Cyclooxygenases and lipoxygenases are used by the fungus Podospora anserina to repel nematodes

Oxylipins are secondary messengers used universally in the living world for communication and defense. The paradigm is that they are produced enzymatically for the eicosanoids and non-enzymatically for the isoprostanoids. They are supposed to be degraded into volatile organic compounds (VOCs) and to participate in aroma production. Some such chemicals composed of eight carbons are also envisoned as alternatives to fossil fuels. In fungi, oxylipins have been mostly studied in Aspergilli and shown to be involved in signalling asexual versus sexual development, mycotoxin production and interaction with the host for pathogenic species. Through targeted gene deletions of genes encoding oxylipin-producing enzymes and chemical analysis of oxylipins and volatile organic compounds, we show that in the distantly-related ascomycete Podospora anserina, isoprostanoids are likely produced enzymatically. We show the disappearance in the mutants lacking lipoxygenases and cyclooxygenases of the production of 10-hydroxy-octadecadienoic acid and that of 1-octen-3-ol, a common volatile compound. Importantly, this was correlated with the inability of the mutants to repel nematodes as efficiently as the wild type. Overall, our data show that in this fungus, oxylipins are not involved in signalling development but may rather be used directly or as precursors in the production of odors against potential agressors.

Queuosine Biosynthesis Is Required for Sinorhizobium meliloti-Induced Cytoskeletal Modifications on HeLa Cells and Symbiosis with Medicago truncatula

Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton.     

A novel pollen-specific α-tubulin in sunflower: structure and characterization

We describe here a new -tubulin isoform from sunflower we named -tubulin. -tubulin is the most divergent higher-plant -tubulin described so far, having an unusual deletion in the H1/B2 loop and a glutamine-rich C-terminus. We constructed a three-dimensional model and discuss its implications. Using specific antibodies, we show that -tubulin expression is restricted to the male gametophyte. -tubulin mRNA represents 90% of -tubulin mRNA and a small percentage of total pollen mRNA. Among the plants tested, -tubulin was only detected in sunflower and in Cosmos. Since both plants are Asteraceae, we propose that -tubulin is specific to this family. Our results suggest that -tubulin can inhibit tubulin assembly in pollen. This hypothesis is reinforced by the fact that -tubulin is found in a complex with -tubulin in mature sunflower pollen.     

Midgut Microbiota of the Malaria Mosquito Vector Anopheles gambiae and Interactions with Plasmodium falciparum Infection

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.     

Domain 3 of NS5A protein from the hepatitis C virus has intrinsic alpha-helical propensity and is a substrate of cyclophilin A

Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and constitutes an attractive target for antiviral drug development. Although structural data for its in-plane membrane anchor and domain D1 are available, the structure of domains 2 (D2) and 3 (D3) remain poorly defined. We report here a comparative molecular characterization of the NS5A-D3 domains of the HCV JFH-1 (genotype 2a) and Con1 (genotype 1b) strains. Combining gel filtration, CD, and NMR spectroscopy analyses, we show that NS5A-D3 is natively unfolded. However, NS5A-D3 domains from both JFH-1 and Con1 strains exhibit a propensity to partially fold into an α-helix. NMR analysis identifies two putative α-helices, for which a molecular model could be obtained. The amphipathic nature of the first helix and its conservation in all genotypes suggest that it might correspond to a molecular recognition element and, as such, promote the interaction with relevant biological partner(s). Because mutations conferring resistance to cyclophilin inhibitors have been mapped into NS5A-D3, we also investigated the functional interaction between NS5A-D3 and cyclophilin A (CypA). CypA indeed interacts with NS5A-D3, and this interaction is completely abolished by cyclosporin A. NMR heteronuclear exchange experiments demonstrate that CypA has in vitro peptidyl-prolyl cis/trans-isomerase activity toward some, but not all, of the peptidyl-prolyl bonds in NS5A-D3. These studies lead to novel insights into the structural features of NS5A-D3 and its relationships with CypA.     

Development of a tetrameric streptavidin mutein with reversible biotin binding capability: engineering a mobile loop as an exit door for biotin

A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop(3-4) functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop(3-4) keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop(7-8). This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop(7-8) is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (k(off) of 4.28×10(-4) s(-1) and K(d) of 1.9×10(-8) M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible.     

Tryptophan metabolism activation by indoleamine 2,3-dioxygenase in adipose tissue of obese women: an attempt to maintain immune homeostasis and vascular tone

Human obesity is characterized by chronic low-grade inflammation in white adipose tissue and is often associated with hypertension. The potential induction of indoleamine 2,3-dioxygenase-1 (IDO1), the rate-limiting enzyme in tryptophan/kynurenine degradation pathway, by proinflammatory cytokines, could be associated with these disorders but has remained unexplored in obesity. Using immunohistochemistry, we detected IDO1 expression in white adipose tissue of obese patients, and we focused on its contribution in the regulation of vascular tone and on its immunoregulatory effects. Concentrations of tryptophan and kynurenine were measured in sera of 36 obese and 15 lean women. The expression of IDO1 in corresponding omental and subcutaneous adipose tissues and liver was evaluated. Proinflammatory markers and T-cell subsets were analyzed in adipose tissue via the expression of CD14, IL-18, CD68, TNFα, CD3ε, FOXP3 [a regulatory T-cell (Treg) marker] and RORC (a Th17 marker). In obese subjects, the ratio of kynurenine to tryptophan, which reflects IDO1 activation, is higher than in lean subjects. Furthermore, IDO1 expression in both adipose tissues and liver is increased and is inversely correlated with arterial blood pressure. Inflammation is associated with a T-cell infiltration in obese adipose tissue, with predominance of Th17 in the omental compartment and of Treg in the subcutaneous depot. The Th17/Treg balance is decreased in subcutaneous fat and correlates with IDO1 activation. In contrast, in the omental compartment, despite IDO1 activation, the Th17/Treg balance control is impaired. Taken together, our results suggest that IDO1 activation represents a local compensatory mechanism to limit obesity-induced inflammation and hypertension.     

Landscape dynamics and evolution of colonizer syndromes: interactions between reproductive effortand dispersal in a metapopulation

The evolutionary consequences of changes in landscape dynamics for the evolution of life history syndromes are studied using a metapopulation model. We consider in turn the long-term effects of a change in the local disturbance rate, in the maximal local population persistence, in habitat productivity, and in habitat fragmentation. We examine the consequences of selective interactions between dispersal and reproductive effort by comparing the outcome of joint evolution to a situation where the species has lost the potential to evolve either its reproductive effort or its dispersal rate. We relax the classical assumption that any occupied site in the metapopulation reaches its carrying capacity immediately after recolonization. Our main conclusions are the following: (1) genetic diversity modifies the range of landscape parameters for which the metapopulation is viable, but it alters very little the qualitative evolutionary trends observed for each trait within this range. Although they are both part of a competition/colonization axis, reproductive effort and dispersal are not substitutable traits: their evolution reflects more directly the change in the landscape dynamics, than a selective interaction among them. (2) no general syndrome of covariation between reproductive effort and dispersal can be predicted: the pattern of association between the two traits depends on the type of change in landscape dynamics and on the saturation level. We review empirical evidence on colonizer syndromes and suggest lines for further empirical work. This revised version was published online in July 2006 with corrections to the Cover Date.