Untersuchungen zur Chromosomen-Segregation und Protein-Sortierung in Staphylococcus aureus

The thesis addressed bacterial cell biological topics of Staphylococcus aureus, one of the most important human pathogens. Insights into essential cellular processes, such as chromosome segregation, could serve as a basis for developing new antibiotic targets. The first part deals with chromosome segregation in S. aureus. In contrast to rod-shaped model bacteria, an S. aureus smc (structural maintenance of chromosomes) mutant was not lethal, and had only mild deficiency in chromosome organization and segregation. Double mutations of smc and spoIIIE encoding an active DNA translocase resulted in severe growth defect and chromosome segregation impairment. Further, the SMC proteins localized as dynamic foci in a cell cycle dependent manner in S. aureus. These results unravel the differences in chromosome dynamics in the spherical staphylococcal cells compared to the model in rods and implies some yet unknown mechanisms. The second part deals with the localization of surface proteins that play key roles in S. aureus pathogenicity. A fluorescence microscopic method was developed to directly monitor the localization of secreted and cell wall anchored surface proteins in S. aureus. Dependent on the presence of signal peptides alone or together with the cell wall sorting sequence the red fluorescent protein mCherry could be targeted into the cytosol, the supernatant and the cell envelope respectively; in all cases mCherry exhibited bright fluorescence. In staphylococci two types of signal peptides (SP) can be distinguished: the +YSIRK motif SPlip and the -YSIRK motif SPsasF. MCherry-hybrids supplied with the SPlip were always expressed higher than those with SPsasF. Further, mCherry-hybrids with SPlip preferentially localized at the cross wall, while those with SPsasF preferentially localized at the peripheral wall. Interestingly, when treated with sub-lethal concentrations of penicillin or moenomycin, both mCherry-fusions with different SPs were concentrated at the cross wall. The effect is most likely due to antibiotic mediated increase of free anchoring sites (Lipid II) at the cross wall, the substrate of Sortase A (SrtA); SrtA was required for this shift. In the last part (in cooperation with the research group of Dr. Proikas-Cezanne), the invasion of S. aureus into non-phagocytic cells (human osteosarcoma U2OS cell line stably expressing GFP-WIPI-1) triggering host autophagic response was investigated. It was found that the invading S. aureus USA300, HG001, SA113 could stimulate autophagy, and became entrapped in intracellular autophagosome-like vesicles. Agr-positive S. aureus strains were more efficiently entrapped than agr-negative cells. The entrapped S. aureus still undergo cell division. Likely, the invading S. aureus become entrapped in autophagosome-like vesicles and are dedicated for lysosomal degradation in non-professional host cells.Die Doktorarbeit behandelt ein Thema der bakteriellen Zellbiologie bei Staphylococcus aureus, einer der wichtigsten humanpathogenen Arten. Einblicke in die grundlegenden zellulären Prozessen, wie z. B. Chromosomensegregation, könnten als Grundlage für die Entwicklung neuer Angriffsorte für Antibiotika dienen. Der erste Teil befasst sich mit der Chromosomen-Segregation in S. aureus. Im Gegensatz zu stäbchenförmigen Modell- Bakterien, war in S. aureus eine SMC-Mutante (structural maintenance of chromosomes) nicht letal, und hatte nur leichte Mängel in der Organisation und Segregation der Chromosomen. Eine Doppel-Mutante von SMC und SpoIIIE, letztere codiert für eine aktive DNA-Translokase, führte zu schwerwiegenden Defekten in Wachstum und Chromosomensegregation. Ferner waren die SMC-Proteine als dynamische Punkte (foci) in einer Zellzyklus-abhängigen Weise in S. aureus lokalisiert. Diese Ergebnisse zeigen, dass es deutliche Unterschiede in der Chromosomen-Dynamik zwischen den sphärischen Staphylokokken den stäbchenförmigen Bakterien gibt und lassen auf noch unbekannte Mechanismen schließen. Der zweite Teil beschäftigt sich mit der Lokalisierung der Oberfläche Proteine, die in S. aureus eine Schlüsselrolle in der Pathogenität spielen. Es wurde ein fluoreszenzmikroskopisches Verfahren entwickelt, um direkt die Lokalisierung von sekretierten und Zellwand verankerten Oberflächenproteinen bei S. aureus verfolgen zu können. Je nach dem, ob das rot fluoreszierendes Protein mCherry allein, mit dem Signalpeptide, oder zusammen mit der Zellwand-Sortierungssequenz fusioniert wurde, war mCherry etweder im Cytosol, dem Überstand oder der Zellwand lokalisiert; in allen Fällen zeigte mCherry helle Fluoreszenz. In Staphylokokken können zwei Arten von Signalpeptiden (SP) unterschieden werden: mit einem (+YSIRK) Motiv (SPlip) und ohne einem (-YSIRK) Motiv (SPsasF). mCherry-Hybride mit (+YSIRK) Motiv waren immer höher exprimiert als als jene ohne (-YSIRK) Motiv (SPsasF). Ferner waren mCherry-Hybride mit SPlip bevorzugt in der Querwand, während diejenigen mit SPsasF bevorzugt in der peripheren Wand lokalisiert. Bei Behandlung mit subletalen Konzentrationen von Penicillin oder Moenomycin, waren beide SP-mCherry Fusionen in der Querwand konzentriert. Der Effekt ist höchstwahrscheinlich auf die Antibiotika-vermittelte Erhöhung der freien Ankerplätze (Lipid II), dem Substrat der Sortase A (SrtA), an der Querwand zurückzuführen. SrtA war für diesen Effekt notwendig. Im letzten Teil (in Zusammenarbeit mit der Arbeitsgruppe Dr. Proikas-Cezanne), wurde die Invasion von S. aureus in nicht-phagozytierenden Zellen (human osteosarcoma U2OS cell line stably expressing GFP-WIPI-1) und die Auslösung von Autophagocytose-Reaktionen untersucht. Es stellte sich heraus, dass die eingedrungenen S. aureus USA300, HG001, SA113 Zellen Autophagie auslösten und in und in intrazellulären Autophagosom-ähnlichen Vesikeln eingeschlossen waren. Agr-positive S. aureus Stämme wurden effizienter als agr-negativen Zellen eingeschlossen. Die eingeschlossenen S. aureus Zellen teilten sich noch. Wahrscheinlich werden die eindringenden S. aureus Zellen in Autophagosom-ähnliche Vesikel eingefangen und dem lysosomalen Abbau in die nichtprofessionellen Wirtszellen zugeführt

