Devising a Bacteriophage-Mediated Prevention Method for the Clostridium Difficile Associated

Devising a bacteriophage-mediated prevention method for the Clostridium difficile associated diarrhea (Biol 2002-01, Team 1, Spr. 11)

Background & Significance

When a broad-spectrum antibiotic is administered to a patient in a hospital setting, it is done with the intent to kill all of the harmful bacteria. However, there is one bacterium that is very resistant to broad-spectrum treatments—Clostridium difficile. These hardy bacteria can withstand broad-spectrum antibiotics, heating, and drying (Mayer et al. 2008). Once the antibiotic is administered to the patient, the C. difficile remains potent, due to its extreme antibiotic resistance, while the other bacteria within the intestinal tract are killed. This results in an extensive proliferation of C. difficile over the lining of the intestinal tract because there is less competition for nutrients and space. In other words, the entire bowel is subjected to colonization by the C. difficile. The colonization of C. difficile leads to development of pseudomembranous colitis, an infection of the intestinal tract, in many patients. Pseudo-membranous colitis causes explosive diarrhea, abdominal pain, fever, and at times can develop into life- threatening issues such as toxic megacolon. Toxic megacolon is “a life-threatening complication of other intestinal conditions that causes rapid dilation of the large intestine within one to a few days and may also occurs as a complication of inflammatory bowel disease” (Schroeder 2005). The aim of this project is to prevent C. difficile from taking over the intestinal tracts of hospital patients, because it becomes extremely difficult to cure the disease once it has already taken over the bowel. Patients and hospitals alike will benefit from the prevention of pseudomembranous colitis. The recuperation time of infected patients will be shorter, therefore opening up space in hospitals which leads to prosperity within health care facilities. Our proposal will involve the use of a bacteriophage, a virus that infects bacteria. Bacteriophages are either one of two types: lytic or lysogenic. During the lysogenic stage, the virus inserts its genome into the bacterial genome and then enters a dormant state called the prophage. During this stage the virus and the host bacterium live harmoniously. However, when the host bacterium is subjected to a stress factor, the lysogenic virus enters a lytic stage in which it copies and reassembles itself using the bacterial enzymatic machinery. This action leads to the production of many viral copies and ultimately leads to the death of the host bacterial cell. The cell dies because the virus produces enzymes that lyse the bacterium. Then, all the copies of the virus are released and are ready to infect new cells and start a new lysogenic cycle within them. If the lytic cycle of a lysogenic virus could be activated at will to kill the bacterium into which it is inserted and releasing many new infective viruses into its surroundings, then, this virus could be successfully used to our advantage to kill a large population of target bacteria at our discretion and in a specific time range. In order to cause a lysogenic virus to become lytic at our desire, we have devised a method using our gene of interest, the fst toxin gene. This gene, first found in the pAD1 plasmid of the bacterium Enterococcus faecalis, codes for a protein called fst toxin. This protein causes harm to the cell as it binds to the cell membrane and creates channels that make the cell membrane permeable and induce the loss of electrolytes (Christoph et. al 2010). This action ultimately leads to the death of the host cell.

