Characterization of the structure-function relationship of Vibrio cholerae toxin ParE2

Student thesis: Master's Thesis

Abstract

Toxin-antitoxin (TA) systems are prevalent present in bacteria and archaea. TA systems are small operons that contain an antitoxin gene and a toxin gene. The antitoxin gene encodes an antitoxin that neutralizes the toxin’s activity. Whereas toxin gene encodes for a toxin that inhibits cell growth by binding to its cellular target. Type II TA system is the most studied one, in which both toxin and antitoxin are proteins. Antitoxin neutralizes the toxin by forming a complex.
Suppression of the operator region mainly happens by the binding of TA complex. This is not always the case, in parDE of RK2 plasmid the N- terminal domain of antitoxin on its own can suppress the operator region. Three homologue parDE loci of RK2 plasmid can be found in the superintegron of Vibrio cholerae (Vc) chromosome II. Both RK2 ParE2 and VcParE2 are in vitro Gyrase poisoners. But the mechanism by which VcParE2 inhibits Gyrase is still unknown.
71 single alanine mutants are screened to find which residues of VcParE2 might be important in its toxicity. Unfortunately, only W25A is not toxic in NEB5-a competent cells, but is still toxic in BL21(DE3). Therefore larger fragments of VcParE2 are mutated. α1-helix, loop (WGK25-27>AA), loop loop (ENPDIGKSC46-54> KAIAE) and loop (GSH67-69> AA) are the only four mutants that are not toxic in NEB5-a and BL21(DE3) competent cells. These mutants are attempted to be purified, only loop (WGK25-27>AA) was successfully purified.
The results suggest that W25 might be an essential amino acid in VcParE2 toxicity which is also predicted by ConSurf analysis. This amino acid is located in α1-α2 hairpin and might be the interaction region of VcParE2. This hairpin is shorter in RelE toxins, which are members of the RelE/ParE superfamily. This probably explains why both toxins of the same superfamily have distinct functions.
Date of Award1 Feb 2023
Original languageEnglish
Awarding Institution
  • Department of Bio-engineering Sciences
  • Vrije Universiteit Brussel
SupervisorRemy Loris (Promotor) & Yana Girardin (Advisor)

Keywords

  • Toxin-antitoxin
  • Gyrase
  • gyrase poison
  • Structural biology

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