Optimized enrichment for the detection of Escherichia coli O26 in French raw milk cheeses

Aims: Our main objective was to optimize the enrichment of Escherichia coli O26 in raw milk cheeses for their subsequent detection with a new automated immunological method. Methods and Results: Ten enrichment broths were tested for the detection of E. coli O26. Two categories of experimentally inoculated raw milk cheeses, semi‐hard uncooked cheese and ‘Camembert’ type cheese, were initially used to investigate the relative efficacy of the different enrichments. The enrichments that were considered optimal for the growth of E. coli O26 in these cheeses were then challenged with other types of raw milk cheeses. Buffered peptone water supplemented with cefixim–tellurite and acriflavin was shown to optimize the growth of E. coli O26 artificially inoculated in the cheeses tested. Despite the low inoculum level (1–10 CFU per 25 g) in the cheeses, E. coli O26 counts reached at least 5·10⁴ CFU ml^?1 after 24‐h incubation at 41·5°C in this medium. Conclusions: All the experimentally inoculated cheeses were found positive by the immunological method in the enrichment broth selected. Significance and Impact of the Study: Optimized E. coli O26 enrichment and rapid detection constitute the first steps of a complete procedure that could be used in routine to detect E. coli O26 in raw milk cheeses.     

Cutting Edge: A dual role for type I IFNs during polyinosinic-polycytidylic acid-induced NK cell activation

NK cells are cytotoxic lymphocytes that are most efficient at fulfilling their functions after a phase of priming provided by cytokines and/or accessory cells. Although type I IFNs are known to be important in this process, it remains unclear whether they act directly on NK cells or indirectly on accessory cells. We used adoptive transfer experiments and mixed bone marrow chimeras to dissect the requirement for type I IFN signaling in response to the dsRNA analog polyinosinic-polycytidylic acid. We demonstrate that optimal NK cell priming requires type I IFNs to signal on both NK cells and accessory cells. In the absence of IL-15, the residual NK cell activation was strictly dependent on cell-intrinsic IFNAR signaling in NK cells. Our results suggest that type I IFNs produced following viral infection simultaneously target accessory cells for IL-15 transpresentation and NK cells themselves and that these two pathways cooperate for NK cell priming.     

Adenovirus E3-19K proteins of different serotypes and subgroups have similar, yet distinct, immunomodulatory functions toward major histocompatibility class I molecules

Our understanding of the mechanism by which the E3-19K protein from adenovirus (Ad) targets major histocompatibility complex (MHC) class I molecules for retention in the endoplasmic reticulum is derived largely from studies of Ad serotype 2 (subgroup C). It is not well understood to what extent observations on the Ad2 E3-19K/MHC I association can be generalized to E3-19K proteins of other serotypes and subgroups. The low levels of amino acid sequence homology between E3-19K proteins suggest that these proteins are likely to manifest distinct MHC I binding properties. This information is important as the E3-19K/MHC I interaction is thought to play a critical role in enabling Ads to cause persistent infections. Here, we characterized interaction between E3-19K proteins of serotypes 7 and 35 (subgroup B), 5 (subgroup C), 37 (subgroup D), and 4 (subgroup E) and a panel of HLA-A, -B, and -C molecules using native gel, surface plasmon resonance (SPR), and flow cytometry. Results show that all E3-19K proteins exhibited allele specificity toward HLA-A and -B molecules; this was less evident for Ad37 E3-19K. The allele specificity for HLA-A molecules was remarkably similar for different serotypes of subgroup B as well as subgroup C. Interestingly, all E3-19K proteins characterized also exhibited MHC I locus specificity. Importantly, we show that Lys(91) in the conserved region of Ad2 E3-19K targets the C terminus of the α2-helix (MHC residue 177) on MHC class I molecules. From our data, we propose a model of interaction between E3-19K and MHC class I molecules.     

