Modified VapBC Toxin-Antitoxin System for Plasmid Stabilization
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Solution Overview
Problem
Shigella sonnei and Shigella flexneri virulence plasmids, essential for virulence, are prone to high rates of loss during in vitro growth, complicating studies on host-pathogen interactions and vaccine development due to the instability of the plasmid pINV.
Innovation Solution
A modified VapBC toxin-antitoxin system with specific amino acid substitutions in VapB, such as Q12, F6, F51, and F60, is introduced to enhance plasmid stability in bacterial hosts, preventing pINV loss and maintaining virulence plasmid retention.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the wild-type VapBC toxin-antitoxin system is used, then the plasmid can be maintained through post-segregational killing, but the plasmid is still lost at high rates during in vitro growth
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the VapB antitoxin protein. Specific residues (Q12, F6, F51, F60) are substituted to alter the stability characteristics of the toxin-antitoxin system. These parameter changes in the protein structure enhance plasmid retention by modifying how the system responds to cellular conditions during growth, thereby reducing plasmid loss without changing the fundamental PSK mechanism.
2Reliability
If the VapB antitoxin is made more stable against Lon protease degradation, then plasmid retention is improved, but the toxin suppression becomes too strong and may prevent necessary plasmid dynamics
Solution Approach 1:
The patent applies local quality by making targeted modifications at specific locations within the VapB protein structure. Rather than uniformly stabilizing the entire protein, only specific amino acid residues (Q12, F6, F51, F60) are modified to enhance resistance to Lon protease. This localized approach allows the protein to maintain appropriate stability dynamics - sufficiently stable to suppress toxin and maintain plasmid, yet capable of controlled degradation when needed for plasmid loss and bacterial adaptation.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The modified VapBC system significantly stabilizes the virulence plasmid, ensuring its retention in bacterial populations at both environmental and host temperatures, facilitating studies on host-pathogen interactions and vaccine design by reducing plasmid loss.
Implementation Method 1
The antitoxin VapB is labile to degradation by Lon protease and less stable than the toxin VapC. The modification to VapB comprises or consists of a substitution of at least one of Q12, F6, F51 and F60 residues relative to Shigella sonnei wild-type VapB
Implementation Method 2
The toxin VapC is characterised by a PIN (PilT N-terminus) domain, which is a compact RNA-binding domain that co-ordinates one or more divalent metal ions and is essential for endoribonuclease activity. Once released from suppression by VapB, the Shigella spp. VapC specifically cleaves the prokaryote initiator tRNA, tRNA formyl methionine (tRNAfMet), thus inhibiting protein translation and halting cell growth.
Data Source
AI summary
The invention related to an isolated plasmid encoding a bacterial VapBC toxin-antitoxin system, wherein the VapBC toxin-antitoxin system comprises a modified VapB amino acid sequence arranged to increase the maintenance of the plasmid in a bacterial host, wherein the modification to VapB comprises or consists of a substitution of at least one of Q12, F6, F51 and F60 residues relative to Shigella sonnei wild-type VapB or Shigella flexneri wild-type VapB, or structurally equivalent residues of Shigella sonnei wild-type VapB or Shigella flexneri wild-type VapB in a homologue thereof; and associated methods, uses and host cells.