Définition du mécanisme de localisation des ARNm cen et ik2 aux centrosomes chez la Drosophile

L’organisation cellulaire repose sur une distribution organisée des macromolécules dans la cellule. Deux ARNm, cen et ik2, montrent une colocalisation parfaite aux centrosomes. Ces deux gènes font partie du même locus sur le chromosome 2L de Drosophila melanogaster et leur région 3’ non traduite (3’UTR) se chevauchent. Dans le mutant Cen, le transport de Ik2 est perturbé, mais dans le mutant Ik2, la localisation de cen n’est aucunement affectée. Ces résultats suggèrent que cen est le régulateur principal de la co-localisation de cen et ik2 aux centrosomes et que cette co-localisation se produit par un mécanisme impliquant la région complémentaire au niveau du 3’UTR des deux transcrits. La localisation de cen au niveau des centrosomes dans les cellules épithéliales de l’embryon est conservée dans différentes espèces de Drosophile : D. melanogater, D. simulans, D. virilis et D. mojavensis. Cependant, la localisation de ik2 n’est pas conservée dans D. virilis et D. mojavensis, deux espèces dont les gènes cen et ik2 sont dissociés dans le génome. Ces résultats suggèrent que la proximité de Cen et Ik2 dans le génome est importante afin d’avoir un événement de co-localisation de ces deux transcrits. J’ai généré différentes lignées de mouches transgéniques dans lesquelles un transgène contenant la séquence GFP fusionnée à différentes partie de Cen (partie codante, 3’UTR, Cod+3’UTR) qui sont sous le contrôle du promoteur UAS et qui sont gal4 inductibles. La région codante de l’ARNm cen était suffisante pour avoir un ciblage précis du transcrit aux centrosomes.

Amino acid substitutions in σ1 and μ1 outer capsid proteins are selected during mammalian reovirus adaptation to Vero cells

Establishment of viral persistence in cell culture has previously led to the selection of mammalian reovirus mutants, although very few of those have been characterized in details. In the present study, reovirus was adapted to Vero cells that, in contrast to classically-used L929 cells, are inefficient in supporting the early steps of reovirus uncoating and are also unable to produce interferon as an antiviral response once infection occurs. The Vero cell-adapted reovirus exhibits amino acids substitutions in both the σ1 and μ1 proteins. This contrasts with uncoating mutants from persistently-infected L929 cells, and various other cell types, that generally harbor amino acids substitutions in the σ3 outer capsid protein. The Vero cell-adapted virus remained sensitive to an inhibitor of lysosomal proteases; furthermore, in the absence of selective pressure for its maintenance, t he virus has partially lost its ability to resist interferon. The positions of the amino acids substitutions on the known protein structures suggest an effect on binding of the viral σ1 protein to the cell surface and on μ1 disassembly from the outer capsid.

Étude du rôle des régions variables 4 et 5 dans les changements de conformation de la gp120 du VIH-1

Le VIH infecte les cellules par fusion de sa membrane avec la membrane de la cellule cible. Cette fusion est effectuée par les glycoprotéines de l'enveloppe (Env) qui sont synthétisées en tant que précurseur, gp160, qui est ensuite clivé en gp120 et gp41. La protéine gp41 est la partie transmembranaire du complexe de l'enveloppe et l’ancre à la particule virale alors que la gp120 assure la liaison au récepteur cellulaire CD4 et corécepteur CCR5 ou CXCR4. Ces interactions successives induisent des changements de conformation d’Env qui alimentent le processus d'entrée du virus conduisant finalement à l'insertion du peptide de fusion de la gp41 dans la membrane de la cellule cible. La sous-unité extérieure gp120 contient cinq régions variables (V1 à V5), dont trois (V1, V2 et V3) étant capables d’empêcher l’adoption spontanée de la conformation liée à CD4. Cependant, le rôle de régions variables V4 et V5 vis-à-vis de ces changements de conformation reste inconnu. Pour étudier leur effet, des mutants de l'isolat primaire de clade B YU2, comprenant une délétion de la V5 ou une mutation au niveau de tous les sites potentiels de N-glycosylation de la V4 (PNGS), ont été générés. L'effet des mutations sur la conformation des glycoprotéines d'enveloppe a été analysé par immunoprécipitation et résonance de plasmon de surface avec des anticorps dont la liaison dépend de la conformation adopté par la gp120. Ni le retrait des PNGS de la V4 ni la délétion de V5 n’a affecté les changements conformationnels d’Env tels que mesurés par ces techniques, ce qui suggère que les régions variables V1, V2 et V3 sont les principaux acteurs dans la prévention de l’adoption de la conformation lié de CD4 d’Env.

Modifications post-traductionnelles des canaux calciques cardiaques de type L : identification des résidus asparagine qui participent à la glycosylation de la sous-unité auxiliaire CaVα2δ1

