Analyse der Signalfunktion von Trehalose-6-Phosphat in Kartoffelknollen

Trehalose-6-Phosphate (T6P) is an essential signalling metabolite in plants. It is necessary for the regulation of carbohydrate metabolism and for the control of development and growth. In planta manipulation of the T6P content, either through insertion of a transgene into the TPS/TPP way or by knocking out a component of the endogenous trehalose biosynthetic pathway, allowed to identify T6P dependent mechanisms in embryogenesis, sucrose signal transduction and in the course of that, starch synthesis and tissue specific modulation of the central metabolic regulator SnRK1. To investigate the mode of action of T6P in a heterotrophic plant tissue, transgenic potato plants were engineered, that overexpressed the E. coli derived isoenzymes of the TPS/TPP pathway OtsA and OtsB under the control of the tuber specific B33 promoter to increase or decrease T6P content, respectively. In the context of this work, it could be shown that a modulated content of T6P massively impaired the development and the carbohydrate metabolism in transgenic potato plants. A reduced content of T6P led to accumulation of hexose-phosphates and interfered with the utilisation of sucrose just as in autotrophic tissue and mediated the accumulation of the latter by induced SPS activity. The total tuber biomass yielded from the B33-TPP plants was massively reduced. On the other hand it could be shown that, in contrast to photoautotrophic leaf tissue, an increased content of T6P did not activate the AGPase and subsequently did not bring about boosted starch synthesis. While the starch synthesis in B33-TPS lines was not negatively affected, an increased respiration rate as consequence of increased flux through the citrate cycle constituted a reduced starch yield per freshweight. In both transgenic approaches the ratio of ATP to ADP, and therefore probably the adenylate energy charge, was reduced. It could be shown that the sprouting properties of the B33-TPP and B33-TPS tubers strongly differed from the wildtype. While B33-TPP tubers were hypersensitive towards treatment with Gibberellic acid and Cytokinin and sprouted after a syncopated period of dormancy, it appeared that B33-TPS tubers sprouted with delay and were insensitive for treatment with these phytohormones. The total content of Abscisic acid (ABA) in tubers with reduced T6P content was considerably lower than in the wildtype and ABA catabolism was induced. In B33-TPS tubers an ABA catabolic enzyme was indeed repressed, but the total content of free ABA was not modulated. T6P could as well be an endogenous inhibitor of tuber sprouting. In B33-TPP tubers the SnRK1 kinase was probably de-repressed. The SnRK1 is the plant homologue of the conserved energy and nutrient sensor AMPK which is indispensable for the regulation of metabolic fluxes, energy homeostasis and most probably growth of the organism in accordance to present conditions. It could be shown that T6P is an in vitro inhibitor of the potato tuber SnRK1 complex. In tubers with reduced content of T6P a bulk of target genes of the SnRK1 kinase were regulated in the direction expected for T6P repression of SnRK1. A major part of the modulated transcripts seemed to be regulated symmetrically in B33-TPS tubers. On the basis of feeding wildtype tubers it could be shown that application of exogenous sucrose and trehalose increased the content of T6P in tuber material and brought upon changes comparable to overexpression of TPS in SnRK1 responsive transcripts. Derived from the data obtained in the transcriptome analysis a promising candidate gene for a T6P responsive transcription factor could be identified. A zinc-finger transcription factor of the GATA family was found to be repressed by T6P in Arabidopsis thaliana as well as in Solanum tuberosum and induced upon depletion of this metabolite. The physiological and molecular surveys indicated that T6P is a metabolite signalling the availability of sucrose in potato tubers. In the course of this, it regulates growth and organ development through altering cell division, at least partially by modulation of SnRK1 activity. Therefore, T6P is an integrator of metabolism and growth. The events of T6P signal transduction differ from those previously observed in source tissue to some extent. Even though the analysis of transgenic potato tubers generated new insights, the underlying receptors and signalling cascades of T6P signal transduction must be further investigated.Trehalose-6-Phosphat (T6P) ist ein essentieller Signalmetabolit in Pflanzen. Es ist notwendig für die Regulation des Kohlenhydratmetabolismus und für die Steuerung der Entwicklung und des