The N-Glycan Cluster from Xanthomonas campestris pv. campestris: A TOOLBOX FOR SEQUENTIAL PLANT N-GLYCAN PROCESSING*

N-Glycans are widely distributed in living organisms but represent only a small fraction of the carbohydrates found in plants. This probably explains why they have not previously been considered as substrates exploited by phytopathogenic bacteria during plant infection. Xanthomonas campestris pv. campestris, the causal agent of black rot disease of Brassica plants, possesses a specific system for GlcNAc utilization expressed during host plant infection. This system encompasses a cluster of eight genes (nixE to nixL) encoding glycoside hydrolases (GHs). In this paper, we have characterized the enzymatic activities of these GHs and demonstrated their involvement in sequential degradation of a plant N-glycan using a N-glycopeptide containing two GlcNAcs, three mannoses, one fucose, and one xylose (N₂M₃FX) as a substrate. The removal of the α-1,3-mannose by the α-mannosidase NixK (GH92) is a prerequisite for the subsequent action of the β-xylosidase NixI (GH3), which is involved in the cleavage of the β-1,2-xylose, followed by the α-mannosidase NixJ (GH125), which removes the α-1,6-mannose. These data, combined to the subcellular localization of the enzymes, allowed us to propose a model of N-glycopeptide processing by X. campestris pv. campestris. This study constitutes the first evidence suggesting N-glycan degradation by a plant pathogen, a feature shared with human pathogenic bacteria. Plant N-glycans should therefore be included in the repertoire of molecules putatively metabolized by phytopathogenic bacteria during their life cycle.     

Treatment with a sphingosine analog after the inception of house dust mite-induced airway inflammation alleviates key features of experimental asthma

Background

In vivo phosphorylation of sphingosine analogs with their ensuing binding and activation of their cell-surface sphingosine-1-phosphate receptors is regarded as the main immunomodulatory mechanism of this new class of drugs. Prophylactic treatment with sphingosine analogs interferes with experimental asthma by impeding the migration of dendritic cells to draining lymph nodes. However, whether these drugs can also alleviate allergic airway inflammation after its onset remains to be determined. Herein, we investigated to which extent and by which mechanisms the sphingosine analog AAL-R interferes with key features of asthma in a murine model during ongoing allergic inflammation induced by Dermatophagoides pteronyssinus.

Methods

BALB/c mice were exposed to either D. pteronyssinus or saline, intranasally, once-daily for 10 consecutive days. Mice were treated intratracheally with either AAL-R, its pre-phosphorylated form AFD-R, or the vehicle before every allergen challenge over the last four days, i.e. after the onset of allergic airway inflammation. On day 11, airway responsiveness to methacholine was measured; inflammatory cells and cytokines were quantified in the airways; and the numbers and/or viability of T cells, B cells and dendritic cells were assessed in the lungs and draining lymph nodes.

Results

AAL-R decreased airway hyperresponsiveness induced by D. pteronyssinus by nearly 70%. This was associated with a strong reduction of IL-5 and IL-13 levels in the airways and with a decreased eosinophilic response. Notably, the lung CD4⁺ T cells were almost entirely eliminated by AAL-R, which concurred with enhanced apoptosis/necrosis in that cell population. This inhibition occurred in the absence of dendritic cell number modulation in draining lymph nodes. On the other hand, the pre-phosphorylated form AFD-R, which preferentially acts on cell-surface sphingosine-1-phosphate receptors, was relatively impotent at enhancing cell death, which led to a less efficient control of T cell and eosinophil responses in the lungs.

Conclusion

Airway delivery of the non-phosphorylated sphingosine analog, but not its pre-phosphorylated counterpart, is highly efficient at controlling the local T cell response after the onset of allergic airway inflammation. The mechanism appears to involve local induction of lymphocyte apoptosis/necrosis, while mildly affecting dendritic cell and T cell accumulation in draining lymph nodes.     

Transformed hairy roots of Discaria trinervis: a valuable tool for studying actinorhizal symbiosis in the context of intercellular infection

Among infection mechanisms leading to root nodule symbiosis, the intercellular infection pathway is probably the most ancestral but also one of the least characterized. Intercellular infection has been described in Discaria trinervis, an actinorhizal plant belonging to the Rosales order. To decipher the molecular mechanisms underlying intercellular infection with Frankia bacteria, we set up an efficient genetic transformation protocol for D. trinervis based on Agrobacterium rhizogenes. We showed that composite plants with transgenic roots expressing green fluorescent protein can be specifically and efficiently nodulated by Frankia strain BCU110501. Nitrogen fixation rates and feedback inhibition of nodule formation by nitrogen were similar in control and composite plants. In order to challenge the transformation system, the MtEnod11 promoter, a gene from Medicago truncatula widely used as a marker for early infection-related symbiotic events in model legumes, was introduced in D. trinervis. MtEnod11::GUS expression was related to infection zones in root cortex and in the parenchyma of the developing nodule. The ability to study intercellular infection with molecular tools opens new avenues for understanding the evolution of the infection process in nitrogen-fixing root nodule symbioses.     