Les canaux calciques de type L CaV1.2 sont principalement responsables de l’entrée des ions calcium pendant la phase plateau du potentiel d’action des cardiomyocytes ventriculaires. Cet influx calcique est requis pour initier la contraction du muscle cardiaque. Le canal CaV1.2 est un complexe oligomérique qui est composé de la sous-unité principale CaVα1 et des sous-unités auxiliaires CaVβ et CaVα2δ1. CaVβ joue un rôle déterminant dans l’adressage membranaire de la sous-unité CaVα1. CaVα2δ1 stabilise l’état ouvert du canal mais le mécanisme moléculaire responsable de cette modulation n’a pas été encore identifié. Nous avons récemment montré que cette modulation requiert une expression membranaire significative de CaVα2δ1 (Bourdin et al. 2015). CaVα2δ1 est une glycoprotéine qui possède 16 sites potentiels de glycosylation de type N. Nous avons donc évalué le rôle de la glycosylation de type-N dans l’adressage membranaire et la stabilité de CaVα2δ1. Nous avons d’abord confirmé que la protéine CaVα2δ1 recombinante, telle la protéine endogène, est significativement glycosylée puisque le traitement à la PNGase F se traduit par une diminution de 50 kDa de sa masse moléculaire, ce qui est compatible avec la présence de 16 sites Asn. Il s’est avéré par ailleurs que la mutation simultanée de 6/16 sites (6xNQ) est suffisante pour 1) réduire significativement la densité de surface de! CaVα2δ1 telle que mesurée par cytométrie en flux et par imagerie confocale 2) accélérer les cinétiques de dégradation telle qu’estimée après arrêt de la synthèse protéique et 3) diminuer la modulation fonctionnelle des courants générés par CaV1.2 telle qu’évaluée par la méthode du « patch-clamp ». Les effets les plus importants ont toutefois été obtenus avec les mutants N663Q, et les doubles mutants N348Q/N468Q, N348Q/N812Q, N468Q/N812Q. Ensemble, ces résultats montrent que Asn663 et à un moindre degré Asn348, Asn468 et Asn812 contribuent à la biogenèse et la stabilité de CaVα2δ1 et confirment que la glycosylation de type N de CaVα2δ1 est nécessaire à la fonction du canal calcique cardiaque de type L.

Déficience dans la réparation par excision de nucléotides en phase S induite par la séquestration du facteur de réplication RPA

Introduction Les lésions induites par les rayons UV peuvent causer des blocages dans la réplication de l'ADN. Ces dommages sont éliminés par le processus moléculaire très conservé de réparation par excision de nucléotides (NER). Nous avons précédemment démontré que la protéine ATR, une kinase majeure impliquée dans le stress réplicatif, est requise pour une NER efficace, et ce exclusivement durant la phase S. Des résultats subséquents ont suggéré que ce prérequis n’était pas lié à la réponse induite par ATR, mais plutôt d’une conséquence globale causée par la présence de stress réplicatif. En ce sens, nous mettons l’emphase qu’après irradiation UV, le complexe RPA joue un rôle crucial dans l'activation des mécanismes de NER ainsi que dans le redémarrage des fourches de réplication bloquées. Hypothèses: En général, les mutations qui confèrent une augmentation du stress réplicatif engendrent une séquestration excessive du facteur RPA aux fourches de réplication bloquées ce qui réduit son accessibilité pour le NER. Méthodes et résultats: Le modèle de la levure a été choisi pour vérifier cette hypothèse. Nous avons développé un essai de NER spécifique à chacune des phases du cycle cellulaire pour démontrer que les cellules déficientes en Mec1, l’homologue d’ATR, sont défectives dans la réparation par excision de nucléotides spécifiquement en phase S. De plus, plusieurs autres mutants de levure, caractérisés par un niveau de dommages spontanés élevé, ont aussi exhibé un défaut similaire. Ces mutants ont démontré une fréquence et une intensité de formation de foyers de RPA plus élevée. Finalement, une diminution partielle de RPA dans les levures a induit un défaut significatif dans le NER spécifiquement durant la phase S. Conclusion: Nos résultats supportent la notion que la séquestration de RPA aux fourches de réplication endommagées durant la phase S prévient son utilisation pour la réparation par excision de nucléotides ce qui inhibe fortement l'efficacité de réparation. Cette étude chez la levure facilite l’élucidation du phénomène analogue chez l’humain et, ultimement, comprend des implications majeures dans la compréhension du mécanisme de développement des cancers UV-dépendants.

Molecular Epidemiology of Escherichia coli isolates from Environmental, Food and Clinical Sources

In the present study diversity of E. coli in the water samples of Cochin estuary were studied for a period of 3 years ranging from January 2010- December 2012. The stations were selected based on the closeness to satellite townships and waste input. Two of the stations (Chitoor and Thevara) were fixed upstream, two in the central part of the estuary namely Bolgatty and Off Marine Science Jetty, and one at the Barmouth. Diversity was assessed in terms of serotypes, phylogenetic groups and genotypes. Two groups of seafood samples such as fish and shellfish collected from the Cochin estuary were used for isolation of E. coli. One hundred clinical E. coli isolates were collected from one public health centre, one hospital and five medical labs in and around Cochin City, Kerala. From our results it was clear that pathogen cycling is occurring through food, water and clinical sources. Pathogen cycling through food is very common and fish and shellfish that harbour these strains might pose potential health risk to consumer. Estuarine environment is a melting pot for various kinds of wastes, both organic and inorganic. Mixing up of waste water from various sources such as domestic, industries, hospitals and sewage released into these water bodies resulting in the co-existence of E. coli from various sources thus offering a conducive environment for horizontal gene transfer. Opportunistic pathogens might acquire genes for drug resistance and virulence turning them to potential pathogens. Prevalence of ExPEC in the Cochin estuary, pose threat to people who use this water for fishing and recreation. Food chain also plays an important role in the transit of virulence genes from the environments to the human. Antibiotic resistant E. coli are widespread in estuarine water, seafood and clinical samples, for reasons well known such as indiscriminate use of antibiotics in animal production systems, aquaculture and human medicine. Since the waste water from these sources entering the estuary provides selection pressure to drug resistant mutants in the environment. It is high time that the authorities concerned should put systems in place for monitoring and enforcement to curb such activities. Microbial contamination can limit people’s enjoyment of coastal waters for contact recreation or shellfish-gathering. E. coli can make people sick if they are present in high levels in water used for contact recreation or shellfish gathering. When feeding, shellfish can filter large volumes of seawater, so any microorganisms present in the water become accumulated and concentrated in the shellfish flesh. If E. coli contaminated shellfish are consumed the impact to human health includes gastroenteritis, urinary tract infections (UTIs), and bacteraemia. In conclusion, the high prevalence of various pathogenic serotypes and phylogenetic groups, multidrug-resistance, and virulence factor genes detected among E. coli isolates from stations close to Cochin city is a matter of concern, since there is a large reservoir of antibiotic resistance genes and virulence traits within the community, and that the resistance genes and plasmid-encoded genes for virulence were easily transferable to other strains. Given the severity of the clinical manifestations of the disease in humans and the inability and/or the potential risks of antibiotic administration for treatment, it appears that the most direct and effective measure towards prevention of STEC and ExPEC infections in humans and ensuring public health may be considered as a priority.