1 The expression of the fst toxin gene can become a stress factor within a bacterium carrying a prophage and thus, it could most likely induce the lytic stage of this virus, which will ultimately kill the cell. This could be achieved by attaching a promoter of a gene whose expression we can control next to the fst toxin gene so that we can induce its expression. Similar mechanisms work by inserting the lac promoter from the lac operon next to a gene of interest and then introducing this construct into an E. coli. The expression of the gene can be mediated by adding lactose to the growth media the E. coli is growing in as this sugar induces the expression of the lac operon. This prompts the lac promoter to initiate the transcription of the gene next to it, which in this case is the gene of interest. However, no evidence indicating the possession of the lac operon by C. difficile was found, therefore, another promoter from an operon used by C. difficile was needed. The xylose operon, which is the analogue of the lactose promoter but for the sugar known as xylose, was found suitable to be used, as it is present in the Clostridium genera. We could join the xylose promoter next to the fst toxin gene and take that gene sequence and introduce it to C. difficile. Then, we could expose these bacteria to xylose so that the fst toxin gene expression is triggered. This expression of the fst toxin gene will harm the bacteria and act as a stressor that ultimately will kill them. Therefore, this combination of the xylose promoter and the fst toxin gene could be considered a suicide construct. Furthermore, if this construct was inserted into the genome of a lysogenic bacteriophage specific for C. difficile, we would have a way to deliver this construct into the bacterial DNA and control its expression. The stress caused by the toxin will trigger the lytic stage of the lysogenic virus and thus the cell will be killed. Since we can trigger the cell death at our own desire, this method can successfully be used to prevent the infection and disease by C. difficile on patients undergoing a broad-spectrum antibiotic therapy. This virus could be administered together with the intake of the antibiotics, infecting any C. difficile they come in contact with inside the intestinal tract. These cells will be killed by the virus after the prophage stage is induced by the deliberate intake of xylose. Many methods could be employed to deliver this suicide construct-carrying virus to the intestinal tract. We propose the use of a non-virulent strain of C. difficile which could be inserted into the intestinal tract in many ways, even inside a pill. This C. difficile will already be carrying the prophage of our suicide construct-carrying virus. Then, by administering xylose, the death of this cell and the release of many infective viruses will be triggered. Such viruses will be ready to infect any wild type C. difficile found within the intestinal tract and will lyse the bacteria after repeated administrations of xylose following the same mechanism. This prevention treatment has many advantages as the use of a few non-virulent cells of C. difficile infected with the prophage of the aforementioned virus could generate many infective copies of such virus. These viruses can, in turn, kill many wild type C. difficile present in the intestinal tract through the mechanism mediated by the addition of only one cheap, non-absorbable and non-toxic reagent: xylose. Many other genes were considered besides the fst toxin gene. We initially considered using the hok gene, which causes similar effects in bacteria but only occurs in gram negative bacterium. Since the fst toxin gene is present on a gram positive bacterium, Enterococcus faecalis, its use is plausible, as C. difficile is also a gram positive. Furthermore, the lambda phage, which infects E. coli, was firstly considered to be the bacteriophage that would carry the suicide construct. However, no evidence was

2 found to conclude that the lambda phage would infect C. difficile, so we chose to use the bacteriophage phi CD119 due to its recent DNA sequencing.

Research Plan

The long-term goal of this research is to devise a prevention method for the infection by Clostridium difficile (and subsequent Clostridium difficile-associated diarrhea, CDAD) in patients undergoing a broad spectrum antibiotic therapy. Our work will center on modifying a lysogenic virus specific for C. difficile by inserting a construct made by joining the xylose promoter of the xylose operon and the fst toxin gene. We hypothesize that if the prophage of this modified virus is already inside C. difficile when such cell is exposed to xylose, the expression of the fst toxin gene will be triggered by this exposure. The fst toxin produced by such exposure to xylose will become a stress factor in the bacterium that will cause the former prophage to enter its lytic cycle, killing the C. difficile as a result. This process will release many infective viruses carrying the aforementioned construct that will attack other C. difficile and lyse them in the presence of xylose. As implied by this procedure, by introducing the aforementioned construct within the genome of this lysogenic bacteriophage, a way of triggering the lytic cycle of this virus at our discretion will be obtained. This could be advantageous as we could safely deliver the prophage of this virus into the intestinal tract inside non-virulent C. difficile and then, trigger the lytic cycle of such virus by exposing the bacteria to xylose. The virus will then replicate, lyse the bacteria and be released into the intestinal tract, ready to infect other C. difficile. Any virulent C. difficle present in the gut at that time will be infected by the virus and due to the exposition of these bacteria to xylose; the lytic cycle of the virus will be triggered, killing these C.dificile. This process gives us a way to prevent or counter the infection of C. difficile inside the intestinal tract without using antibiotics. A well-known lysogenic virus specific for C. difficile is needed to carry out this proposal. Because the bacteriophage phi CD 119 has already been sequenced and analyzed by Govind et al. (2006) and such information about its genome sequence is available, we will use this virus to bring about our proposal. A restriction site to insert the xylose promoter – fst toxin gene construct that does not disrupt the viral ability to replicate, reassemble and lyse its host has to be found. After analyzing the phi CD119 viral genome with the software NEBcutter (Vincze et al. 2003), a unique restriction site for BlpI was found that was within a non-coding region of this genome. Therefore, this restriction site was selected to be the one in which the construct will be inserted as this insertion would not disrupt the viral ability to replicate and assemble itself. Our general hypothesis has been broken-up into the particular postulates stated below that need to be proved in order for our proposal to be successful: 1) The expression of the fst toxin gene can be induced when attached to the xylose promoter in the presence of xylose when this whole construct is inserted into the genome of C. difficile. 2) The fst toxin will effectively act as a stress factor that will trigger the lytic cycle of the lysogenic bacteriophage phi CD119 if this virus is already a prophage within the bacterial genome of C. difficile. 3) A construct containing the xylose promoter sequence and the fst toxin gene sequence can be assembled and inserted into the phage phi CD119 at a specific site without disrupting its replication and assembly.

