Flagellin/TLR5 signalling activates renal collecting duct cells and facilitates invasion and cellular translocation of uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) colonizing kidneys is the main cause of acute pyelonephritis. TLR5 that senses flagellin was shown to be highly expressed in the bladder and to participate in host defence against flagellated UPEC, although its role in kidneys still remains elusive. Here we show that TLR5 is expressed in renal medullary collecting duct (MCD) cells, which represent a preferential site of UPEC adhesion. Flagellin, like lipopolysaccharide, stimulated the production of the chemoattractant chemokines CXCL1 and CXCL2, and subsequent migration capacity of neutrophils in cultured wild‐type (WT) and Tlr4^?/? MCDs, but not in Tlr5^?/? MCDs. UPEC can translocate across intact MCD layers without altering tight junctions. Strikingly, the invasion capacity and transcellular translocation of the UPEC strain HT7 were significantly lower in Tlr5^?/? than in WT MCDs. The non‐motile HT7ΔfliC mutant lacking flagellin also exhibited much lower translocation capacities than the HT7 isolates. Finally, Tlr5^?/? kidneys exhibited less infiltrating neutrophils than WT kidneys one day after the transurethral inoculation of HT7, and greater delayed renal bacterial loads in the day 4 post‐infected Tlr5^?/? kidneys. Overall, these findings indicate that the epithelial TLR5 participates to renal antibacterial defence, but paradoxically favours the translocation of UPEC across intact MCD cell layers.     

A deubiquitylating complex required for neosynthesis of a yeast mitochondrial ATP synthase subunit

The ubiquitin system is known to be involved in maintaining the integrity of mitochondria, but little is known about the role of deubiquitylating (DUB) enzymes in such functions. Budding yeast cells deleted for UBP13 and its close homolog UBP9 displayed a high incidence of petite colonies and slow respiratory growth at 37°C. Both Ubp9 and Ubp13 interacted directly with Duf1 (DUB-associated factor 1), a WD40 motif-containing protein. Duf1 activates the DUB activity of recombinant Ubp9 and Ubp13 in vitro and deletion of DUF1 resulted in the same respiratory phenotype as the deletion of both UBP9 and UBP13. We show that the mitochondrial defects of these mutants resulted from a strong decrease at 37°C in the de novo biosynthesis of Atp9, a membrane-bound component of ATP synthase encoded by mitochondrial DNA. The defect appears at the level of ATP9 mRNA translation, while its maturation remained unchanged in the mutants. This study describes a new role of the ubiquitin system in mitochondrial biogenesis.     

Molecular genetics of the transcription factor GLIS3 identifies its dual function in beta cells and neurons

The GLIS family zinc finger 3 isoform (GLIS3) is a risk gene for Type 1 and Type 2 diabetes, glaucoma and Alzheimer's disease endophenotype. We identified GLIS3 binding sites in insulin secreting cells (INS1) (FDR q < 0.05; enrichment range 1.40–9.11 fold) sharing the motif wrGTTCCCArTAGs, which were enriched in genes involved in neuronal function and autophagy and in risk genes for metabolic and neuro-behavioural diseases. We confirmed experimentally Glis3-mediated regulation of the expression of genes involved in autophagy and neuron function in INS1 and neuronal PC12 cells. Naturally-occurring coding polymorphisms in Glis3 in the Goto-Kakizaki rat model of type 2 diabetes were associated with increased insulin production in vitro and in vivo, suggestive alteration of autophagy in PC12 and INS1 and abnormal neurogenesis in hippocampus neurons. Our results support biological pleiotropy of GLIS3 in pathologies affecting β-cells and neurons and underline the existence of trans?nosology pathways in diabetes and its co-morbidities.     