Funktionelle Analyse der LC-FACS in Dictyostelium discoideum

"Funktionelle Analyse der LC-FACS in Dictyostelium discoideum" Das Dictyostelium discoideum Gen fcsA kodiert für ein 75 kDa großes Protein. Es kann durch Homologieanyalysen der Amino-säuresequenz zu den "long-chain fatty acyl-CoA"-Synthetasen ge-rechnet werden, die lang-kettige Fettsäuren durch die kovalente Bindung von Coenzym A akti-vie-ren und damit für diverse Reak-tionen in Stoffwechsel und Molekül-Synthese der Zelle verfügbar machen. Die hier untersuchte D. discoideum LC-FACS lokalisiert als peripher assoziiertes Protein an der cytosolischen Seite der Membran von Endo-somen und kleiner Vesikel. Bereits kurz nach der Bildung in der frühen sauren Phase kann die Lokalisation der LC-FACS auf Endosomen ge-zeigt werden. Sie dissoziiert im Laufe ihrer Neutra-li-sierung und kann auf späten Endosomen, die vor ihrer Exocytose stehen nicht mehr nach-gewiesen werden. Ein Teil der kleinen die in der gesamte Zelle verteilten kleinen Vesikel zeigt eine Kolokalisation mit lysosomalen Enzymen. Trotz des intrazellulären Verteilungs-mus-ters, das eine Beteiligung dieses Pro-teins an der Endocytose nahe-legt, konnte kein signifikanter Rückgang der Pino- und Phagocytose-Rate in LC-FACS Nullmutanten beobachtet werden. Der endo-cy-to-ti-sche Transit ist in diesen Zellen etwas verlängert, außerdem zeigen die Endosomen einen deutlich erhöhten pH-Wert, was zu einer weniger effektiven Prozessierung eines lysosomalen Enzyms führt (a-Mannosidase). Die Funktion der LC-FACS ist die Aufnahme von langkettigen Fettsäuren aus dem Lumen der Endosomen.

Molekularbiologische Funktionsanalysen von Thioredoxinen aus Dictyostelium discoideum und Identifizierung neuer Interaktionspartner durch das Two-Hybrid-System

Thioredoxins are small, regulatory proteins with a mass of approximately 12 kDa and a characteristic conserved active center, which is represented in the pentapeptide trp-cys-gly-pro-cys. Up to now it is not possible to present a complete list of thioredoxin interaction partners because there is no predictable sequence in the target enzymes where thioredoxins can interact with. To get closer information about the functions and possible interaction partners of the three thioredoxins from the social soil amoeba Dictyostelium discoideum (DdTrx1 - 3) we have chosen two different strategies. In the first one the thioredoxin levels in the cell should changed by different mutants. But both the antisense technique as well as the creation of knock out mutants were not appropiate strategies in this case. Just an thioredoxin overexpressing mutant results in a developmental phenotype which allows some conclusions for possible functions of the thioredoxin in Dictyostelium discoideum. The second strategie was the two hybrid system where thioredoxin interactions partners can identified systematically. After a screening with a cDNA library from Dictyostelium 13 potential interaction partners could be detected, among them a ribonucleotid reductase, TRFA, two different cytochrome c oxidase subunits, filopodin, three ribosomal proteins, the elongationfactor 1a and the alcohol dehydrogenase from yeast. The verification of the interaction between thioredoxin and these two hybrid clones happened indirectly by a dobble mutant of thioredoxin 1, where the cysteines in the active center were replaced by redox-inactive serins. Further examinations of two choosen candidates resulted that the alcohol dehydrogenase from yeast is a thioredoxin-modululated enzym and that there is an interaction between the elongationfactor 1a and the thioredoxin 1 from Dictyostelium discoideum.

