Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Biochemistry
Non Technical Summary
Despite technological advances, microbial contamination is constant threat to the food industry. Over 50 million cases of food poisoning occurs in the United States annually, resulting in over a 100,000 hospital visits and several thousand fatalities. Cooking usually kills most pathogens. However, changes in lifestyle have increased demand for processed and ready-to-eat food, increasing the chances of food-borne pathogens. Processed meat, poultry, vegetables and dairy are carriers of common food-borne pathogens such as Salmonella, Listeria, Campylobacter and Escherichia and have been subject to product recalls several times in recent years. Besides health and well-being, microbial contamination also plays a major role in spoilage of food which is estimated at a staggering 40% of all food produced, adding a huge financial burden. As food travels increasing longer distances from farm to fork, the food industry is facing the challenge of ensuring product quality and shelf life over longer time frames. The threat of microbial contamination is highly exacerbated by the emergence of antibiotic resistant strains. Solving the antibiotic resistance crisis is one of the grand challenges of our time.We need to fundamentally rethink our approach to treating bacterial contamination. Ideally, we seek an antimicrobial solution that transcends these limitations and co-evolves with bacteria to remain effective as bacteria eventually evolve resistance to antibiotic drugs. Nature provides an elegant solution: bacteriophages (or 'phages'). Phages are viruses that specifically invade and destroy bacteria without harming eukaryotic cells. Discovered in 1915, phages were immediately recognized for their therapeutic potential and used to fight infectious diseases (i.e., 'phage therapy'), most notably in Eastern Europe. The arrival of penicillin--the first safe antibiotic in the late 1930's--was followed by a series of other molecular antibiotic discoveries that dampened interest in phage-based treatment of bacterial infections in humans. The availability of multiple new classes of antibiotics in the following decades enabled physicians to treat infections with a new class of antibiotics when the current drug was deemed ineffective. However, this is no longer sustainable. This research to bring phages to the forefront of antimicrobials agents against food-borne pathogens. By doing so, we will develop a broad technology platform for designing customized phages to kill pathogenic microbes using synthetic biology tools.
Animal Health Component
60%
Research Effort Categories
Basic
20%
Applied
60%
Developmental
20%
Goals / Objectives
The objective of this research is to bring phages to the forefront of antimicrobials agents against food-borne pathogens. We will develop a broad technology platform for designing customized phages to kill pathogenic microbes using synthetic biology tools. As a proof-of-principle, we will design phages specifically against E. coli O157:H7 which is a enterohemorrhagic strain resident in ruminant animals. The beauty of our approach is that it can be broadly applied to kill other pathogens as well. Engineered phages can also be used for microbiome editing, pathogen detection, gene delivery and biofilm disruption.Aims: 1) To develop a yeast-based system to assemble recombinant phage genomes2) To modulate T7 phage host range by high-throughput tail fiber protein redesign3) To improve efficacy of engineered T7 phages and minimize bacterial resistance
Project Methods
Aim 1) To develop a yeast-based system to assemble recombinant phage genomesPhage infection is initiated by the phage adsorbing on to the surface of the host bacteria. The tail fiber protein at the distal tip of a phage plays a central role in host recognition and specificity. To engineer phages, we need a facile method to assemble new phage genomes. Bacteria are an unsuitable host for assembling obligate lytic phage genomes because the assembled phage particles would lyse the host. Homologous recombination in yeast has been used to assemble large pathways, and phage genomes. We are developing yeast as a non-infectable cloning host to assemble phage genomic libraries. Using a yeast-bacteria shuttle vector we assemble and store the genomic libraries in yeast. Phages can be subsequently "rebooted" by transfer into a susceptible host, like E. coli. Our initial results suggest that we can assemble a T7 phage genome (~40kb) by assembling eight 5kb fragments in yeast. Our preliminary data shows that approximately 40% of the yeast colonies (out 30 screened colonies) are positive based on a PCR screen of a single junction of the fragments.Aim 2) To modulate T7 phage host range by high-throughput tail fiber protein redesignWe show that a T7 phage whose native tail fiber is replaced with that of a T3 phage, adopts the host range of a T3 phage. Having established that the tail fiber is necessary and sufficient to switch host specificity (for the strains tested), we hypothesize that T7 could be made to infect non-native hosts by redesigning or replacing its tail fiber with variants that can adsorb on other hosts. We propose to develop a process we call host-agnostic propagation to facilitate creation of large amounts of designer phage with alternative tail fibers. Designer phages will be genetically engineered to include an operator to allow in trans repression. The genome of the designer phage will then be electroporated into 10G E. coli and will be capable of rebooting and initiating a normal replication cycle in the cell.During replication, the designer tail fiber is repressed in trans, while a wild type T7 tail fiber will be provided in trans. Progeny phage assemble with the wild type T7 tail fiber while maintaining the designer tail fiber in the genome. These phages will be propagated repeatedly using this in trans scheme to high titer. In the final replication cycle phages are propagated a single time without in trans repression or the T7 wild type tail fiber, resulting in a high titer of progeny designer phage expressing the new tail fiber. With this process, we expect to generate large amounts of designer phage agnostic of the targeted host for the phage toward the creation of unbiased libraries of phage variants. To change T7 specificity by high-throughput tail fiber engineering, we will build a large library of T7 variants by evolutionary recombination of thousands of natural tail fibers. Since tail fibers of natural phages sample large microbial diversity, we expect that by 'mixing-and-matching' them with T7 tail fiber, we can expand the host range of T7. Shuffling sequence homologs has been highly successful in changing the specificity of enzymes and protein-protein interactions. Only phage variants that can infect and lyse the host amplify. This is a simple and robust selection that can be linked to deep sequencing of the unselected and selected libraries to identify successful design variants.Aim 3) To improve efficacy of engineered T7 phages and minimize bacterial resistanceOnce we find initial 'hits', we will further evolve efficacy in order for the phage to be able to kill the pathogen at low phage dosage. The evolved phages will be efficacious at a lower multiplicity of infection (fewer phages required per bacterial cells) and absorb faster. As an empirical estimate, we will evolve engineered T7 phages through additional rounds of mutagenesis and selection to kill E. coli O157:H7 at 100-to-1000 fold greater efficacy than initial hits. As with traditional antibiotics, bacteria develop resistance to phage. Bacteria reduce their susceptibility to phage attack by mutating bacterial genes required for phage propagation. Phages typically bind to bacterial receptors or lipopolysaccharides expressed on cell surface. We hypothesize that bacterial resistance could be fully suppressed by the use of multiple phages each targeting a different receptor. We will build phage cocktails comprising three T7 variants and determine the frequency.