Bile salts modulate expression of the CmeABC multidrug efflux pump in Campylobacter jejuni

Abstract

CmeABC, a multidrug efflux pump, is involved in the resistance of Campylobacter jejuni to a broad spectrum of antimicrobial agents and is essential for Campylobacter colonization in animal intestine by mediating bile resistance. Previously, we have shown that expression of this efflux pump is under the control of a transcriptional repressor named CmeR. Inactivation of CmeR or mutation in the cmeABC promoter (PcmeABC) region derepresses cmeABC, leading to overexpression of this efflux pump. However, it is unknown if the expression of cmeABC can be conditionally induced by the substrates it extrudes. In this study, we examined the expression of cmeABC in the presence of various antimicrobial compounds. Although the majority of the antimicrobials tested did not affect the expression of cmeABC, bile salts drastically elevated the expression of this efflux operon. The induction was observed with both conjugated and unconjugated bile salts and was in a dose- and time-dependent manner. Experiments using surface plasmon resonance demonstrated that bile salts inhibited the binding of CmeR to PcmeABC, suggesting that bile compounds are inducing ligands of CmeR. The interaction between bile salts and CmeR likely triggers conformational changes in CmeR, resulting in reduced binding affinity of CmeR to PcmeABC. Bile did not affect the transcription of cmeR, indicating that altered expression of cmeR is not a factor in bile-induced overexpression of cmeABC. In addition to the CmeR-dependent induction, some bile salts (e.g., taurocholate) also activated the expression of cmeABC by a CmeR-independent pathway. Consistent with the elevated production of CmeABC, the presence of bile salts in culture media resulted in increased resistance of Campylobacter to multiple antimicrobials. These findings reveal a new mechanism that modulates the expression of cmeABC and further support the notion that bile resistance is a natural function of CmeABC.

FIG. 1.

Induction of cmeABC expression by bile salts in C. jejuni 81-176. (A) Effects of various bile salts on the transcription of cmeABC in 81-176, as measured by transcriptional fusion (JL110). For the LacZ assay, JL110 was grown in the absence (control) of bile salt or presence of the following bile salts at the concentrations sublethal to Campylobacter: 2 mg/ml of cholate (CA), 2 mg/ml of deoxycholate (DOC), 2.5 mg/ml of taurodeoxycholate (TDC), 5 mg/ml of glycocholate (GCA), 3 mg/ml of chenodeoxycholate (CDC), 25 mg/ml of taurocholate (TCA), and 2.5 mg/ml of choleate. The bars represent the means ± standard deviations of triplicate samples from a single representative experiment. (B) Immunoblot analysis of CmeB and CmeC production in 81-176 grown in the absence (lane 2) or presence of cholate with final concentrations of 2 mg/ml (lane 3) and 1 mg/ml (lane 4). The same number of bacterial cells (based on optical density at 600 nm) was loaded in each lane. Prestained protein molecular mass markers (Bio-Rad) are shown in lane 1. The positions of CmeB and CmeC are indicated by arrows. Two different blots, immunostained with anti-CmeB (top) and anti-CmeC (bottom) antibodies, are shown.

FIG. 2.

Dose-dependent (A) and time-dependent (B) induction of the cmeABC operon by cholate. The data are presented as the ratios of LacZ activity between JL110 with cholate and the same strain grown without bile salts. Each bar represents the average LacZ ratio from triplicate samples.

FIG.3.

SPR analysis of CmeR interaction with immobilized P_cmeABC. (A) CmeR (104 nM) was preincubated with sodium choleate at various concentrations, including 0 μg/ml (- -), 2 μg/ml (•), 16 μg/ml (▪), and 125 μg/ml (×). The preincubated mixture (30 μl) was injected at a flow rate of 20 μl/min. (B) Sodium choleate at 0 μg/ml (- -), 2 μg/ml (•), 16 μg/ml (▪), and 125 μg/ml (×) was injected just after the association phase by using the “co-inject” Biacore procedure. The arrow indicates the time of injection of sodium choleate. Each experiment was replicated at least three times, and all yielded similar results. This figure shows the results from one representative experiment.

FIG. 4.

CmeR-dependent and -independent induction of cmeABC by bile salts. JL110 and JL111 grown in MH broth only or with 1 mg/ml cholate (CA) or 12.5 mg/ml taurocholate (TCA) were used for the LacZ assay. The genetic backgrounds and the culture conditions are illustrated at the bottom. Each bar represents the mean LacZ activity ± standard deviation of triplicate samples.