Charakterisierung einer bakteriellen Gemeinschaft aus dem Süßwasser mit dem Schwerpunkt auf dem horizontalen Gentransfer durch Konjugation und Transduktion

Beilstein F. Charakterisierung einer bakteriellen Gemeinschaft aus dem Süßwasser mit dem Schwerpunkt auf dem horizontalen Gentransfer durch Konjugation und Transduktion. Bielefeld (Germany): Bielefeld University; 2005.Innerhalb dieser Arbeit wurde aus den drei Bielefelder Teichen im Oetkerpark, am Schlosshof und am Meierteich eine bakterielle Gemeinschaft isoliert und charakterisiert. Von 848 verschiedenen Isolaten konnten 367 Bakterienstämme durch Vergleiche ihrer Gesamtproteinmuster und anschließender Sequenzanalyse ihrer 16S-rDNA 14 verschiedenen Bakteriengattungen zugeordnet werden. Die abundantesten Gattungen sind hierbei Pseudomonas und Aeromonas. Kleinere Gruppen konnten den Gattungen Flexibacter, Flavobacterium, Bacillus, Arthrobacter, Escherichia, Hydrogenophaga, Brevundimonas, Shigella, Chromobacterium, Streptomyces, Pantoea und Rahnella zugeordnet werden. Unter 600 auf ihren Plasmidgehalt untersuchten Bakterienstämmen wurden 75 plasmidhaltige detektiert, was einem Plasmidgehalt von 12.5 Prozent entspricht. Besonders auffällig erwies sich hierbei die mit 12 Isolaten zahlenmäßig sehr kleine E. coli-Gruppe. Alle Isolate dieser Art sind ausnahmslos plasmidhaltig und beinhalten bis zu 11 verschiedene Plasmide. Ein Großteil dieser Plasmide ist kleiner als 4 kb und aufgrund fehlender Funktion als cryptisch zu bezeichnen. Die durchgeführten Antibiotika- und Schwermetallresistenztests ergaben keine Hinweise auf plasmidkodierte Resistenzen. Die isolierten Plasmide entsprechen größtenteils nicht den bisher bekannten Inkompatibilitätsgruppen, welche unter Verwendung klinischer Plasmide erstellt wurden. Lediglich zwei Plasmide aus den E. coli-Isolaten konnten den Inkompatibilitätsgruppen IncY und IncI zugeordnet werden. Mit Mobilisierungsexperimenten wurde einerseits der Transfer des Plasmides pSunny durch endogene Plasmide der Isolate aus dem Süßwasser oder andererseits der Transfer des Plasmides selbst analysiert. Von insgesamt 25 untersuchten Isolaten (der Bakteriengattungen Aeromonas, Pseudomonas, Rahnella, Escherichia sowie nicht näher bestimmten Gattungen) oder Plasmiden waren 15 in der Lage, pSunny zu mobilisieren oder selbst mobilisiert zu werden. Insgesamt betrachtet sind 60 Prozent aller untersuchten Plasmide mobilisierbar oder können pSunny mobilisieren und sind daraus folgend potentiell konjugativ. Sieben Plasmide konnten mit Hilfe eines in vitro Transposonmutagenesesystems mit einem Kanamycinresistenzgen markiert werden. Vier dieser Plasmide kamen allerdings aufgrund ihrer zu geringen Größe für eine selbstständige Konjugation nicht in Frage. Für die übrigen drei transposonmarkierten Plasmide aus den Gruppen Aeromonas (pO50), Rahnella (pO60) und E. coli (pM101.7) konnte ein konjugativer Transfer gezeigt werden. Bei dem Plasmid pM101.7 handelt es sich um ein broad host range Plasmid, für das ein Transfer innerhalb verschiedener Proteobakteriengruppen gezeigt werden konnte. Die Genorganisation der Tral- und Trall-Region von pM101.7 zeigt Homologien zu der des Plasmides pBi1063 aus Stenotrophomonas maltophilia. Die Sequenzierung des kleinsten cryptischen Plasmides des E. coli-Isolates pM101 ergab, dass dieses 1832 kb große Plasmid nur für sein eigenes Rep-Protein kodiert. Aus Wasserproben der beschriebenen Teiche sowie anliegender Gewässer konnten 39 Bakteriophagen für Wirte der Gattungen Aeromonas, Brevundimonas, Escherichia, Pseudomonas, Arthrobacter sowie nicht näher spezifizierten Gattungen isoliert werden. Durch UV-Induktion wurden aus zwei Aeromonas Isolaten sowie aus zwei E. coli Isolaten je ein Prophage induziert. Mittels Elektronenmikroskopie konnten 19 Bakteriophagen der Ordnung der Caudalovirales zugeordnet werden. Hiervon entfallen auf die Familie der Siphoviridae 42 Prozent, auf die Familie der Podoviridae 32 Prozent und 26 Prozent aller Phagen konnten der Familie der Myoviridae zugeordnet werden. Alle Phagen enthalten lineare DNA. Die DNA-Größe von 31 Bakteriophagen wurde durch Pulsfeldgelelektrophorese bestimmt und liegt zwischen 27 kb (Aeromonas [Phi]18P) und 180 kb (Aeromonas [Phi]O238), wobei die meisten DNAs in ihrer Größe ungefähr der von [lambda] entsprechen. Bei fünf der untersuchten Bakteriophagen-DNAs konnten kohäsive Enden detektiert werden. Durch [lambda]-Exonuklease Verdau konnten bei weiteren 16 Phagen-DNAs terminal redundante Endstrukturen nachgewiesen werden. Die DNAs von vier dieser Phagen, der Gattungen Aeromonas ([Phi]S32D), Escherichia ([Phi]M101.11 und [Phi]O221.15) sowie Arthrobacter ([Phi]O262), sind zusätzlich zyklisch permutiert. Die sieben Bakteriophagen der Brevundimonas vesicularis Gruppe wurden nach morphologischen und molekularbiologischen Charakteristika eingehender untersucht und in zwei Gruppen eingeteilt. Die erste Gruppe enthält drei genetisch sehr eng miteinander verwandte Phagen. Die zweite Gruppe beinhaltet drei Phagen, die genetisch sehr unterschiedlich sind, aber gleiche Plaque- und Partikelmorphologie zeigen. 17 Bakteriophagen wurden auf die Fähigkeit zur Transduktion von Plasmiden getestet. Es konnte hierbei für fünf Phagen mit Wirten aus den Gruppen E. coli und Pseudomonas eine Transduktion des Plasmides pSunny gezeigt werden. Die Transduktion des Kanamycin-Markergens aphA2 wurde für zwölf Phagen getestet. Hierbei konnte gezeigt werden, dass alle Bakteriophagen, die Plasmide transduzieren können, auch in der Lage sind, das verwendete Markergen weiterzugeben