Wachstums. In planta Manipulationen des T6P Gehalts, sei es durch Einbringen eines Transgens in den TPS/TPP Weg oder durch Ausschalten einer der Komponenten des endogenen Trehalose Synthesewegs, erlaubten die Identifikation von T6P abhängigen Mechanismen in der Embryogenese, der Saccharosesignaltransduktion und im Zuge dessen der Stärkesynthese und der gewebsspezifischen Modulation des zentralen Stoffwechsel-regulators SnRK1. Um die Funktionen von T6P in einem heterotrophen Pflanzengewebe zu untersuchen wurden transgene Kartoffelpflanzen hergestellt, die unter der Kontrolle des knollenspezifischen B33 Promotors die E. coli Isoenzyme des TPS/TPP Weges OtsA und OtsB überexprimieren um den T6P Gehalt zu erhöhen bzw. zu reduzieren. Im Rahmen dieser Arbeit konnte gezeigt werden, dass ein modulierter Gehalt an T6P die Entwicklung und den Kohlenhydratmetabolismus von transgenen Kartoffelknollen massiv beeinflusst. Ein reduzierter Gehalt an T6P führte, wie in autotrophem Gewebe, zu einer Akkumulation von Hexose-Phosphaten und zu einer Störung in der Verwertung von Saccharose, bei gleichzeitiger Akkumulation derselben aufgrund von induzierter SPS Aktivität. Die Gesamtbiomasse der Knollen von B33-TPP Pflanzen war massiv reduziert. Auf der anderen Seite konnte gezeigt werden, dass ein erhöhter Gehalt an T6P, anders als in photoautotrophem Blattmaterial, nicht zu einer Aktivierung der AGPase und infolgedessen zu einer erhöhten Stärkesynthese führte. Während die Stärkesynthese in den B33-TPS Linien nicht betroffen war, führte eine erhöhte Respirationsrate infolge eines erhöhten Flusses durch den Zitratzyklus zu einer reduzierten Stärkeausbeute bezogen auf das Frischgewicht. In beiden transgenen Ansätzen war das Verhältnis von ATP zu ADP und damit vermutlich die ‚Adenylat Energieladung’ reduziert. Es konnte weiterhin gezeigt werden, dass das Keimungsverhalten von B33-TPP und B33-TPS Knollen stark von dem im Wildtyp verschieden war. Während B33-TPP Knollen hypersensitiv gegenüber Behandlung mit Gibberellinsäure und Cytokinin waren und nach stark verkürzter Dormanz keimten, zeigte sich, dass B33-TPS Knollen verzögert keimten und gegenüber Behandlung mit den Phytohormonen insensitiv waren. Der Gesamtgehalt an Abscisinsäure (ABA) in den Knollen mit reduziertem T6P war geringer als im Wildtyp und der ABA Katabolismus induziert. In den B33-TPS Knollen war zwar ein ABA abbauendes Enzym reprimiert, jedoch der Gehalt an ABA über die ganze Knolle nicht verändert. T6P könnte ein endogener Inhibitor der Knollenkeimung sein. In B33-TPP Knollen ist vermutlich die Aktivität der SnRK1 Kinase dereprimiert. Die SnRK1 Kinase ist das pflanzliche Homolog des konservierten Energie- und Nährstoffsensors AMPK und reguliert Stoffwechselflüsse, die Energiehomöostase und höchstwahrscheinlich das Wachstum des Organismus, angepasst auf die vorliegenden Bedingungen. Es konnte gezeigt werden, dass T6P ein in vitro Inhibitor des SnRK1 Komplexes aus Kartoffelknolle ist. In den Knollen mit reduziertem T6P Gehalt waren eine Reihe von Zielgenen der SnRK1 Kinase in die für eine Repression der SnRK1 durch T6P erwartete Richtung reguliert. Ein Großteil der modulierten Transkripte schien in B33-TPS Knollen symmetrisch reguliert. Es konnte anhand von Fütterung in Wildtypknollen gezeigt werden, dass eine Zufuhr von exogener Saccharose oder Trehalose den Gehalt an T6P im Knollenmaterial erhöht und vergleichbare Änderungen in den SnRK1 responsiven Transkripten wie Überexpression von TPS hervorruft. Aus den Daten der Transkriptomanalyse konnte ein viel versprechendes Kandidatengen für einen T6P responsiven Transkriptionsfaktor identifiziert werden. Ein Zink Finger Transkriptionsfaktor der GATA Familie wurde als T6P reprimiertes Transkript sowohl in Arabidopsis thaliana als auch in Solanum tuberosum bei Modulation des T6P Gehalts identifiziert. Die physiologischen und molekularen Untersuchungen deuten darauf hin, dass T6P in Kartoffelknollen ein Signal für die Verfügbarkeit von Saccharose darstellt. Es reguliert in diesem Zusammenhang, zumindest teilweise durch Modulation der Aktivität des SnRK1 Komplexes, Wachstumsprozesse und die Entwicklung von Organen durch Steuerung der Zellteilungsaktivität. Damit ist T6P ein Integrator des Metabolismus und des Wachstums. Die T6P Signaltransduktion in Kartoffelknolle unterscheidet sich teilweise von den bisher im Source-Blatt beschriebenen Funktionen. Obwohl aus den Analysen von transgenen Kartoffelknollen neue Erkenntnisse gewonnen wurden, müssen die Rezeptoren und Signalkaskaden der T6P Signaltransduktion eingehender untersucht werden

The association between histamine 2 receptor antagonist use and Clostridium difficile infection: a systematic review and meta-analysis.

