Virulence of an emerging pathogenic lineage of Vibrio nigripulchritudo is dependent on two plasmids

Vibrioses are the predominant bacterial infections in marine shrimp farms. Vibrio nigripulchritudo is an emerging pathogen of the cultured shrimp Litopenaeus stylirostris in New Caledonia and other regions in the Indo‐Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have revealed that recent pathogenic V. nigripulchritudo isolates from New Caledonia all cluster into a monophyletic clade and contain a small plasmid, pB1067. Here, we report that a large plasmid, pA1066 (247 kb), can also serve as a marker for virulent V. nigripulchritudo, and that an ancestral version of this plasmid was likely acquired prior to other virulence‐linked markers. Additionally, we demonstrate that pA1066 is critical for the full virulence of V. nigripulchritudo in several newly developed experimental models of infection. Plasmid pB1067 also contributes to virulence; only strains containing both plasmids induced the highest level of shrimp mortality. Thus, it appears that these plasmids, which are absent from non‐pathogenic isolates, may be driving forces, as well as markers, for the emergence of a pathogenic lineage of V. nigripulchritudo.     

An epigenetic switch involving overlapping fur and DNA methylation optimizes expression of a type VI secretion gene cluster

Type VI secretion systems (T6SS) are macromolecular machines of the cell envelope of Gram-negative bacteria responsible for bacterial killing and/or virulence towards different host cells. Here, we characterized the regulatory mechanism underlying expression of the enteroagregative Escherichia coli sci1 T6SS gene cluster. We identified Fur as the main regulator of the sci1 cluster. A detailed analysis of the promoter region showed the presence of three GATC motifs, which are target of the DNA adenine methylase Dam. Using a combination of reporter fusion, gel shift, and in vivo and in vitro Dam methylation assays, we dissected the regulatory role of Fur and Dam-dependent methylation. We showed that the sci1 gene cluster expression is under the control of an epigenetic switch depending on methylation: fur binding prevents methylation of a GATC motif, whereas methylation at this specific site decreases the affinity of Fur for its binding box. A model is proposed in which the sci1 promoter is regulated by iron availability, adenine methylation, and DNA replication.     

Rodent-Borne Hantaviruses in Cambodia, Lao PDR, and Thailand

In order to evaluate the circulation of hantaviruses present in southeast Asia, a large scale survey of small mammal species was carried out at seven main sites in the region (Cambodia, Lao People's Democratic Republic, and Thailand). Small scale opportunistic trapping was also performed at an eighth site (Cambodia). Using a standard IFA test, IgG antibodies reacting to Hantaan virus antigens were detected at six sites. Antibody prevalence at each site varied from 0 to 5.6% with antibodies detected in several rodent species (Bandicota indica, B. savilei, Maxomys surifer, Mus caroli, M. cookii, Rattus exulans, R. nitidius, R. norvegicus, and R. tanezumi). When site seroprevalence was compared with site species richness, seropositive animals were found more frequently at sites with lower species richness. In order to confirm which hantavirus species were present, a subset of samples was also subjected to RT-PCR. Hantaviral RNA was detected at a single site from each country. Sequencing confirmed the presence of two hantavirus species, Thailand and Seoul viruses, including one sample (from Lao PDR) representing a highly divergent strain of Seoul virus. This is the first molecular evidence of hantavirus in Lao PDR and the first reported L segment sequence data for Thailand virus.     

Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells

Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.     

Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells

Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.     

Programming an in vitro DNA oscillator using a molecular networking strategy

Living organisms perform and control complex behaviours by using webs of chemical reactions organized in precise networks. This powerful system concept, which is at the very core of biology, has recently become a new foundation for bioengineering. Remarkably, however, it is still extremely difficult to rationally create such network architectures in artificial, non‐living and well‐controlled settings. We introduce here a method for such a purpose, on the basis of standard DNA biochemistry. This approach is demonstrated by assembling de novo an efficient chemical oscillator: we encode the wiring of the corresponding network in the sequence of small DNA templates and obtain the predicted dynamics. Our results show that the rational cascading of standard elements opens the possibility to implement complex behaviours in vitro. Because of the simple and well‐controlled environment, the corresponding chemical network is easily amenable to quantitative mathematical analysis. These synthetic systems may thus accelerate our understanding of the underlying principles of biological dynamic modules.     

Structural bases for heme binding and diatomic ligand recognition in truncated hemoglobins

Truncated hemoglobins (trHbs) are low-molecular-weight oxygen-binding heme-proteins distributed in eubacteria, cyanobacteria, unicellular eukaryotes, and in higher plants, constituting a distinct group within the hemoglobin (Hb) superfamily. TrHbs display amino acid sequences 20-40 residues shorter than classical (non)vertebrate Hbs and myoglobins, to which they are scarcely related by sequence similarity. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which represents a striking editing of the highly conserved 3-on-3 alpha-helical globin fold, achieved through deletion/truncation of alpha-helices and specific residue substitutions. Despite their 'minimal' polypeptide chain span, trHbs display an inner tunnel/cavity system held to support ligand diffusion to/from the heme distal pocket, accumulation of heme ligands within the protein matrix, and/or multiligand reactions. Moreover, trHbs bind and effectively stabilize the heme and recognize diatomic ligands (i.e., O2, CO, NO, and cyanide), albeit with varying thermodynamic and kinetic parameters. Here, structural bases for heme binding and diatomic ligand recognition by trHbs are reviewed.