Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: Changes in cell structure, numbers and adherence

Host-pathogen interaction models in aquatic species are useful tools for understanding the pathogenicity of diseases in cultured and wild populations. In this study we report the differential in vivo response of soft-shell clam (Mya arenaria) hemocytes against two strains of Vibrio splendidus. Responses were measured 24 h after injecting into the posterior adductor muscle either an endemic wild-type strain (7SHRW) or a strain associated with oyster mortalities (LGP32-GFP). Changes in hemocyte structure (percentage of rounded cells) were assessed microscopically. Changes in adherence and hemocyte numbers were analyzed by flow-cytometric cell counting. Increased percentages of rounded cells were found in response to both strains. However, values from the group infected with LGP32-GFP were significantly higher (p < 0.01) than with 7SHRW. The cell adherence was markedly diminished (p < 0.001) by LGP32-GFP whereas 7SHRW did not change it significantly. Increased numbers of hemocytes (p < 0.001) were induced by LGP32-GFP, while no significant changes were found after infection with 7SHRW. These results show the regulatory capacity of soft-shell clams hemocytes to perform specific responses against different strains of V. splendidus.     

The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity

Nucleotide-binding and oligomerization domain (NOD)-like receptors constitute a first line of defense against invading bacteria. X-linked Inhibitor of Apoptosis (XIAP) is implicated in the control of bacterial infections, and mutations in XIAP are causally linked to immunodeficiency in X-linked lymphoproliferative syndrome type-2 (XLP-2). Here, we demonstrate that the RING domain of XIAP is essential for NOD2 signaling and that XIAP contributes to exacerbation of inflammation-induced hepatitis in experimental mice. We find that XIAP ubiquitylates RIPK2 and recruits the linear ubiquitin chain assembly complex (LUBAC) to NOD2. We further show that LUBAC activity is required for efficient NF-κB activation and secretion of proinflammatory cytokines after NOD2 stimulation. Remarkably, XLP-2-derived XIAP variants have impaired ubiquitin ligase activity, fail to ubiquitylate RIPK2, and cannot facilitate NOD2 signaling. We conclude that XIAP and LUBAC constitute essential ubiquitin ligases in NOD2-mediated inflammatory signaling and propose that deregulation of NOD2 signaling contributes to XLP-2 pathogenesis.     

Phylogenetic study and identification of Vibrio splendidus-related strains based on gyrB gene sequences

Different strains related to Vibrio splendidus have been associated with infection of aquatic animals. An epidemiological study of V. splendidus strains associated with Crassostrea gigas mortalities demonstrated genetic diversity within this group and suggested its polyphyletic nature. Recently 4 species, V. lentus, V. chagasii, V. pomeroyi and V. kanaloae, phenotypically related to V. splendidus, have been described, although biochemical methods do not clearly discriminate species within this group. Here, we propose a polyphasic approach to investigate their taxonomic relationships. Phylogenetic analysis of V. splendidus-related strains was carried out using the nucleotide sequences of 16S ribosomal DNA (16S rDNA) and gyrase B subunit (gyrB) genes. Species delineation based on 16S rDNA-sequencing is limited because of divergence between cistrons, roughly equivalent to divergence between strains. Despite a high level of sequence similarity, strains were separated into 2 clades. In the phylogenetic tree constructed on the basis of gyrB gene sequences, strains were separated into 5 independent clusters containing V. splendidus, V. lentus, V. chagasii-type strains and a putative new genomic species. This phylogenetic grouping was almost congruent with that based on DNA-DNA hybridisation analysis. V. pomeroyi, V. kanaloae and V. tasmaniensis-type strains clustered together in a fifth clade. The gyrB gene-sequencing approach is discussed as an alternative for investigating the taxonomy of Vibrio species.     

Validation of in situ hybridisation and histology assays for the detection of the oyster parasite Marteilia refringens

An in situ hybridisation technique has been developed for the detection of infection in oysters with Marteilia refringens with particular emphasis on light infections or confirmation of suspected cases by means of histology. Although validation of new diagnostic methods is usually achieved by comparison with standard techniques, in our case the sensitivity and specificity of the standard (histology) had not previously been established. Another point to consider is that surveillance and monitoring frequently target populations displaying different levels of prevalence under different field conditions. The objective of our study was to evaluate the sensitivity and specificity values of in situ hybridisation and histology for the detection of M. refringens, based on 3 populations of flat oysters, free of the disease and with mild and high levels of prevalence. A blind assay of 200 individuals from each population was performed using both techniques. Results were analysed by means of the classical approach and latent models (maximum likehood and Bayesian approach). Assumptions and results were found to vary slightly with the different statistical approaches. The more realistic estimate by the Bayesian approach shows a link between the level of prevalence and the sensitivity of the techniques. Values of sensitivity and specificity for histology were 0.7 and 0.99 respectively, and 0.9 and 0.99 respectively in the case of in situ hybridisation. Some uncertainty remains regarding these values because the study does not take into account the severity of infection or the developmental stages of the parasite actually present in each individual. This work provides valuable information with regard to the choice and potential use of those 2 diagnostic methods currently recommended by international standards.     