Background Clostridium difficile infection (CDI) is a major health problem. Epidemiological evidence suggests that there is an association between acid suppression therapy and development of CDI. Purpose We sought to systematically review the literature that examined the association between histamine 2 receptor antagonists (H2RAs) and CDI. Data source We searched Medline, Current Contents, Embase, ISI Web of Science and Elsevier Scopus from 1990 to 2012 for all analytical studies that examined the association between H2RAs and CDI. Study selection Two authors independently reviewed the studies for eligibility. Data extraction Data about studies characteristics, adjusted effect estimates and quality were extracted. Data synthesis Thirty-five observations from 33 eligible studies that included 201834 participants were analyzed. Studies were performed in 6 countries and nine of them were multicenter. Most studies did not specify the type or duration of H2RAs therapy. The pooled effect estimate was 1.44, 95% CI (1.22–1.7), I2 = 70.5%. This association was consistent across different subgroups (by study design and country) and there was no evidence of publication bias. The pooled effect estimate for high quality studies was 1.39 (1.15–1.68), I2 = 72.3%. Meta-regression analysis of 10 study-level variables did not identify sources of heterogeneity. In a speculative analysis, the number needed to harm (NNH) with H2RAs at 14 days after hospital admission in patients receiving antibiotics or not was 58, 95% CI (37, 115) and 425, 95% CI (267, 848), respectively. For the general population, the NNH at 1 year was 4549, 95% CI (2860, 9097). Conclusion In this rigorous systematic review and meta-analysis, we observed an association between H2RAs and CDI. The absolute risk of CDI associated with H2RAs is highest in hospitalized patients receiving antibiotics

Functional analysis of PsbS transmembrane domains through base editing in Physcomitrium patens

: Plants exposed to light fluctuations are protected from photodamage by non-photochemical quenching (NPQ), a reversible mechanism that enables dissipation of excess absorbed energy as heat, which is essential for plant fitness and crop productivity. In plants NPQ requires the presence of the membrane protein PsbS that, upon activation, interacts with antenna proteins, inducing their dissipative conformation. Here, we exploited base editing in the moss Physcomitrium patens to introduce in vivo specific amino acid changes and assess their impact on PsbS activity, targeting transmembrane regions to investigate their role in essential protein-protein interactions. This approach enabled the recognition of residues essential for protein stability and the identification of a hydrophobic cluster of amino acids impacting PsbS activity. This work provides new information on the PsbS molecular mechanism while also demonstrating the potential of base editing approaches for in planta gene function analysis

A SNP associated with alternative splicing of RPT5b causes unequal redundancy between RPT5a and RPT5b among Arabidopsis thaliana natural variation

<p>Abstract</p> <p>Background</p> <p>The proteasome subunit RPT5, which is essential for gametophyte development, is encoded by two genes in <it>Arabidopsis thaliana</it>; <it>RPT5a </it>and <it>RPT5b</it>. We showed previously that <it>RPT5a </it>and <it>RPT5b </it>are fully redundant in the Columbia (Col-0) accession, whereas in the Wassilewskia accession (Ws-4), <it>RPT5b </it>does not complement the effect of a strong <it>rpt5a </it>mutation in the male gametophyte, and only partially complements <it>rpt5a </it>mutation in the sporophyte. <it>RPT5b^Col-0</it>and <it>RPT5b^Ws-4</it>differ by only two SNPs, one located in the promoter and the other in the seventh intron of the gene.</p> <p>Results</p> <p>By exploiting natural variation at <it>RPT5b </it>we determined that the SNP located in <it>RPT5b </it>intron seven, rather than the promoter SNP, is the sole basis of this lack of redundancy. In Ws-4 this SNP is predicted to create a new splicing branchpoint sequence that induces a partial mis-splicing of the pre-mRNA, leading to the introduction of a Premature Termination Codon. We characterized 5 accessions carrying this A-to-T substitution in intron seven and observed a complete correlation between this SNP and both a 10 to 20% level of the <it>RPT5b </it>pre-mRNA mis-splicing and the lack of ability to complement an <it>rpt5a </it>mutant phenotype.</p> <p>Conclusion</p> <p>The accession-dependent unequal redundancy between <it>RPT5a </it>and <it>RPT5b </it>genes illustrates an example of evolutionary drifting between duplicated genes through alternative splicing.</p

