Marie-Christine Maurel

Bile Salts Modulate Expression of the CmeABC Multidrug Efflux Pump in Campylobacter jejuni

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.

Novel pathways in gonadotropin receptor signaling and biased agonism

Gonadotropins play a central role in the control of male and female reproduction. Selective agonists and antagonists of gonadotropin receptors would be of great interest for the treatment of infertility or as non steroidal contraceptive. However, to date, only native hormones are being used in assisted reproduction technologies as there is no pharmacological agent available to manipulate gonadotropin receptors. Over the last decade, there has been a growing perception of the complexity associated with gonadotropin receptors’ cellular signaling. It is now clear that the Gs/cAMP/PKA pathway is not the sole mechanism that must be taken into account in order to understand these hormones’ biological actions. In parallel, consistent with the emerging paradigm of biased agonism, several examples of ligand-mediated selective signaling pathway activation by gonadotropin receptors have been reported. Small molecule ligands, modulating antibodies interacting with the hormones and glycosylation variants of the native glycoproteins have all demonstrated their potential to trigger such selective signaling. Altogether, the available data and emerging concepts give rise to intriguing opportunities towards a more efficient control of reproductive function and associated disorders.

Partially Deglycosylated Equine LH Preferentially Activates β-Arrestin-Dependent Signaling at the Follicle-Stimulating Hormone Receptor

Deglycosylated FSH is known to trigger poor Galphas coupling while efficiently binding its receptor. In the present study, we tested the possibility that a deglycosylated equine LH (eLHdg) might be able to selectively activate beta-arrestin-dependent signaling. We compared native eLH to an eLH derivative [i.e. truncated eLHbeta (Delta121-149) combined with asparagine56-deglycosylated eLHalpha (eLHdg)] previously reported as an antagonist of cAMP accumulation at the FSH receptor (FSH-R). We confirmed that, when used in conjunction with FSH, eLHdg acted as an antagonist for cAMP accumulation in HEK-293 cells stably expressing the FSH-R. Furthermore, when used alone at concentrations up to 1 nM, eLHdg had no detectable agonistic activity on cAMP accumulation, protein kinase A activity or cAMP-responsive element-dependent transcriptional activity. At higher concentrations, however, a weak agonistic action was observed with eLHdg, whereas eLH led to robust responses whatever the concentration. Both eLH and eLHdg triggered receptor internalization and led to beta-arrestin recruitment. Both eLH and eLHdg triggered ERK and ribosomal protein (rp) S6 phosphorylation at 1 nM. The depletion of endogenous beta-arrestins had only a partial effect on eLH-induced ERK and rpS6 phosphorylation. In contrast, ERK and rpS6 phosphorylation was completely abolished at all time points in beta-arrestin-depleted cells. Together, these results show that eLHdg has the ability to preferentially activate beta-arrestin-dependent signaling at the FSH-R. This finding provides a new conceptual and experimental framework to revisit the physiological meaning of gonadotropin structural heterogeneity. Importantly, it also opens a field of possibilities for the development of selective modulators of gonadotropin receptors.

Topography of equine chorionic gonadotropin epitopes relative to the luteinizing hormone and follicle-stimulating hormone receptor interaction sites

In order to localize the epitopes of equine chorionic gonadotropin (eCG) involved in interaction with luteinizing hormone (LH) and follicle-stimulating hormone (FSH) receptors, we used 14 monoclonal anti-eCG antibodies (mAbs). Different effects of these mAbs on the bioactivities of eCG were observed in in vitro bioassays, but the effects of each mAb on the two bioactivities were similar for all but four mAbs. All mAbs were found to inhibit the binding of eCG to LH receptors except 3A3 mAb, in radioreceptor assay. Six mAbs, which were strong inhibitors of eCG binding to LH receptors and of both bioactivities, recognized the same area on the alpha subunit of eCG. All others, except 3A3, recognized epitopes close to the former, and close to each other. 3A3 mAb had a hyperstimulatory effect on FSH bioactivity, and was the only mAb that did not inhibit binding. It appeared to recognize a different epitopic area. These observations suggest that there is a main antigenic area on eCG, which corresponds to the interaction site of eCG with both receptors. It mostly involves the alpha subunit and to a lesser extent the beta subunit.