The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics

Background information. Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization

Characterization of the Interaction between the Matrix Protein of Vesicular Stomatitis Virus and the Immunoproteasome Subunit LMP2

The matrix protein (M) of vesicular stomatitis virus (VSV) is involved in virus assembly, budding, gene regulation, and cellular pathogenesis. Using a yeast two-hybrid system, the M globular domain was shown to interact with LMP2, a catalytic subunit of the immunoproteasome (which replaces the standard proteasome catalytic subunit PSMB6). The interaction was validated by coimmunoprecipitation of M and LMP2 in VSV-infected cells. The sites of interaction were characterized. A single mutation of M (I96A) which significantly impairs the interaction between M and LMP2 was identified. We also show that M preferentially binds to the inactive precursor of LMP2 (bearing an N-terminal propeptide which is cleaved upon LMP2 maturation). Furthermore, taking advantage of a sequence alignment between LMP2 and its proteasome homolog, PSMB6 (which does not bind to M), we identified a mutation (L45R) in the S1 pocket where the protein substrate binds prior to cleavage and a second one (D17A) of a conserved residue essential for the catalytic activity, resulting in a reduction of the level of binding to M. The combination of both mutations abolishes the interaction. Taken together, our data indicate that M binds to LMP2 before its incorporation into the immunoproteasome. As the immunoproteasome promotes the generation of major histocompatibility complex (MHC) class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells, we suggest that M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. IMPORTANCE: The immunoproteasome promotes the generation of MHC class I-compatible peptides, a feature which favors the recognition and the elimination of infected cells by CD8 T cells. Here, we report on the association of vesicular stomatitis virus (VSV) matrix protein (M) with LMP2, one of the immunoproteasome-specific catalytic subunits. M preferentially binds to the LMP2 inactive precursor. The M-binding site on LMP2 is facing inwards in the immunoproteasome and is therefore not accessible to M after its assembly. Hence, M binds to LMP2 before its incorporation into the immunoproteasome. We suggest that VSV M, by interfering with the immunoproteasome assembly, has evolved a mechanism that allows infected cells to escape detection and elimination by the immune system. Modulating this M-induced immunoproteasome impairment might be relevant in order to optimize VSV for oncolytic virotherapy