Role of Rab5 in synaptic vesicle recycling

During synaptic transmission, NT-filled synaptic vesicles are released by Ca2+-triggered exocytosis at the active zone. Following exocytosis, SV membrane is immediately re-internalized and synaptic vesicles (SVs) are regenerated by a local recycling mechanism within the presynaptic terminal. It is debated whether an endosomal compartment is involved in this recycling process. In contrast, it is well known from cultured mammalian cells, that endocytic vesicles fuse to the early sorting endosome. The early endosome is a major sorting station of the cell where cargo is send into the degradative pathway to late endosome and lysosome or towards recycling. Each trafficking step is mediated by a certain protein of the Rab family. Rab proteins are small GTPases belonging to the Ras superfamily. They accumulate at their target compartments and have thereby been used as markers for the different endocytic organelles in cultured mammalian cells. Rab5 controls trafficking from the PM to the early endosome and has thereby been used as marker for this compartment. A second marker is based on the specific binding of the FYVE zinc finger protein domain to the lipid PI(3)P that is specifically generated at the early endosomal membrane. This study used the Drosophila NMJ as a model system to investigate the SV recycling process. In particular, three questions were addressed: First, is an endosomal compartment present at the synapse? Second, do SVs recycle through an endosome? Third, is Rab5 involved in SV recycling? We used GFP fusions of Rab5 and 2xFYVE to visualize endosomal compartments at the presynaptic terminal of Drosophila third instar larval NMJs. Furthermore, the endosomes are located within the pool of recycling SVs, labeled with the styryl-dye FM5-95. Using the temperature-sensitive mutation in Dynamin, shibirets, we showed that SV recycling involves trafficking through an intermediate endosomal compartment. In cultured mammalian cells, interfering with Rab5 function by expressing the dominant negative version, Rab5SN causes the fragmentation of the endosome and the accumulation of endocytic vesicles. In contrast, when Rab5 is overexpressed enlarged endosomal compartments were observed. In Drosophila, the endosomal compartment was disrupted when loss of function and dominant negative mutants of Rab5 were expressed. In addition, at the ultrastructural we observed an accumulation of endocytic vesicles in Rab5S43N expressing terminals and enlarged endosomes when Rab5 was overexpressed. Furthermore, interfering with Rab5 function using the dominant negative Rab5S43N caused a decrease in the SV recycling kinetics as shown by FM1-43 experiments. In contrast, overexpression of Rab5 or GFP-Rab5 caused an increase in the FM1-43 internalization rate. Finally, standard electrophysiological techniques were used to measure synaptic function. We found that the Rab5-mediated endosomal SV recycling pathway generates vesicles with a higher fusion efficacy during Ca2+-triggered release, compared to SVs recycled when Rab5 function was impaired. We therefore suggest a model in which the endosome serves as organelle to control the SV fusion efficacy and thereby the synaptic strength. Since changes in the synaptic strength are occuring during learning and memory processes, controlling endosomal SV recycling might be a new molecular mechanism involved in learning and memory.

The role of Ken&Barbie in JAK/STAT signalling during development of Drosophila melanogaster

Cell-cell interactions during embryonic development are crucial in the co-ordination of growth, differentiation and maintenance of many different cell types. To achieve this co-ordination each cell must properly translate signals received from neighbouring cells, into spatially and temporally appropriate developmental responses. A surprisingly limited number of signal pathways are responsible for the differentiation of enormous variety of cell types. As a result, pathways are frequently 'reused' during development. Thus, in mammals the JAK/STAT pathway is required during early embryogenesis, mammary gland formation, hematopoiesis and, finally, plays a pivotal role in immune response. In the canonical way, the JAK/STAT pathway is represented by a transmembrane receptor associated with a Janus kinase (JAK), which upon stimulation by an extra-cellular ligand, phosphorylates itself, the receptor and, finally, the signal transducer and activator of transcription (STAT) molecules. Phosphorylated STATs dimerise and translocate to the nucleus where they activate transcription of target genes. The JAK/STAT pathway has been conserved throughout evolution, and all known components are present in the genome of Drosophila melanogaster. Besides hematopoietic and immunity functions, the pathway is also required during development for processes including embryonic segmentation, tracheal morphogenesis, posterior spiracle formation etc. This study describes Drosophila Ken&Barbie (Ken) as a selective regulator of JAK/STAT signalling. ken mutations identified in a screen for modulators of an eye overgrowth phenotype, caused by over-expression of the pathway ligand unpaired, also interact genetically with the pathway receptor domeless (dome) and the transcription factor stat92E. Over-expression of Ken can phenocopy developmental defects known to be caused by the loss of JAK/STAT signalling. These genetic interactions suggest that Ken may function as a negative regulator of the pathway. Ken has C-terminal Zn-finger domain, presumably for DNA binding, and N-terminal BTB/POZ domain, often found in transcriptional repressors. Using EGFP-fused construct expressed in vivo revealed nuclear accumulation of Ken. Therefore, it is proposed that Ken may act as a suppresser of STAT92E target genes. An in vitro assay, termed SELEX, determined that Ken specifically binds to a DNA sequence, with the essential for DNA recognition core overlapping that of STAT92E. This interesting observation suggests that not all STAT92E sites may also allow Ken binding. Strikingly, when effects of ectopic Ken on the expression of putative JAK/STAT pathway target genes were examined, only a subset of the genes tested, namely vvl, trh and kni, were down-regulated by Ken, whereas some others, such as eve and fj, appeared to be unresponsive. Further analysis of vvl, one of the genes susceptible to ectopic Ken, was undertaken. In the developing hindgut, expression of vvl is JAK/STAT pathway dependent, but remains repressed in the posterior spiracles, despite the stimulation of STAT92E by Upd in their primordia. Importantly, ken is also expressed in the developing posterior spiracles. Strikingly, up-regulation of vvl is observed in these tissues in ken mutant embryos. These imply that while ectopic Ken is sufficient to repress the expression of vvl in the hindgut, endogenous Ken is also necessary to prevent its activation in the posterior spiracles. It is therefore conceivable that ectopic vvl expression in the posterior spiracles of the ken mutants may be the result of de-repression of endogenous STAT92E activity. Another consequence of these observations is a fine balance that must exist between STAT92E and Ken activities. Apparently, endogenous level of Ken is sufficient to repress vvl, but not other, as yet unidentified, JAK/STAT pathway targets, whose presumable activation by STAT92E is required for posterior spiracle development as the embryos mutant for dome, the receptor of the pathway, show severe spiracle defects. These defects are also observed in the embryos mis-expressing Ken. Though it is possible that the posterior spiracle phenotype caused by higher levels of Ken results from a JAK/STAT pathway independent activity, it seems to be more likely that Ken acts in a dosage dependent manner, and extra Ken is able to further antagonise JAK/STAT pathway target genes. While STAT92E binding sites required for target gene expression have been poorly characterised, the existence of genome data allows the prediction of candidate STAT92E sites present in target genes promoters to be attempted. When a 6kb region containing the putative regulatory domains flanking the vvl locus are examined, only a single potential STAT92E binding site located 825bp upstream of the translational start can be detected. Strikingly, this site also includes a perfect Ken binding sequence. Such an in silico observation, though consistent with both Ken DNA binding assay in vitro and regulation of STAT92E target genes in vivo, however, requires further analysis. The JAK/STAT pathway is implicated in a variety of processes during embryonic and larval development as well as in imago. In each case, stimulation of the same transcription factor results in different developmental outcomes. While many potential mechanisms have been proposed and demonstrated to explain such pleiotropy, the present study indicates that Ken may represent another mechanism, with which signal transduction pathways are controlled. Ken selectively down-regulates a subset of potential target genes and so modifies the transcriptional profile generated by activated STAT92E - a mechanism, which may be partially responsible for differences in the morphogenetic processes elicited by JAK/STAT signalling during development.

