage can only deliver DNA sequences into bacteria. Anything the phage needs is then created utilizing
bacterial ‘machinery’. A distinct lack of ability to deliver antibiotics to resistant bacterial cells is ever
looming: why not reverse modern antibiotic resistance in bacteria by inserting sensitive genes? The
antibiotic streptomycin binds to a highly conserved region in 30S small ribosomal subunit in bacteria,
this inhibits translation of mRNA into proteins. Point mutations in resistant bacteria mean that
streptomycin can no longer bind. The 30S small ribosomal subunit is encoded by rspL gene, the most
common mutations in the gene are K88R and R68S but rpsL carries dominant sensitivity over its
homolog; mutation results in resistance but also generates a recessive gene. This knowledge can be
used to reinstate streptomycin sensitivity via phage insertion of a dominant gene (Edgar et al. 2012).
In this instance, phage λGT11 was used as a delivery vector. This phage can be directed to specific cell
types under particular conditions and enters the lysogenic (as opposed to lytic) cycle at 32°C – phage
genes form into circular plasmids and can be transferred through bacterial populations by conjugation.
Phage mediated insertion of the original, dominant gene infers sensitivity of resistant bacteria to
streptomycin. Bacteriophage which can be used as adjuvants for antibiotic therapy, like the above
example, provide great potential as a strategy for the delivery of antimicrobials to bacterial cells.
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Reversing resistance relies on a previous understanding of antibiotic targets. Bactericidal antibiotics
such as quinolones induce hydroxyl radical formation that leads to DNA damage, which induces the
SOS response in bacteria for DNA repair. Increased resistance to these compounds arises from
evolution of a more efficient DNA repair response and it has been shown that bacterial killing from
quinolones can be enhanced by knocking out recA – a critical gene in the SOS response pathway
(Kohanski et al. 2007). Lu and Collins (2009) used non-lytic filamentous phage M13 which can
accommodate DNA insertions into the genome to deliver the lexA3 gene which disrupted the SOS
response and in turn made even resistant bacteria sensitive to bactericidal antibiotics. Furthermore,
the team show that engineering phage to target other gene networks and overexpress multiple factors
can also produce effective antibiotic adjuvants. The work as a whole establishes a synthetic biology
method for the rapid production of modified phage which target specified gene networks and puts
forward a valid argument for the creation of a bacteriophage genomic library. Current prices for a
bacteriophage genome carrying multiple constructs to target different gene networks are decreasing
rapidly, making the idea more feasible as time passes and prices drop (Baker 2011).
With my final note on phage therapy, I believe the technology will compensate for unavoidable
complications of antimicrobial therapy with antibiotics such as the appearance of multi-drug
resistance or substituted microbism. Moreover, traditional antibiotic drugs typically do not take
advantage of targets that need to be upregulated to achieve antimicrobial activity. Phage therapy has
at least the potential to inhibit up regulated targets as demonstrated by Lu and Collins (2009). In
addition, phage therapy in the traditional sense (non-GM phage) has been improved vastly thanks to
the arrival of the genomic era. The two technologies should evolve in conjunction with one another
as phage studies have already provided us with invaluable insights into bacterial DNA/cell biology
processes (Birge 2000).
Phage may be difficult to administer for internal infections because of protease susceptibility but
provide a valid argument for them to be considered as disinfectants and for external application. I
suggest that genomic isolation and synthesis of the ‘hole punching’ and delivery mechanisms from
phage could yield an important tool in the delivery of antimicrobials to bacterial cells.
Pyocins
Sharing a similar morphology and delivery mechanism with phage, and greatly understood with the
aid of genome sequencing, pyocins represent another technology with potential to deliver
antimicrobial compounds into even Gram-negative bacteria. Pyocins are produced by Psuedomonas
aeruginosa, look much like a bacteriophage tail (particularly R-type pyocins) (Figure 5) and portray a
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similar recognition mechanism. In many respects, R-type pyocins are viewed as defective prophages
that produce noninfective particles containing only the tail region without the capsid head or DNA,
which have been adapted by the host as defensive antibacterial agents. Neither the host nor its
offspring are affected by the pyocins, although one exception is noted (Goodwin et al. 1972). The
potential of pyocins as antimicrobial agents has only recently been exposed, because at the time of
discovery, antimicrobial resistance was not as prominent and so there was no need for new
antimicrobial therapies. However, in the face of rising antimicrobial resistance, particularly in Gram-
negative bacteria, this avenue must now be explored.
The lipopolysaccharide tail fibres of pyocins are able to recognise competing P. aeruginosa strains as
well as many other Gram-negative pathogens, indicating a conserved target. Once adsorption occurs
between pyocins and target, penetration of the core through the outer membrane, cell wall and
cytoplasmic membrane immediately ensues, this is a process critical to bactericidal activity. After
adsorption, pyocins halt critical bacterial processes and promote the release of intercellular material,
indicating cell wall permeabilization. Upon permeabilization of the bacterial cell wall, the targets of
many antimicrobials would become available making pyocins valid for use as adjuvants with antibiotics,
providing another strategy for the delivery of antimicrobials. Protein sequences for pyocins can be
deduced from the genome sequencing of P. aeruginosa and by comparing these sequences with the
structure of similar phage, it is possible to assign functions to ORF’s coding for these proteins (Michel-
Briand and Baysse 2002). In theory, the components of the ‘hole punching’ mechanism could be
produced under the control of a strong constitutive promoter. In addition, R type pyocins are protease
resistant providing an added benefit over phage therapy.