Retroviral Oligonucleotide Distributions Correlate with Biased Nucleotide Compositions of Retrovirus Sequences,Suggesting a Duplicative Stepwise Molecular Evolution

A computer-assisted analysis was made of 24 complete nucleotide sequences selected from the vertebrate retroviruses to represent the ten viral groups. The conclusions of this analysis extend and strengthen the previously made hypothesis on the Moloney murine leukemia virus: The evolution of the nucleotide sequence appears to have occurred mainly through at least three overlapping levels of duplication: (1) The distributions of overrepresented (3–6)-mers are consistent with the universal rule of a trend toward TG/CT excess and with the persistence of a certain degree of symmetry between the two strands of DNA. This suggests one or several original tandemly repeated sequences and some inverted duplications. (2) The existence of two general core consensuses at the level of these (3–6)-mers supports the hypothesis of a common evolutionary origin of vertebrate retroviruses. Consensuses more specific to certain sequences are compatible with phylogenetic trees established independently. The consensuses could correspond to intermediary evolutionary stages. (3) Most of the (3–6)-mers with a significantly higher than average frequency appear to be internally repeated (with monomeric or oligomeric internal iterations) and seem to be at least partly the cause of the bias observed by other researchers at the level of retroviral nucleotide composition. They suggest a third evolutionary stage by slippage-like stepwise local duplications. Received: 3 January 1996 / Accepted: 27 March 1996     

The "prismane" protein resolved: X-ray structure at 1.7 Å and multiple spectroscopy of two novel 4Fe clusters

The three-dimensional structure of the native "putative prismane" protein from Desulfovibrio vulgaris (Hildenborough) has been solved by X-ray crystallography to a resolution of 1.72?Å. The molecule does not contain a [6Fe-6S] prismane cluster, but rather two 4Fe clusters some 12?Å apart and situated close to the interfaces formed by the three domains of the protein. Cluster 1 is a conventional [4Fe-4S] cubane bound, however, near the N-terminus by an unusual, sequential arrangement of four cysteine residues (Cys 3, 6, 15, 21). Cluster 2 is a novel 4Fe structure with two μ₂-sulfido bridges, two μ₂-oxo bridges, and a partially occupied, unidentified μ₂ bridge X. The protein ligands of cluster 2 are widely scattered through the second half of the sequence and include three cysteine residues (Cys 312, 434, 459), one persulfido-cysteine (Cys 406), two glutamates (Glu 268, 494), and one histidine (His 244). With this unusual mixture of bridging and external type of ligands, cluster 2 is named the "hybrid" cluster, and its asymmetric, open structure suggests that it could be the site of a catalytic activity. X-ray absorption spectroscopy at the Fe K-edge is readily interpretable in terms of the crystallographic model when allowance is made for volume contraction at 10?K; no Fe··Fe distances beyond 3.1?Å could be identified. EPR, Mössbauer and MCD spectroscopy have been used to define the oxidation states and the magnetism of the clusters in relation to the crystallographic structure. Reduced cluster 1 is a [4Fe-4S]¹⁺ cubane with S?=?3/2; it is the first biological example of a "spin-admixed" iron-sulfur cluster. The hybrid cluster 2 has four oxidation states from (formally) all Fe^III to three Fe^II plus one Fe^III. The four iron ions are exchange coupled resulting in the system spins S?=?0, 9/2, 0 (and 4), 1/2, respectively, for the four redox states. Resonance Raman spectroscopy suggests that the bridging ligand X which could not be identified unambiguously in the crystal structure is a solvent-exchangeable oxygen.     

Agroinjection of tomato fruits. A tool for rapid functional analysis of transgenes directly in fruit

Transient expression of foreign genes in plant tissues is a valuable tool for plant biotechnology. To shorten the time for gene functional analysis in fruits, we developed a transient methodology that could be applied to tomato (Solanum lycopersicum cv Micro Tom) fruits. It was found that injection of Agrobacterium cultures through the fruit stylar apex resulted in complete fruit infiltration. This infiltration method, named fruit agroinjection, rendered high levels of 35S Cauliflower mosaic virus-driven beta-glucuronidase and yellow fluorescence protein transient expression in the fruit, with higher expression levels around the placenta and moderate levels in the pericarp. Usefulness of fruit agroinjection was assayed in three case studies: (1) the heat shock regulation of an Arabidopsis (Arabidopsis thaliana) promoter, (2) the production of recombinant IgA antibodies as an example of molecular farming, and (3) the virus-induced gene silencing of the carotene biosynthesis pathway. In all three instances, this technology was shown to be efficient as a tool for fast transgene expression in fruits.