Charakterisierung der Multidrug-Efflux-Transporter NorM und AcrAB in Erwinia amylovora

Pflanzen sind permanent phytopathogenen Mikroorganismen ausgesetzt. Jedoch wird eine Erkrankung meistens durch die pflanzliche Abwehr vereitelt. Ein wichtiger Abwehrmechanismus ist die Synthese toxischer Sekundärmetabolite, die an der Infektionsstelle gebildet und freigesetzt werden. In einer kompatiblen Pathogen-Pflanzen-Interaktion muss das Pathogen der Wirkung dieser Toxine widerstehen. Dabei kann eine Akkumulation der Toxine in der Zelle durch aktiven Efflux verhindert werden. Dieser erfolgt beispielsweise durch Multidrug-Efflux-Proteine, die unspezifisch ein breites Spektrum an Substanzen aus der Zelle schleusen. Dass Multidrug-Efflux-Pumpen für die Virulenz von Bedeutung sind, konnte bisher nur bei pilzlichen Pathogenen gezeigt werden. Im Rahmen dieser Arbeit wurden zwei Tranporter, AcrAB und NorM, in dem Gram-negativen Bakterium E. amylovora, dem Erreger des Feuerbrandes an Rosengewächsen, identifiziert sowie genetisch und biochemisch charakterisiert. AcrAB gehört der RND-Familie und NorM der MATE-Familie sekundärer Transporter an. Das AcrAB-Transportsystem aus E. amylovora exportiert unterschiedliche lipophile und amphiphile Substanzen. Im Gegensatz zu NorM, für das hydrophobe Kationen als Substrat nachgewiesen wurden, besitzt AcrAB ein breiteres Substratspektrum. Virulenztests an Apfelpflanzen zeigten eine verminderte Virulenz der acrB-Mutante im Vergleich zum Wildtyp. Erstmals deutet dies daraufhin, dass ein Multidrug-Efflux-Protein bei einer Bakteriose eine Rolle spielt. Apfelblattextrakte besaßen eine Hemmwirkung auf die acrB-Mutante, die weder beim Wildtyp noch bei der norM-Mutante auftrat. Zugabe der Phytoalexine Phloretin, Quercetin, Naringenein und (+)-Catechin bewirkten ein deutlich vermindertes Wachstum der acrB-Mutante. Weiterhin konnte eine ~3fache Induktion der acrAB-Transkription durch Phloretin nachgewiesen werden. Mit AcrAB wurde somit ein Multidrug-Efflux-Tranporter als Virulenzfaktor eines bakteriellen Phytopathogens identifiziert, der durch ein Phytoalexin induzierbar ist. Im Gegensatz dazu scheint NorM eine Funktion während der epiphytischen Besiedlung der Wirtspflanze in der Konkurrenz mit anderen Mikroorganismen zu besitzen. Es konnte eine deutliche Hemmung der norM-Mutante durch Antibiotika des epihytischen Bakteriums P. agglomerans nachgewiesen werden. Da der Epiphyt und das Pathogen im selben Habitat vorkommen, hat sich möglicherweise in E. amylovora durch koevolutive Prozesse ein Resistenzmechanismus gegen die Toxine von P. agglomerans in Form des Multidrug-Effluxes entwickelt. Des weiteren wurde eine ~2fach induzierte Genexpression von norM bei 18°C im Vergleich zu 28°C ermittelt. In gemäßigten Klimazonen korreliert niedrige Temperatur mit hoher Luftfeuchte und der Bildung von Wasserfilmen auf Pflanzenoberflächen, die eine Bewegung des Pathogens zu natürlichen Öffnungen oder Verwundungen ermöglicht. Durch niedrige Temperaturen werden somit günstige Bedingungen für die Besiedlung der Pflanze durch E. amylovora, gleichzeitig aber auch die Notwendigkeit zur Konkurrenz mit anderen Mikroorganismen, wie z. B. P. agglomerans, erzeugt

Thioglycosides as inhibitors of hSGLT1 and hSGLT2: Potential therapeutic agents for the control of hyperglycemia in diabetes