Microbiological Characterization and Clinical Outcomes After Extended-Pulsed Fidaxomicin Treatment for Clostridioides difficile Infection in the EXTEND Study

Background: Clostridioides (Clostridium) difficile infection (CDI) is diagnosed using clinical signs and symptoms plus positive laboratory tests. Recurrence of CDI after treatment is common, and coinfection with other enteric pathogens may influence clinical outcomes. Methods: We aimed to assess rates of C difficile positivity, by enzyme-linked immunosorbent assay (ELISA) toxin A/B and BioFire FilmArray, and the effect of enteric coinfection on clinical outcomes, using samples from the EXTEND study of extended-pulsed fidaxomicin (EPFX) versus standard vancomycin. Results: All 356 randomized and treated patients tested positive for C difficile toxin A/B by local tests; a majority (225 of 356, 63.2%) also tested positive by both ELISA and BioFire. Most stool samples taken at screening tested positive for C difficile only using BioFire (EPFX: 112 of 165, 69.7%; vancomycin: 118 of 162, 72.8%). Of the 5 patients who failed treatment and had stool samples available, all (1) had tested negative for C difficile by BioFire at screening and (2) were negative by ELISA at time of treatment failure. When analyzed by BioFire results at screening, rates of sustained clinical cure at 30 days after end of treatment were numerically higher with EPFX than with vancomycin for almost all patients, except for those who tested negative for C difficile but positive for another pathogen. However, these outcome differences by presence of coinfection did not reach statistical significance. Whole-genome sequencing analysis determined that 20 of 26 paired samples from patients with recurrence were reinfections with the same C difficile strain. Conclusions: Testing for presence of copathogens in clinical trials of antibiotics could help to explain clinical failures

Clostridium difficile infection.

Infection of the colon with the Gram-positive bacterium Clostridium difficile is potentially life threatening, especially in elderly people and in patients who have dysbiosis of the gut microbiota following antimicrobial drug exposure. C. difficile is the leading cause of health-care-associated infective diarrhoea. The life cycle of C. difficile is influenced by antimicrobial agents, the host immune system, and the host microbiota and its associated metabolites. The primary mediators of inflammation in C. difficile infection (CDI) are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), and, in some bacterial strains, the binary toxin CDT. The toxins trigger a complex cascade of host cellular responses to cause diarrhoea, inflammation and tissue necrosis - the major symptoms of CDI. The factors responsible for the epidemic of some C. difficile strains are poorly understood. Recurrent infections are common and can be debilitating. Toxin detection for diagnosis is important for accurate epidemiological study, and for optimal management and prevention strategies. Infections are commonly treated with specific antimicrobial agents, but faecal microbiota transplants have shown promise for recurrent infections. Future biotherapies for C. difficile infections are likely to involve defined combinations of key gut microbiota

A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens

CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation. CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens. Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing

Clostridium: Transmission difficile?

Stephan Harbarth and Matthew Samore discuss the implications, and the limitations, of new research that might indicate that most Clostridium difficile cases are imported into hospitals

Mechanisms behind variation in the Clostridium difficile 16S–23S rRNA intergenic spacer region

Clostridium difficile infection is an increasing problem in hospitals worldwide, mainly due to the recent emergence of a hypervirulent C. difficile strain. C. difficile PCR ribotyping, based on size variation of the 16S–23S rRNA intergenic spacer region (16S–23S ISR), is widely used in Europe for molecular epidemiological investigation. The mechanism underlying the 16S–23S ISR size variations in the genome of C. difficile is currently not completely understood. To elucidate this mechanism, isolates of six different PCR ribotypes were analysed by cloning and sequencing the 16S–23S ISR. A direct repeat, IB, of 9 bp was detected up to five times in the 16S–23S ISR in all 47 clones investigated. Thirty-five clones displayed differences either by ribotype or by nucleotide sequence. The sequences of the 16S–23S ISR of C. difficile showed a uniformly organized structure, composed of a tRNAAla gene and spacers of 33 and 53 bp separated by the 9 bp direct repeat IB. The results of the study support the hypothesis that this composition is responsible for the length variations seen in the 16S–23S ISR, and indicate that these length variations result from slipped-strand mispairing and intra- and possibly interchromosomal homologous recombination