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<h2 class="tb-post-title tb-f48-lg tb-m0">Mutagenesis Protocol</h2>
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<p>The promise of phage engineering is growing as it has the potential to enhance a phage’s antimicrobial activity for treatment while being able to overcome some of the more fundamental problems with phage practicality such as its rate of replication, host-range, and binding-receptor domains. The need for synthetic, low-cost DNA sequencing methods offers much promise in solving many of these problems in years to come, but have not been able to successfully provide a simple method for engineering phages with desirable traits. </p>
<br>
<p>This hasn’t stopped the use of phage therapy for special, chronic infections in the form of “alternative medicine” as the first use of engineered phage was utilized to treat disseminated Mycobacterium abscessus infection in a 15 year old cystic fibrosis patient. The phage killed the pathogen by eliminating the bacteria’s repressor gene. What was profound about this was that the mechanism of phages to destroy the bacteria directly came from targeting their resistance mechanisms directly, which is critical in increasing not just their sensitivity, but also eliciting a better immune response. In fact, the same CF patient saw an increased 32 weeks of higher tolerance to bacterial infections in that species cluster. </p>
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<p>Thanks to a new process called mutagenesis, we now don’t need to identify and engineer our desirable traits into phages, but we can now let the phages build these traits by applying constraints to them. Mutagenesis is the natural tendency of an organism to change its genetic code in response to stressors in the environment, and this is the same inherent system that bacteria and phages have used to fight each other for generations, and is also the same thing that antibiotics lack: the ability to adapt and change. Mutagenesis is especially key in increasing the phage’s host range. Let’s see how this fits in with our phage vaccine product. </p>
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<p>We’re trying to solve a phage’s host range, and this is quite tricky because phages are tied to their host primarily because of their binding agents that are on the cell surface receptors in order to recognize hosts and initiate an infection. Since bacteria are easily able to mutate the sites that phages latch onto, phages undergo mutagenesis to make changes in their binding agents. As a result, most phages are composed of multiple, unrelated phages that collectively target a range of receptors and distribute the selective pressure away from any individual phage receptor. This makes it harder for bacteria to defend against phage infections, and is synonymous to the faster rate of mutation that bacteriophages are known for compared to bacteria. Despite this, the wide array of targets that these phages have naturally are not as effective at actually targeting and stopping a wide variety of pathogenic bacterial infections. </p>
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<p> This prompts the question of whether we can change or alter a phage to bind to a desired host.A study used targeted mutagenesis on the minor coat of a phage (essential for host recognition) and found this to be a successful approach. The researchers used something similar for an antibody’s epitope of binding regions that they could engineer in vast amounts of host-diversity for antigens. </p>
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<p> The place of interest for using mutagenesis is the phage’s tail fibers. These tails give the phage their iconic shape and are capable of specific recognition of bacterial surfaces during the first step of viral infection.</p>
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<p> The epitope recognition region of phages is directed by three regions called complementary determining regions on the tail proteins. Mutations in these regions can alter the target specificity of the antibody. Several phages like the T7 have tail fibers with similar regions which can be directly engineered through mutagenesis to increase their host-range, and this has already been done quite successfully for treating E. Coli patients. </p>
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<p> This prevents the need for expensive phage cocktails, and allows a single phage to become a library for multiple phage host targets. What this means is that for some phages like the T3 phage, you can increase the host range so that the phage can target naturally occuring phage-resistant bacteria mutations and prevent the onset of phage resistance. </p>
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<p> Identifying these Host Range Determining Regions can be done through simple observation of the shape and homology. This prompts the idea of using homologous recombineering, which uses the structure and shape of a specific target to make genetic modifications on the organism. Through this, we can sequence mutations in these domains and identify their relation to host-range using Phage display. </p>
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<p> A T3 mutation is designed selectively for T3-resistant bacteria only, meaning that the T3 can naturally mutate its host-range by being introduced to resistant bacteria. While this process is occurring, scientists found that introducing wild-type bacteria (a gene or phenotype that represents a bacterial species) will add pressure on the T3 to mutate and eliminate the bacteria in the process. This allows the T3 to now create new pathways that will attach on the bacteria with these wild-types. </p>
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<p> Think of it like sending innocent civilians in between a war. The army that sees the approaching waves of families will now have to change their objective to not just eliminating the enemy army, but also tending for the displaced families. Although grim, it paints a good picture of how selective pressure works in favor of the phage and can expedite the process of increasing a phage’s host range to not just inside of its species, but across a wide variety of species. Although unproven, this could potentially have the ability to make 1 phage a universal cure to many bacterial infections. That’s the goal we’re building up to, and we’re doing so with mutagenesis through homologous recombineering. </p>
<br>
<p> What’s great about recombineering is that it is time efficient. The primary mutations that bacteria use to defend against foreign enemies are structural changes to their LPS (LPS mutations are changes in the receptor on the bacteria’s outside), and these mutations occur just after a few hours. Through random selection, unique mutations can be formed for the individual HRDRs and create more unique phage mutations to increase the host range.</p>
<br>
<p> This can be scaled up for mass production simply by turning the mutated sites into plasmids in order to selectively amplify the gene mutations that are important using PCR. You can now screen certain mutations and make the sequences accessible by contributing to a library and were found to yield productive phage mutants in receptor binding.</p>
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<p> For phages that don’t have this tail, scientists can engineer phages with the tail fiber gene from a library of plasmids encoding these homologs with a known host-range. They will then evolve through chemical mutagenesis where the phages that have the greatest capacity to transduce are used for the host.</p>
<p> This method is also critical in mapping residues for receptor recognition and determining which amino-acid substitutions lead to host-range alterations, providing a rich tool for structural biology of phae receptor recognition. </p>
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<p> Furthermore, homologous recombineering allows for mutagenesis to not just increase host-range, but also possess different inhibiting mechanisms against bacterial resistance mechanisms. This also applies for enzymes like dispersin B in the T7 phage which can deliver biofilm-degradation through the enzyme. The engineered phage T7 expressed DspB gene of Actinobacillus actinomycetemcomitans derived by the T7 φ10 promoter, which can be recognized by T7 RNA polymerase, therefore can significantly reduce bacterial count in a single-species E. coli biofilm than the T7 phage control did. The same T7 was used to interfere with quorum sensing and inhibit biofilm formation, one of the primary resistance mechanisms. </p>
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<p>The problem with this system however is that it is very difficult in many cases to identify desirable bacteriophages that can actually lyse a bacteria when infected. We feel that phage-derived enzymes directly from virion-associated lysins, endolysin, and deploymerace can be used to lyse bacteria and can be used with any phage through intraperitoneal inoculation. The benefit of this is that they can lyse a wide range of species rather than strains. Several studies have exploited the fact that host range is linked to tail fiber composition for some phages. One scientist Yoichi genetically modified a T2 phage by swapping the long tail fiber genes (gp37 and gp38) with those from phage PP01, which specifically targets E. coli O157:H7. The exchange was done by homologous recombination between the genome of phage T2 and a plasmid carrying two regions of homology, flanking the gp37 and gp38 genes of PP01. As DNA synthesis, sequencing, and genome engineering tech will become more efficient, it will significantly expedite the possible host range. </p>
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