Osmoadaptative responses in the rhizobia nodulating Acacia isolated from south-eastern Moroccan Sahara

Four strains of rhizobia nodulating Acacia were isolated from the Moroccan desert soil by trapping with seedlings of Acacia gummifera and Acacia raddiana, and were studied for their ability to tolerate high salinity and dryness conditions. The strains MDSMC 2, MDSMC 18 and MDSMC 50 were halotolerant (they tolerated up to 1 M NaCl) and they accumulated glutamate and mannosucrose. The synthesis of the latter solute, which is the major endogenous osmolyte, is partially repressed in the presence of glycine betaine. The strain MDSMC 34 was less halotolerant (growth inhibited by a concentration greater than 0.5 M NaCl), and accumulated trehalose (as the main endogenous osmolyte) and glutamate. Rhizobia from the Moroccan desert soil were highly resistant to desiccation and their tolerance to dryness was stimulated by osmotic pretreatment. Thus, the accumulation of mannosucrose or trehalose by desert rhizobia represents both an osmoadaptative response and a part of a desiccation tolerance mechanism.     

Transposon-mediated resistance to Bacillus sphaericus in a field-evolved population of Culex pipiens (Diptera: Culicidae)

The binary toxin is the major active component of Bacillus sphaericus, a microbial larvicide used for controlling some vector mosquito-borne diseases. B. sphaericus resistance has been reported in many part of the world, leading to a growing concern for the usefulness of this environmental friendly insecticide. Here we characterize a novel mechanism of resistance to the binary toxin in a natural population of the West Nile virus vector, Culex pipiens. We show that the insertion of a transposable element-like DNA into the coding sequence of the midgut toxin receptor induces a new mRNA splicing event, unmasking cryptic donor and acceptor sites located in the host gene. The creation of the new intron causes the expression of an altered membrane protein, which is incapable of interacting with the toxin, thus providing the host mosquito with an advantageous phenotype. As a large portion of insect genomes is composed of transposable elements or transposable elements-related sequences, this new mechanism may be of general importance to appreciate their significance as potent agents for insect resistance to the microbial insecticides.     

A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd^mdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.     

The size of the plasmacytoid dendritic cell compartment is a multigenic trait dominated by a locus on mouse chromosome 7

Plasmacytoid dendritic cells (pDC) compose one of the many distinct dendritic cell subsets. The primary function of pDC is to potently produce type 1 IFNs upon stimulation, which is highly relevant in antiviral responses. Consequently, the ability to manipulate the size of the pDC compartment in vivo may increase the capacity to clear viral infections. In an attempt to identify genetic loci affecting the size of the pDC compartment, defined by both the proportion and absolute number of pDC, we undertook an unbiased genetic approach. Linkage analysis using inbred mouse strains identified a locus on chromosome 7 (Pdcc1) significantly linked to both the proportion and the absolute number of pDC in the spleen. Moreover, loci on either chromosome 11 (Pdcc2) or 9 (Pdcc3) modified the effect of Pdcc1 on chromosome 7 for the proportion and absolute number of pDC, respectively. Further analysis using mice congenic for chromosome 7 confirmed Pdcc1, demonstrating that variation within this genetic interval can regulate the size of the pDC compartment. Finally, mixed bone marrow chimera experiments showed that both the proportion and the absolute number of pDC are regulated by cell-intrinsic hematopoietic factors. Our findings highlight the multigenic regulation of the size of the pDC compartment and will facilitate the identification of genes linked to this trait.     

Human T lymphotropic virus type 1 increases T lymphocyte migration by recruiting the cytoskeleton organizer CRMP2

Recruitment of virus-infected T lymphocytes into the CNS is an essential step in the development of virus-associated neuroinflammatory diseases, notably myelopathy induced by retrovirus human T leukemia virus-1 (HTLV-1). We have recently shown the key role of collapsin response mediator protein 2 (CRMP2), a phosphoprotein involved in cytoskeleton rearrangement, in the control of human lymphocyte migration and in brain targeting in animal models of virus-induced neuroinflammation. Using lymphocytes cloned from infected patients and chronically infected T cells, we found that HTLV-1 affects CRMP2 activity, resulting in an increased migratory potential. Elevated CRMP2 expression accompanies a higher phosphorylation level of CRMP2 and its more pronounced adhesion to tubulin and actin. CRMP2 forms, a full length and a shorter, cleaved one, are also affected. Tax transfection and extinction strategies show the involvement of this viral protein in enhanced full-length and active CRMP2, resulting in prominent migratory rate. A role for other viral proteins in CRMP2 phosphorylation is suspected. Full-length CRMP2 confers a migratory advantage possibly by preempting the negative effect of short CRMP2 we observe on T lymphocyte migration. In addition, HTLV-1-induced migration seems, in part, supported by the ability of infected cell to increase the proteosomal degradation of short CRMP2. Finally, gene expression in CD69(+) cells selected from patients suggests that HTLV-1 has the capacity to influence the CRMP2/PI3K/Akt axis thus to positively control cytoskeleton organization and lymphocyte migration. Our data provide an additional clue to understanding the infiltration of HTLV-1-infected lymphocytes into various tissues and suggest that the regulation of CRMP2 activity by virus infection is a novel aspect of neuroinflammation.     

