Exploring the role of the iron sulphur cluster regulator IscR in Yersinia

Brooks, Clair Rose (2021) Exploring the role of the iron sulphur cluster regulator IscR in Yersinia. PhD thesis, University of Nottingham.

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Abstract

Yersinia pseudotuberculosis is an enteropathogen that is transmitted through contaminated food or water and results in self-limiting fever and gastroenteritis. Y. pseudotuberculosis is closely related to Yersinia pestis, the cause of bubonic, pneumonic and septicaemic plague. Despite causing vastly different diseases, virulence and in particular, type three secretion (T3S) as well as biofilm formation, motility, and aggregation are controlled in both species by several interrelated regulatory systems including quorum sensing (QS). The latter depends on an N-acylhomoserine lactone (AHL) system that incorporates two AHL synthases (YpsI and YtbI) and two LuxR-type response regulators (YpsR and YtbR).

Recently a novel component of this network has emerged, the iron-sulfur cluster regulator, IscR. IscR is a transcription factor best understood for its role in regulating the formation of Fe-S cluster containing proteins in E.coli. It is now known that in Y. pseudotuberculosis IscR regulates type three secretion, a key virulence mechanism employed by pathogenic Yersinia spp. to inject effector proteins into host cells, which have a range of effects including dampening the immune response and inducing apoptosis.

Considering the links between QS, T3S and other QS mediated phenotypes, this study set out to investigate how IscR contributes to this regulatory network, by creating a series of knock-out mutants in Y. pseudotuberculosis and Y. pestis. Yop secretion assays confirmed that IscR regulates T3S in Y. pseudotuberculosis and using chromosomal promoter:lux this fusions was found to be via action on yscW-lcrF and lcrF specific promoters. Further Yop assays showed that IscR’s effect on Yop secretion was lost in a QS response regulator gene mutant background, suggesting that IscR may further regulate T3S via the QS system. This was further supported by promoter:lux fusion data that showed that IscR positively regulated the expression of ypsR and ytbR. However, IscR did not affect expression of either of the AHL synthase genes (ypsI and ytbI). Whether IscR affects the production of AHL signalling molecules remains inconclusive.

Phenotypic characterisation of the mutants showed that IscR did not affect colony morphology, growth rate or biofilm formation on C. elegans in Y. pseudotuberculosis or Y. pestis. Nor did IscR have an effect on the iron scavenging abilities or motility of Y. pseudotuberculosis. The iscR mutant did show attenuation of biofilm formation on glass at 22oC, which was not influenced by QS-mediated repression. Mutating iscR also resulted in faster killing in a louse infection model which has been linked to dysregulation of biofilm production within the louse gut. A further link between IscR and T3S was identified through auto-aggregation, as this is significantly reduced in an iscR mutant. Interestingly, this trend was also observed in QS mutant backgrounds, which did not correspond to levels of Yops secreted, suggesting an alternative mechanism of regulation of auto-aggregation separate from T3S which is IscR-dependent.

Considering these results, the regulation of T3S by IscR has been confirmed, and there is strong evidence for a regulatory link between IscR and QS. This places IscR as a key regulator of many virulence associated phenotypes, including T3S, QS, biofilm formation and aggregation. As a virulence regulator IscR could be a future target for alternative antimicrobial therapies, a necessity given the threat of multidrug resistance and the classification of Y. pestis as a re-emerging pathogen.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Atkinson, Steve
Williams, Paul
Keywords: Iron sulphur cluster regulator, IscR, Yersinia
Subjects: Q Science > QR Microbiology > QR 75 Bacteria. Cyanobacteria
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 65291
Depositing User: Brooks, Clair
Date Deposited: 04 Aug 2021 04:41
Last Modified: 04 Aug 2021 04:41
URI: http://eprints.nottingham.ac.uk/id/eprint/65291

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