Suppression of integrin activation by activated Ras or Raf does not correlate with bulk activation of ERK MAP kinase

The rapid modulation of ligand-binding affinity ("activation") is a central property of the integrin family of cell adhesion receptors. The Ras family of small GTP-binding proteins and their downstream effectors are key players in regulating integrin activation. H-Ras can suppress integrin activation in fibroblasts via its downstream effector kinase, Raf-1. In contrast, to H-Ras, a closely related small GTP-binding protein R-Ras has the opposite activity, and promotes integrin activation. To gain insight into the regulation of integrin activation by Ras GTPases, we created a series of H-Ras/R-Ras chimeras. We found that a 35-amino acid stretch of H-Ras was required for full suppressive activity. Furthermore, the suppressive chimeras were weak activators of the ERK1/2 MAP kinase pathway, suggesting that the suppression of integrin activation may be independent of the activation of the bulk of ERK MAP kinase. Additional data demonstrating that the ability of H-Ras or Raf-1 to suppress integrin activation was unaffected by inhibition of bulk ERK1/2 MAP kinase activation supported this hypothesis. Thus, the suppression of integrin activation is a Raf kinase induced regulatory event that can be mediated independently of bulk activation of the ERK MAP-kinase pathway.     

Research note: Yeast flora of Haitian rum distilleries

Summary In Haïtian rum distilleries, few modifications have been carried out on the working conditions for some time.Schizosaccharomyces is the main yeast genus which exists in such distilleries. In the distilleries of the French West-Indies, there is a predominance ofSaccharomyces yeasts.Schizosaccharomyces strains allow the production of a light rum which is in great demand.

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Resumen En las destilerias de ron de Haiti los métodos de producción se han mantenido practicamente invariables desde hace tiempo. El género de levaduras más abundante en dichas destilerias esSchizosaccharomyces. En las destilerias de la Guayana francesa las levaduras predominantes son del géneroSaccharomyces. Las cepasde Schizosaccharomyces son las que permiten la producción de ron claro para el que existe una gran demanda.

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Résumé Il y a déjà un certain temps que l'on n'effectue que peu de modifications aux conditions de travail dans les distilleries Haïtiennes de rhum.Schizosaccharomyces est le genre principal de levure qui existe dans ces distilleries. Dans les distilleries des Antilles françaises, il y a prédominance des levuresSaccharomyces. Les souches deSchizosaccharomyces permettent la production de rhum de variété light qui est fort demandé.

     

Interaction of alleles of therelA, relC andspoT genes inEscherichia coli: Analysis of the interconversion of GTP, ppGpp and pppGpp

Summary Mutants in thespoT gene have been isolated as stringent second site revertants of therelC mutation. These show varying degrees of the characteristics associated with thespoT1 gene,viz relative amount and absolute levels of both pppGpp and ppGpp and the decay rate of the latter. The entry of³H-guanosine into GTP and ppGpp pools inspoT ⁺ andspoT1 cells either growing exponentially or during amino acid starvation was determined, and the rate of ppGpp synthesis and its decay constant calculated. During exponential growth the ppGpp pool is 2-fold higher, its decay constant 10-fold lower, and its synthesis rate 5-fold lower inspoT ^- than inspoT ⁺ cells; during amino acid starvation the ppGpp pool is 2-fold higher, its decay constant 20-fold lower, and its synthesis rate 10-fold lower inspoT than inspoT ⁺ cells. In one of the “intermediate”spoT mutants the rate of entry of³H-guanosine into GTP, ppGpp and pppGpp was measured during amino acid starvation. The data form the basis of a model for the interconversion of the guanosine nucleotides in which the flow is:GDP→GTP→pppGpp→ppGpp→Y. Calculations of the rates of synthesis and conversion of pppGpp and ppGpp under various conditions in variousspoT ⁺ andspoT ^- strains indicate that the ppGpp concentration indirectly controls the rate of pppGpp synthesis. ThespoT1 allele was introduced into various relaxed mutants. It was shown that many phenomena associated with the relaxed response ofrelC and “intermediate”relA mutants were phenotypically suppressed when thespoT1 allele was introduced into these mutants. These double mutants exhibit ppGpp accumulation, rate of RNA accumulation, rate of β-galactosidase synthesis, and heat lability of β-galactosidase synthesized during amino acid starvation similar to the stringent wild-type. It is concluded that the relaxed response is due directly to the lack of ppGpp and that the stringest response is due directly to ppGpp.     

Distribution spatio-temporelle de la faune de phlébotomes en zones urbaine et périurbaine de Bamako,Mali

Au Mali la leishmaniose cutanée (LC) est bien décrite en milieu rural, mais sa transmission n’est pas encore établie en milieu urbain. L’objectif de l’étude était de décrire la faune phlébotomienne à Bamako et dans ses environs. Les pièges CDC et adhésifs servaient à collecter les phlébotomes en 2011 et 2012 à Bamako. Les pièges étaient placés dans le district de Bamako répartis en 30 zones (200 m × 200 m) à l’aide des images de satellite SPOT-5. La délimitation des zones était basée sur les facteurs écologiques (relief, hydrographie, couverture végétale) et anthropologiques (nature des maisons, dépôts d’ordure). Les pièges étaient placés en 2011 à Sotuba, zone périurbaine, puis à Donéguébougou et Banambani, deux villages environnants de Bamako. Au total 122 phlébotomes étaient collectés 27 (22,13 %) à Banambani, 12 (9,83 %) à Donéguébougou, 2 (1,63 %) à Sotuba et 81 (66,39 %) à Bamako. La densité des phlébotomes en novembre était plus élevée, avec 48 spécimens capturés (39,3 %), que celle d’avril, mai et décembre (p ≥ 0,001). A Bamako, les zones 6 et 28 étaient associées aux densités les plus élevées de phlébotomes respectivement 32 (26,22 %) et 23 (18,85 %) (p ≤ 0,005). Les résultats suggèrent que le genre Phlebotomus n’était pas présent à Bamako, et que la densité des vecteurs variait en fonction des zones et des mois.