3 4) If the phi CD119 xylose promoter - fst toxin gene construct-carrying virus is already a prophage within the genome of C. difficile, the lytic cycle of such virus can be triggered by having the bacterium use xylose as its sole carbon source. 5) The infection of multiple C. difficile cells and its subsequent lysis can be attained by mixing such cells with a group of C. difficile cells carrying the prophage of the virus mentioned in #4 and making these cells use xylose as the their only carbon source both in a culture and within a hamster disease model. The achievement of the following specific aims will be necessary to prove all of our particular postulates stated above: Specific aim # 1: Design a construct by joining the xylose promoter (xylA) sequence and the fst toxin gene sequence. This construct must have restriction sites on its ends that will allow it to be later introduced into the genome of the lysogenic virus phi CD119.

Methods for specific aim # 1 Two plasmids, one carrying the fst toxin gene and another carrying the xylose promoter sequence have to be obtained. The plasmid pDAK606 (shown in figure 2), that contains the fst toxin gene sequence, will be obtained from Weaver et. al, and used as a template to isolate and amplify such gene using the polymerase chain reaction (PCR). The plasmid pXYLgusA (shown in figure 3), that contains the xylose promoter sequence (xylA), will be obtained from Girbal et al. and used as a template to isolate and amplify by PCR the sequence of the such promoter. The primer sequences that will flank these two genes will be designed so that the xylA amplified product and the fst gene amplified product have an EcoRI site at the 3’ and 5’ end respectively. In addition, these primers will be designed so that the aforementioned amplified products have a BlpI site at the 5’ end of the xylA sequence and at the 3’ end of the fst gene. Both genes will be joined together with DNA ligase to assemble the construct.

Specific aim #2: Insert the aforementioned construct inside the viral genome of the phage phi CD119 without disrupting the ability of the virus to replicate, reassemble and lyse its host. Infect a culture of C. difficile with this modified virus and induce the lysis of the bacteria by growing them in a xylose media. Provided that the construct worked and was successfully inserted into the genome of the phage phi CD119 without disrupting it, collect such virus from the plaques formed. A schematic drawing of the modified phage phi CD119 is shown in figure 1.

Methods for specific aim # 2: The prophage of the phage phi CD119 within its host C. difficile strain 602 will be obtained from Govind et al. These bacteria will be grown in Brain Heart Infusion (BHI) agar and the lytic cycle of the virus will be induced with the antibiotic mytomicin C, following the procedures used by Govind et al in 2006. The virus will be collected from the plaques. The viral genome will be isolated and separated from the viral proteins and then digested with BlpI. The digested viral genome will be combined with the construct and both, the viral genome and the construct, will be joined with DNA ligase. The solution containing the modified viral genome will be combined with cells of C. difficile strain 602 and its entry to the bacterial cell will be mediated by electroporation. Every functional viral genome should be capable of

4 integrating into the bacterial chromosome on its own as it comes from a lysogenic virus. The bacteria will be grown in BHI agar for many generations and then transferred to a xylose containing media. Viruses containing the construct should be collected from the plaques formed. PCR will be performed targeting the construct sequence on the viruses collected to verify the successful insertion of such construct into the viral genome. The modified viruses carrying the construct will be harvested by repeating the aforementioned procedure.

Specific aim # 3: Create a non-virulent C. difficile culture an expose it to the modified phage phi CD119. Insert these non-virulent bacteria into the intestinal tract of a hamster that has had its intestinal flora removed. Challenge this hamster with a virulent strain of C. difficile and administer xylose to the intestinal tract of the hamster. Observe if the virulent C. difficile population decreases over time due to the infection and lysis by the modified virus. After the non-virulent C. difficile carrying the prophage of the modified virus phi CD119 are exposed to xylose, they should be lysed by this virus and release many copies of it into the intestinal lumen. This virus should then infect the virulent C. difficile and due to the continuous exposition of these cells to xylose, they should also be lysed by the virus.

