Quorum sensing and the regulation of multicellular behaviour in Yersinia pseudotuberculosis

Wiechmann, Anja (2016) Quorum sensing and the regulation of multicellular behaviour in Yersinia pseudotuberculosis. PhD thesis, University of Nottingham.

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Abstract

Yersinia pseudotuberculosis is a human enteropathogen and is the direct ancestor of Yersinia pestis, the causative agent of plague. For its pathogenicity Y. pseudotuberculosis harbours a virulence plasmid (pYV) encoding genes for a type-III-secretion system (T3SS) which inhibits host phagocytosis and inflammatory responses.

Y. pseudotuberculosis regulates key phenotypes, including T3S, motility, aggregation and biofilm formation on Caenorhabditis elegans via quorum sensing (QS). This involves two LuxR/I type QS systems termed YpsR/I and YtbR/I and multiple N-acylhomoserine lactone (AHL) signal molecules. Recently, the T3S system was shown to promote biofilm formation on the surface of the nematode Caenorhabditis elegans at 22ºC and in turn QS and flagellar regulatory components were found to repress the T3S system via a regulatory hierarchy.

To further expand this network, this study then set out to investigate the regulation of the aggregation phenotype at 37ºC. Aggregation experiments using the parent and QS mutants revealed that aggregation is downregulated by QS, since aggregation was enhanced in strains lacking ypsI/ytbI and ypsR/ytbR. Cultures lacking pYV or treated with proteinase K did not aggregate, suggesting that QS is regulating aggregation by repressing a pYV encoded and surface located aggregation factor(s). Tn5 transposon mutagenesis of pYV together with targeted mutagenesis of the key structural T3S injectisome components YscF and YscV revealed that aggregation is attenuated when cells are unable to express the injectisome, demonstrating that the aggregation factor is the T3S injectisome and that QS reduces its expression.

N-Acetyl-D-glucosamine (GlcNAc) is known to play diverse roles in bacterial cells for signalling pathways that impact on virulence and is the major component of the extracellular matrix of Yersinia biofilms. The repressor of the GlcNAc metabolism, NagC, may also impact on QS in Y. pseudotuberculosis since it was found to bind to the ypsR promoter in pull-down experiments. By using QS gene and nagC promoter lux-fusions, NagC was revealed as a repressor of both ytbI and ypsI, as an early activator of ypsR and as an activator of ytbR at 37ºC. It was further demonstrated that there is reciprocal regulation by QS. These observations were extended by phenotypic assays showing that NagC represses the aggregation phenotype and may affect T3S and motility by regulating QS. In contrast the biofilm phenotype appears to be directly regulated by NagC. Given that QS promotes biofilm formation, the upregulation of ypsI and ytbI in a nagC mutant should not lead to reduction of biofilm. However, biofilm formation in the nagC mutant was attenuated and addition of 5 mM GlcNAc could restore this phenotype, suggesting that attenuation of biofilm formation is caused by an enhanced degradation of external GlcNAc by the nag-operon.

Addition of GlcNAc or N-acetylmuramic acid to strains containing QS gene promoter lux fusions repressed QS gene expression, revealing that Y. pseudotuberculosis may modify its behaviour in presence of other bacteria by sensing cell wall turnover.

Taken together these results demonstrate that GlcNAc and/or NagC impacts on T3S and injectisome mediated aggregation by regulating QS and that the injectisome of the T3SS is the aggregation factor, which may help and protect the bacteria in the early stages of host infection.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Atkinson, S.
Williams, P.
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 31691
Depositing User: Wiechmann, Anja
Date Deposited: 06 Jul 2016 15:02
Last Modified: 18 Oct 2017 03:14
URI: https://eprints.nottingham.ac.uk/id/eprint/31691

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