The treatment of diabetes has been mainly focused on maintaining normal blood glucose concentrations. Insulin and hypoglycemic agents have been used as standard therapeutic strategies. However, these are characterized by limited efficacy and adverse side effects, making the development of new therapeutic alternatives mandatory. Inhibition of glucose reabsorption in the kidney, mediated by SGLT1 or SGLT2, represents a promising therapeutic approach. Therefore, the aim of the present study was to evaluate the effect of thioglycosides on human SGLT1 and SGLT2. For this purpose, stably transfected Chinese hamster ovary (CHO) cells expressing human SGLT1 and SGLT2 were used. The inhibitory effect of thioglycosides was assessed in transport studies and membrane potential measurements, using α-methyl-glucoside uptake and fluorescence resonance energy transfer, respectively. We found that some thioglycosides inhibited hSGLT more strongly than phlorizin. Specifically, thioglycoside I (phenyl-1'-thio-β-D-glucopyranoside) inhibited hSGLT2 stronger than hSGLT1 and to a larger extent than phlorizin. Thioglycoside VII (2-hydroxymethyl-phenyl-1'-thio-β-D-galacto-pyranoside) had a pronounced inhibitory effect on hSGLT1 but not on hSGLT2. Kinetic studies confirmed the inhibitory effect of these thioglycosides on hSGLT1 or hSGLT2, demonstrating competitive inhibition as the mechanism of action. Therefore, these thioglycosides represent promising therapeutic agents for the control of hyperglycemia in patients with diabetes

Autofluorescence-Based Identification and Functional Validation of Antennal Gustatory Sensilla in a Specialist Leaf Beetle

Herbivorous insects mainly rely on their sense of taste to decode the chemical composition of potential hosts in close range. Beetles for example contact and scan leaves with their tarsi, mouthparts and antennal tips, i.e., appendages equipped with gustatory sensilla, among other sensillum types. Gustatory neurons residing in such uniporous sensilla detect mainly non-volatile compounds that contribute to the behavioral distinction between edible and toxic plants. However, the identification of gustatory sensilla is challenging, because an appendage often possesses many sensilla of distinct morphological and physiological types. Using the specialized poplar leaf beetle (Chrysomela populi, Chrysomelidae), here we show that cuticular autofluorescence scanning combined with electron microscopy facilitates the identification of antennal gustatory sensilla and their differentiation into two subtypes. The gustatory function of sensilla chaetica was confirmed by single sensillum tip-recordings using sucrose, salicin and salt. Sucrose and salicin were found at higher concentrations in methanolic leaf extracts of poplar (Populus nigra) as host plant compared to willow (Salix viminalis) as control, and were found to stimulate feeding in feeding choice assays. These compounds may thus contribute to the observed preference for poplar over willow leaves. Moreover, these gustatory cues benefited the beetle’s performance since weight gain was significantly higher when C. populi were reared on leaves of poplar compared to willow. Overall, our approach facilitates the identification of insect gustatory sensilla by taking advantage of their distinct fluorescent properties. This study also shows that a specialist beetle selects the plant species that provides optimal development, which is partly by sensing some of its characteristic non-volatile metabolites via antennal gustatory sensilla

NorM, an Erwinia amylovora Multidrug Efflux Pump Involved in In Vitro Competition with Other Epiphytic Bacteria

Blossoms are important sites of infection for Erwinia amylovora, the causal agent of fire blight of rosaceous plants. Before entering the tissue, the pathogen colonizes the stigmatic surface and has to compete for space and nutrient resources within the epiphytic community. Several epiphytes are capable of synthesizing antibiotics with which they antagonize phytopathogenic bacteria. Here, we report that a multidrug efflux transporter, designated NorM, of E. amylovora confers tolerance to the toxin(s) produced by epiphytic bacteria cocolonizing plant blossoms. According to sequence comparisons, the single-component efflux pump NorM is a member of the multidrug and toxic compound extrusion protein family. The corresponding gene is widely distributed among E. amylovora strains and related plant-associated bacteria. NorM mediated resistance to the hydrophobic cationic compounds norfloxacin, ethidium bromide, and berberine. A norM mutant was constructed and exhibited full virulence on apple rootstock MM 106. However, it was susceptible to antibiotics produced by epiphytes isolated from apple and quince blossoms. The epiphytes were identified as Pantoea agglomerans by 16S rRNA analysis and were isolated from one-third of all trees examined. The promoter activity of norM was twofold greater at 18°C than at 28°C. The lower temperature seems to be beneficial for host infection because of the availability of moisture necessary for movement of the pathogen to the infection sites. Thus, E. amylovora might employ NorM for successful competition with other epiphytic microbes to reach high population densities, particularly at a lower temperature

Thioglycosides as inhibitors of hSGLT1 and hSGLT2: Potential therapeutic agents for the control of hyperglycemia in diabetes

