RNA-Bindestudien und funktionelle Analysen der chloroplastidären RNA-Bindeproteine CP29A und CP31A in Arabidopsis thaliana

cpRNPs (chloroplastidäre Ribonukleoproteine) sind kernkodierte RNA-Bindeproteine, die jeweils zwei RRM (RNA recognition motif)-Domänen besitzen und in die Chloroplasten höherer Landpflanzen importiert werden. Die Mitwirkung dieser Proteine an der Reifung plastidärer mRNAs in vitro, ihre bemerkenswert hohe Abundanz sowie die Beeinflussung ihrer Funktion durch lichtabhängige posttranslationale Modifikationen weisen auf eine bedeutende Rolle der cpRNPs in der Regulierung der plastidären Genexpression hin. In dieser Arbeit erfolgte erstmals eine Analyse der in vivo bestehenden Interaktionen zwischen cpRNPs und RNA im Stroma von Chloroplasten mittels RIP-Chip-Analysen. Es konnte gezeigt werden, dass ein Großteil der plastidären mRNA-Spezies mit CP31A und CP29A kopräzipitiert. Neben der mehrheitlichen Überlappung der RNA-Interaktionen konnten spezifische Präferenzen von CP29A und CP31A für bestimmte Transkripte identifiziert werden. Um Einblicke in die molekularen Funktionen der cpRNPs zu erhalten, wurden cp29a- und cp31a- Einzel- und Doppelmutanten hinsichtlich der Akkumulation, sowie des Spleiß- und Edierungsstatus plastidärer mRNAs untersucht. Da unter Standard-Wachstumsbedingungen lediglich wenige milde Defekte innerhalb der mRNA-Reifung in den Mutanten detektiert wurden, erfolgten weitere Analysen unter Kältestress-Bedingungen, die zu einer Induktion der CP29A-Expression und zu dem Ausbleichen junger Blattgewebe in cp29a- und cp31a-Mutanten führten. In diesen chlorotischen Geweben wurden reduzierte Akkumulationen plastidärer Proteine sowie PEP-abhängig transkribierter mRNAs detektiert. Darüber hinaus Prozessierungsdefekte für einige Transkripte beobachtet: Die Reifung der 23S-rRNA findet in cp29a- und cp31a-Mutanten nur eingeschränkt statt. In cp31a-Mutanten sind zudem die Prozessierung der ycf3-RNA sowie die Spaltung eines polycistronischen rpl33-Vorläufers gestört. In cp29a-Mutanten ist unter anderem die Prozessierung der rpoC1- und der ycf3-mRNA defizitär.cpRNPs (chloroplast ribonucleoproteins) are nuclear encoded RNA-binding proteins, which contain two RRM (RNA recognition motif) domains and reside within the chloroplasts of higher land plant species. They are involved in plastid mRNA accumulation and processing in vitro. Their involvement in multiple steps of mRNA maturation and their remarkable abundance together with their regulation via posttranslational modifications identified the cpRNPs as potential central regulators of chloroplast gene expression. This work investigated the in vivo interactions of CP29A and CP31A from Arabidopsis with RNA in isolated chloroplasts via RIP-Chip. In this approach the association of both proteins with the majority of plastid mRNA species could be shown. While the interaction profiles of CP29A and CP31A were largely overlapping, also specific preferences for some transcripts were detected. To gain insights in the molecular functions of CP29A and CP31A, null mutants for one or both cpRNPs were analyzed for mRNA accumulation, splicing and editing. Since only few mild mRNA maturation defects were detected at standard growth conditions, analyses at cold stress were performed. Cold stress leads to an increase in CP29A expression and in both cp29a- and cp31a single- and double mutants a bleaching of young leaf tissue was observed. The chlorotic tissues showed a strong decrease in plastid protein accumulation accompanied by reduced levels of PEP-dependent transcripts. In addition some processing defects were observed: The maturation of the 23S-rRNA was reduced in cp29a- and cp31a mutants. In addition the processing of the ycf3-mRNA and a polycistronic rpl33 transcript were defective in cp31a mutants. In cp29a mutants processing defects within the rpoC1- and the ycf3-mRNA were detected

German-Wide Analysis of the Prevalence and the Propagation Factors of the Zoonotic Dermatophyte Trichophyton benhamiae