Stabilization of the integrase‐DNA complex by Mg2+ ions and prediction of key residues for binding HIV‐1 integrase inhibitors

The HIV‐1 integrase is an attractive target for the therapeutics development against AIDS, as no host homologue of this protein has been identified. The integrase strand transfer inhibitors (INSTIs), including raltegravir, specifically target the second catalytic step of the integration process by binding to the DDE motif of the catalytic site and coordinating Mg²⁺ ions. Recent X‐ray crystallographic structures of the integrase/DNA complex from prototype foamy virus allowed to investigate the role of the different partners (integrase, DNA, Mg²⁺ ions, raltegravir) in the complex stability using molecular dynamics (MD) simulations. The presence of Mg²⁺ ions is found to be essential for the stability, whereas the simultaneous presence of raltegravir and Mg²⁺ ions has a destabilizing influence. A homology model of HIV‐1 integrase was built on the basis of the X‐ray crystallographic information, and protein marker residues for the ligand binding were detected by clustering the docking poses of known HIV‐1 integrase inhibitors on the model. Interestingly, we had already identified some of these residues to be involved in HIV‐1 resistance mutations and in the stabilization of the catalytic site during the MD simulations. Classification of protein conformations along MD simulations, as well as of ligand docking poses, was performed by using an original learning method, based on self‐organizing maps. This allows us to perform a more in‐depth investigation of the free‐energy basins populated by the complex in MD simulations on the one hand, and a straightforward classification of ligands according to their binding residues on the other hand. Proteins 2014; 82:466–478. © 2013 Wiley Periodicals, Inc.     

N-Glycan-dependent and -independent Quality Control of Human δ Opioid Receptor N-terminal Variants

Quality control (QC) in the endoplasmic reticulum (ER) scrutinizes newly synthesized proteins and directs them either to ER export or ER-associated degradation (ERAD). Here, we demonstrate that the human δ-opioid receptor (hδOR) is subjected to ERQC in both N-glycan-dependent and -independent manners. This was shown by investigating the biosynthesis and trafficking of wild-type and non-N-glycosylated F27C variants in metabolic pulse-chase assays coupled with flow cytometry and cell surface biotinylation. Both QC mechanisms distinguished the minute one-amino acid difference between the variants, targeting a large fraction of hδOR-Cys²⁷ to ERAD. However, the N-glycan-independent QC was unable to compensate the N-glycan-dependent pathway, and some incompletely folded non-N-glycosylated hδOR-Cys²⁷ reached the cell surface in conformation incompatible with ligand binding. The turnover of receptors associating with the molecular chaperone calnexin (CNX) was significantly slower for the hδOR-Cys²⁷, pointing to an important role of CNX in the hδOR N-glycan-dependent QC. This was further supported by the fact that inhibiting the co-translational interaction of hδOR-Cys²⁷ precursors with CNX led to their ERAD. Opioid receptor pharmacological chaperones released the CNX-bound receptors to ER export and, furthermore, were able to rescue the Cys²⁷ variant from polyubiquitination and retrotranslocation to the cytosol whether carrying N-glycans or not. Taken together, the hδOR appears to rely primarily on the CNX-mediated N-glycan-dependent QC that has the capacity to assist in folding, whereas the N-glycan-independent mechanism constitutes an alternative, although less accurate, system for directing misfolded/incompletely folded receptors to ERAD, possibly in altered cellular conditions.     

Development and Characterization of Pepducins as Gs-biased Allosteric Agonists

The β₂-adrenergic receptor (β₂AR) is a prototypical G protein-coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. β-Agonists used to combat hypercontractility in airway smooth muscle stimulate β₂AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the β₂AR by long acting β-agonists used in the treatment of asthma can promote attenuated responsiveness to agonists and an increased frequency of fatal asthmatic attacks. β₂AR desensitization to β-agonists is primarily mediated by G protein-coupled receptor kinases and β-arrestins that attenuate receptor-G_s coupling and promote β₂AR internalization and degradation. A biased agonist that can selectively stimulate G_s signaling without promoting receptor interaction with G protein-coupled receptor kinases and β-arrestins should serve as an advantageous asthma therapeutic. To identify such molecules, we screened ∼50 lipidated peptides derived from the intracellular loops of the β₂AR, known as pepducins. This screen revealed two classes of G_s-biased pepducins, receptor-independent and receptor-dependent, as well as several β-arrestin-biased pepducins. The receptor-independent G_s-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependent G_s-biased pepducins appear to stabilize a G_s-biased conformation of the β₂AR that couples to G_s but does not undergo G protein-coupled receptor kinase-mediated phosphorylation or β-arrestin-mediated internalization. Functional studies in primary human airway smooth muscle cells demonstrate that G_s-biased pepducins are not subject to conventional desensitization and thus may be good candidates for the development of next generation asthma therapeutics. Our study reports the first G_s-biased activator of the β₂AR and provides valuable tools for the study of β₂AR function.     