On the function of the Dictyostelium Argonaute A protein (AgnA) in epigenetic gene regulation

With molecular biology methods and bioinformatics, the Argonaute proteins in Dictyostelium discoideum were characterized, and the function of the AgnA protein in RNAi and DNA methylation was investigated, as well as cellular features. Also interaction partners of the PAZ-Piwi domain of AgnA (PAZ-PiwiAgnA) were discovered. The Dictyostelium genome encodes five Argonaute proteins, termed AgnA/B/C/D/E. The expression level of Argonaute proteins was AgnB/D/E > AgnA > AgnC. All these proteins contain the characteristic conserved of PAZ and Piwi domains. Fluorescence microscopy revealed that the overexpressed C-terminal GFP-fusion of PAZ-PiwiAgnA (PPWa-GFP) localized to the cytoplasm. Overexpression of PPWa-GFP leaded to an increased gene silencing efficiency mediated by RNAi but not by antisense RNA. This indicated that PAZ-PiwiAgnA is involved in the RNAi pathway, but not in the antisense pathway. An analysis of protein-protein interactions by a yeast-two-hybrid screen on a cDNA library from vegetatively grown Dictyostelium revealed that several proteins, such as EF2, EF1-I, IfdA, SahA, SamS, RANBP1, UAE1, CapA, and GpdA could interact with PAZ-PiwiAgnA. There was no interaction between PAZ-PiwiAgnA and HP1, HelF and DnmA detected by direct yeast-two-hybrid analysis. The fluorescence microscopy images showed that the overexpressed GFP-SahA or IfdA fusion proteins localized to both cytoplasm and nuclei, while the overexpressed GFP-SamS localized to the cytoplasm. The expression of SamS in AgnA knock down mutants was strongly down regulated on cDNA and mRNA level in, while the expression of SahA was only slightly down regulated. AgnA knock down mutants displayed defects in growth and phagocytosis, which suggested that AgnA affects also cell biological features. The inhibition of DNA methylation on DIRS-1 and Skipper retroelements, as well as the endogenous mvpB and telA gene, observed for the same strains, revealed that AgnA is involved in the DNA methylation pathway. Northern blot analysis showed that Skipper and DIRS-1 were rarely expressed in Ax2, but the expression of Skipper was upregulated in AgnA knock down mutants, while the expression of DIRS-1 was not changed. A knock out of the agnA gene failed even though the homologous recombination of the disruption construct occurred at the correct site, which indicated that there was a duplication of the agnA gene in the genome. The same phenomenon was also observed in ifdA knock out experiments.

Heterochromatin und epigenetische Kontrolle der Centromere in Dictyostelium discoideum

