Montfort-Gardeazabal, Jorge M.
(2017)
Investigation of the effect of insertional mutation in agr and sigB loci in Clostridium difficile R20291.
PhD thesis, University of Nottingham.
Abstract
Clostridium difficile has become the major cause of healthcare acquired diarrhoea in the world. The disease caused by this pathogen is largely mediated by the production of toxins. The C. difficile genome contains an incomplete accessory gene regulator (agr) locus, designated agr1. In Staphylococcus aureus, it has been shown that the agr locus regulates virulence factors through Quorum Sensing (QS). In addition to the incomplete agr1 locus found in all of the C. difficile strains sequenced to date, the strains belonging to the BI/NAP1/027 group possess an additional agr operon, designated agr2. These strains have been found to produce higher toxin levels and a more severe disease in patients. The studies described in this thesis have shown that the two agr systems present in the Clostridium difficile BI/NAP1/027 strain R20291 are involved in the regulation of spore formation and toxin production. The agr1 locus (agrB1, agrD1) is responsible for the production of the autoinducing peptide (AIP) AgrD1, while the second agr2 locus (agrC, agrA, agrB2, agrD2) mediates production of the AgrD2. Initial mutational analysis using a University of Nottingham isolate (R20291 NM) suggested that AgrD1 both positively regulates sporulation and negatively regulates toxin production, whereas AgrD2 positively regulates toxin production, but negatively regulates spore formation.
It was subsequently discovered that strain R20291 NM exhibited significantly different phenotypes to R20291 BW. The former possessed a single polar flagellum, whereas R20291 BW was peritrichously flagellated. The NM strain exhibited impaired motility and increased biofilm formation, demonstrated different growth rates, produced greater quantities of toxins and exhibited a relative delay in the onset of sporulation compared to R20291 BW. The equivalent agr mutants made in R20291 BW indicated that the regulatory control exerted by AgrD1 in sporulation was broadly the same, while AgrD2 does not seem to play any role in the regulation of spores, contrasting with observations made in the agrB2 mutant created in R20291 NM. Surprisingly, the effects of the agr mutants made in R20291 BW on toxin production were opposite to those observed in the NM strain.
The subtle differences in the behaviour of the two R20291 isolates was most likely due to the presence of mutations in R20291 NM revealed by whole genome sequencing. Of the four genes affected, a mutation in the anti-sigma factor RsbW, was considered the most likely culprit. An investigation of its possible role in toxin production and sporulation was therefore undertaken through complete ClosTron-mediated inactivation of rbsW in both R20291 isolates and the creation of a sigB ClosTron mutant in R20291 BW. The sporulation and toxin production phenotypes of the rbsW mutants of the two strains mirrored that of their respective agr2 mutants. Thus, inactivation of rbsW brought forward the onset of sporulation in R20291 NM, but had no effect on the initiation of sporulation in R20291 BW, while toxin production in R20291 NM was reduced, but increased in R20291 BW. Motility and biofilm formation in the rbsW mutants of both strains was unaffected. These data suggest that interference with the RbsW/SigB interaction preferentially affects AgrD2-mediated regulatory processes. In contrast, however, inactivation of SigB in R20291 BW caused a delay in the initiation of sporulation, but did not affect toxin production. Mutation of sigB, however, did not affect motility or biofilm formation, although, in common with other bacteria, the resistance of the R20291 BW sigB mutant to oxidative stress was reduced.
The picture that emerges is of a complex regulatory interrelationship between the two agr QS systems and SigB in this important nosocomial pathogen, a relationship that has been subtly subverted in strain R20291 NM. These findings emphasise the importance of knowing the genome sequence of strains under investigation if valid conclusions are to be drawn.
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