30 research outputs found
Structural and Biochemical Characterization of SrcA, a Multi-Cargo Type III Secretion Chaperone in Salmonella Required for Pathogenic Association with a Host
Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 Ă… revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS
No-Slip Boundary Conditions for Electron Hydrodynamics and the Thermal Casimir Pressure
We derive modified reflection coefficients for electromagnetic waves in the THz and far infrared range. The idea is based on hydrodynamic boundary conditions for metallic conduction electrons. The temperature-dependent part of the Casimir pressure between metal plates is evaluated. The results should shed light on the “thermal anomaly,” where measurements deviate from the standard fluctuation electrodynamics for conducting metals
Reifung von
Schnelle synaptische ReizĂĽbertragung wird
durch die gesteuerte Freisetzung von Neurotransmittern aus
synaptischen Vesikeln (SV) vermittelt. Um synaptische
Reizübertragung und neuronale Aktivität zu regulieren, setzen
Neurone zusätzlich Neuropeptide und Hormone aus „Dense-Core“
Vesikeln (DCV) frei. Während SV recycelt werden können, müssen DCV
nach ihrer Freisetzung in den neuronalen Zellkörpern neu generiert
werden. Es wird angenommen, dass DCV am trans-Golgi-Netzwerk (TGN)
als unreife DCV entstehen, die nachfolgend einen Reifungsprozess
durchlaufen, der fĂĽr eine effiziente Prozessierung der Neuropeptide
innerhalb der DCV erforderlich ist. Des Weiteren werden bei diesem
Prozess Faktoren entfernt, die die Freisetzung der DCV behindern
wĂĽrden. Im Vorfeld konnten wir bereits zeigen, dass die kleine Rab
GTPase, RAB-2, und deren Effektor, RIC-19/ICA69, an der Reifung von
neuronalen DCV in Caenorhabditis elegans involviert sind. In rab-2
Mutanten geht spezifisches Cargo aus den reifenden DCV verloren, da
es fälschlicherweise in den endosomalen-lysosomalen Abbauweg
geleitet wird. Interessanterweise konnte dieser Cargo-Verlust durch
eine Blockade des endosomalen Abbauweges verhindert werden. Dies
weist darauf hin, dass RAB-2 an der ZurĂĽckhaltung von DCV
Komponenten während des Sortierungsprozesses im Übergangsbereich
zwischen Golgi und Endosomen involviert ist. Um zu verstehen wie
die Aktivität von RAB-2 am Golgi reguliert wird, entschieden wir
uns dazu, nach RAB-2 spezifischen GTPase Aktivierungsproteinen
(GAP) zu suchen. Wir identifizierten TBC-8 als einen potentiellen
RAB-2 GAP, der exklusiv in Neuronen exprimiert wird. WĂĽrmer, die
kein tbc-8 exprimieren, zeigten ähnliche DCV Reifungsdefekte wie
rab-2 Mutanten. Zudem konnten wir nachweisen, dass RAB-2 an TBC-8
bindet. Weiterhin identifizierten wir neue Faktoren, die an der
Reifung von DCV beteiligt sind. RUND-1 zum Beispiel ist wie RIC-19
ein RAB-2 Effektor und interessanterweise interagierten beide
Effektoren mit dem GAP, TBC-8, in Bindestudien. Daher ist es
möglich, dass RAB-2 sein GAP mit Hilfe des eigenen
Effektorkomplexes rekrutiert, was zu einer beschleunigten
Deaktivierung von RAB-2 führt. Diese negative Rückkopplung könnte
einen neuen Mechanismus zur Regulation von Rab-Funktionen
darstellen und spricht für eine äußerst dynamische Regulierung von
RAB-2 am Golgi während der Reifung von DCV. Des Weiteren haben wir
zum ersten Mal gezeigt, dass auch retrograder Proteintransport,
welcher durch den GARP-Komplex gesteuert wird, während der Reifung
von DCV benötigt wird: Wir vermuten, dass aktives RAB-2 den Empfang
von retrograden Transportvesikeln, die zurĂĽck zu dem reifenden
DCV-Kompartiment transportiert werden, ermöglicht. All diese
Ergebnisse zeigen, dass die Reifung von DCV ein stark regulierter
Prozess ist, der auf das Zusammenspiel verschiedener Proteinen wie
Rab GTPasen und groĂźen Proteinkomplexen angewiesen ist
No-Slip Boundary Conditions for Electron Hydrodynamics and the Thermal Casimir Pressure
We derive modified reflection coefficients for electromagnetic waves in the THz and far infrared range. The idea is based on hydrodynamic boundary conditions for metallic conduction electrons. The temperature-dependent part of the Casimir pressure between metal plates is evaluated. The results should shed light on the “thermal anomaly,” where measurements deviate from the standard fluctuation electrodynamics for conducting metals.Peer Reviewe
