Population structure of invasive Streptococcus pneumoniae in The Netherlands in the pre-vaccination era assessed by MLVA and capsular sequence typing

The introduction of nationwide pneumococcal vaccination may lead to serotype replacement and the emergence of new variants that have expanded their genetic repertoire through recombination. To monitor alterations in the pneumococcal population structure, we have developed and utilized Capsular Sequence Typing (CST) in addition to Multiple-Locus Variable number tandem repeat Analysis (MLVA).To assess the serotype of each isolate CST was used. Based on the determination of the partial sequence of the capsular wzh gene, this method assigns a capsular type of an isolate within a single PCR reaction using multiple primersets. The genetic background of pneumococcal isolates was assessed by MLVA. MLVA and CST were used to create a snapshot of the Dutch pneumococcal population causing invasive disease before the introduction of the 7-valent pneumococcal conjugate vaccine in The Netherlands in 2006. A total of 1154 clinical isolates collected and serotyped by the Netherlands Reference Laboratory for Bacterial Meningitis were included in the snapshot. The CST was successful in discriminating most serotypes present in our collection. MLVA demonstrated that isolates belonging to some serotypes had a relatively high genetic diversity whilst other serotypes had a very homogeneous genetic background. MLVA and CST appear to be valuable tools to determine the population structure of pneumococcal isolates and are useful in monitoring the effects of pneumococcal vaccination.     

Structural determination of novel lacto-N-decaose and its monofucosylated analogue from human milk by electrospray tandem mass spectrometry and 1H NMR spectroscopy

We have isolated and characterised two neutral oligosaccharides, one nonfucosylated and the other monofucosylated, from human milk that are based on the doubly branched lacto-N-decaose core. Their structures have been determined by a combined use of electrospray tandem mass spectrometry (ES-MS/MS) and NMR spectroscopy. The sequences of the three branches resulted from the double-branching, including the identity and location of the blood-group-related Lewis determinant and partial linkages, were elucidated by the unique method of high sensitivity negative-ion ES-MS/MS analysis. Their full structure assignment was completed by methylation analysis and 1H NMR. The monofucosylated lacto-N-decaose, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4Glc is a novel sequence, whereas the nonfucosylated lacto-N-decaose, Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-6(Galbeta1-3GlcNAcbeta1-3)Galbeta1-4Glc, has not been isolated and identified as an individual oligosaccharide.     

Structure and mechanism of the magnesium-independent aromatic prenyltransferase CloQ from the clorobiocin biosynthetic pathway

CloQ is an aromatic prenyltransferase from the clorobiocin biosynthetic pathway of Streptomyces roseochromogenes var. oscitans. It is involved in the synthesis of the prenylated hydroxybenzoate moiety of the antibiotic, specifically catalyzing the attachment of a dimethylallyl moiety to 4-hydroxyphenylpyruvate. Herein, we report the crystal structure of CloQ and use it as a framework for interpreting biochemical data from both wild-type and variant proteins. CloQ belongs to the aromatic prenyltransferase family, which is characterized by an unusual core fold comprising five consecutive ααββ elements that form a central 10-stranded anti-parallel β-barrel. The latter delineates a solvent-accessible cavity where substrates bind and catalysis takes place. This cavity has well-defined polar and nonpolar regions, which have distinct roles in substrate binding and facilitate a Friedel-Crafts-type mechanism. We propose that the juxtaposition of five positively charged residues in the polar region circumvents the necessity for a Mg²⁺, which, by contrast, is a strict requirement for the majority of prenyltransferases characterized to date. Our structure of CloQ complexed with 4-hydroxyphenylpyruvate reveals the formation of a covalent link between the substrate and Cys215 to yield a thiohemiketal species. Through site-directed mutagenesis, we show that this link is not essential for enzyme activity in vitro. Furthermore, we demonstrate that CloQ will accept alternative substrates and, therefore, has the capacity to generate a range of prenylated compounds. Since prenylation is thought to enhance the bioactivity of many natural products, CloQ offers considerable promise as a biocatalyst for the chemoenzymatic synthesis of novel compounds with therapeutic potential.     

A simple equation for describing the temperature dependent growth of free-floating macrophytes

Temperature is one of the most important factors determining growth rates of free-floating macrophytes in the field. To analyse and predict temperature dependent growth rates of these pleustophytes, modelling may play an important role. Several equations have been published for describing temperature responses of macrophytes and algae. But they are often complex or are only applicable in a limited range of temperatures. In this paper, we present a simple three-parameter equation for describing the temperature dependent growth rates of pleustophytes. The equation that we developed is tested using results from laboratory growth experiments conducted with three different species of pleustophytes (Lemna minor, Salvinia molesta and Azolla filiculoides). The equation is simple and demonstrates reliable fits (adjusted R² reaching from 0.89 to 0.95). Additionally, our equation primarily uses parameters of biological significance, resulting in estimates of useful cardinal temperatures (minimum and maximum).     

The function of neurofascin155 in oligodendrocytes is regulated by metalloprotease-mediated cleavage and ectodomain shedding

Formation of the paranodal axo-glial junction requires the oligodendrocyte-specific 155-kDa isoform of neurofascin (NF155). Here, we report the presence of two peptides in cultured oligodendrocytes, which are recognized by distinct NF155-specific antibodies and correspond to a membrane anchor of 30 kDa and a 125 kDa peptide, which is shed from the cells, indicating that it consists of the NF155 ectodomain. Transfection of OLN-93 cells with NF155 verified that both peptides originate from NF155 cleavage, and we present evidence that metalloproteases mediate NF155 processing. Interestingly, metalloprotease activity is required for NF155 transport into oligodendrocyte processes supporting the functional significance of NF155 cleavage. To further characterize NF155 cleavage and function, we transfected MDCK cells with NF155. Although ectodomain shedding was observed in polarized and non-polarized MDCK cells, surface localization of NF155 was restricted to the lateral membrane of polarized cells consistent with a role in cell-cell adhesion. Aggregation assays performed with OLN-93 cells confirmed that NF155 accelerates cell-cell adhesion in a metalloprotease-dependent manner. The physiological relevance of NF155 processing is corroborated by the presence of NF155 cleavage products in heavy myelin, suggesting a role of NF155 ectodomain shedding for the generation and/or stabilization of the nodal/paranodal architecture.