Fabrice Neiers

Functional roles of the sweet taste receptor in oral and extraoral tissues

Purpose of review This review summarizes and discusses the current knowledge about the physiological roles of the sweet taste receptor in oral and extraoral tissues. Recent findings The expression of a functional sweet taste receptor has been reported in numerous extragustatory tissues, including the gut, pancreas, bladder, brain and, more recently, bone and adipose tissues. In the gut, this receptor has been suggested to be involved in luminal glucose sensing, the release of some satiety hormones, the expression of glucose transporters, and the maintenance of glucose homeostasis. More recently, the sweet taste receptor was proposed to regulate adipogenesis and bone biology. Summary The perception of sweet taste is mediated by the T1R2/T1R3 receptor, which is expressed in the oral cavity, wherein it provides input on the caloric and macronutrient contents of ingested food. This receptor recognizes all the chemically diverse compounds perceived as sweet by human beings, including natural sugars and sweeteners. Importantly, the expression of a functional sweet taste receptor has been reported in numerous extragustatory tissues, wherein it has been proposed to regulate metabolic processes. This newly recognized role of the sweet taste receptor makes this receptor a potential novel therapeutic target for the treatment of obesity and related metabolic dysfunctions, such as diabetes and hyperlipidemia.

Disulfide Bond Formation and Cysteine Exclusion in Gram-positive Bacteria

Most secretion pathways in bacteria and eukaryotic cells are challenged by the requirement for their substrate proteins to mature after they traverse a membrane barrier and enter a reactive oxidizing environment. For Gram-positive bacteria, the mechanisms that protect their exported proteins from misoxidation during their post-translocation maturation are poorly understood. To address this, we separated numerous bacterial species according to their tolerance for oxygen and divided their proteomes based on the predicted subcellular localization of their proteins. We then applied a previously established computational approach that utilizes cysteine incorporation patterns in proteins as an indicator of enzymatic systems that may exist in each species. The Sec-dependent exported proteins from aerobic Gram-positive Actinobacteria were found to encode cysteines in an even-biased pattern indicative of a functional disulfide bond formation system. In contrast, aerobic Gram-positive Firmicutes favor the exclusion of cysteines from both their cytoplasmic proteins and their substantially longer exported proteins. Supporting these findings, we show that Firmicutes, but not Actinobacteria, tolerate growth in reductant. We further demonstrate that the actinobacterium Corynebacterium glutamicum possesses disulfide-bonded proteins and two dimeric Dsb-like enzymes that can efficiently catalyze the formation of disulfide bonds. Our results suggest that cysteine exclusion is an important adaptive strategy against the challenges presented by oxidative environments.

Two Crystal Structures of Pneumococcal Pilus Sortase C Provide Novel Insights into Catalysis and Substrate Specificity

The respiratory tract pathogen Streptococcus pneumoniae is a primary cause of morbidity and mortality worldwide. Pili enhance initial adhesion as well as the capacity of pneumococci to cause pneumonia and bacteremia. Pilus-associated sortases (SrtB, SrtC, and SrtD) are involved in the biogenesis of pneumococcal pili, composed of repeating units of RrgB that create the stalk to which the RrgA adhesin and the preferential pilus tip subunit RrgC are covalently associated. Using single sortase-expressing strains, we demonstrate that both pilin-polymerizing sortases SrtB and SrtC can covalently link pili to the peptidoglycan cell wall, a property shared with the non-pilus-polymerizing enzyme SrtD and the housekeeping sortase SrtA. Comparative analysis of the crystal structures of S. pneumoniae SrtC and SrtB revealed structural differences explaining the incapacity of SrtC, but not of SrtB, to incorporate RrgC into the pilus. Accordingly, site-directed mutagenesis of Thr(160) in SrtB to an arginine as in SrtC (Arg(160)) partially converted its substrate specificity into that of SrtC. Solving two crystal structures for SrtC suggests that an opening of a flexible lid and a concomitant cysteine rotation are important for catalysis and the activation of the catalytic cysteine of pilus-associated sortases.

