Engineering MS2 Phage Lysis Protein for DnaJ Independence and Enhanced Lytic Efficiency Against E. coli

Abstract

Phage therapy offers promising advantages over antibiotics, but bacterial resistance remains a significant hurdle. In the evolutionary arms race between bacteria and phages, synthetic biology provides tools to give phages a strategic advantage. MS2, a single-stranded RNA phage that infects Escherichia coli, relies on host proteins like the DnaJ chaperone to properly process its lysis protein (L protein). E. coli may mutate this chaperone to avoid lysis, rendering the phage ineffective. This project aims to engineer the L protein of MS2 with two main goals: (1) to eliminate its dependence on the host chaperone DnaJ, and (2) to increase the speed and efficiency of bacterial lysis. Using structural modeling, protein engineering, and selection of beneficial mutations, we will attempt to evolve a phage that is more resilient to bacterial resistance and more efficient in killing its host.

Introduction

The MS2 bacteriophage is a small RNA virus that infects E. coli via attachment to the F-pilus. After infection, its RNA is translated directly into viral proteins, including the lysis protein (L), which is essential for bacterial cell wall breakdown and phage release. However, the L protein requires correct folding, potentially facilitated by the bacterial chaperone DnaJ. Mutations in DnaJ can interfere with this process, allowing E. coli to resist phage infection.

Given this challenge, we propose to enhance the MS2 phage's ability to kill E. coli by mutating the L protein. Our approach focuses on two directions: (1) redesigning the L protein so that it no longer depends on DnaJ or other host chaperones, and (2) engineering the L protein for faster pore formation or membrane integration, thereby increasing the lysis rate. These strategies aim to prevent E. coli from acquiring resistance through chaperone mutations and to improve the overall efficacy of phage lysis.

Goal:

Our goal for this part of the homework is to create mutants of L-protein that affect its lysis activity and/or its interaction with DNAj. Making a mutation for L-protein without a way to computationally predict what happens to lysis or its interaction with DNAj is hard. So we are going to try various hypotheses on how to use the models from last week and also try a few other tools. These mutants will be tested in the lab.

OPTION 2: Follow the below pipeline to engineer the L-protein

1. Mutagenesis using Protein Language Models [Easiest One]
   1. Designing these mutants with good computational confidence is hard. It will show you limitations of some of the structure based models. Ultimately you can pick various combinations of mutations and get lab results and then decide to pick the next round of mutations. But this assay won’t be easy to run at scale in this class. So using the information below you can either make a best guess or you can use the strategy Allan was talking about during recitation. Contact Manu or Allan if you need one on one help.
   2. Run this notebook to generate for each position in the amino acid sequence, a “score” for what would happen to the protein if you mutated into another amino acid. It can be positive or negative for the protein. We want to identify possible mutations that are “positive” If you run this notebook - you will see a .CSV file in the sidebar. You can download it and look at it in the google sheets if that’s easier

TOP MUTATION

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The mutated sequence is: ['M', 'E', 'T', 'R', 'F', 'P', 'Q', 'Q', 'S', 'Q', 'Q', 'T', 'P', 'A', 'S', 'T', 'N', 'R', 'R', 'R', 'P', 'F', 'K', 'H', 'E', 'D', 'Y', 'P', 'C', 'R', 'R', 'Q', 'Q', 'R', 'S', 'S', 'T', 'L', 'Y', 'V', 'L', 'I', 'F', 'L', 'A', 'I', 'F', 'L', 'S', 'K', 'F', 'T', 'N', 'Q', 'L', 'L', 'L', 'S', 'L', 'L', 'E', 'A', 'V', 'I', 'R', 'T', 'V', 'T', 'T', 'L', 'Q', 'Q', 'L', 'L', 'T']

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