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There is growing concern over the emergence of bacteria becoming antibiotic resistant; for instance Pseudomonas aeruginosa, Enterococcus faecalis, Acinetobacter baumannii, Mycobacterium tuberculosis, and Staphylococcus aureus. In more recent years, the most notable pathogen to come to the forefront of public concern and the media is the methicillin-resistant strain of Staphylococcus aureus, or simply better recognised from its' abbreviation; MRSA. Other multiresistant bacteria include Acinetobacter; the most frequently isolated bacterial species from clinical samples and is a cause of hospital acquired infections. This poses treatment difficulties as Acinetobacter is resistant to carbapenems, fluoroquinolones and aminoglycosides. The emergence of multidrug resistant bacteria obviously indicates the importance for the development of new antimicrobials and treatment strategies. According to and evaluation to assess the current antimicrobial agents in development, by Spellberg et al., concludes that the development rate of antibiotics is not sufficient enough to outweigh the rate of antibiotics becoming less effective. They had also found a 56% decrease, over a 20 year period, of the United States Food and Drug Administration (FDA) in the approval of antimicrobial agents. They had also found that only five antibacterial agents were being developed, across 15 of the largest pharmaceutical companies. If this development rate continues, there is a possibility that antibiotics may become redundant as more resistant bacteria emerge. This highlights the need for an alternative to antibiotics to treat antibiotic resistant bacterial infections. One such treatment strategy is the use of bacteriophages; a bacterial virus.
Bacteriophages, or phages, are viruses which infect bacteria that can either have a detrimental affect on its' host but can also influence the hosts' biological functions; contributing to the pathogenicity of the bacteria. Phages are believed to be the most abundant life form on Earth as they are estimated to be 10 times more abundant than bacteria; their numbers are estimated to be 1032. They are more frequently present wherever bacteria are found but particularly reside in water environments. It is estimated that the number of isolated phages, and have been characterised, are scarcely a fraction of the phages believed to be in existence. Bacteriophages are specific to bacteria i.e. they only infect bacteria and, in turn, do not infect mammalian cells. To highlight further specificity, phages will only target one bacterial species and even only show specificity to one particular bacterial strain.
The morphological features of phages do vary but, however, the most typical example of a bacteriophage is shown in figure 1.
Figure 1 shows the structure of a tailed. There are at least 4950 phages which are tailed and make up 96% of the total phages described to date. These tailed phages make up the order Caudovirales. They are comprised of a head, neck, collar, sheath, and a tail. The head is sometimes referred to as the capsid, which is an icosahedron shell made up of protein. This capsid houses either single stranded DNA (ssDNA) or double stranded DNA (dsDNA) which makes up the phages' viral genome. Other phages may contain either single stranded RNA (ssRNA) or double stranded RNA (dsRNA). There are typically six tail fibres present on each phage and function as receptors. These are capable of attachment to specific sites on the surface of a target bacterial cell and may also exhibit contractile properties. Phages with noncontractile tails make up 61% of tailed phages including the Siphoviridae. There are other phages that do not have tails as part of their morphology and therefore other mechanisms are in place required for attachment. The remainder of phages make up 4% of total bacteriophages including polydedral, filamentous, and pleomorphic phages.
Table 1 outlines the characteristics and morphology of the order and families of bacteriophages.
Life cycle of phages
There is one of two life cycles in which a phage undergoes. This can be either virulent or temperate phages. The virulent phages undergo a process known as the lytic cycle as once a bacterium is infected; it results in lysis of the host cell and causes death. Conversely, temperate phages undergo the lysogenic life cycle in which a phage will integrate its nucleic acid into the bacteria and becomes dormant within the host genome; this results in the formation of a prophage. During the lysogenic cycle, the phage DNA may be inserted into the bacterial host DNA or within bacterial plasmids. As the DNA becomes integrated in the host genome (prophage DNA) it will, in turn, be replicated into daughter cells during host replication. Therefore each generation of daughter cells will inherit the prophage DNA. Figure 2 shows both (A) lytic and (B) lysogenic life cycles of bacteriophages.
Bacteriophages become in contact its bacterial host through random motion whereby attachment occurs; this is achieved via specific receptor sites present on the host. The receptors are specific epitopes of cell surface molecules that may consist of protein, teichoic acid, lipopolysaccharide, oligosaccharide or peptidoglycan. It has also been noted that attachment may occur between the phage and the hosts' flagella, cell capsule, or the conjugative pili. Following attachment, the genetic material of the phage needs to be transferred into the bacterial host cell. Insertion of the phage genetic material into the host can be achieved by a variety of mechanisms, but is largely governed by the morphology of the infecting bacteriophage. Tailed phages are able to contract which results in the formation of a hole in the host cell wall (Figure 3). Phage DNA has many chemically modified bases which prevent host cellular restriction and nuclease enzymes from degrading the phage DNA. The mechanism for the bacterial metabolic processes is taken over by mRNA that is produced when the host cell RNA polymerase transcribes the viral genome. This manipulates the metabolic machinery resulting in the production of new viral components. Complete virions can then be completed when the viral components have been assembled together. The newly synthesised phage particles have to be released from the host cell into the surrounding environment in order to go on to infect other bacteria.
