Team:Nottingham/Project
Project description
Clostridium difficile
Clostridium difficile is a Gram-positive, rod-shaped, anaerobic bacterium and is the most common causative agent of hospital-acquired diarrhoea in the Western world. The symptoms of C. difficile infection (CDI) can range from watery diarrhoea to pseudomembranous colitis, toxic megacolon and in severe cases death. Most C. difficile strains produce two major toxins, TcdA and TcdB, which are responsible for causing the characteristic symptoms of CDI.
C. difficile is estimated to be present in the natural gut microbiota of around 4% of the healthy adult population however, exposure to broad-spectrum antibiotics, such as cephalosporins, can cause disruption to the microbiota. This disruption can promote the colonisation of toxigenic strains allowing infection to persist. It is thought that non-toxigenic strains of C. difficile can act as a probiotic by outcompeting toxigenic strains in the gut and reducing the likelihood of disease. Currently, CDI is treated using two main antibiotics, metronidazole and vancomycin however, raised concerns over the emergence of antibiotic resistance has led to a desire for alternative treatments.
Phage and phage therapy
Bacteriophages (phages) are viruses which infect bacteria and can exist anywhere bacteria are located. Phages are highly specific, only infecting a single species or strain of bacteria and can be defined as either lytic or temperate depending on the life cycle they follow.
Lytic phages exclusively follow the lytic life cycle, following infection these phages will hijack the host cell machinery to produce multiple copies of the phage proteins. These proteins are then assembled into multiple phage progeny which burst out of the host cell and go onto infect other bacterial cells. Temperate phages can follow the lytic life cycle but are also able to follow the lysogenic life cycle. These phages can integrate their genome in the host cell chromosome upon infection where they can remain dormant for long periods of time as prophages. When conditions are favourable, usually due to host cell stress, these prophages can excise from the host cell chromosome and enter the lytic life cycle where progeny phage particles are produced. To date, all phages found to infect C. difficile are temperate phages.
Ever since the first phage was isolated their use as a potential therapeutic agent has been explored, such as in the treatment of wound infections. Traditionally such therapy relies on strictly lytic phages to wipe out the problematic/problem causing bacterial populations. Phage therapy would be an ideal alternative treatment for CDI as their highly specific nature would mean they would not disrupt the natural gut microbiota, only targeting C. difficile cells.
Figure 1: Lytic and lysogenic phage life cycle. After a bacterium has been infected by a lytic bacteriophage, the viral genome material is transcribed, translated and replicated using the bacterial cellular machinery to produce viral proteins. The proteins are assembled to make viral particles and the genomic material is packaged into the virions. Once the bacterial cell reaches capacity, the host cell lyse, resulting in the release of the viral particles. These viruses can go off to infect other uninfected bacterial cells. Bacteria can also be infected by lysogenic bacteria. However, transcription and translation of the viral genetic material is repressed. It is instead integrated into the host genome where it remains and is replicated with the host genome. Upon induction, however, the lysogenic life cycle is switched to the lytic life cycle.
This could reduce the incidence of relapse by allowing the gut microbiota to remain in its protective role against future colonisation. In comparison to antibiotics, the impact of resistance to phage therapy would be minimal due to phages and bacteria co-evolving. As bacteria gain resistance to overcome phage infection, the phages can evolve to evade these systems resulting in susceptible bacterial populations which can be treated. Although phage therapy would be the ideal alternative treatment for CDI the major roadblock is that no strictly lytic phages currently exist.
Project proposal
The main aim of this project was to create a therapeutic for CDI that would allow the natural gut microbiota to remain unchanged and reduce the reliance on antibiotics. To achieve this goal phage therapy was selected as an appropriate alternative due to its highly specific nature. It has been shown that non-toxigenic strains of C. difficile can act as probiotics to reduce the colonisation of toxigenic C. difficile in the gut therefore, by silencing the toxin gene expression in C. difficile, non-toxigenic probiotic strains are created.
Figure 2: Toxin repression in C. difficile using antisense RNA. (starting with the image on the bottom left and going clockwise) Once the therapy is administered and in the gut, the genetically modified bacteriophage binds to a toxin-producing C. difficile cell and injects its genetic material (DNA containing our antisense RNA constructs) into the cell. The phage genome is subsequently integrated into the host (C. difficile) chromosome. Later, the DNA is transcribed along with the host genome, resulting in the expression of antisense RNA. The antisense RNA binds the toxin mRNA hence preventing translation of the toxin mRNA into the protein. The RNA-RNA duplex is degraded in the cell. As a result, the C. difficile cell is no longer producing toxins, converting to into a non-toxigenic cell.
Figure 3: Toxin repression in C. difficile using dead-Cas9. (starting with the image on the bottom left and going clockwise) Once the therapy is administered and in the gut, the genetically modified bacteriophage binds to a toxin-producing C. difficile cell and injects its genetic material (DNA containing dCas9 gene and sgRNA coding region) into the cell. The phage genome is subsequently integrated into the host (C. difficile) chromosome. Later, the dCas9 gene and sgRNA coding region are transcribed along with the host genome, resulting in the expression of dCas9 proteins and sgRNAs. dCas9 binds to the sgRNA forming a complex which binds to the promoter region upstream of the toxin gene. As a result, the toxin gene is not transcribed so the C. difficile cell is no longer producing toxins, converting it into a non-toxigenic cell.
To achieve this two strategies of gene silencing are investigated, the use of anti-sense RNA and dead Cas9. In order to ensure the therapeutic is specific for C. difficile these “silencers” will be delivered to the gut using a phage. This ensures that only the C. difficile cells are targeted and due to the specificity of the “silencers” only strains capable of producing the toxins will be silenced allowing the now non-toxigenic strains to remain part of the gut microbiota to protect against other opportunistic toxic bacteria. In addition, with this approach the lack of a lytic phage is no longer an issue as using the ability of temperate phage to integrate into the host cell chromosome to express the “silencers” results in stable repression of the toxin while keeping the cells alive which allows the strains to become part of the gut microbiota. The presence of toxin silenced C. difficile strains in the gut microbiota can have a protective effect to reduce the likelihood of toxic strains colonising and causing future infections.
Abstract
Antibiotics serve a critical role in remedying bacterial infections, however a major disadvantage to their use is the non-specificity of broad spectrum antibiotics that drastically kills off beneficial bacteria reducing the diversity of the gut flora. The use of antibiotics allows opportunistic pathogens like Clostridium difficile to take advantage of the dysbiosis caused.
A consequence of antibiotic misuse and the capability of bacteria to readily adapt to versatile conditions, has allowed antibiotic resistance in bacteria to become a major dilemma. Each year in the United States alone 2 million people are subject to infection from antibiotic resistant bacteria. Phage therapy is an alternative to antibiotics. The goal of our project was to engineer a bacteriophage which will infect C. difficile and express genetic constructs designed to suppress toxin production. We will pursue two strategies to achieve this; asRNA and dCAS-9, both of which will target the toxin genes tcdB and tcdA. Ultimately, we aim to produce a phage therapy which will reduce toxigenicity of resident strains of C. difficile without significantly affecting the native gastrointestinal microbiota.
