Major primary bile salts repress Salmonella enterica serovar Typhimurium invasiveness partly via the efflux regulatory locus ramRA

Abstract

Bile represses Salmonella enterica serovar Typhimurium (S. Typhimurium) intestinal cell invasion, but it remains unclear which bile components and mechanisms are implicated. Previous studies reported that bile inhibits the RamR binding to the ramA promoter, resulting in ramA increased transcription, and that ramA overexpression is associated to decreased expression of type III secretion system 1 (TTSS-1) invasion genes and to impaired intestinal cell invasiveness in S. Typhimurium. In this study, we assessed the possible involvement of the ramRA multidrug efflux regulatory locus and individual bile salts in the bile-mediated repression of S. Typhimurium invasion, using Caco-2 intestinal epithelial cells and S. Typhimurium strain ATCC 14028s. Our results indicate that (i) major primary bile salts, chenodeoxycholate and its conjugated-derivative salts, cholate, and deoxycholate, activate ramA transcription in a RamR-dependent manner, and (ii) it results in repression of hilA, encoding the master activator of TTSS-1 genes, and as a consequence in the repression of cellular invasiveness. On the other hand, crude ox bile extract and cholate were also shown to repress the transcription of hilA independently of RamR, and to inhibit cell invasion independently of ramRA. Altogether, these data suggest that bile-mediated repression of S. Typhimurium invasion occurs through pleiotropic effects involving partly ramRA, as well as other unknown regulatory pathways. Bile components other than the bile salts used in this study might also participate in this phenomenon.

Keywords: RamR; Salmonella; Typhimurium; bile; intestinal; invasion; ramA; regulation.

Figure 1

Effects of crude bile extract at 25.6 g/L or of individual bile salts at 5 mM on ramA and hilA expression and on adhesion to/invasion of Caco-2 intestinal epithelial cells. Transcript levels of ramA (A) and hilA (B) were determined using qRT-PCR for the WT S. Typhimurium strain 14028s. Values were normalized by those obtained for control (LB alone). Bars represent the standard deviation from three independent replicates. (C) Adhesion to and (D) invasion of Caco-2 cells by the WT strain 14028s. Results are expressed relative to values obtained for control (LB alone), arbitrarily set at 100%. Bars indicate the percentage of attached/internalized bacteria ± standard error of the mean. In all panels, asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001). □ Controls (LB alone), crude bile extract, chenodeoxycholic acid-derived salts (CDC, chenodeoxycholate; TCDC, taurochenodeoxycholate; GCDC, glycochenodeoxycholate) and cholic acid-derived salts (C, cholate; DC, deoxycholate; TC, taurocholate; GC, glycocholate).

Figure 2

qRT-PCR analysis of the dependence on ramR of bile and individual bile salts effects on ramA and hilA expression. Transcript levels of ramA (A) and hilA (B) were determined for the WT S. Typhimurium strain 14028s strain and for its ramR deletion mutant, complemented or not with a pramR plasmid, after growth in the presence of crude bile extract at 25.6 g/L or of individual bile salts at 5 mM. Bars represent the standard deviation from three independent replicates. □ Controls (LB alone), crude bile extract, chenodeoxycholate (CDC), cholate (C), and deoxycholate (DC). Asterisks indicate significant differences (NS, non-significant; *P < 0.05, **P < 0.01, ***P < 0.001). ND, not determined.

Figure 3

In vitro analysis of the dependence on the ramRA locus of bile effects on adhesion to/invasion of intestinal epithelial cells. Adhesion to and invasion of Caco-2 cells was analyzed after growth, in the absence (□, –) or presence (, +) of crude bile extract at 25.6 g/L, of the WT S. Typhimurium strain 14028s and its ramRA::kan deletion mutant complemented with a pramA plasmid. Bars indicate the percentage of attached/internalized bacteria ± standard error of the mean. Asterisks indicate significant differences (***P < 0.001).

Figure 4

Bile-mediated regulatory network controlling SPI-1 gene expression. Only the key regulators and relevant proteins in this context are shown. Bile acids (C, cholic acid; CDC, chenodeoxycholic acid) directly bind to RamR alleviating its transcriptional repression of ramA. This leads to ramA overexpression and concomitantly repression of hilA and overexpression of the AcrAB-TolC efflux pump. The precise regulatory pathway between ramA n and SPI-1 remains unknown. See main text for further details and Lou et al. (2019) for a review.