There are a number of enzymes that have been developed by almost all dsDNA phages; all of which are directed to the bacterial peptidoglycan. Peptide linkages can be targeted by endopeptidases, sugar bonds can be targeted by lysozymes, or amidases that target amide bonds. All of these enzymes are lytic, specifically termed endolysins or muralytic enzymes. As these enzymes are synthesised in the cell cytoplasm, they require another enzyme to allow passage across cytoplasmic membrane to reach their target substrate. Holin is the enzyme which allows the lysin to degrade the peptidoglycan by disrupting the cytoplasmic membrane. This ultimately leads to host cell lysis. Some other phages, however, such as filamentous phages do not lyse cells. These are released from host cells via extrusion; this leaves the host cell intact and functional. Bacteriophages that do not lyse cells have not been described as useful in phage therapy. The latent period is a term used from the time when a bacteriophage infects the host cell to the time when the viral progeny is released. It has been estimated that over 100 new viral particles can be released from each infected host cell which, in turn, can infect others. This process has been referred to as the single-step growth curve; which was first described to show this process of phage replication. This process may signify that bacterial infections with phages would continue perpetually until all bacteria have been destroyed. Because of this, it would seem that lytic phages have the potential to be used to treat bacterial infections.
Conversely, temperate phages are lysogenic. These phages integrate their DNA into the host genome and reside there as prophages. The drawback for temperate phages in therapy is governed upon that they do not automatically enter the lytic cycle. Instead they are replicated with the bacterial DNA and, in turn, are integrated into each daughter cell. Each of these cells are able to undergo a number of division cycles without releasing a progeny phage. However, spontaneous lysis can occur to one or a few cells during a period of cell division. This factor would suggest that temperate phages would not be suitable in phage therapy. However, the lysis of host cells infected with temperate phages can be induced with the use of mutagenic agents or exposure to ultraviolet light. Other temperate phages can be influenced to switch from the lysogeny to lytic cycle under high temperature and stationary phase.
The genome of bacteria that are infected by temperate phages may be significantly changed to such a point whereby the new genome encodes increased pathogenicity or virulence. The prophage DNA may become excised from the bacterial DNA when repressor genes are down regulated. However, when the prophage DNA is excised, the bacterial genes can be left incorporated along with it. These can then be transferred to other host cells; through a process of transduction whereby genes are transferred from one host cell to another.
There are many genes that are described for bacterial virulence. These are genes which are involved for host attachment, invasion and survival. Genes have also been described which are involved in the production of toxins. Transduction has provided the acquirement of toxin genes such as the diphtheria toxin of Corynebacterium diphtheriae, the neurotoxin Clostridium botulinum, the Shiga toxins found in Escherichia coli O157 and the cholera toxin in Vibrio cholerae. There are many other bacterial phages that have contributed to bacterial pathogenicity mechanisms; these are highlighted in the table 2.
Genome sequencing has brought to light that bacteriophage and bacteriophage elements are in excess as it has shown sequence diversity and also has implied that these elements have played a substantial role to the evolution of bacteria.
Due to the increasing knowledge of bacteriophage biology, the use of phage therapy for bacterial infections is more demanding as antibiotic resistant strains of bacteria is emerging. One particular area of phage therapy development is through the use of small acid-soluble spore proteins (SASP).
Small acid-soluble spore proteins (SASP)
Bacillus ssp. endospores contain low water content within the spore core which shows significant resistance to heat, chemicals, and radiation. There are also α/β-type small, acid-soluble spore proteins (SASPs), which also contributes to the protection of the spore DNA. Three subtypes of SASP have been identified; including α, β and γ. The α/β-type SASP has been shown to function as DNA binding proteins which prevent DNA damage. The γ-type SASP serves to provide amino acid for outgrowth. The gene which encodes α/β-type SASP has been demonstrated to cause a vegetative cell to share characteristics of a spore, when inserted into a plasmid under the influence of an inducible promoter. An antimicrobial system has been developed which exploits bacteriophage specificity that targets SASP genes into pathogenic bacteria. By inserting α/β-type SASP gene into bacteria, with the use of genetically engineered phages, the lysis gene is subsequently removed. DNA is injected to the target bacteria by the phage and the SASP and viral genes become expressed. SASP are then produced, which are toxic to the bacteria cell; all cellular activity is stopped as SASP binds irreversibly to the bacterial DNA. There is no multiplication of virus in the bacteria and ultimately results in rapid death.
Phage therapy is no new concept. However with the emergence of resistant strains of bacteria, it is essential that other treatment strategies for infection are explored. The SASP mechanism is one area of phage therapy which is being developed to treat such bacteria as C. difficile and MRSA.