The Metal Ion-Dependent Adhesion Site Motif of the Enterococcus faecalis EbpA Pilin Mediates Pilus Function in Catheter-Associated Urinary Tract Infection

ABSTRACT Though the bacterial opportunist Enterococcus faecalis causes a myriad of hospital-acquired infections (HAIs), including catheter-associated urinary tract infections (CAUTIs), little is known about the virulence mechanisms that it employs. However, the endocarditis- and biofilm-associated pilus (Ebp), a member of the sortase-assembled pilus family, was shown to play a role in a mouse model of E. faecalis ascending UTI. The Ebp pilus comprises the major EbpC shaft subunit and the EbpA and EbpB minor subunits. We investigated the biogenesis and function of Ebp pili in an experimental model of CAUTI using a panel of chromosomal pilin deletion mutants. A nonpiliated pilus knockout mutant (EbpABC− strain) was severely attenuated compared to its isogenic parent OG1RF in experimental CAUTI. In contrast, a nonpiliated ebpC deletion mutant (EbpC− strain) behaved similarly to OG1RF in vivo because it expressed EbpA and EbpB. Deletion of the minor pilin gene ebpA or ebpB perturbed pilus biogenesis and led to defects in experimental CAUTI. We discovered that the function of Ebp pili in vivo depended on a predicted metal ion-dependent adhesion site (MIDAS) motif in EbpA’s von Willebrand factor A domain, a common protein domain among the tip subunits of sortase-assembled pili. Thus, this study identified the Ebp pilus as a virulence factor in E. faecalis CAUTI and also defined the molecular basis of this function, critical knowledge for the rational development of targeted therapeutics. IMPORTANCE Catheter-associated urinary tract infections (CAUTIs), one of the most common hospital-acquired infections (HAIs), present considerable treatment challenges for physicians. Inherently resistant to several classes of antibiotics and with a propensity to acquire vancomycin resistance, enterococci are particularly worrisome etiologic agents of CAUTI. A detailed understanding of the molecular basis of Enterococcus faecalis pathogenesis in CAUTI is necessary for the development of preventative and therapeutic strategies. Our results elucidated the importance of the E. faecalis Ebp pilus and its subunits for enterococcal virulence in a mouse model of CAUTI. We further showed that the metal ion-dependent adhesion site (MIDAS) motif in EbpA is necessary for Ebp function in vivo. As this motif occurs in other sortase-assembled pili, our results have implications for the molecular basis of virulence not only in E. faecalis CAUTI but also in additional infections caused by enterococci and other Gram-positive pathogens. Catheter-associated urinary tract infections (CAUTIs), one of the most common hospital-acquired infections (HAIs), present considerable treatment challenges for physicians. Inherently resistant to several classes of antibiotics and with a propensity to acquire vancomycin resistance, enterococci are particularly worrisome etiologic agents of CAUTI. A detailed understanding of the molecular basis of Enterococcus faecalis pathogenesis in CAUTI is necessary for the development of preventative and therapeutic strategies. Our results elucidated the importance of the E. faecalis Ebp pilus and its subunits for enterococcal virulence in a mouse model of CAUTI. We further showed that the metal ion-dependent adhesion site (MIDAS) motif in EbpA is necessary for Ebp function in vivo. As this motif occurs in other sortase-assembled pili, our results have implications for the molecular basis of virulence not only in E. faecalis CAUTI but also in additional infections caused by enterococci and other Gram-positive pathogens.

Evidence for a New Sub-class of Methionine Sulfoxide Reductases B with an Alternative Thioredoxin Recognition Signature