Geometrisch bestimmte offenzellige Feingussstrukturen
Für die Herstellung von offenzelligen Feingussstrukturen, kurz Metallschwamm genannt, werden in der Regel Kunststoff-Urmodelle verwendet. Die Kunststoffe werden in einem chemischen Prozess aufgeschäumt und sind dadurch in ihrem Aufbau nicht exakt definiert. Gerade für Wärmetauscher werden jedoch geometrisch bestimmte Strukturen dringend benötigt, um Wärmefluss und Strömung präzise leiten zu können. Mittels CAD lassen sich 3D-Wachsmodelle definiert drucken und damit an den jeweiligen Anwendungsfall anpassen. Im internen Forschungsprojekt wurde die Herstellung von Feingussstrukturen mittels 3D-Wachsmodellen untersucht
The Challenge of Open Cellular Metal Foam Production: Presentation held at 11th International Conference on Porous Metals and Metallic Foams, MetFoam 2019, August 20-23, 2019, Dearborn, Michigan, USA
Five years ago Fraunhofer-IWU started research in open cellular structures as the market is growing caused by their large surface. Heat exchangers, filters and battery electrodes are examples for potential applications. Casted Aluminum foams are in serial production and we cooperate with the manufacturer but for prototyping of individual foams especially in combination with solid regions for fixing, encasing or sub-division new parameters and technological processes were developed. Advantageous, limitations and potentials will be shown. Alternatively, galvanic coating of PUR foams with Copper, Nickel and other metals is common, again buyable and state of the art. R&D activities related to application development and adapted geometries not yet realizable by conventional mass production will be shown. Investigations for graded strut-structures and cylindrical tube-like parts were intensified to reach serial production level. The lecture represents investigations, results, dropped and new approaches of all routes
The Legionella longbeachae Icm/Dot substrate SidC selectively binds phosphatidylinositol 4-phosphate with nanomolar affinity and promotes pathogen vacuole-endoplasmic reticulum interactions
Legionella spp. cause the severe pneumonia Legionnaires' disease. The environmental bacteria replicate intracellularly in free-living amoebae and human alveolar macrophages within a distinct, endoplasmic reticulum (ER)-derived compartment termed the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS) that translocates into host cells a plethora of different "effector" proteins, some of which anchor to the pathogen vacuole by binding to phosphoinositide (PI) lipids. Here, we identified by unbiased pulldown assays in Legionella longbeachae lysates a 111-kDa SidC homologue as the major phosphatidylinositol 4-phosphate [PtdIns(4)P]-binding protein. The PI-binding domain was mapped to a 20-kDa P4C [PtdIns(4)P binding of SidC] fragment. Isothermal titration calorimetry revealed that SidC of L. longbeachae (SidC(Llo)) binds PtdIns(4)P with a K(d) (dissociation constant) of 71 nM, which is 3 to 4 times lower than that of the SidC orthologue of Legionella pneumophila (SidC(Lpn)). Upon infection of RAW 264.7 macrophages with L. longbeachae, endogenous SidC(Llo) or ectopically produced SidC(Lpn) localized in an Icm/Dot-dependent manner to the PtdIns(4)P-positive LCVs. An L. longbeachae ΔsidC deletion mutant was impaired for calnexin recruitment to LCVs in Dictyostelium discoideum amoebae and outcompeted by wild-type bacteria in Acanthamoeba castellanii. Calnexin recruitment was restored by SidC(Llo) or its orthologues SidC(Lpn) and SdcA(Lpn). Conversely, calnexin recruitment was restored by SidC(Llo) in L. pneumophila lacking sidC and sdcA. Together, biochemical, genetic, and cell biological data indicate that SidC(Llo) is an L. longbeachae effector that binds through a P4C domain with high affinity to PtdIns(4)P on LCVs, promotes ER recruitment to the LCV, and thus plays a role in pathogen-host interactions
Divergent Evolution of Legionella RCC1 Repeat Effectors Defines the Range of Ran GTPase Cycle Targets
