Quorum sensing has an unexpected role in virulence in the model pathogen Citrobacter rodentium

Abstract

The bacterial mouse pathogen Citrobacter rodentium causes attaching and effacing (AE) lesions in the same manner as pathogenic Escherichia coli, and is an important model for this mode of pathogenesis. Quorum sensing (QS) involves chemical signalling by bacteria to regulate gene expression in response to cell density. E. coli has never been reported to have N-acylhomoserine lactone (AHL) QS, but it does utilize luxS-dependent signalling. We found production of AHL QS signalling molecules by an AE pathogen, C. rodentium. AHL QS is directed by the croIR locus and a croI mutant is affected in its surface attachment, although not in Type III secretion. AHL QS has an important role in virulence in the mouse as, unexpectedly, the QS mutant is hypervirulent; by contrast, we detected no impact of luxS inactivation. Further study of QS in Citrobacter should provide new insights into AE pathogenesis. As the croIR locus might have been horizontally acquired, AHL QS might exist in some strains of pathogenic E. coli.

Figure 1

Wild-type Citrobacter rodentium, but not a croI mutant, produces N-acylhomoserine lactone signalling molecules. (A) AHL production by strains of C. rodentium: (i) DBS100 (wild type); (ii) IF232 (croI); (iii) DBS100 (pBluescript; vector control); (iv) IF232 (pBluescript); (v) DBS100 (pSJC45; croI in trans); (vi) IF232 (pSJC45), as detected using LIS and CV026 biosensor strains. (B) Production of AHL activity throughout growth by DBS100 (WT) and IF232 (croI). AHL activity was detected using LIS or CV026 (reported as area of pigmentation induced in the sensor) and growth was measured as OD₆₀₀. Results for one representative culture are shown; those from duplicate cultures were essentially identical. (C) Analysis of different AHL-like signalling molecules produced by C. rodentium. Cell-free supernatant samples from DBS100 (wild type), IF232 (croI), Escherichia coli DH5α (pBluescript) and E. coli DH5α (pSJC45) were subjected to ethyl acetate extraction and separation by thin-layer chromatography. AHL-like signalling molecules were detected using LIS and CV026 overlays. The ‘standards' sample contained 50 μM BHL, HHL and OHHL, and the positions of these standards are indicated. (Note that twice the amount of sample was loaded for the DBS100 and IF232 samples on the CV026 plate compared with the other samples and the LIS plate.) AHL, N-acylhomoserine lactone; BHL, N-butanoyl-L-homoserine lactone; HHL, N-hexanoyl-L-homoserine lactone; OD, optical density; OHHL, N-3-oxohexanoyl-L-homoserine lactone.

Figure 2

Schematic representation of the croIR locus of Citrobacter rodentium DBS100. Numbers refer to lengths in base pairs, and the position of the transposon insertion in IF232 (after base pair 280 of croI) is indicated with an asterisk. Orf, open reading frame.

Figure 3

Surface attachment of Citrobacter rodentium is modulated by quorum sensing. (A) The ability of C. rodentium strains DBS100 (wild type, WT), IF232 (croI mutant) and TJE2 (luxS mutant) to adhere to the wells of a 96-well microtitre plate was determined in the presence and absence of 2 μM BHL, after incubation for either 40 h at 25°C (grey bars) or 20 h at 37°C (black bars). (B) Attachment of DBS100 (WT), KJR025 (croR mutant), IF232 (croI) and KJR026 (croI, croR) after incubation for 20 h at 37°C. Attachment was quantified using crystal violet staining and is expressed as A595; bars show mean±s.d. (n=3). BHL, N-butanoyl-L-homoserine lactone.

Figure 4

Morbidity curves of C3H/heJ mice infected with wild-type Citrobacter rodentium (DBS100) or the croI mutant (IF232).