University of Cambridge
Investigating the DNA damage response induced by Typhoid Toxin
Abstract
dc:description.abstractTyphoid toxin is produced by the antimicrobial resistant human pathogen Salmonella Typhi that causes 27 million cases of typhoid fever per year and chronic infections, which retain the pathogen in the population. The toxin is secreted by intracellular Salmonella into the extracellular milieu from where it enters bystander cells and causes DNA damage through its putative nickase activity. Purified typhoid toxin causes typhoid fever symptoms and mortality in animal models, and in animal infection models the toxin facilitates chronic infections by Salmonella. However, it is neither understood how the toxin manipulates the cellular DNA damage response (DDR) nor how this promotes infection. The kinases ATM and ATR are the master regulators of DNA damage response and both lead to the phosphorylation of Histone 2AX at S139 (γH2AX). ATM initiates repair of double strand breaks mainly in G1 phase of the cell cycle, while the ATR repair pathway is activated by single stranded DNA (ssDNA) occurring mainly in S phase due to stalled replication forks. The ssDNA sensor RPA binds to ssDNA to activate the ATR pathway marked by phosphorylation of ATR substrates H2AX, CHK1 and RPA itself. This study found that typhoid toxin induces a canonical DDR with γH2AX accumulating in foci and functioning as a hub for recruiting downstream DNA repair proteins. Strikingly however, a large proportion of cells did not contain γH2AX foci. Instead, novel γH2AX localisation was observed at the nuclear periphery (γH2AX rings) that failed to recruit DNA repair factors. This signified a non-canonical DDR that became the main focus of this project. This toxin-induced γH2AX ring pathway was shown to be replication-dependent: it was exacerbated in S phase, marked by an activated ATR pathway and persistent hyper-phosphorylation of RPA. RPA foci accumulated in cells with γH2AX rings indicating that the cellular pool of RPA might be overwhelmed by toxin-induced damage. Indeed, depletion of RPA by siRNA- mediated knock-down was sufficient to induce ring formation. Conversely, ring formation induced by the toxin was impeded by over-expression of RPA. Thus, the results suggest that toxin-induced damage causes replicative stress by exhausting the cellular pool of RPA. The γH2AX ring pathway was found to drive cells into a senescence-like state, a phenomenon associated with ageing. Induction of senescence via the toxin increased the burden of infection by Salmonella. This study provides evidence of a new virulence mechanism by revealing a novel DDR that leads to cellular senescence, which promotes infection. The work suggests that S. Typhi deploys the toxin to induce local ageing that primes cells for infection, which may be relevant to chronic disease caused by a globally important pathogen.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2019
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Ibler, Angela Erna Maria
- Advisor dc:contributor.advisor
-
- Humphreys, Daniel
Subjects
dc:subject × 7Rights
dc:rightsIdentifiers
dc:identifier.*- Author Identifier
-
0000-0003-1492-9972
0000-0002-1038-2538 - OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/292064