Binding of the RamR Repressor to Wild-Type and Mutated Promoters of the ramA Gene Involved in Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium

ABSTRACT The transcriptional activator RamA is involved in multidrug resistance (MDR) by increasing expression of the AcrAB-TolC RND-type efflux system in several pathogenic Enterobacteriaceae. In Salmonella enterica serovar Typhimurium (S. Typhimurium), ramA expression is negatively regulated at the local level by RamR, a transcriptional repressor of the TetR family. We here studied the DNA-binding activity of the RamR repressor with the ramA promoter (PramA). As determined by high-resolution footprinting, the 28-bp-long RamR binding site covers essential features of PramA, including the −10 conserved region, the transcriptional start site of ramA, and two 7-bp inverted repeats. Based on the RamR footprint and on electrophoretic mobility shift assays (EMSAs), we propose that RamR interacts with PramA as a dimer of dimers, in a fashion that is structurally similar to the QacR-DNA binding model. Surface plasmon resonance (SPR) measurements indicated that RamR has a 3-fold-lower affinity (KD [equilibrium dissociation constant] = 191 nM) for the 2-bp-deleted PramA of an MDR S. Typhimurium clinical isolate than for the wild-type PramA (KD = 66 nM). These results confirm the direct regulatory role of RamR in the repression of ramA transcription and precisely define how an alteration of its binding site can give rise to an MDR phenotype.

Repeated superovulation using a simplified FSH/eCG treatment for in vivo embryo production in sheep

This study investigated the efficacy of a simplified repeated superovulation treatment (eCG plus FSH in a single dose, rather than the usual protocol of six decreasing doses of FSH) in the in vivo embryo production in Ojalada donor ewes during the breeding season. In vitro viability after vitrification and warming of embryos recovered from both treatments was also assessed. In addition, the study examined the effects of the concentration of anti-eCG antibodies before each eCG/FSH treatment on in vivo embryo production. Thirty-eight females at the end of their reproductive lives were given the decreasing (n = 19) or simplified (n = 19) superovulatory treatment up to three times at intervals of ≥ 50 d. The onset of estrus was 5 h earlier (P < 0.05) among ewes that received the eCG/FSH protocol (25.2 ± 0.80 h) than it was among those that received the decreasing superovulatory treatment (30.1 ± 1.0 h), but the two treatments did not differ significantly in ovulation rates or the number and viability of embryos recovered. Both of the superovulatory protocols were significantly (P < 0.05 to P < 0.01) less effective after the first application. After three superovulatory treatments, the average number of viable embryos per ewe was 14.1 ± 2.3 and 13.7 ± 2.5 in the decreasing and simplified protocols, respectively. High anti-eCG antibody concentrations just before the superovulatory treatment with eCG/FSH were associated with a significant decrease (P < 0.05) in the rates of fertilization, viability, and freezability, especially in the second and third recoveries. Repeated superovulatory treatments with eCG/FSH can provide an efficient means of producing high quality embryos in the ewes of endangered breeds at the end of their reproductive lives, although further studies are needed to characterize the response associated with high concentrations of anti-eCG antibodies.

Bile-mediated activation of the acrAB and tolC multidrug efflux genes occurs mainly through transcriptional derepression of ramA in Salmonella enterica serovar Typhimurium

OBJECTIVES In Salmonella Typhimurium, the genes encoding the AcrAB-TolC multidrug efflux system are mainly regulated by the ramRA locus, composed of the divergently transcribed ramA and ramR genes. The acrAB and tolC genes are transcriptionally activated by RamA, the gene for which is itself transcriptionally repressed by RamR. Previous studies have reported that bile induces acrAB in a ramA-dependent manner, but none provided evidence for an induction of ramA expression by bile. Therefore, the objective of this study was to clarify the regulatory mechanism by which bile activates acrAB and tolC. METHODS qRT-PCR was used to address the effects of bile (using choleate, an ox-bile extract) on the expression of ramA, ramR, acrB and tolC. Electrophoretic mobility shift assays and surface plasmon resonance experiments were used to measure the effect of bile on RamR binding to the ramA promoter (PramA) region. RESULTS We show that ramA is transcriptionally activated by bile and is strictly required for the bile-mediated activation of acrB and tolC. Additionally, bile is shown to specifically inhibit the binding of RamR to the PramA region, which overlaps the putative divergent ramR promoter, thereby explaining our observation that bile also activates ramR transcription. CONCLUSIONS We propose a regulation model whereby the bile-mediated activation of the acrAB and tolC multidrug efflux genes occurs mainly through the transcriptional derepression of the ramA activator gene.