Proteomic Analysis of Lipid Droplets from Caco-2/TC7 Enterocytes Identifies Novel Modulators of Lipid Secretion

International audienceIn enterocytes, the dynamic accumulation and depletion of triacylglycerol (TAG) in lipid droplets (LD) during fat absorption suggests that cytosolic LD-associated TAG contribute to TAG-rich lipoprotein (TRL) production. To get insight into the mechanisms controlling the storage/secretion balance of TAG, we used as a tool hepatitis C virus core protein, which localizes onto LDs, and thus may modify their protein coat and decrease TRL secretion. We compared the proteome of LD fractions isolated from Caco-2/TC7 enterocytes expressing or not hepatitis C virus core protein by a differential proteomic approach (isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with liquid chromatography and tandem mass spectrometry). We identified 42 proteins, 21 being involved in lipid metabolism. Perilipin-2/ADRP, which is suggested to stabilize long term-stored TAG, was enriched in LD fractions isolated from Caco-2/TC7 expressing core protein while perilipin-3/TIP47, which is involved in LD synthesis from newly synthesized TAG, was decreased. Endoplasmic reticulum-associated proteins were strongly decreased, suggesting reduced interactions between LD and endoplasmic reticulum, where TRL assembly occurs. For the first time, we show that 17b-hydroxysteroid dehydrogenase 2 (DHB2), which catalyzes the conversion of 17-keto to 17 b-hydroxysteroids and which was the most highly enriched protein in core expressing cells, is localized to LD and interferes with TAG secretion, probably through its capacity to inactivate testosterone. Overall, we identified potential new players of lipid droplet dynamics, which may be involved in the balance between lipid storage and secretion, and may be altered in enterocytes in pathological conditions such as insulin resistance, type II diabetes and obesity

Characteristics and functions of lipid droplets and associated proteins in enterocytes

International audienceCytosolic lipid droplets (LDs) are observed in enterocytes of jejunum during lipid absorption. One important function of the intestine is to secrete chylomicrons, which provide dietary lipids throughout the body, from digested lipids in meals. The current hypothesis is that cytosolic LDs in enterocytes constitute a transient pool of stored lipids that provides lipids during interprandial period while lowering chylomicron production during the post-prandial phase. This smoothens the magnitude of peaks of hypertriglyceridemia. Here, we review the composition and functions of lipids and associated proteins of enterocyte LDs, the known physiological functions of LDs as well as the role of LDs in pathological processes in the context of the intestine

Mutational Analysis of the Herpes Simplex Virus Type 1 DNA Packaging Protein UL33 ▿

The UL33 protein of herpes simplex virus type 1 (HSV-1) is thought to be a component of the terminase complex that mediates the cleavage and packaging of viral DNA. In this study we describe the generation and characterization of a series of 15 UL33 mutants containing insertions of five amino acids located randomly throughout the 130-residue protein. Of these mutants, seven were unable to complement the growth of the UL33-null virus dlUL33 in transient assays and also failed to support the cleavage and packaging of replicated amplicon DNA into capsids. The insertions in these mutants were clustered between residues 51 and 74 and between 104 and 116, within the most highly conserved regions of the protein. The ability of the mutants to interact with the UL28 component of the terminase was assessed in immunoprecipitation and immunofluorescence assays. All four mutants with insertions between amino acids 51 and 74 were impaired in this interaction, whereas two of the three mutants in the second region (with insertions at positions 111 and 116) were not affected. These data indicate that the ability of UL33 to interact with UL28 is probably necessary, but not sufficient, to support viral growth and DNA packaging

Protein analysis of sucrose gradient fractions prepared from Caco-2/TC7 and Caco-2/TC7 GFP-CP cells.