Advances in the synthesis and pharmacological activity of lupane-type triterpenoid saponins

Lupeol, betulin and betulinic acid are members of the so-called lupane-type triterpenoids. These natural products found worldwide in quite of lot of vegetables, fruits and plant species exhibit promising pharmacological activities including anti-inflammatory, anti-HIV and antitumor activities. Nevertheless, the poor pharmacokinetic properties of these cholesterol-like triterpenoids hampered further pharmaceutical developments. The synthesis of lupane-type saponins, i.e., sugar-derived lupanes, seems to be a good avenue to improve both their water solubility and pharmacological activity. The aims of this review are twofold: first, to describe the biological activity of naturally occurring lupane-type saponins, and second, report the different methodologies employed for the elaboration of glycosidic linkages at the C-3 and/or C-28 positions on the lupane core. The synthesis of both natural and unnatural lupane-type saponins is discussed with an emphasis on molecules exhibiting relevant biological activities.     

Ddc1 checkpoint protein and DNA polymerase ɛ interact with nick-containing DNA repair intermediate in cell free extracts of Saccharomyces cerevisiae

To characterize proteins that interact with base excision/single-strand interruption repair DNA intermediates in cell free extracts of Saccharomyces cerevisiae, we used a combination of photoaffinity labeling with the protein identification by MALDI-TOF-MS peptide mapping. Photoreactive analogue of dCTP, namely exo-N-[4-(4-azido-2,3,5,6,-tetrafluorobenzylidenehydrazinocarbonyl)-butylcarbamoyl]-2'-deoxycytidine-5'-triphosphate, and [(32)P]-labeled DNA duplex containing one nucleotide gap were used to generate nick-containing DNA with a photoreactive dCMP residue at the 3'-margin of the nick. This photoreactive DNA derivative was incubated with the yeast cell extract and after UV irradiation a number of proteins were labeled. Two of the crosslinked proteins were identified as the catalytic subunit of DNA polymerase ? and Ddc1 checkpoint protein. Labeling of DNA polymerase ? catalytic subunit with the nick-containing DNA repair intermediate indicates that the DNA polymerase is involved in the DNA repair synthesis in yeast, at least at DNA single-strand interruptions. Crosslinking of Ddc1 to DNA nicks took place independently of the other components of checkpoint clamp, Mec3 and Rad17, suggesting that the protein alone is able to recognize DNA single-strand breaks. Indeed, purified GST-tagged Ddc1 protein was efficiently crosslinked to nick-containing DNA. The interaction of Ddc1 with DNA nicks may provide a link between the DNA damage checkpoint and DNA base excision/single-strand breaks repair pathways in yeast. In addition, we found that absence of Ddc1 protein greatly influences the overall pattern of other proteins crosslinked to DNA nick. We suggested that this last effect of Ddc1 is at least partially due to its capacity to prevent proteolytic degradation of the DNA-protein adducts.     

Transposable elements in yeasts

With the development of new sequencing technologies in the past decade, yeast genomes have been extensively sequenced and their structures investigated. Transposable elements (TEs) are ubiquitous in eukaryotes and constitute a limited part of yeast genomes. However, due to their ability to move in genomes and generate dispersed repeated sequences, they contribute to modeling yeast genomes and thereby induce plasticity. This review assesses the TE contents of yeast genomes investigated so far. Their diversity and abundance at the inter- and intraspecific levels are presented, and their effects on gene expression and genome stability is considered. Recent results concerning TE-host interactions are also analyzed.     

Ralstonia syzygii, the Blood Disease Bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles

The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical evolution than to the acquisition of DNA by horizontal transfer.     

Overexpression of beta2-adrenergic receptors in mouse liver alters the expression of gluconeogenic and glycolytic enzymes

In the livers of humans and many other mammalian species, beta2-adrenergic receptors (beta2-ARs) play an important role in the modulation of glucose production by glycogenolysis and gluconeogenesis. In male mice and rats, however, the expression and physiological role of hepatic beta2-ARs are rapidly lost with development under normal physiological conditions. We previously described a line of transgenic mice, F28 (Andre C, Erraji L, Gaston J, Grimber G, Briand P, and Guillet JG. Eur J Biochem 241: 417-424, 1996), which carry the human beta2-AR gene under the control of its own promoter. In these mice, hepatic beta2-AR levels are shown to increase rapidly after birth and, as in humans, be maintained at an elevated level in adulthood. F28 mice display strongly enhanced adenylyl cyclase responses to beta-AR agonists in their livers and, compared with normal mice, have increased basal hepatic adenylyl cyclase activity. In this report we demonstrate that, under normal physiological conditions, this increased beta2-AR activity affects the expression of the gluconeogenic and glycolytic key enzymes phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and l-pyruvate kinase and considerably decreases hepatic glycogen levels. Furthermore, we show that the effects of beta-adrenergic ligands on liver glycogen observed in humans are reproduced in these mice: liver glycogen levels are strongly decreased by the beta2-AR agonist clenbuterol and increased by the beta-AR antagonist propranolol. These transgenic mice open new perspectives for studying in vivo the hepatic beta2-AR system physiopathology and for testing the effects of beta-AR ligands on liver metabolism.     

Multiple-mutation reaction: a method for simultaneous introduction of multiple mutations into the glpK gene of Mycoplasma pneumoniae

In Mycoplasma pneumoniae, the UGA opal codon specifies tryptophan rather than a translation stop site. This often makes it difficult to express Mycoplasma proteins in E. coli isolates. In this work, we developed a strategy for the one-step introduction of several mutations. This method, the multiple-mutation reaction, is used to simultaneously replace nine opal codons in the M. pneumoniae glpK gene.