The treatment of diabetes has been mainly focused on maintaining normal blood glucose concentrations. Insulin and hypoglycemic agents have been used as standard therapeutic strategies. However, these are characterized by limited efficacy and adverse side effects, making the development of new therapeutic alternatives mandatory. Inhibition of glucose reabsorption in the kidney, mediated by SGLT1 or SGLT2, represents a promising therapeutic approach. Therefore, the aim of the present study was to evaluate the effect of thioglycosides on human SGLT1 and SGLT2. For this purpose, stably transfected Chinese hamster ovary (CHO) cells expressing human SGLT1 and SGLT2 were used. The inhibitory effect of thioglycosides was assessed in transport studies and membrane potential measurements, using &#945;-methyl-glucoside uptake and fluorescence resonance energy transfer, respectively. We found that some thioglycosides inhibited hSGLT more strongly than phlorizin. Specifically, thioglycoside I (phenyl-1'-thio-&#946;-D-glucopyranoside) inhibited hSGLT2 stronger than hSGLT1 and to a larger extent than phlorizin. Thioglycoside VII (2-hydroxymethyl-phenyl-1'-thio-&#946;-D-galacto-pyranoside) had a pronounced inhibitory effect on hSGLT1 but not on hSGLT2. Kinetic studies confirmed the inhibitory effect of these thioglycosides on hSGLT1 or hSGLT2, demonstrating competitive inhibition as the mechanism of action. Therefore, these thioglycosides represent promising therapeutic agents for the control of hyperglycemia in patients with diabetes.</p

Effect on sodium-dependent CAMG-uptake obtained in hSGLT1 or hSGLT2 treated with thioglycosides (10 µM each) or phlorizin (10 µM)

<p><b>Copyright information:</b></p><p>Taken from "Thioglycosides as inhibitors of hSGLT1 and hSGLT2: Potential therapeutic agents for the control of hyperglycemia in diabetes"</p><p></p><p>International Journal of Medical Sciences 2007;4(3):131-139.</p><p>Published online 5 May 2007</p><p>PMCID:PMC1868657.</p><p>© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.</p> Results are expressed as percent of inhibition based on uptake in CHO cells expressing hSGLT1 or hSGLT2 not exposed to thioglycosides (control cells). Blue and red bars represent hSGLT1 and hSGLT2, respectively. Results are the mean of six different experiments. Error bars represents standard deviations. * < 0.01 shows significantly higher inhibition of sodium-dependent AMG uptake in treated cells as compared to control cells. Control uptake in CHO cells expressing hSGLT1 was 735 pmol/mg/h ± 22 pmol/mg/h and in CHO cells expressing hSGLT2 was 342 pmol/mg/h ± 15 pmol/mg/h

Independently recruited oxidases from the glucose-methanol-choline oxidoreductase family enabled chemical defences in leaf beetle larvae (subtribe Chrysomelina) to evolve

Larvae of the leaf beetle subtribe Chrysomelina sensu stricto repel their enemies by displaying glandular secretions that contain defensive compounds. These repellents can be produced either de novo (iridoids) or by using plant-derived precursors (e.g. salicylaldehyde). The autonomous production of iridoids, as in Phaedon cochleariae, is the ancestral chrysomeline chemical defence and predates the evolution of salicylaldehyde-based defence. Both biosynthesis strategies include an oxidative step of an alcohol intermediate. In salicylaldehyde-producing species, this step is catalysed by salicyl alcohol oxidases (SAOs) of the glucose-methanol-choline (GMC) oxidoreductase superfamily, but the enzyme oxidizing the iridoid precursor is unknown. Here, we show by in vitro as well as in vivo experiments that P. cochleariae also uses an oxidase from the GMC superfamily for defensive purposes. However, our phylogenetic analysis of chrysomeline GMC oxidoreductases revealed that the oxidase of the iridoid pathway originated from a GMC clade different from that of the SAOs. Thus, the evolution of a host-independent chemical defence followed by a shift to a host-dependent chemical defence in chrysomeline beetles coincided with the utilization of genes from different GMC subfamilies. These findings illustrate the importance of the GMC multi-gene family for adaptive processes in plant–insect interactions

Changes in cell membrane potential induced by D-glucose, thioglycosides I and VII (10

<p><b>Copyright information:</b></p><p>Taken from "Thioglycosides as inhibitors of hSGLT1 and hSGLT2: Potential therapeutic agents for the control of hyperglycemia in diabetes"</p><p></p><p>International Journal of Medical Sciences 2007;4(3):131-139.</p><p>Published online 5 May 2007</p><p>PMCID:PMC1868657.</p><p>© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.</p> Blue and red bars represent hSGLT1 and hSGLT2, respectively. The change in FRET signal was normalized to the values obtained from non-transfected CHO cells (controls). Results are the mean of six different experiments. Error bars represents standard deviations. * < 0.01 shows significantly higher induction of cell membrane depolarization in treated cells as compared to control cells (not exposed to thioglycosides)