Source: CORNELL UNIVERSITY submitted to
ENGINEERING BACTERIOPHAGES TO OVERCOME HOST RESISTANCE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
Reporting Frequency
Annual
Accession No.
1014314
Grant No.
2018-67030-27383
Project No.
NYC-143532
Proposal No.
2016-11603
Multistate No.
(N/A)
Program Code
A1801
Project Start Date
Dec 15, 2017
Project End Date
Dec 31, 2020
Grant Year
2018
Project Director
Nugen, S. R.
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Food Science
Non Technical Summary
Phages have been used for the decontamination of foods such as meat and produce in order to reduce foodborne illness. While phages have evolved to become near-perfect predators of bacterial pathogens, the pathogens themselves rely on evolution to avoid infection form the phages.Our central hypothesis is that the scales of the evolutionary arms race between phages and their bacterial hosts can be tipped by human intervention using genetic engineering.We will engineer the E. coli-specific bacteriophage T4 to avoid two host resistance mechanisms.1) Multiple tail fibers: While T4 naturally contains six identical tail fibers, a variant containing mixed tail fibers from other phages targeting different epitopes on the same E. coli will be engineered. This will then require multiple surface mutations on E. coli to avoid adsorption.2) Engineering a T4 gol mutant to avoid the Lit abortive infection system: While T4 can evade most of the E. coli resistance mechanisms, the abortive infection mechanism Lit can prevent expression of T4 genes during late-stage infection. This has been shown to be facilitated an interaction between the Lit protease and gol site in the T4 capsid gene.At the conclusion of this project we will have demonstrated the potential to engineer bacteriophages to avoid host resistance. The result will significantly improve our ability to detect, remediate, and treat specific bacteria.
Animal Health Component
0%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7115010104034%
7115010108033%
7115010110133%
Goals / Objectives
Overall hypothesis and goal: Our working hypothesis is that bacteriophages can be genetically engineered to evade several of the host resistance mechanisms. Many of the methods bacteriophages have developed to evade the host resistance have been identified in numerous phage isolates. In this project, we will genetically engineer the E. coli specific bacteriophage T4 with two strategies to overcome resistance. The result will be a bacteriophage with super-virulence towards E. coli. The phage can then be further tailored for a bacteriophage-based detection system, or to decontaminate E. coli from processing equipment or surfaces. Advances in genetic engineering will allow rapid progress and testing of our hypothesis.Specific objectives: In order to test our hypothesis, we engineer a T4 bacteriophage to resist two of the primary host resistance mechanisms. While the natural mutations of the surface epitopes can prevent a bacteriophage from binding, the Lit protease system can prevent T4 late-stage gene expression resulting in an abortive infection.Multiple tail fibers: While T4 naturally contains six identical tail fibers, a variant containing mixed tail fibers from other phages targeting different epitopes on the same E. coli will be engineered. This will then require multiple surface mutations on E. coli to avoid adsorption.Engineering a T4 gol mutant to avoid the Lit abortive infection system: While T4 can evade most of the E. coli resistance mechanisms, the abortive infection mechanism Lit can prevent expression of T4 genes during late-stage infection. This has been shown to be facilitated an interaction between the Lit protease and gol site in the T4 capsid gene.
Project Methods
Genetic engineering of T4 phage: The genetic engineering of T4 will follow the procedures outlined by Ando et. al, 2015 (Figure 1). Briefly, the entire genome of T4 will be PCR amplified (10kb - 12kb fragments) including overlapping regions of homology (50-100 bp). The portion of the genome where the gene insertion/edit will take place will not be included in the PCR fragments. That fragment will be replaced with a synthetically derived construct (Genscript®, Piscataway, NJ) containing our gene of interest and overlapping regions of homology. The PCR fragments along with the synthetic fragment, will be co-transformed into yeast along with a Yeast Artificial Chromosome (YAC) with a tryptophan selectable marker. Using homologous recombination, the yeast will assemble the fragments and YAC which can then be isolated following growth. The purified YAC (including T4 genome) will then be purified and transformed into E. coli 10G which will initiate the infection and replication cycle of the engineered T4. Although, the missing wild type PCR fragment at the location of the gene insertion results in 100% mutants,selected plaques will be PCR-verified for the gene of interest.Mixed Tail fibers: Tail fibers from three E. coli O157:H7-infecting phages will be introduced into T4, replacing its current tail fiber which does not allow adsorption onto O157:H7. Constructs containing tail fiber genes (gp37 equivalent) from phages RB27,AR1,and TP7will be designed and then synthesized commercially. We will engineer and test T4 phages with the following tail fiber configurations: (1) 100% AR1, (2) 100% TP7, (3) 100% RB27, (4) 50% AR1; 50% TP7, (5) 50% AR1; 50% RB27, (6) 50% TP7; 50% RB27, and (7) 33% AR1; 33%TP7; 33% RB27. The genes will use the same promoter and the ribosome binding site will be designed such that the cumulative expression of tail fiber genes is similar to wild type T4, and the individual expressions are all equivalent.Determination of E. coli O157:H7 mutation rates for resistance: We will determine the rate at which the host is able to mutate from susceptible to resistant during prolonged exposure to the engineered T4 phages. Methods for this determination were first introduced in the 1940's and have had only slight modifications.Similar methods have been adapted for determining the mutation rates for antimicrobial resistance. We will use the Fluctuation Test to determine the host mutation rate.Briefly, a culture of known concentration is infected with the bacteriophage and the infection is allowed to proceed. The culture is then plated and visible colonies (resistant bacteria) are then quantified. The overall mutation rate can be calculated by the po method which analyses the proportion of cultures without mutants, or the mean method where the mean number of mutants is determined. Both values will be calculated and compared.Genetic engineering of T4 phage: T4 will be genetically engineered using the same technique as described in Specific Aim 1. In this aim, the gol region of the T4 capsid gene will be engineered to avoid the E. coli abortive infection (Abi) system's Lit protease. An engineered gol site have previously been demonstrated on a E. coli transformed plasmid to avoid the Lit protease activation. Unfortunately, this has not been demonstrated in an engineered T4 and therefore its performance in a phage infection has not been demonstrated.Therefore, we will engineer the T4 capsid gene with the identified mutation. A genetic construct will be designed and commercially obtained. The construct will consist of the engineered gol site with 50-100 bp flanking regions. The T4 genome will be PCR amplified (10-12kb fragments) and will exclude the wild type gol site which then requires the incorporation of the engineered construct for full recombination. The yeast-based recombination and verification will then be performed as described in Specific Aim #1.Avoidance of the Lit protease: Then engineered and verified T4 phage will then be shown to avoid the abortive-infection Lit activation in specified E. coli. This will be performed using the wilt type T4 as a control. E. coli known to contain the Lit protease defense, and a control E. coli which does not contain this defense will be used in the validation.

Progress 12/15/17 to 12/14/18

Outputs
Target Audience:The target audience for this reporting period is food safety professionals in academia, industry, and government. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Students were trained in genetic engineering methods and hoe to present their projects to a scientific audience. Additionally, students received training on science communication by presenting to a broader audience including non-technical listeners. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We plan to have completed the genetic engineering of T4 using a combination of homologous recombination and CRISPR Cas9 engineering. We will then have demonstrated a change in host range using a panel of E. coli.

Impacts
What was accomplished under these goals? The objective of the project was to overcome host resistance and allow bacteriaophages to infect a broader range of bacteria. The project started by selecting the ideal tail fibers to use given the latest research. Here we decided to engineer T2 and T6 tail fibers (adhesins) into a T4 bacteriophage. The genetic designs were made and the students began their project design and training.

Publications