For about 10 years, a new variant of the pathogen Trichophyton (T.) benhamiae has appeared in Germany, characterized by a previously unobserved culture phenotype with a strong yellow reverse. A few studies suggest that this new variety is now the most common zoophilic dermatophyte in Germany. The guinea pig is the main carrier. Exact prevalence measurements are not yet available. Thus, the aim of our ongoing study was to collect data on the frequency and geographic distribution of the pathogen and its phenotypes (white and yellow) in humans and guinea pigs throughout Germany. Our former studies have already shown that animals from large breeding farms are particularly heavily affected. In contrast to this, 21 small, private breedings were sampled and husbandry conditions recorded. This placed us in a position to identify propagation factors and to give recommendations for containment. For animals from private breedings, we detected T. benhamiae with a prevalence of 55.4%, which is a reduction of nearly 40% compared with animals from large breeding farms. As risk factors, we identified the type of husbandry and the contact to other breedings. Furthermore, certain animal races, like Rex guinea pigs and races with long hair in combination with curls were predestined for colonization with T. benhamiae due to their phenotypic coat characteristics. A prevalence for infections with T. benhamiae of 36.2% has been determined for symptomatic pet guinea pigs suspected of having dermatophytosis and is comparable to the study of Kraemer et al. showing a prevalence of 34.9% in 2009 in Germany. The prevalence in humans is stable with about 2-3% comparing the data of 2010-2013 and 2018 in Thuringia. The new type of T. benhamiae was by far the most frequent cause in all settings

Chloroplast ribonucleoprotein CP31A is required for editing and stability of specific chloroplast mRNAs

Chloroplast ribonucleoproteins (cpRNPs) are nuclear-encoded, highly abundant, and light-regulated RNA binding proteins. They have been shown to be involved in chloroplast RNA processing and stabilization in vitro and are phylogenetically related to the well-described heterogeneous nuclear ribonucleoproteins (hnRNPs). cpRNPs have been found associated with mRNAs present in chloroplasts and have been regarded as nonspecific stabilizers of chloroplast transcripts. Here, we demonstrate that null mutants of the cpRNP family member CP31A exhibit highly specific and diverse defects in chloroplast RNA metabolism. First, analysis of cp31a and cp31a/cp31b double mutants uncovers that these 2 paralogous genes participate nonredundantly in a combinatorial fashion in processing a subset of chloroplast editing sites in vivo. Second, a genome-wide analysis of chloroplast transcript accumulation in cp31a mutants detected a virtually complete loss of the chloroplast ndhF mRNA and lesser reductions for specific other mRNAs. Fluorescence analyses show that the activity of the NADH dehydrogenase complex, which also includes the NdhF subunit, is defective in cp31a mutants. This indicates that cpRNPs are important in vivo for calibrating the expression levels of specific chloroplast mRNAs and impact chloroplast physiology. Taken together, the specificity and combinatorial aspects of cpRNP functions uncovered suggest that these chloroplast proteins are functional equivalents of nucleocytosolic hnRNPs

Host glycoprotein Gp96 and scavenger receptor SREC interact with PorB of disseminating Neisseria gonorrhoeae in an epithelial invasion pathway

Neisseria gonorrhoeae expresses numerous surface proteins that mediate bacterial adherence and invasion during infection. Gonococci expressing serotype A of the major outer membrane porin PorB (PorB(IA)) are frequently isolated from patients with severe disseminating infections. PorB(IA) triggers efficient adherence and invasion under low phosphate conditions mimicking systemic bloodstream infections. Here, we identify the human heat shock glycoprotein Gp96 and the scavenger receptor SREC as PorB(IA)-specific receptors. Gonococci expressing PorB(IA), but not those expressing PorB serotype B instead, bind to purified native or recombinant Gp96. Depletion of Gp96 from host cells prevented adherence but significantly triggered gonococcal invasion. Furthermore, such invasion was blocked by chemical inhibitors of scavenger receptors, and we identified SREC as the scavenger receptor involved in PorB(IA)-dependent invasion. Thus, we establish Gp96 as an anti-invasion factor and SRECs as receptors mediating host cell entry of highly invasive disseminating gonococci