Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae

The benzylisoquinoline alkaloids (BIAs) are a diverse class of metabolites that exhibit a broad range of pharmacological activities and are synthesized through plant biosynthetic pathways comprised of complex enzyme activities and regulatory strategies. We have engineered yeast to produce the key intermediate reticuline and downstream BIA metabolites from a commercially available substrate. An enzyme tuning strategy was implemented that identified activity differences between variants from different plants and determined optimal expression levels. By synthesizing both stereoisomer forms of reticuline and integrating enzyme activities from three plant sources and humans, we demonstrated the synthesis of metabolites in the sanguinarine/berberine and morphinan branches. We also demonstrated that a human P450 enzyme exhibits a novel activity in the conversion of (R)-reticuline to the morphinan alkaloid salutaridine. Our engineered microbial hosts offer access to a rich group of BIA molecules and associated activities that will be further expanded through synthetic chemistry and biology approaches

Helicobacter pylori Perturbs Iron Trafficking in the Epithelium to Grow on the Cell Surface

Helicobacter pylori (Hp) injects the CagA effector protein into host epithelial cells and induces growth factor-like signaling, perturbs cell-cell junctions, and alters host cell polarity. This enables Hp to grow as microcolonies adhered to the host cell surface even in conditions that do not support growth of free-swimming bacteria. We hypothesized that CagA alters host cell physiology to allow Hp to obtain specific nutrients from or across the epithelial barrier. Using a polarized epithelium model system, we find that isogenic ΔcagA mutants are defective in cell surface microcolony formation, but exogenous addition of iron to the apical medium partially rescues this defect, suggesting that one of CagA's effects on host cells is to facilitate iron acquisition from the host. Hp adhered to the apical epithelial surface increase basolateral uptake of transferrin and induce its transcytosis in a CagA-dependent manner. Both CagA and VacA contribute to the perturbation of transferrin recycling, since VacA is involved in apical mislocalization of the transferrin receptor to sites of bacterial attachment. To determine if the transferrin recycling pathway is involved in Hp colonization of the cell surface, we silenced transferrin receptor expression during infection. This resulted in a reduced ability of Hp to colonize the polarized epithelium. To test whether CagA is important in promoting iron acquisition in vivo, we compared colonization of Hp in iron-replete vs. iron-deficient Mongolian gerbils. While wild type Hp and ΔcagA mutants colonized iron-replete gerbils at similar levels, ΔcagA mutants are markedly impaired in colonizing iron-deficient gerbils. Our study indicates that CagA and VacA act in concert to usurp the polarized process of host cell iron uptake, allowing Hp to use the cell surface as a replicative niche

Occurrence des amibes libres en réseaux d’eau intérieurs

Les amibes libres sont des protozoaires (unicellulaires) très présents dans l’environnement et notamment dans les milieux hydriques et ont la capacité à s’y multiplier sans l’aide d’un hôte. Certaines espèces sont directement pathogènes pour l’homme mais d’autres peuvent également être responsables, de façon indirecte, d’infections en jouant le rôle de réservoir de bactéries pathogènes comme Legionella pneumophila. Afin d’évaluer l’occurrence des amibes libres en eau chaude sanitaire, une campagne d’analyse a été réalisée sur 36 réseaux intérieurs réels (eau froide et eau chaude). La campagne d’analyse sur sites a montré que les amibes libres thermophiles sont retrouvées dans 19 % des prélèvements effectués et les amibes mésophiles dans 46 %. Le genre amibien le plus retrouvé est Hartmanella. A priori, ce genre d’amibe n’est pas intrinsèquement pathogène pour l’homme. Sur quelques échantillons, des amibes du genre Acanthamoeba ont été retrouvées. Les points les plus fréquemment contaminés par les amibes libres sont l’eau froide et les points d’usage. Les caractéristiques des sites ont été mis au regard de la contamination par les amibes totales. Aucune corrélation n’a pu être trouvée entre la présence d’amibes et le type d’établissement, le type de production d’eau chaude, la qualité du bouclage, le type de matériaux. Très peu d’amibes sont retrouvées lorsque la température de l’eau est supérieure à 60 °C

Detection of Escherichia coli in biofilms from pipe samples and coupons in drinking water distribution networks

Fluorescence in situ hybridization (FISH) was applied for direct detection of Escherichia coli on pipe surfaces and coupons in drinking water distribution networks. Old cast iron main pipes were removed from water distribution networks in France, England, Portugal and Latvia and E. coli was analyzed in the biofilm. In addition, 44 flat coupons made of cast iron, PVC or stainless steel were placed into and continuously exposed to water on 15 locations of 6 distribution networks in France and Latvia and examined after one to six months exposure to the drinking water. In order to increase signal intensity a peptide nucleic acid (PNA) 15-mer probe was used in the FISH screening for the presence or absence of E. coli on the surface of pipes and coupons, thus reducing occasional problems of autofluorescence and low fluorescence of the labeled bacteria. For comparison, cells were removed from the surfaces and examined with culture-based or enzymatic (detection of -D-glucuronidase) methods. An additional verification was made using PCR. Culture method indicated presence of E.coli in 1 out of 5 pipes whereas all pipes were positive with FISH methods. E.coli were detected in 56% of coupons using PNA FISH but no E.coli were detected using culture or enzymatic methods. PCR analyses confirmed the presence of E. coli in samples which were negative according to the culture-based and enzymatic methods. The viability of E. coli cells in the samples was demonstrated by the cell elongation after resuscitation in low nutrient medium supplemented with pipemidic acid, suggesting that the cells were present in an active but non-culturable state (ABNC), unable to grow on agar media. E. coli contributed to approximately 0.001 - 0.1 % of the total bacterial number in the samples. The presence and number of E. coli did not correlate with any of physical and/or chemical characteristic of the drinking water (e.g. temperature, chlorine or biodegradable organic matter concentration).The study showed that E. coli is present in the biofilms of drinking water networks in Europe. Some of the cells are metabolically active but are often not detected due to limitations of traditionally used culture-based methods, indicating that biofilm should be considered as a reservoir that must be investigated further in order to evaluate the risk for human health.<br/

Detection of Escherichia coli in Biofilms from Pipe Samples and Coupons in Drinking Water Distribution Networks

Fluorescence in situ hybridization (FISH) was applied for direct detection of Escherichia coli on pipe surfaces and coupons in drinking water distributio