Microevolution of S-allele frequencies in wild cherry populations: respective impacts of negative frequency dependent selection and genetic drift

Negative frequency dependent selection (NFDS) is supposed to be the main force controlling allele evolution at the gametophytic self-incompatibility locus (S-locus) in strictly outcrossing species. Genetic drift also influences S-allele evolution. In perennial sessile organisms, evolution of allelic frequencies over two generations is mainly shaped by individual fecundities and spatial processes. Using wild cherry populations between two successive generations, we tested whether S-alleles evolved following NFDS qualitative and quantitative predictions. We showed that allelic variation was negatively correlated with parental allelic frequency as expected under NFDS. However, NFDS predictions in finite population failed to predict more than half S-allele quantitative evolution. We developed a spatially explicit mating model that included the S-locus. We studied the effects of self-incompatibility and local drift within populations due to pollen dispersal in spatially distributed individuals, and variation in male fecundity on male mating success and allelic frequency evolution. Male mating success was negatively related to male allelic frequency as expected under NFDS. Spatial genetic structure combined with self-incompatibility resulted in higher effective pollen dispersal. Limited pollen dispersal in structured distributions of individuals and genotypes and unequal pollen production significantly contributed to S-allele frequency evolution by creating local drift effects strong enough to counteract the NFDS effect on some alleles.     

Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors

Hematopoietic progenitors from murine fetal liver efficiently differentiate in culture into proplatelet-producing megakaryocytes and have proved valuable to study platelet biogenesis. In contrast, megakaryocyte maturation is far less efficient in cultured bone marrow progenitors, which hampers studies in adult animals. It is shown here that addition of hirudin to media containing thrombopoietin and serum yielded a proportion of proplatelet-forming megakaryocytes similar to that in fetal liver cultures (approximately 50%) with well developed extensions and increased the release of platelet particles in the media. The effect of hirudin was maximal at 100 U/ml, and was more pronounced when it was added in the early stages of differentiation. Hirugen, which targets the thrombin anion binding exosite I, and argatroban, a selective active site blocker, also promoted proplatelet formation albeit less efficiently than hirudin. Heparin, an indirect thrombin blocker, and OTR1500, a stable heparin-like synthetic glycosaminoglycan generated proplatelets at levels comparable to hirudin. Heparin with low affinity for antithrombin was equally as effective as standard heparin, which indicates antithrombin independent effects. Use of hirudin and heparin compounds should lead to improved culture conditions and facilitate studies of platelet biogenesis in adult mice.     

HLA-DQA2 and HLA-DQB2 genes are specifically expressed in human Langerhans cells and encode a new HLA class II molecule

The precise role of human epidermal Langerhans cells (LCs) in immune response is highly controversial. While studying the gene expression profile of these cells, we were intrigued to identify the HLA-DQB2 gene as potentially expressed in LCs. Despite a strong evolutionary conservation of their sequences, the concomitant expression of the poorly polymorphic HLA-DQA2/HLA-DQB2 genes, paralogous to the HLA-DQA1/HLA-DQB1 genes, has never been detected in any cell type. We confirmed by RT-PCR that the HLA-DQA2 and -DQB2 genes are both expressed in LCs, but not in monocyte-derived dendritic cells, or in blood CD1c(+) or plasmacytoid dendritic cells. The presence of the HLA-DQβ2 chain in LCs could be demonstrated by Western blotting, whereas immunofluorescence revealed its localization in early endosomes. As in the case of other HLA class II molecules, the HLA-DQα2 and -DQβ2 chains formed heterodimers that had to associate with the invariant chain to reach endosomal compartments. HLA-DQα2/β2 heterodimers were expressed at the cell surface, where they could mediate staphylococcal superantigen stimulation of T cells. Interestingly, HLA-DQα2 and HLA-DQβ1 chains formed mixed heterodimers which efficiently left the endoplasmic reticulum. These observations strongly suggest that the poorly polymorphic HLA-DQA2 and -DQB2 genes should be considered to be of immunological importance. The HLA-DQα2/β2 molecules could influence the complexity of the repertoire of Ags presented by LCs.     

The nucleoid‐associated protein Fis directly modulates the synthesis of cellulose,an essential component of pellicle–biofilms in the phytopathogenic bacterium Dickeya dadantii

Bacteria use biofilm structures to colonize surfaces and to survive in hostile conditions, and numerous bacteria produce cellulose as a biofilm matrix polymer. Hence, expression of the bcs operon, responsible for cellulose biosynthesis, must be finely regulated in order to allow bacteria to adopt the proper surface‐associated behaviours. Here we show that in the phytopathogenic bacterium, Dickeya dadantii, production of cellulose is required for pellicle–biofilm formation and resistance to chlorine treatments. Expression of the bcs operon is growth phase‐regulated and is stimulated in biofilms. Furthermore, we unexpectedly found that the nucleoid‐associated protein and global regulator of virulence functions, Fis, directly represses bcs operon expression by interacting with an operator that is absent from the bcs operon of animal pathogenic bacteria and the plant pathogenic bacterium Pectobacterium. Moreover, production of cellulose enhances plant surface colonization by D. dadantii. Overall, these data suggest that cellulose production and biofilm formation may be important factors for surface colonization by D. dadantii and its subsequent survival in hostile environments. This report also presents a new example of how bacteria can modulate the action of a global regulator to co‐ordinate basic metabolism, virulence and modifications of lifestyle.