TextureGAN: Controlling Deep Image Synthesis with Texture Patches

In this paper, we investigate deep image synthesis guided by sketch, color, and texture. Previous image synthesis methods can be controlled by sketch and color strokes but we are the first to examine texture control. We allow a user to place a texture patch on a sketch at arbitrary locations and scales to control the desired output texture. Our generative network learns to synthesize objects consistent with these texture suggestions. To achieve this, we develop a local texture loss in addition to adversarial and content loss to train the generative network. We conduct experiments using sketches generated from real images and textures sampled from a separate texture database and results show that our proposed algorithm is able to generate plausible images that are faithful to user controls. Ablation studies show that our proposed pipeline can generate more realistic images than adapting existing methods directly.Comment: CVPR 2018 spotligh

Strain-induced semiconductor to metal transition in MA2Z4 bilayers

Very recently, a new type of two-dimensional layered material MoSi2N4 has been fabricated, which is semiconducting with weak interlayer interaction, high strength, and excellent stability. We systematically investigate theoretically the effect of vertical strain on the electronic structure of MA2Z4 (M=Ti/Cr/Mo, A=Si, Z=N/P) bilayers. Taking bilayer MoSi2N4 as an example, our first principle calculations show that its indirect band gap decreases monotonically as the vertical compressive strain increases. Under a critical strain around 22%, it undergoes a transition from semiconductor to metal. We attribute this to the opposite energy shift of states in different layers, which originates from the built-in electric field induced by the asymmetric charge transfer between two inner sublayers near the interface. Similar semiconductor to metal transitions are observed in other strained MA2Z4 bilayers, and the estimated critical pressures to realize such transitions are within the same order as semiconducting transition metal dichalcogenides. The semiconductor to metal transitions observed in the family of MA2Z4 bilayers present interesting possibilities for strain-induced engineering of their electronic properties

Experimental study on water evaporation from sand using environmental chamber

International audienceLarge-scale evaporation experiments were conducted on bare sand using an environmental chamber. Four different atmospheric conditions and various drying durations were imposed to soil sample. Both the atmospheric parameters (air flow rate, relative humidity and temperature) and the response of soil (volumetric water content, temperature and soil suction) were monitored simultaneously. Notably, the temperature and matric suction at soil surface were monitored using infrared thermometer and high-capacity tensiometer, respectively. The results show that the air and soil temperatures depend on the evaporation process and atmospheric conditions. In addition, volumetric water content in the near-surface zone is strongly affected by the evaporation process and changes linearly over depth. The evaporation rate is strongly dependent on the air conditions