Crystal Structure and Pathophysiological Role of the Pneumococcal Nucleoside-binding Protein PnrA

Nucleotides are important for RNA and DNA synthesis and, despite a de novo synthesis by bacteria, uptake systems are crucial. Streptococcus pneumoniae, a facultative human pathogen, produces a surface-exposed nucleoside-binding protein, PnrA, as part of an ABC transporter system. Here we demonstrate the binding affinity of PnrA to nucleosides adenosine, guanosine, cytidine, thymidine and uridine by microscale thermophoresis and indicate the consumption of adenosine and guanosine by ¹H NMR spectroscopy. In a series of five crystal structures we revealed the PnrA structure and provide insights into how PnrA can bind purine and pyrimidine ribonucleosides but with preference for purine ribonucleosides. Crystal structures of PnrA:nucleoside complexes unveil a clear pattern of interactions in which both the N- and C- domains of PnrA contribute. The ribose moiety is strongly recognized through a conserved network of H-bond interactions, while plasticity in loop 27–36 is essential to bind purine- or pyrimidine-based nucleosides.Further, we deciphered the role of PnrA in pneumococcal fitness in infection experiments. Phagocytosis experiments did not show a clear difference in phagocytosis between PnrA-deficient and wild-type pneumococci. In the acute pneumonia infection model the deficiency of PnrA attenuated moderately virulence of the mutant, which is indicated by a delay in the development of severe lung infections. Importantly, we confirmed the loss of fitness in co-infections, where the wild-type out-competed the pnrA-mutant. In conclusion, we present the PnrA structure in complex with individual nucleosides and show that the consumption of adenosine and guanosine under infection conditions is required for virulence.     

Extracts and sesquiterpene derivatives from the red alga Laurencia chondrioides with antibacterial activity against fish and human pathogenic bacteria

During screening of seaweeds from different places in Europe for antimicrobial activities against human and fish pathogenic bacteria, Laurencia chondrioides was identified as a promising species. By bioassay-guided isolation, followed by structure elucidation by mass spectrometry and 1H- and 13C-NMR spectrometry, two sesquiterpenoides of the chamigrene-type from the selected red seaweed Laurencia chondrioides were identified. Both compounds inhibit the growth of some fish and human pathogenic bacteria.     

Laccase-catalyzed cross-linking of amino acids and peptides with dihydroxylated aromatic compounds

In order to design potential biomaterials, we investigated the laccase-catalyzed cross-linking between l-lysine or lysine-containing peptides and dihydroxylated aromatics. l-Lysine is one of the major components of naturally occurring mussel adhesive proteins (MAPs). Dihydroxylated aromatics are structurally related to 3,4-dihydroxyphenyl-l-alanine, another main component of MAPs. Mass spectrometry and nuclear magnetic resonance analyses show that the ε-amino group of l-lysine is able to cross-link dihydroxylated aromatics. Additional oligomer and polymer cross-linked products were obtained from di- and oligopeptides containing l-lysine. Potential applications in medicine or industry for biomaterials synthesised via the three component system consisting of the oligopeptide [Tyr-Lys]₁₀, dihydroxylated aromatics and laccase are discussed.     

Fed-batch process for the psychrotolerant marine bacterium <Emphasis Type="Italic">Pseudoalteromonas haloplanktis</Emphasis>

Background  

Pseudoalteromonas haloplanktis is a cold-adapted γ-proteobacterium isolated from Antarctic sea ice. It is characterized by remarkably high growth rates at low temperatures. P. haloplanktis is one of the model organisms of cold-adapted bacteria and has been suggested as an alternative host for the soluble overproduction of heterologous proteins which tend to form inclusion bodies in established expression hosts. Despite the progress in establishing P. haloplanktis as an alternative expression host the cell densities obtained with this organism, which is unable to use glucose as a carbon source, are still low. Here we present the first fed-batch cultivation strategy for this auspicious alternative expression host.     

Nuclear amination catalyzed by fungal laccases: Comparison of laccase catalyzed amination with known chemical routes to aminoquinones

Laccases are able to initiate nuclear amination of p-hydroquinones with primary aromatic amines, resulting in the formation of the corresponding monoaminated and diaminated quinones. Two laccase catalyzed reactions are compared with established synthetic routes to aminoquinones, showing that formation of products from laccase catalyzed reaction is comparable with reaction using sodium iodate as oxidant. Advantages and disadvantages of laccase catalyzed amination are discussed. It is concluded that laccase catalysis is less suitable than sodium iodate oxidation for the amination of simple p-hydroquinones with simple amines.     

Staphylococcus aureus Alpha-Toxin Mediates General and Cell Type-Specific Changes in Metabolite Concentrations of Immortalized Human Airway Epithelial Cells

Staphylococcus aureus alpha-toxin (Hla) is a potent pore-forming cytotoxin that plays an important role in the pathogenesis of S. aureus infections, including pneumonia. The impact of Hla on the dynamics of the metabolome in eukaryotic host cells has not been investigated comprehensively. Using ¹H-NMR, GC-MS and HPLC-MS, we quantified the concentrations of 51 intracellular metabolites and assessed alterations in the amount of 25 extracellular metabolites in the two human bronchial epithelial cell lines S9 and 16HBE14o⁻ under standard culture conditions and after treatment with sub-lethal amounts (2 µg/ml) of recombinant Hla (rHla) in a time-dependent manner. Treatment of cells with rHla caused substantial decreases in the concentrations of intracellular metabolites from different metabolic pathways in both cell lines, including ATP and amino acids. Concomitant increases in the extracellular concentrations were detected for various intracellular compounds, including nucleotides, glutathione disulfide and NAD⁺. Our results indicate that rHla has a major impact on the metabolome of eukaryotic cells as a consequence of direct rHla-mediated alterations in plasma membrane permeability or indirect effects mediated by cellular signalling. However, cell-specific changes also were observed. Glucose consumption and lactate production rates suggest that the glycolytic activity of S9 cells, but not of 16HBE14o⁻ cells, is increased in response to rHla. This could contribute to the observed higher level of resistance of S9 cells against rHla-induced membrane damage.     

Biotransformation and reduction of estrogenicity of bisphenol A by the biphenyl-degrading Cupriavidus basilensis

The biphenyl-degrading Gram-negative bacterium Cupriavidus basilensis (formerly Ralstonia sp.) SBUG 290 uses various aromatic compounds as carbon and energy sources and has a high capacity to transform bisphenol A (BPA), which is a hormonally active substance structurally related to biphenyl. Biphenyl-grown cells initially hydroxylated BPA and converted it to four additional products by using three different transformation pathways: (a) formation of multiple hydroxylated BPA, (b) ring fission, and (c) transamination followed by acetylation or dimerization. Products of the ring fission pathway were non-toxic and all five products exhibited a significantly reduced estrogenic activity compared to BPA. Cell cultivation with phenol and especially in nutrient broth (NB) resulted in a reduced biotransformation rate and lower product quantities, and NB-grown cells did not produce all five products in detectable amounts. Thus, the question arose whether enzymes of the biphenyl degradation pathway are involved in the transformation of BPA and was addressed by proteomic analyses.

     

Synthesis of indirubin-N'-glycosides and their anti-proliferative activity against human cancer cell lines

The first indirubin-N'-glycosides were prepared based on reactions of isatin-N'-glycosides with indoxyls. The products show a significant anti-proliferative activity against various human cancer cell lines. Good results were observed for an indirubin-N'-mannoside which was shown to have medium to high anti-proliferative activity against all investigated cell lines. The highest activities and selectivities against the MCF-7 breast cancer cell line were observed for indirubin-N'-rhamnosides.