Methionine sulfoxide reductases catalyze the reduction of protein-bound methionine sulfoxide back to methionine via a thioredoxin-recycling process. Two classes of methionine sulfoxide reductases, called MsrA and MsrB, exist that display opposite stereoselectivities toward the sulfoxide function. Although they are structurally unrelated, they share a similar chemical mechanism that includes three steps with 1) formation of a sulfenic acid intermediate with a concomitant release of 1 mol of methionine per mole of enzyme; 2) formation of an intradisulfide Msr bond; and 3) reduction of the oxidized Msr by thioredoxin. In the MsrBs that have been biochemically, enzymatically, and structurally characterized so far, the cysteine involved in the regeneration of the catalytic Cys-117 is Cys-63. Cys-117 is located on a β strand, whereas the recycling Cys-63 is on a loop near Cys-117. The distance between the two cysteines is compatible with formation of the Cys-117/Cys-63 intradisulfide bond. Analyses of MsrB sequences show that at least 37% of the MsrBs do not possess the recycling Cys-63. In the present study, it is shown that Cys-31 in the Xanthomonas campestris MsrB, which is located on another loop, can efficiently substitute for Cys-63. Such a result implies flexibility of the MsrB structures, at least of the loops on which Cys-31 or Cys-63 are located. The fact that about 25% of the putative MsrBs have no recycling cysteine supports other recycling processes in which thioredoxin is not operative.

The N-terminal Domain of PILB from Neisseria meningitidis Is a Disulfide Reductase That Can Recycle Methionine Sulfoxide Reductases

The PilB protein of the Neisseria genus comprises three domains. Two forms have been recently reported to be produced in vivo. One form, containing the three domains, is secreted from the bacterial cytoplasm to the outer membrane, whereas the second form, which is cytoplasmic, only contains the central and the C-terminal domains. The secreted form was shown to be involved in survival under oxidative conditions. Although previous studies indicated that the central and the C-terminal domains display methionine sulfoxide reductase A and B activities, respectively, no function was described so far for the N-terminal domain. In the present study, the N-terminal domain of the PilB of Neisseria meningitidis was produced as a folded entity, and its biochemical and enzymatic properties have been determined. The data show that the N-terminal domain possesses a disulfide redox-active site with a redox potential in the range of that of thioredoxin. Moreover, the N-terminal domain, either as an isolated form or included in PilB, recycles the oxidized forms of the methionine sulfoxide reductases like thioredoxin. These results, which show that the N-terminal domain exhibits a disulfide reductase activity and probably has a thioredoxin-fold, are discussed in relation to its possible functional role in Neisseria.

Methionine sulfoxide reductase B displays a high level of flexibility.

Methionine sulfoxide reductases (Msrs) are enzymes that catalyze the reduction of methionine sulfoxide back to methionine. In vivo, Msrs are essential in the protection of cells against oxidative damage to proteins and in the virulence of some bacteria. Two structurally unrelated classes of Msrs, named MsrA and MsrB, exist. MsrB are stereospecific to R epimer on the sulfur of sulfoxide. All MsrB share a common reductase step with the formation of a sulfenic acid intermediate. For the subclass of MsrB whose recycling process passes through the formation of an intradisulfide bond, the recycling reducer is thioredoxin. In the present study, X-ray structures of Neisseria meningitidis MsrB have been determined. The structures have a fold based on two beta-sheets, similar to the fold already described for other MsrB, with the recycling Cys63 located in a position favorable for disulfide bond formation with the catalytic Cys117. X-ray structures of Xanthomonas campestris MsrB have also been determined. In the C117S MsrB structure with a bound substrate, the recycling Cys31 is far from Ser117, with Trp65 being essential in the reductase step located in between. This positioning prevents the formation of the Cys31-Cys117 disulfide bond. In the oxidized structure, a drastic conformational reorganization of the two beta-sheets due to withdrawal of the Trp65 region from the active site, which remains compatible with an efficient thioredoxin-recycling process, is observed. The results highlight the remarkable structural malleability of the MsrB fold.