Genetic Resistance to Scrapie Infection in Experimentally Challenged Goats

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M₁₄₂, N/D₁₄₆, S/D₁₄₆, R/Q₂₁₁, and Q/K₂₂₂) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I₁₄₂R₁₅₄R₂₁₁Q₂₂₂) goats and homozygous and/or heterozygous I/M₁₄₂, R/H₁₅₄, R/Q₂₁₁, and Q/K₂₂₂ goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M₁₄₂ PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H₁₅₄, Q₂₁₁, and K₂₂₂ PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H₁₅₄ and Q₂₁₁ allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K₂₂₂ and a small proportion of homozygous K₂₂₂ goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K₂₂₂ goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie.     

Localization of NADPH-protochlorophyllide reductase in plastids of barley at different greening stages

The localization of protochorophyllide (Pchlide) and of NADPH-protochlorophyllide oxidoreductase (POR, EC 1.6.99.1) within (etio)chloroplasts has been investigated at selected stages of greening of barley seedlings. Pchlide pigment and POR protein contents were evaluated in different plastid membrane fractions by fluorescence spectroscopy and immunoblot analysis using a monospecific polyclonal antibody raised against the purified enzyme. Fluorescence analysis showed the presence of Pchlide in both the envelope and thylakoid membranes. During greening, the Pchlide content, expressed on a total protein basis, decreased in thylakoid membranes, whereas it increased in the envelope membranes. POR proteins were detected mainly in thylakoid membranes at early greening stages. In contrast, the weak amount of POR proteins was associated more specifically with envelope membranes of mature chloroplasts. Whatever the greening stage, thylakoid-bound Pchlide and POR proteins were more abundant in the thylakoid regions which remained unsolubilized after mild Triton treatment used as standard procedure to prepare PS II particles. This suggests the preferential association of Pchlide and POR to the appressed regions of thylakoids. This revised version was published online in June 2006 with corrections to the Cover Date.     

Unraveling G protein-coupled receptor endocytosis pathways using real-time monitoring of agonist-promoted interaction between beta-arrestins and AP-2

The most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves beta-arrestin and clathrin-coated pits. However, both beta-arrestin- and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that beta-arrestin binding to the beta2 subunit of the clathrin adaptor AP-2 (beta2-adaptin) is needed for the beta-arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrin-independent pathways, agonist stimulation promoted beta-arrestin 1 and 2 interaction with beta2-adaptin, indicating a beta-arrestin- and clathrin-dependent endocytic process. Detailed analyses of beta-arrestin interactions with both the receptor and beta2-adaptin also allowed us to demonstrate that recruitment of beta-arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between beta-arrestins and beta2-adaptin. However, both receptors recruited beta-arrestins upon agonist stimulation, suggesting a beta-arrestin-dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based beta-arrestin/beta2-adaptin interaction assay represents a novel biosensor to assess receptor activation.     

Recovery of intracellular recombinant proteins from the yeast Pichia pastoris by cell permeabilization

A cell permeabilization method for the release of intracellular proteins from microbial cells was developed. The method was applied to the recovery of recombinant botulinum neurotoxin fragments, expressed intracellularly in the yeast Pichia pastoris, by suspending the cells in an aqueous solution containing N,N-dimethyltetradecylamine. For the botulinum neurotoxin serotype B C-terminal heavy chain fragment, 1.8 mg g(-1) biomass were recovered. For the botulinum neurotoxin serotype A C-terminal heavy chain fragment, 3.7 mg g(-1) biomass were recovered. The concentration of recombinant protein in the cell extracts remained stable for up to 48 and 24 h for the serotype B and serotype A fragments, respectively. The permeabilization method was compared with high-pressure homogenization; the permeabilization method proved to be both more selective and more efficient.