Methods for specific aim # 3: Produce a non-virulent culture of C. difficile strain 602, by following the methods explained by Kuehne et al. (2010), that is, by using the ClosTron gene knockout system to insert bacterial group II introns within the tcdA and tcdB genes of C. difficile to knock them out. Expose this strain to the modified phage phi CD119 so that this virus can integrate into to the bacterial genome of this mutant C. difficile. Follow the procedure used by Vijayashree Ramesh (1998) to create a hamster disease model in which we will be able to perform the experiment described in our particular postulate #5; that is, setting up a sterile environment for the hamster to live: sterile cage, food, water and bedding. Select a group of these hamsters and remove their intestinal flora from their intestinal tracts with 1ml of clyndamicin (3g/100mg of body weight) intragastrically, that is, by using a line inserted into the hamsters’ stomach, as described by V. Ramesh (1998). Inoculate the intestinal tract of these hamsters with the non- virulent C. difficile strain 602 carrying the modified phi CD119 at different concentrations. Collect the stools of these hamsters and dilute them in 1 ml of saline solution. Analyze these stool samples with real-time polymerase chain reaction (qPCR) targeting the viral genome to determine the correct concentration of administered bacteria at which the colonization the hamsters’ intestinal tract is successful. Once the right concentration is found, administer xylose at different concentrations to these hamsters, collect their stools after each inoculation and do a qPCR to these stools targeting the viral genome to find the concentration of xylose that induces the lysis of the bacteria and the viral release. Repopulate the intestinal tract of the hamsters with the non-virulent C. difficile, challenge these hamsters with virulent C. difficile strain 602 and continuously administer xylose to them intragastrically. Quantify the concentration of the virulent strain of C. difficile against that of the non-virulent strain of C. difficile over time in the hamsters’ stools to evaluate if, after the addition of xylose, the concentration of virulent C. difficile in the hamster’s

5 intestinal tract decreases as expected. At the same time, evaluate the challenged hamster for signs corresponding to the C. difficile associated diarrhea. A flow chart of the research plan is shown in figure 5.

Discussion

Because of its financial and life costs, pseudomembranous colitis caused by C. difficile has been researched extensively. The results of past research projects have given us the confidence that our project proposal will succeed. Though we are positive in our outlook there are several possible issues that we must first address. The vector we choose must successfully lyse C. difficile in an environment (the human intestinal lumen) that has already been subjected to broad spectrum antibiotics. The fst toxin gene is part of a toxin- antitoxin system that is known to be capable of lysing gram-positive bacteria (Christoph et. al 2010). As C. difficile is a gram-positive bacterium, we can safely assume that the fst toxin gene expression will cause harm to C. difficile. We need a procedure that will introduce the bacteriophage to the digestive system without harming the patient or destroying the function of the bacteriophage. Research has been done using Salmonella carrying RNase P ribozyme into the body of a mouse in order to kill Cytomegalovirus. They were able to successfully deliver the RNase P ribozyme into the body using Salmonella that was administered via oral inoculation. Once in the body, enzymes within the bacteria were released into the human macrophage, removing the temperate virus from the genome (Bai et. al 2011). We will imitate this procedure and administer non-virulent C. difficile (carrying the prophage of the virus with the suicidal construct) orally. We can be confident that the bacteria will then arrive safely into the intestine without harming the patient. Once the phage is introduced into the body, we need to induce it so that it enters the lytic cycle and lyses the C. difficile cells. The use of xylose ensures that the lytic cycle will be induced by activating the promoter that controls expression of the fst toxin gene. This will increase the amount of fst toxin, which will then stress the cell (Kok. 1996; Hu et al 2010). Through this stress, the phage will enter its lytic cycle and kill C.difficile. One problem that arises when working with temperate phages is transduction. Normally the prophage is cut from the bacterial genome and is inserted into its already assembled capsid where it becomes infective and can attack another cell (Novick 2010). However, it is possible for a mistake to happen in this process, leading to a portion of bacterial DNA to be cut off from the bacterial genome and inserted inside a viral capsid. The capsid is still infective and transfers this piece of DNA which may carry a beneficial gene into another bacterium when it tries to infect it. This could be detrimental because if this piece of DNA codes for a virulence factor, that factor can be transferred among bacteria making them more virulent or antibiotic resistant. If this were to happen, the transduction process would generate many strains of bacteria that will carry antibiotic resistant or virulence-factor coding genes. Another problem when working with bacteria is the phenomenon of transformation. In this case, with the elimination of many bacteria, the DNA of these bacteria is released into the surrounding area and other bacteria can pick up these DNA fragments and incorporate them into their genomes (Ochman. 2000). If again, these genes code for antibiotic resistance or virulence, this could create harmful bacteria that will be more of a problem than a solution.