The limited bacterial spectra of natural R-type pyocins ultimately compromises their ability for clinical
use. However, replacing pyocins tail fibres with phage PS17 tail fibres changes the specificity of the
pyocins to target the P. aeruginosa strain that which PS17 predates. Natural and retargeted pyocins
exhibit very narrow bactericidal action and thus yield little in the way of collateral damage to healthy
microbiotae, blocking the rise of antibiotic resistance through horizontal gene transfer. This is
achieved by “swapping tail fibres or fusing the N-terminal portion of the tail fibres of bacteriophages
that infect hosts other than R2-sensitive P. aeruginosa.” (Williams et al. 2008). In their experiments,
Williams et al. (2008) showed that fusing of the C terminus of the P2 (phage 2) tail fibre to the R2
pyocin protein Prf15 (tail fibre protein), changed pyocins killing spectrum to target Escherichia coli
strain C and multiple uropathogenic E. coli strains as well as creating pyocins that kill Yersinia pestis.
More evidence that the plasticity observed among bacteriophage tail genes can, with modern
molecular techniques, be exploited to produce non-natural, targeted microbial agents is detailed in a
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separate study (Scholl et al. 2009). This team emphasised pyocins potential for killing clinically relevant
Gram-negative bacteria by switching pyocins tails with tails from phage ΦV10, the natural virus
predator of food-borne pathogen E.coli 0157:H7. Pyocin production is initiated by the SOS response
in bacteria so treatment with UV light is needed, but after this, bacteria produce around 100-200
particles per cell; a viable option for large scale production. These examples outline pyocin potential,
highlighting especially the new found ability to target any bacterial strain, provided a relevant phage
has been identified. Through genomic identification of the pyocin mechanism - which is rightly likened
to that of the bacteriophage tail – in combination with a bacteriophage capsid head containing
antimicrobial compounds I believe an efficient vehicle for the delivery of antimicrobial compounds
can be achieved. In absence of this technology at present, pyocins can provide entry to antimicrobial
targets via permeabilization of the bacterial cell membrane or exhibit antibacterial action themselves.
Conclusion
Finally, resistant bacteria continue to pose a threat to human health after the relative early success of
antibiotics. It is in these difficult times, where antimicrobial compounds lack entry to bacterial cells,
when we must look to novel, innovative research for answers and properly educate those who will be
treated to overcome any reluctance about potentially lifesaving therapies such as phage. New
technology aiding in the delivery of antimicrobials to bacterial cells currently captures much interest
and funding in the struggle to halt the ongoing rise of antimicrobial resistance. The most promising
ideas stem from old knowledge thought to be exhausted. Phage technology is a case in point, allowing
researchers to deliver any small DNA fragments, helping to permeabilize the Gram-negative cell wall.
As bacteria evolve more efficient resistance mechanisms we must endure to explore novel strategies
for the delivery of antimicrobials into those bacteria which are causing most incurable and deadly
infections. By further investigating technologies like phage and pyocin therapy, researchers have
discovered specific and powerful weapons in the fight against the rise of resistant bacteria. These
include the natural ability of these molecules to attack specific bacterial targets of our choosing.
However, I believe that an important aspect of these technologies has been overlooked and that is
the ability to use these advances in combination therapies, providing entry to bacterial cells for
antimicrobials. Indeed, the antibacterial effectiveness of combination chemotherapies has already
been well documented; clavulanic acid, which inhibits β-lactamases is proven to increase the
antimicrobial effects of penicillin through β-lactamase inhibition (Heerema et al. 1979) and brand
name products adhering to this combination chemotherapy are prescribed all over the world.
Using cell wall permeabilizing compounds like phage, pyocins or membrane active peptides provides
effective, novel strategies for the delivery of antimicrobials into bacterial cells, mostly through
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disintegration of the bacterial cell wall and I believe that in the future, designs based on this
technology will be used to deliver antimicrobial compounds directly into the cytoplasm of bacteria,
taking advantage of the phage/pyocin ‘hole punching’ mechanism. For example the T7 phage virus
has already been used to implement a biotemplating technique for the creation of magnetic
nanoparticles which imitate phage morphology and biochemical activity (Lui et al. 2006).
Nanotechnology, phage and pyocin technologies, membrane active peptides and hybrid molecules
which can overcome the bacterial OM represent major advances in strategies for the delivery of
antimicrobials into bacterial cells. Future research should focus on the identification and isolation of
compounds which bind to conserved targets on the pathogenic Gram-negative bacterial OM with
emphasis on pore targets to aid in the efficient transport of antimicrobial compounds to the Gram-
negative cytoplasm. In addition, an international bacteriophage genomic library should be constructed
so that bacteriophage legs specific to problematic bacteria can be integrated on the pyocin body.
Unlike the original studies and other reviews which outline and describe strategies for the delivery of
antimicrobials into resistance bacterial cells singularly, this work hopes to encompass all of the most
important work, helping the reader to understand new technologies. Whilst some other delivery
strategies are described in the literature, this work only focuses on the most promising strategies
published in respected journals and papers. This review also suggests ways in which future research
should be directed to complement existing strategies, like the identification of conserved targets on
pathogenic bacteria for use with phage/pyocin tail recognition domains. To conclude, I hope that this
work outlines the importance of discovering novel strategies for the delivery of antimicrobials and the
danger of drug resistant pathogenic bacteria and can go some way to encourage use of these therapies
in conjunction, now, to slow the occurrence of new resistant bacteria.
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