Heterochromatin Protein 1 (HP1) is an evolutionarily conserved protein required for formation of a higher-order chromatin structures and epigenetic gene silencing. The objective of the present work was to functionally characterise HP1-like proteins in Dictyostelium discoideum, and to investigate their function in heterochromatin formation and transcriptional gene silencing. The Dictyostelium genome encodes three HP1-like proteins (hcpA, hcpB, hcpC), from which only two, hcpA and hcpB, but not hcpC were found to be expressed during vegetative growth and under developmental conditions. Therefore, hcpC, albeit no obvious pseudogene, was excluded from this study. Both HcpA and HcpB show the characteristic conserved domain structure of HP1 proteins, consisting of an N-terminal chromo domain and a C-terminal chromo shadow domain, which are separated by a hinge. Both proteins show all biochemical activities characteristic for HP1 proteins, such as homo- and heterodimerisation in vitro and in vivo, and DNA binding activtity. HcpA furthermore seems to bind to K9-methylated histone H3 in vitro. The proteins thus appear to be structurally and functionally conserved in Dictyostelium. The proteins display largely identical subnuclear distribution in several minor foci and concentration in one major cluster at the nuclear periphery. The localisation of this cluster adjacent to the nucleus-associated centrosome and its mitotic behaviour strongly suggest that it represents centromeric heterochromatin. Furthermore, it is characterised by histone H3 lysine-9 dimethylation (H3K9me2), which is another hallmark of Dictyostelium heterochromatin. Therefore, one important aspect of the work was to characterise the so-far largely unknown structural organisation of centromeric heterochromatin. The Dictyostelium homologue of inner centromere protein INCENP (DdINCENP), co-localized with both HcpA and H3K9me2 during metaphase, providing further evidence that H3K9me2 and HcpA/B localisation represent centromeric heterochromatin. Chromatin immunoprecipitation (ChIP) showed that two types of high-copy number retrotransposons (DIRS-1 and skipper), which form large irregular arrays at the chromosome ends, which are thought to contain the Dictyostelium centromeres, are characterised by H3K9me2. Neither overexpression of full-length HcpA or HcpB, nor deletion of single Hcp isoforms resulted in changes in retrotransposon transcript levels. However, overexpression of a C-terminally truncated HcpA protein, assumed to display a dominant negative effect, lead to an increase in skipper retrotransposon transcript levels. Furthermore, overexpression of this protein lead to severe growth defects in axenic suspension culture and reduced cell viability. In order to elucidate the proteins functions in centromeric heterochromatin formation, gene knock-outs for both hcpA and hcpB were generated. Both genes could be successfully targeted and disrupted by homologous recombination. Surprisingly, the degree of functional redundancy of the two isoforms was, although not unexpected, very high. Both single knock-out mutants did not show any obvious phenotypes under standard laboratory conditions and only deletion of hcpA resulted in subtle growth phenotypes when grown at low temperature. All attempts to generate a double null mutant failed. However, both endogenous genes could be disrupted in cells in which a rescue construct that ectopically expressed one of the isoforms either with N-terminal 6xHis- or GFP-tag had been introduced. The data imply that the presence of at least one Hcp isoform is essential in Dictyostelium. The lethality of the hcpA/hcpB double mutant thus greatly hampered functional analysis of the two genes. However, the experiment provided genetic evidence that the GFP-HcpA fusion protein, because of its ability to compensate the loss of the endogenous HcpA protein, was a functional protein. The proteins displayed quantitative differences in dimerisation behaviour, which are conferred by the slightly different hinge and chromo shadow domains at the C-termini. Dimerisation preferences in increasing order were HcpA-HcpA << HcpA-HcpB << HcpB-HcpB. Overexpression of GFP-HcpA or a chimeric protein containing the HcpA C-terminus (GFP-HcpBNAC), but not overexpression of GFP-HcpB or GFP-HcpANBC, lead to increased frequencies of anaphase bridges in late mitotic cells, which are thought to be caused by telomere-telomere fusions. Chromatin targeting of the two proteins is achieved by at least two distinct mechanisms. The N-terminal chromo domain and hinge of the proteins are required for targeting to centromeric heterochromatin, while the C-terminal portion encoding the CSD is required for targeting to several other chromatin regions at the nuclear periphery that are characterised by H3K9me2. Targeting to centromeric heterochromatin likely involves direct binding to DNA. The Dictyostelium genome encodes for all subunits of the origin recognition complex (ORC), which is a possible upstream component of HP1 targeting to chromatin. Overexpression of GFP-tagged OrcB, the Dictyostelium Orc2 homologue, showed a distinct nuclear localisation that partially overlapped with the HcpA distribution. Furthermore, GFP-OrcB localized to the centrosome during the entire cell cycle, indicating an involvement in centrosome function. DnmA is the sole DNA methyltransferase in Dictyostelium required for all DNA(cytosine-)methylation. To test for its in vivo activity, two different cell lines were established that ectopically expressed DnmA-myc or DnmA-GFP. It was assumed that overexpression of these proteins might cause an increase in the 5-methyl-cytosine(5-mC)-levels in the genomic DNA due to genomic hypermethylation. Although DnmA-GFP showed preferential localisation in the nucleus, no changes in the 5-mC-levels in the genomic DNA could be detected by capillary electrophoresis.

Untersuchungen zum Vorkommen und molekularen Mechanismus der Biofilm-Bildung bei Enterokokken aus verschiedenen klinischen Bereichen