Legionella pneumophila governs its interactions with host cells by secreting >300 different "effector" proteins. Some of these effectors contain eukaryotic domains such as the RCC1 (regulator of chromosome condensation 1) repeats promoting the activation of the small GTPase Ran. In this report, we reveal a conserved pattern of L. pneumophila RCC1 repeat genes, which are distributed in two main clusters of strains. Accordingly, strain Philadelphia-1 contains two RCC1 genes implicated in bacterial virulence, legG1 (Legionella eukaryotic gene 1), and ppgA, while strain Paris contains only one, pieG The RCC1 repeat effectors localize to different cellular compartments and bind distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself, and yet they all promote the activation of Ran. The pieG gene spans the corresponding open reading frames of legG1 and a separate adjacent upstream gene, lpg1975legG1 and lpg1975 are fused upon addition of a single nucleotide to encode a protein that adopts the binding specificity of PieG. Thus, a point mutation in pieG splits the gene, altering the effector target. These results indicate that divergent evolution of RCC1 repeat effectors defines the Ran GTPase cycle targets and that modulation of different components of the cycle might fine-tune Ran activation during Legionella infection.IMPORTANCELegionella pneumophila is a ubiquitous environmental bacterium which, upon inhalation, causes a life-threatening pneumonia termed Legionnaires' disease. The opportunistic pathogen grows in amoebae and macrophages by employing a "type IV" secretion system, which secretes more than 300 different "effector" proteins into the host cell, where they subvert pivotal processes. The function of many of these effector proteins is unknown, and their evolution has not been studied. L. pneumophila RCC1 repeat effectors target the small GTPase Ran, a molecular switch implicated in different cellular processes such as nucleocytoplasmic transport and microtubule cytoskeleton dynamics. We provide evidence that one or more RCC1 repeat genes are distributed in two main clusters of L. pneumophila strains and have divergently evolved to target different components of the Ran GTPase activation cycle at different subcellular sites. Thus, L. pneumophila employs a sophisticated strategy to subvert host cell Ran GTPase during infection
TBC-8, a Putative RAB-2 GAP, Regulates Dense Core Vesicle Maturation in <em>Caenorhabditis elegans</em>
<div><p>Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in <em>Caenorhabditis elegans</em> motoneurons. In <em>rab-2</em> mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2–specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as <em>rab-2</em> mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.</p> </div
TBC-8 is a putative RAB-2 GAP.
<p>(A) TBC-8 contains two predicted domains: an N-terminal RUN domain (96 to 230 aa) (blue) and a C-terminal TBC-domain (621 to 862 aa) (purple). Mutation of the catalytically active arginine (R697) to alanine within the TBC-domain is indicated. Please see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002722#pgen.1002722.s004" target="_blank">Figure S4</a> for full protein sequence. (B) Alignment of the catalytic motif of TBC-8 and its orthologs in humans (SGSM1) and in <i>Drosophila melanogaster</i> (CG32506-PC) are shown. The arrow indicates the catalytic arginine residue necessary for GAP activity. (C) In a yeast two-hybrid assay, all <i>C. elegans</i> Rabs in their constitutively GTP-bound form were tested against wild type TBC-8 (upper panel) and a catalytically inactive form of TBC-8 (R697A) (lower panel), respectively. Strikingly, RAB-2 (Q65L) interacted with TBC-8 (R697A) but not with wild type TBC-8, suggesting that TBC-8 is the GAP for RAB-2. Unlike RAB-2, RAB-19 (Q69L) interacted weakly with both forms of TBC-8. (D) Interactions of RAB-2 and RAB-19 with TBC-8 occurred in a GTP-dependent manner. Constitutively active RAB-2 (Q65L) and RAB-19 (Q69L) interacted with TBC-8 whereas their dominant inactive forms [RAB-2 (S20N), RAB-19 (T24N)] did not. The closest paralog of RAB-2, RAB-14, did not show interaction with TBC-8 wild type or R697A in a yeast two-hybrid analysis. AD: Gal4p DNA activation domain fusion, BD: Gal4p DNA binding domain fusion, His: histidine, RAB<sub>GTP</sub>: constitutively GTP-bound RAB GTPase, “–”: empty vector pGADT7 was used for testing self-activation.</p