Regulation of fructooligosaccharide metabolism in an extra‐intestinal pathogenic Escherichia coli strain

A gene cluster involved in the metabolism of prebiotic short‐chain fructooligosaccharides (scFOS) has recently been identified in the extra‐intestinal avian pathogenic Escherichia coli strain BEN2908. This gene cluster, called the fos locus, plays a major role in the initiation stage of chicken intestinal colonization. This locus is composed of six genes organized as an operon encoding a sugar transporter and enzymes involved in scFOS metabolism, and of a divergently transcribed gene encoding a transcriptional regulator, FosR, belonging to the LacI/GalR family. To decipher the regulation of scFOS metabolism, we monitored the fos operon promoter activity using a luciferase reporter gene assay. We demonstrated that the expression of fos genes is repressed by FosR, controlled by catabolite repression and induced in the presence of scFOS. Using electrophoretic mobility shift assays and surface plasmon resonance experiments, we showed that FosR binds to two operator sequences of the fos operon promoter region. This binding to DNA was inhibited in the presence of scFOS, especially by GF2. We then propose a model of scFOS metabolism regulation in a pathogenic bacterium, which will help to identify the environmental conditions required for fos gene expression and to understand the role of this locus in intestinal colonization.

Assessment LOPU-IVF in Japanese sika deer (Cervus nippon nippon) and application to Vietnamese sika deer (Cervus nippon pseudaxis) a related subspecies threatened with extinction

In mammals, recovery of oocytes by laparoscopic ovum pick-up (LOPU) coupled with in vitro production (IVP) of embryos represents a promising strategy for both amplification and genetic management of sparse animals from captive endangered wild species. As integrated technique developed mainly for domestic livestock, LOPU-IVP requires several studies to set up protocols for follicular stimulation or optimization of IVP before envisaging successful transposition to wild species. In deer, many endangered subspecies would be potentially concerned by applying such an approach using common subspecies for protocols optimization. The aim of the present study was to assess efficiency of follicle stimulation using ovine FSH (oFSH) for recovery of oocytes by LOPU in common sika deer (Cervus nippon nippon) before transposition of an optimized methodology for IVP of embryos from endangered Vietnamese sika deer hinds (Cervus nippon pseudaxis). In common sika deer, two doses of oFSH (0.25 and 0.5 U) and two frequencies of administration (12 and 24 h) were compared by monitoring of subsequent ovarian response, quality of oocytes recovered by LOPU, and in vitro developmental competence. In a first experiment, the dose of oFSH administered did not significantly affect the total number of follicles aspirated per hind per session (8.6 ± 1.0 vs. 8.2 ± 1.6 with 0.5 vs. 0.25 U oFSH, respectively; not significant). In a second experiment, frequency of 0.25 U oFSH administration did not affect ovarian response. Efficiency of IVP determined on blastocysts rates after in vitro maturation, fertilization, and development in oviduct epithelial cells coculture was increased when FSH was administered at 12-h intervals. Immune response after several follicular stimulations was detected against exogenous oFSH in plasma from the majority of sika deer hinds but was not associated with decreased ovarian response. When 0.25 U oFSH was administered at 12-h intervals to Vietnamese sika deer (N = 4), good quality cumulus oocyte complexes with complete and compact cumulus investments were recovered allowing a high cleavage rate after in vitro maturation and fertilization. Development to the blastocyst stage occurred in a high proportion (30% of oocytes) after coculture with ovine epithelial cells allowing cryobanking of transferable embryos from Vietnamese sika deer. These results confirm that LOPU-IVF after ovarian stimulation with oFSH may be a successful tool for cryobanking transferable embryos from endangered sika deer subspecies.