Pilin and Sortase Residues Critical for Endocarditis- and Biofilm-Associated Pilus Biogenesis in Enterococcus faecalis

Enterococci commonly cause hospital-acquired infections, such as infective endocarditis and catheter-associated urinary tract infections. In animal models of these infections, a long hairlike extracellular protein fiber known as the endocarditis- and biofilm-associated (Ebp) pilus is an important virulence factor for Enterococcus faecalis. For Ebp and other sortase-assembled pili, the pilus-associated sortases are essential for fiber formation as they create covalent isopeptide bonds between the sortase recognition motif and the pilin-like motif of the pilus subunits. However, the molecular requirements governing the incorporation of the three pilus subunits (EbpA, EbpB, and EbpC) have not been investigated in E. faecalis. Here, we show that a Lys residue within the pilin-like motif of the EbpC subunit was necessary for EbpC polymerization. However, incorporation of EbpA into the pilus fiber only required its sortase recognition motif (LPXTG), while incorporation of EbpB only required its pilin-like motif. Only the sortase recognition motif would be required for incorporation of the pilus tip subunit, while incorporation of the base subunit would only require the pilin recognition motif. Thus, these data support a model with EbpA at the tip and EbpB at the base of an EbpC polymer. In addition, the housekeeping sortase, SrtA, was found to process EbpB and its predicted catalytic Cys residue was required for efficient cell wall anchoring of mature Ebp pili. Thus, we have defined molecular interactions involved in fiber polymerization, minor subunit organization, and pilus subcellular compartmentalization in the E. faecalis Ebp pilus system. These studies advance our understanding of unique molecular mechanisms of sortase-assembled pilus biogenesis.

Characterization of the amino acids from Neisseria meningitidis methionine sulfoxide reductase B involved in the chemical catalysis and substrate specificity of the reductase step.

Methionine sulfoxide reductases (Msrs) are antioxidant repair enzymes that catalyze the thioredoxin-dependent reduction of methionine sulfoxide back to methionine. The Msr family is composed of two structurally unrelated classes of enzymes named MsrA and MsrB, which display opposite stereoselectivities toward the S and R isomers of the sulfoxide function, respectively. Both classes of Msr share a similar three-step chemical mechanism involving first a reductase step that leads to the formation of a sulfenic acid intermediate. In this study, the invariant amino acids of Neisseria meningitidis MsrB involved in the reductase step catalysis and in substrate binding have been characterized by the structure-function relationship approach. Altogether the results show the following: 1) formation of the MsrB-substrate complex leads to an activation of the catalytic Cys-117 characterized by a decreased pKapp of ∼2.7 pH units; 2) the catalytic active MsrB form is the Cys-117-/His-103+ species with a pKapp of 6.6 and 8.3, respectively; 3) His-103 and to a lesser extent His-100, Asn-119, and Thr-26 (via a water molecule) participate in the stabilization of the polarized form of the sulfoxide function and of the transition state; and 4) Trp-65 is essential for the catalytic efficiency of the reductase step by optimizing the position of the substrate in the active site. A scenario for the reductase step is proposed and discussed in comparison with that of MsrA.

Binding of Divalent Cations to Polygalacturonate: A Mechanism Driven by the Hydration Water.

We have investigated the interactions between polygalacturonate (polyGal) and four divalent cations (M(2+) = Ba(2+), Ca(2+), Mg(2+), Zn(2+)) that differ in size and affinity for water. Our results evidence that M(2+)-polyGal interactions are intimately linked to the affinity of M(2+) for water. Mg(2+) interacts so strongly with water that it remains weakly bound to polyGal (polycondensation) by sharing water molecules from its first coordination shell with the carboxylate groups of polyGal. In contrast, the other cations form transient ionic pairs with polyGal by releasing preferentially one water molecule (for Zn(2+)) or two (for Ca(2+) and Ba(2+)), which corresponds to monodentate and bidentate binding modes with carboxylates, respectively. The mechanism for the binding of these three divalent cations to polyGal can be described by two steps: (i) monocomplexation and formation of point-like cross-links between polyGal chains (at low M(2+)/Gal molar ratios, R) and (ii) dimerization (at higher R). The threshold molar ratio, R*, between these two steps depends on the nature of divalent cations and is lower for calcium ions (R* < 0.1) than for zinc and barium ions (R* > 0.3). This difference may be explained by the intermediate affinity of Ca(2+) for water with respect to those of Zn(2+) and Ba(2+), which may induce the formation of cross-links of intermediate flexibility. By comparison, the lower and higher flexibilities of the cross-links formed by Zn(2+) and Ba(2+), respectively, may shift the formation of dimers to higher molar ratios (R*).