Im Rahmen der vorliegenden Arbeit wurde eine größere Anzahl an E. faecalis Isolaten aus Vaginalabstrichen, erstmals insbesondere von Patientinnen, die an Bakterieller Vaginose litten, untersucht und mit E. faecalis Stämme aus verschiedenen anderen klinischen Bereichen auf das Vorkommen von Virulenzfaktoren verglichen. Weiterhin wurden Korrelationen zwischen bestimmten Faktoren und der Menge an produziertem Biofilm erstellt, um mögliche Zusammenhänge zum Mechanismus der Biofilm-Bildung zu erfassen. Mittels statistischer Analysen konnte hinsichtlich der 150 untersuchten E. faecalis Isolate nachgewiesen werden, dass keine signifikanten Unterschiede der Inzidenzen von Virulenzfaktoren (esp, asa1, gelE, GelE, cylA, β-Hämolyse) zwischen den Stämmen der verschiedenen Herkunftsbereiche bestanden. In Bezug auf das Auftreten von Biofilm-Bildung zeigte sich ein erhöhtes Vorkommen bei Stämmen aus Urin sowie invasiver Herkunft (insgesamt jeweils ca. 70 % mäßige und starke Biofilm-Bildner) im Vergleich zu E. faecalis Isolaten aus Wunden oder Faeces (je ca. 40 %). Statistische Auswertungen bzgl. des Zusammenhangs eines oder einer Kombination von Virulenzfaktoren mit der Menge an gebildetem Biofilm wiesen darauf hin, dass Isolate, die das esp Gen besaßen, in erhöhtem Maße zur Biofilm-Bildung befähigt waren. Dies zeigte einen gewissen Einfluss des Zellwandproteins auf die Fähigkeit zur Biofilm-Bildung bei E. faecalis. Allerdings wurden stets auch Stämme identifiziert, die die Fähigkeit zur Biofilm-Bildung trotz des Fehlens der jeweils untersuchten genetischen Determinante bzw. der Determinanten aufwiesen, so dass auf das Vorhandensein weiterer, unbekannter Einflussfaktoren auf den Mechanismus der Biofilm-Bildung bei E. faecalis geschlossen werden konnte. Unter 78 untersuchten E. faecium Isolaten aus verschiedenen klinischen Bereichen konnte lediglich ein Stamm (1,3 %) als mäßiger Biofilm-Bildner charakterisiert werden, so dass die Fähigkeit bei dieser Spezies in der hier untersuchten Region unter diesen Bedingungen kaum nachgewiesen werden konnte. Einen Schwerpunkt dieser Arbeit bildete die Untersuchung zum Vorkommen von Virulenzfaktoren und Biofilm-Bildung bei E. faecalis Isolaten aus Vaginalabstrichen. Bzgl. des Auftretens von Virulenzfaktoren und Biofilm-Produktion konnte kein Unterschied zwischen Stämmen assoziiert mit Bakterieller Vaginose und Isolaten einer Vergleichsgruppe festgestellt werden. Allerdings zeigte eine Gegenüberstellung mit den untersuchten E. faecalis Stämmen aus anderen klinischen Bereichen, dass die 80 Isolate aus Vaginalabstrichen eine ähnlich hohe Inzidenz bestimmter Virulenzfaktoren wie Stämme aus Faeces, Urin, Wunden oder invasiver Herkunft sowie eine mit den Isolaten aus Urin und invasiver Herkunft vergleichbar hohe Fähigkeit zur Biofilm-Bildung aufwiesen (ca. 75 % mäßige und starke Biofilm-Bildner). Dies deutete auf eine Verbreitung der Biofilm-Bildungsfähigkeit bei E. faecalis Stämmen der Vaginalflora und somit auf eine große Bedeutung der Eigenschaft für Isolate dieser Herkunft hin. Die statistische Auswertung der Korrelationen von Virulenzfaktoren mit der Menge an gebildetem Biofilm lieferte ähnliche Ergebnisse wie die Analysen bzgl. der 150 E. faecalis Isolate aus anderen klinischen Bereichen und untermauerte die Annahme, dass zusätzliche Faktoren zu den hier untersuchten Determinanten bei E. faecalis vorhanden sein müssen, die Einfluss auf den Mechanismus der Biofilm-Bildung nehmen. Deshalb konzentrierte sich ein weiterer Teil der vorliegenden Arbeit auf die Herstellung und Charakterisierung von E. faecalis Biofilm-Spontanmutanten, um bisher noch ungeklärte Mechanismen oder neue Faktoren zu erkennen, die Einfluss auf die Biofilm-Bildung bei E. faecalis nehmen. Die Untersuchung einer Mutante (1.10.16) und ihres Wildtypstamms lieferte erstmals den phänotypischen Nachweis des HMW-Komplexes der drei Bee-Proteine sowie die Identifizierung konservierter Pili-Motive dieser Proteine. Des Weiteren schien die in diesem Cluster ebenfalls codierte Sortase-1 dasjenige Enzym zu sein, das höchstwahrscheinlich die Bindung des Proteins Bee-2 innerhalb dieses HMW-Komplexes katalysiert. Insofern lieferten diese Untersuchungen neue, konkrete Hinweise zur Rolle des bee Genclusters bei der Pili-Biogenese und Biofilm-Bildung von E. faecalis. Darüber hinaus stellen Erkenntnisse aus der Charakterisierung von zwei weiteren hergestellten Biofilm-Spontanmutanten viel versprechende Ausgangspunkte für zukünftige Untersuchungen dar, die ein weitergehendes Verständnis der molekularen Mechanismen der Biofilm-Bildung bei E. faecalis erzielen könnten.

Unveiling the components of the signaling pathway between endosomes and the phagocytic uptake machinery in Dictyostelium discoideum

The soil amoebae Dictyostelium discoideum take up particles from their environment in order to obtain nutrition. The particle transits through the cell within a phagosome that fuses with organelles of different molecular compositions, undergoing a gradual degradation by different sets of hydrolytic enzymes. Griffiths’ concept of “phagosome individuality” predicts signaling from phagosomes into the cytoplasm, which might regulate many aspects of cell physiology. The finding that Dictyostelium cells depleted of the lysozyme AlyA or over-expressing the esterase Gp70 exhibit increased uptake of food particles, led to the postulation of a signaling cascade between endocytic compartments and the cytoskeletal uptake machinery at the plasma membrane. Assuming that Gp70 acts downstream of AlyA, gene-expression profiling of both mutants revealed different and overlapping sets of misregulated genes that might participate in this signaling cascade. Based on these results, we analyzed the effects of the artificial misregulation of six candidate genes by over-expression or negative genetic interference, in order to reconstruct at least part of the signaling pathway. SSB420 and SSL793 were chosen as candidates for the first signaling step, as they were up-regulated in AlyA-null cells and remained unaltered in the Gp70 over-expressing cells. The over-expression of SSB420 enhanced phagocytosis and raised the expression levels of Gp70, supporting its involvement in the signaling pathway between AlyA and Gp70 as a positive regulator of phagocytosis. However, this was not the case of cells over-expressing SSL793, as this mutation had no effects on phagocytosis. For the signaling downstream of Gp70, we studied four commonly misregulated genes in AlyA-depleted and Gp70 over-expressing cells. The expression levels of SLB350, SSB389 and TipD were lower in both mutants and therefore these were assumed as possible candidates for the negative regulation of phagocytosis. Cells depleted of SLB350 exhibited an increased phagocytic activity and no effect on Gp70 expression, proving its participation in the signaling pathway downstream of Gp70. Unlike SLB350, the disruption of the genes coding for SSB389 and TipD had no effects on particle uptake, excluding them from the pathway. The fourth candidate was Yipf1, the only gene that was commonly up-regulated in both mutants. Yet, the artificial over-expression of this protein had no effects on phagocytosis, so this candidate is also not included in the signaling pathway. Furthermore, localizing the products of the candidate genes within the cell helped unveiling several cellular organelles that receive signals from the phagosome and transduce them towards the uptake machinery.

