Protein Glycosylation in Helicobacter pylori: Beyond the Flagellins?

Glycosylation of flagellins by pseudaminic acid is required for virulence in Helicobacter pylori. We demonstrate that, in H. pylori, glycosylation extends to proteins other than flagellins and to sugars other than pseudaminic acid. Several candidate glycoproteins distinct from the flagellins were detected via ProQ-emerald staining and DIG- or biotin- hydrazide labeling of the soluble and outer membrane fractions of wild-type H. pylori, suggesting that protein glycosylation is not limited to the flagellins. DIG-hydrazide labeling of proteins from pseudaminic acid biosynthesis pathway mutants showed that the glycosylation of some glycoproteins is not dependent on the pseudaminic acid glycosylation pathway, indicating the existence of a novel glycosylation pathway. Fractions enriched in glycoprotein candidates by ion exchange chromatography were used to extract the sugars by acid hydrolysis. High performance anion exchange chromatography with pulsed amperometric detection revealed characteristic monosaccharide peaks in these extracts. The monosaccharides were then identified by LC-ESI-MS/MS. The spectra are consistent with sugars such as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Ac7Ac) previously described on flagellins, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Am7Ac), bacillosamine derivatives and a potential legionaminic acid derivative (Leg5AmNMe7Ac) which were not previously identified in H. pylori. These data open the way to the study of the mechanism and role of protein glycosylation on protein function and virulence in H. pylori

Evaluating the pathogenic potential of environmental Escherichia coli by using the caenorhabditis elegans infection model

The detection and abundance of Escherichia coli in water is used to monitor and mandate the quality of drinking and recreational water. Distinguishing commensal waterborne E. coli isolates from those that cause diarrhea or extraintestinal disease in humans is important for quantifying human health risk. A DNA microarray was used to evaluate the distribution of virulence genes in 148 E. coli environmental isolates from a watershed in eastern Ontario, Canada, and in eight clinical isolates. Their pathogenic potential was evaluated with Caenorhabditis elegans, and the concordance between the bioassay result and the pathotype deduced by genotyping was explored. Isolates identified as potentially pathogenic on the basis of their complement of virulence genes were significantly more likely to be pathogenic to C. elegans than those determined to be potentially nonpathogenic. A number of isolates that were identified as nonpathogenic on the basis of genotyping were pathogenic in the infection assay, suggesting that genotyping did not capture all potentially pathogenic types. The detection of the adhesin-encoding genes sfaD, focA, and focG, which encode adhesins; of iroN2, which encodes a siderophore receptor; of pic, which encodes an autotransporter protein; and of b1432, which encodes a putative transposase, was significantly associated with pathogenicity in the infection assay. Overall, E. coli isolates predicted to be pathogenic on the basis of genotyping were indeed so in the C. elegans infection assay. Furthermore, the detection of C. elegans-infective environmental isolates predicted to be nonpathogenic on the basis of genotyping suggests that there are hitherto-unrecognized virulence factors or combinations thereof that are important in the establishment of infection. \ua9 2013, American Society for Microbiology.Peer reviewed: YesNRC publication: Ye

Engineering xylose metabolism in thraustochytrid T18

Abstract Background Thraustochytrids are heterotrophic, oleaginous, marine protists with a significant potential for biofuel production. High-value co-products can off-set production costs; however, the cost of raw materials, and in particular carbon, is a major challenge to developing an economical viable production process. The use of hemicellulosic carbon derived from agricultural waste, which is rich in xylose and glucose, has been proposed as a sustainable and low-cost approach. Thraustochytrid strain T18 is a commercialized environmental isolate that readily consumes glucose, attaining impressive biomass, and oil production levels. However, neither thraustochytrid growth capabilities in the presence of xylose nor a xylose metabolic pathway has been described. The aims of this study were to identify and characterize the xylose metabolism pathway of T18 and, through genetic engineering, develop a strain capable of growth on hemicellulosic sugars. Results Characterization of T18 performance in glucose/xylose media revealed diauxic growth and copious extracellular xylitol production. Furthermore, T18 did not grow in media containing xylose as the only carbon source. We identified, cloned, and functionally characterized a xylose isomerase. Transcriptomics indicated that this xylose isomerase gene is upregulated when xylose is consumed by the cells. Over-expression of the native xylose isomerase in T18, creating strain XI 16, increased xylose consumption from 5.2 to 7.6 g/L and reduced extracellular xylitol from almost 100% to 68%. Xylose utilization efficiency of this strain was further enhanced by over-expressing a heterologous xylulose kinase to reduce extracellular xylitol to 20%. Moreover, the ability to grow in media containing xylose as a sole sugar was dependent on the copy number of both xylose isomerase and xylulose kinase present. In fed-batch fermentations, the best xylose metabolizing isolate, XI-XK 7, used 137 g of xylose versus 39 g by wild type and produced more biomass and fatty acid. Conclusions The presence of a typically prokaryotic xylose isomerase and xylitol production through a typically eukaryotic xylose reductase pathway in T18 is the first report of an organism naturally encoding enzymes from two native xylose metabolic pathways. Our newly engineered strains pave the way for the growth of T18 on waste hemicellulosic feedstocks for biofuel production

Jasmonate-dependent depletion of soluble sugars compromises plant resistance to Manduca sexta

Jasmonates regulate plant secondary metabolism and herbivore resistance. How they influence primary metabolites and how this may affect herbivore growth and performance are not well understood. We profiled sugars and starch of jasmonate biosynthesis-deficient and jasmonate-insensitive Nicotiana attenuata plants and manipulated leaf carbohydrates through genetic engineering and in vitro complementation to assess how jasmonate-dependent sugar accumulation affects the growth of Manduca sexta caterpillars. We found that jasmonates reduce the constitutive and herbivore-induced concentration of glucose and fructose in the leaves across different developmental stages. Diurnal, jasmonate-dependent inhibition of invertase activity was identified as a likely mechanism for this phenomenon. Contrary to our expectation, both in planta and in vitro approaches showed that the lower sugar concentrations led to increased M. sexta growth. As a consequence, jasmonate-dependent depletion of sugars rendered N. attenuata plants more susceptible to M. sexta attack. In conclusion, jasmonates are important regulators of leaf carbohydrate accumulation and this determines herbivore growth. Jasmonate-dependent resistance is reduced rather than enhanced through the suppression of glucose and fructose concentrations, which may contribute to the evolution of divergent resistance strategies of plants in nature