<p>Caco-2/TC7 cells (TC7, left panel) and Caco-2/TC7 GFP-CP cells (TC7 GFP-CP, right panel) were cultured on filters for 17 days then supplied with lipid micelles for 24 h. Cell homogenates were centrifuged for 10 min at 1000×g and the supernatants were fractionated onto sucrose gradients. Top to bottom fractions (1 to 11) and pellets were analyzed by western blot using antibodies specific for (a) perilipin-2/ADRP, (b) protein disulfide isomerase (PDI), (c) calnexin, (d) heat shock protein 60 (HSP 60), (e) 78 kDa glucose-regulated protein (GRP78) and (f) apolipoprotein B48 (ApoB48). The same percentage of each fraction of the sucrose gradient was loaded on gels except for fraction 1 which was 2-fold loaded in order to evaluate the presence of organelle markers with greater sensitivity.</p

Lysophosphatidylcholine acyltransferase 1 is downregulated by hepatitis C virus: impact on production of lipo-viro-particles

International audienceObjective: HCV is intimately linked with the liver lipid metabolism, devoted to the efflux of triacylglycerols stored in lipid droplets (LDs) in the form of triacylglycerol-rich very-low-density lipoproteins (VLDLs): (i) the most infectious HCV particles are those of lowest density due to association with triacylglycerol-rich lipoproteins and (ii) HCV-infected patients frequently develop hepatic steatosis (increased triacylglycerol storage). The recent identification of lysophosphatidylcholine acyltransferase 1 (LPCAT1) as an LD phospholipid-remodelling enzyme prompted us to investigate its role in liver lipid metabolism and HCV infectious cycle.Design Huh-7.5.1 cells and primary human hepatocytes (PHHs) were infected with JFH1-HCV. LPCAT1 depletion was achieved by RNA interference. Cells were monitored for LPCAT1 expression, lipid metabolism and HCV production and infectivity. The density of viral particles was assessed by isopycnic ultracentrifugation.Results: Upon HCV infection, both Huh-7.5.1 cells and PHH had decreased levels of LPCAT1 transcript and protein, consistent with transcriptional downregulation. LPCAT1 depletion in either naive or infected Huh-7.5.1 cells resulted in altered lipid metabolism characterised by LD remodelling, increased triacylglycerol storage and increased secretion of VLDL. In infected Huh-7.5.1 cells or PHH, LPCAT1 depletion increased production of the viral particles of lowest density and highest infectivity.Conclusions: We have identified LPCAT1 as a modulator of liver lipid metabolism downregulated by HCV, which appears as a viral strategy to increase the triacylglycerol content and hence infectivity of viral particles. Targeting this metabolic pathway may represent an attractive therapeutic approach to reduce both the viral titre and hepatic steatosis

List of proteins identified in lipid droplet fractions isolated from Caco-2/TC7 GFP-CP cells compared to that of Caco-2/TC7 cells, ranked by decreasing iTRAQ labelling ratios.

<p>The two samples of peptides generated by trypsin digestion of the proteins present in lipid droplets fractions were labelled with two different iTRAQ labels then analyzed by LC-MS/MS. The proteins listed were identified in at least three out of four independent experiments performed in duplicate. An iTRAQ ratio above one indicates that this protein was more abundant in the lipid droplet fraction of Caco-2/TC7 GFP-CP cells than in that isolated from Caco-2/TC7 cells. Conversely, a ratio below one indicates that this protein is less abundant in the lipid droplet fraction of Caco-2/TC7 GFP-CP cells than in that isolated from Caco-2/TC7 cells. Stars highlight proteins whose amounts are significantly different between the two cell lines (P<0.05).</p

Western blot analysis of some proteins identified by the differential proteomic approach in the lipid droplet fractions isolated from Caco-2/TC7 (TC7) and Caco-2/TC7 GFP-CP (TC7 GFP-CP) cells.

<p>The lipid droplet fractions (fraction 1) were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053017#pone-0053017-g003" target="_blank">figure 3</a>, freeze-dried for concentration and analyzed by western blot using antibodies against 17β-hydroxysteroid dehydrogenase type 2 (DHB2), perilipin-2/ADRP (PLIN2), 3-beta-hydroxysteroid dehydrogenase (3BHS1), lysophosphatidylcholine acyltransferase 2 (PCAT2), FAS-associated factor 2/UBXD8 (FAF2), Na+/K+ATPase α1 (AT1B1), perilipin-3/TIP47 (PLIN3) and protein disulfide isomerase (PDI). The amount of material loaded per well was 10 times higher than in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053017#pone-0053017-g003" target="_blank">Fig.3</a>, fraction 1.</p