Biologische Funktionsanalyse und Identifizierung neuer Substrate der Methyltransferase Dnmt2

Obwohl die DNA Methyltransferase 2 (Dnmt2) hoch konserviert ist und zu der am weitesten verbreiteten eukaryotischen MTase-Familie gehört, ist ihre biologische Funktion nach wie vor unklar. Nachdem lange Zeit keine DNA Methylierungsaktivität nachgewiesen werden konnte, wurde vor einigen Jahren über geringe Mengen an 5-Methylcytosin (5mC) in Retroelementen der “Dnmt2-only”-Organismen D. melanogaster, D. discoideum und E. histolytica berichtet (Kunert et al. 2003; Fisher et al. 2004; Kuhlmann et al. 2005; Phalke et al. 2009). Als kurze Zeit später robuste Methylierung der tRNAAsp durch humane Dnmt2 gezeigt wurde (Goll et al. 2006), wurde zunächst eine Dualspezifität des Enzyms vorgeschlagen (Jeltsch et al. 2006). Neuere Daten zum 5mC-Status verschiedener „Dnmt2-only“-Organismen bilden Anlass für kontroverse Diskussionen über Ausmaß und Bedeutung der DNA Methyltransferaseaktivität von Dnmt2 (Schaefer et al. 2010a; Krauss et al. 2011). Die vorliegende Arbeit konzentriert sich auf die Identifizierung neuer RNA Substrate des Dnmt2-Homologs DnmA aus D. discoideum sowie die biologische Bedeutung der tRNA-Methylierung durch Dnmt2. Wie in anderen Organismen beschrieben, fungiert auch DnmA als tRNAAsp(GUC) MTase in vitro und in vivo. Zusätzlich konnte in vitro tRNAGlu(UUC) als neues Substrat der Dnmt2-Homologe aus D. discoideum und dem Menschen identifiziert werden. In einem Kooperationsprojekt wurde außerdem auch tRNAAsp-Methylierungsaktivität für das Dnmt2-Homolog aus S. pombe (Pmt1) nachgewiesen. Crosslink-RNA-Immunopräzipitationen (RNA-CLIP) mit anschließender Next-Generation-Sequenzierung der mit DnmA assoziierten RNAs zeigen, dass DnmA mit tRNA Fragmenten interagiert, die sich vom Anticodonloop bis in den T-loop erstrecken. Neben der tRNAAsp(GUC) und tRNAGlu(UUC/CUC) sind Fragmente der tRNAGly(GCC) verstärkt angereichert. Inwiefern diese Fragmente eine biologische Funktion haben oder spezifische Degradationsprodukte darstellen, ist noch ungeklärt. Interessanterweise sind von einigen tRNAs wenige Sequenzen von antisense-Fragmenten in den RNA-CLIP Daten zu finden, die etwas kürzer, jedoch exakt komplementär zu den genannten sense-Fragmenten sind. Besonders stark sind diese Fragmente der tRNAGlu(UUC) vertreten. In einem weiteren RNA-CLIP Experiment wurden U-snRNAs, snoRNA und intergenische Sequenzen mit DnmA angereichert. Bei nachfolgenden in vitro Methylierungsstudien konnte ausschließlich die U2-snRNA als potentielles Nicht-tRNA-Substrat der hDnmt2 und DnmA identifiziert werden. Da tRNA Modifikationen im Anticodonloop die Codonerkennung beeinflussen können, wurde ein System etabliert um die Translationseffizienz eines GFP-Reportergens in Wildtyp- und dnmAKO-Zellen zu messen. In D. discoideum wird das Aspartat-Codon GAU ca. zehnmal häufiger genutzt als das GAC Codon, allerdings ist nur eine tRNAAsp(GUC) im Genom der Amöbe kodiert. Aus diesem Grund wurde zusätzlich die Frage adressiert, inwiefern die DnmA-abhängige Methylierung dieser tRNA das „Wobbling“ beeinflusst. Dazu wurde dem Reportergen jeweils eine (GAU)5- und (GAC)5-Leadersequenz vorgeschaltet. Entgegen der Annahme wurde der (GAC)5-Leader in beiden Stämmen etwas effizienter translatiert. Insgesamt zeigte der dnmAKO-Stamm eine leicht erhöhte Translationseffizienz der Reportergene. Vergleichende Analysen zur Aufnahme von Fremd-DNA zeigten signifikant reduzierte Transformationseffizienzen mit einem integrierenden Plasmid in dnmAKO-Zellen. Ein weiterer dnmAKO-Stamm zeigte diesen Effekt jedoch nicht, wobei bei derselben Mutante eine deutlich reduzierte Aufnahme eines extrachromosomalen Plasmids zu verzeichnen war. Untersuchungen zum Einfluss von DnmA auf die Regulation des Retroelements skipper ergaben keinen Zusammenhang zwischen der Generierung kleiner RNAs und der erhöhten Transkription des Retrotransposons in dnmAKO-Zellen (Kuhlmann et al. 2005). Durch Kompensationsversuche sowie Experimente mit einer weiteren dnmAKO-Mutante konnte die Mobilisierung des Retrotransposons nicht eindeutig als DnmA-Funktion eingeordnet werden. In einem weiteren Projekt wurden die Bindung des m5C-bindenden Proteins EhMLBP aus E. histolytica an DNA mittels Rasterkraftmikroskopie abgebildet (Lavi et al. 2006). Neben vermutlich unspezifischen Endbindungsereignissen konnte eine bevorzugte Bindungsstelle des Proteins an LINE DNA (long intersperesed nuclear element) identifiziert werden. Möglicherweise fällt diese mit einem von zwei A/T-reichen Bereichen der LINE DNA zusammen, von denen vermutet wird, dass diese für die Bindung von EhMLBP an DNA von Bedeutung sind. Insgesamt bestätigen die Ergebnisse dieser Arbeit die tRNAAsp Methylierungsaktivität als konservierte Dnmt2-Funktion. Darüber hinaus erweitern sie das Substratspektrum der Dnmt2-Methyltransferasen im Bereich der tRNA. Außerdem wird erstmals ein potentielles Nicht-tRNA Substrat vorgeschlagen. Zusätzlich geben neu entdeckte Phänotypen Hinweise auf vielfältige zelluläre Dnmt2-Funktionen.