6 Using the Basic Local Alignment Search Tool, BLAST, primary biological sequences such as amino acids can be compared to a database of known sequences. A search was performed with the fst toxin and Enterococcus faecalis. Only one other organism besides E. faecalis was found with a similar protein. As seen in figure 4, these two bacteria species are closely related to one another. The ortholog found in Lactobacillus gasseri was found to code for a hypothetical protein similar to the product of the fst toxin gene found in E. faecalis with a score of 42% and 14/33 identities matching. L. gasseri is an anaerobic, gram-positive bacterium found in the gastrointestinal tract of animals and humans. While this score appears low, the hypothetical protein coding sequence is 35 base pairs long, and it is possible that this sequence could match another portion of the genome. Because this is such a small segment and because L. gasseri resides in the gut, gene transfer between the two organisms is possible. L. gasseri is a probiotic commonly found in yogurt. While these bacteria are beneficial to the organism, if such a gene transfer occurs, there is a chance that the bacteria will be destroyed and the lactose cannot be broken down. Such an event could lead to lactose intolerance in the patient to whom the treatment is being administered, and could greatly affect such patient’s diet. This possible outcome would have to be investigated and further research attention must focus on L. gasseri. Since there are multiple strains of disease-causing C. difficile and each is infected by a narrow array of bacteriophages, this treatment will have to be repeated using many viruses (instead of phiCD119) that are lysogenic and specific to C. difficile and several strains of non-virulent C. difficile to carry them. Since we will be testing our genetically modified organism in a hamster, there is the possibility that it will react differently in the human body or not work at all. If the treatment is viable in humans we would have to do further research on how often patients would need to take the treatment to make it effective. A risk for the patients involved in this experiment is the rate at which the C. difficile is killed. If it is lysed too quickly, toxins contained inside the bacteria will be released into the intestinal tract, causing a harmful build-up of toxins in the intestinal tract. Such a problem would amplify the symptoms the patient exhibits from the original infection. Killing all of the bacteria at once could be disastrous and cause numerous complications for the patients. This issue could be addressed by combining the treatment with “developing therapies that aim to neutralize the toxins, including the use of antibodies and toxin-absorbing agents such as Tolevamer.” (Mayer et. al 2008) However, if the C. difficile is not killed quickly enough, there will be time for the C. difficile to repopulate the gut, making it very difficult to cure the patient. Nevertheless, if the treatment is administered early as a preventative measure, no infection should arise, leaving the patient unharmed. If our project does not work as a prevention method, we could clone and harvest the products of the genes that code for the proteins holin and endolysin. These are enzymes that permeabilize the bacterial cell wall and cause cell lysis. These genes will be isolated from a virus lytic to C. difficile and cloned inside E. coli, following the procedure described by Mayer et. al (2008). We will isolate the holin/endolysin genes from the viral genome and insert them into a plasmid. This plasmid would then be inserted into E. coli and the expression of the genes of interest will be induced. This expression will lead to the harvest of products of these genes. These products will then be isolated and collected and turned it into a pill to be used as a preventive treatment method. When a patient takes the pill, the endolysin and holin proteins would be released inside the intestinal tract. If there were C. difficile in the patient’s digestive

7 tract, the holins and endolysins would bind to the bacterial cell walls, permeabilizing them and causing the cell death. Since we are using living organisms, hamsters, we will need to ensure the well- being of these animals. We will monitor the health of the hamsters as a result of our experiment both before and after the procedure. If the experiment were successful, and the hamsters were able to survive after being subjected to C. difficile, they will be exposed again to the pathogen some time later in absence of the treatment to confirm that it was the treatment what saved them. Had the treatment been successful, these hamsters would most likely die due to a second infection of C. difficile. Although this raises ethical questions, the benefit from these studies is worth the death of the test subjects; many lives and dollars can be saved if we can find a prevention method for the CDAD. Assuming this project is successful, the next step would be to start testing of this procedure on humans. Future studies should investigate the management of potential treatment complications, such as the buildup of harmful toxins or recolonization of C. difficile. Additionally, if this project can prevent the infection of one species of harmful bacteria, more work could be done to decide if this method could be used on other bacterial infections. Perhaps we could use this method to effectively kill other harmful bacteria species in the intestine (or elsewhere in the body) in a safe and cost-effective way.

References

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9 Figures

Figure 1. Modified phi CD119 virus carrying the xylA promoter – fst toxin gene construct. As seen from figure 1, the xylA promoter sequence (1166 bp) and the fst toxin gene (400bp) have been joined together using an EcoRI site. BlpI sites were inserted by PCR to both ends of the suicide construct so that it could be inserted within one of the many BlpI sites present in the viral genome.

10 Figure 2. Schematic drawing of the plasmid pDAK606. This plasmid is going to serve as a template to obtain the fst toxin gene. It has spectinomycin resistance gene as a selectable marker.

Figure 3. Schematic drawing of the plasmid pXYLgusA. This plasmid is going to serve as a template to obtain the xylose promoter-operator sequence (xylA). It has two selectable markers, one for ampicillin and another for erythromycin

Figure 4. Phylogenetic tree of various bacteria species found in the intestinal tract. This shows the genomic relationship between these species and serves as an divergent evolutionary map.

12 Figure 5. Flow chart of the research plan.