764 research outputs found
Localization of protein kinase C ε to macrophage vacuoles perforated by Listeria monocytogenes cytolysin
Three proteins secreted by Listeria monocytogenes facilitate escape from macrophage vacuoles: the cholesterol-dependent cytolysin listeriolysin O (LLO), a phosphoinositide-specific phospholipase C (PI-PLC) and a broad-range phospholipase C (PC-PLC). LLO and PI-PLC can activate several members of the protein kinase C (PKC) family during infection. PKCε is a novel PKC that contributes to macrophage activation, defence against bacterial infection, and phagocytosis; however, a role for PKCε in Lm infections has not been described. To study PKCε dynamics, PKCε-YFP chimeras were visualized in macrophages during Lm infection. PKCε-YFP was recruited to forming vacuoles during macrophage phagocytosis of Lm and again later to fully formed Lm vacuoles. The PKCε-YFP localization to the fully formed Lm vacuole was LLO-dependent but independent of PI-PLC or PC-PLC. PKCε-YFP recruitment often followed LLO perforation of the membrane, as indicated by localization of PKCε-YFP to Lm vacuoles after they released small fluorescent dyes into the cytoplasm. PKCε-YFP recruitment to vesicles also followed phagocytosis of LLO-containing liposomes or osmotic lysis of endocytic vesicles, indicating that vacuole perforation by LLO was the chief cause of the PKCε response. These studies implicate PKCε in a cellular mechanism for recognizing damaged membranous organelles, including the disrupted vacuoles created when Lm escapes into cytoplasm.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73267/1/j.1462-5822.2007.00903.x.pd
Characterisation of a new VUV beamline at the Daresbury SRS using a dispersed fluorescence apparatus incorporating CCD detection
The design and performance of a new normal incidence monochromator at the Daresbury Synchrotron Radiation Source, optimised for experiments requiring high flux of vacuum-UV radiation, are described. The re-developed beamline 3.1, based on the Wadsworth design of monochromator, is the source of tunable vacuum-UV photons in the range 4 – 31 eV, providing over two orders of magnitude more flux than the vacuum-UV, Seya monochromator in its previous manifestation. The undispersed and dispersed fluorescence spectra resulting from photoexcitation of N, CO, CF and CF are presented. Emitting species observed were N B - X, CO A - X and B - X, CF CT - XT and CT - AT, CF* A - A, and CF BA - XE. A CCD multi-channel detector has significantly reduced the time period needed to record dispersed fluorescence spectra with a comparable signal-to-noise ratio
Comparative transcriptomics of pathogenic and non-pathogenic Listeria species
Comparative RNA-seq analysis of two related pathogenic and non-pathogenic bacterial strains reveals a hidden layer of divergence in the non-coding genome as well as conserved, widespread regulatory structures called ‘Excludons', which mediate regulation through long non-coding antisense RNAs
Studies of Diffuse Interstellar Bands. V. Pairwise Correlations of Eight Strong DIBs and Neutral Hydrogen, Molecular Hydrogen, and Color Excess
We establish correlations between equivalent widths of eight diffuse
interstellar bands (DIBs), and examine their correlations with atomic hydrogen,
molecular hydrogen, and EB-V . The DIBs are centered at \lambda\lambda 5780.5,
6204.5, 6283.8, 6196.0, 6613.6, 5705.1, 5797.1, and 5487.7, in decreasing order
of Pearson\^as correlation coefficient with N(H) (here defined as the column
density of neutral hydrogen), ranging from 0.96 to 0.82. We find the equivalent
width of \lambda 5780.5 is better correlated with column densities of H than
with E(B-V) or H2, confirming earlier results based on smaller datasets. We
show the same is true for six of the seven other DIBs presented here. Despite
this similarity, the eight strong DIBs chosen are not well enough correlated
with each other to suggest they come from the same carrier. We further conclude
that these eight DIBs are more likely to be associated with H than with H2, and
hence are not preferentially located in the densest, most UV shielded parts of
interstellar clouds. We suggest they arise from different molecules found in
diffuse H regions with very little H (molecular fraction f<0.01). Of the 133
stars with available data in our study, there are three with significantly
weaker \lambda 5780.5 than our mean H-5780.5 relationship, all of which are in
regions of high radiation fields, as previously noted by Herbig. The
correlations will be useful in deriving interstellar parameters when direct
methods are not available. For instance, with care, the value of N(H) can be
derived from W{\lambda}(5780.5).Comment: Accepted for publication in The Astrophysical Journal; 37 pages, 11
figures, 6 table
The Rotational Excitation Temperature of the 6614 Diffuse Interstellar Band Carrier
Analysis of high spectral resolution observations of the 6614
diffuse interstellar band (DIB) line profile show systematic variations in the
positions of the peaks in the substructure of the profile. These variations --
shown here for the first time -- can be understood most naturally in the
framework of rotational contours of large molecules, where the variations are
caused by changes in the rotational excitation temperature. We show that the
rotational excitation temperature for the DIB carrier is likely significantly
lower than the gas kinetic temperature -- indicating that for this particular
DIB carrier angular momentum buildup is not very efficient.Comment: Accepted by ApJ Letters; 16 pages, 2 figure
Deciphering interplay between Salmonella invasion effectors
Bacterial pathogens have evolved a specialized type III secretion system (T3SS) to translocate virulence effector proteins directly into eukaryotic target cells. Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells. Six effectors (SipC, SipA, SopE/2, SopB, SptP) can individually manipulate actin dynamics at the plasma membrane, which acts as a ‘signaling hub’ during Salmonella invasion. The extent of crosstalk between these spatially coincident effectors remains unknown. Here we describe trans and cis binary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell. The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen–host interaction
NS1 Specific CD8(+) T-Cells with Effector Function and TRBV11 Dominance in a Patient with Parvovirus B19 Associated Inflammatory Cardiomyopathy
Background: Parvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the role of B19V specific T-cells in this entity.
Methodology and Principal Findings: An exceptionally high B19V viral load in EMBs (115,091 viral copies/mg nucleic acids), peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8(+) T-cell responses were elicited to the 10-amico-acid peptides SALKLAIYKA (19.7% of all CD8(+) cells) and QSALKLAIYK (10%) and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFN gamma secretion-assay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFN gamma, IL2, IL27 and Tbet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4, GATA3) and regulatory T-cells (FoxP3) was low.
Conclusions: Our results indicate that B19V Ag-specific CD8(+) T-cells with effector function are involved in B19V associated DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of CD8(+) T-cell responses to the identified epitopes
Three-dimensional architecture of actin filaments in Listeria monocytogenes comet tails
The intracellular bacterial pathogen Listeria monocytogenes is capable of remodelling the actin cytoskeleton of its host cells such that "comet tails" are assembled powering its movement within cells and enabling cell-to-cell spread. We used cryo-electron tomography to visualize the 3D structure of the comet tails in situ at the level of individual filaments. We have performed a quantitative analysis of their supramolecular architecture revealing the existence of bundles of nearly parallel hexagonally packed filaments with spacings of 12-13 nm. Similar configurations were observed in stress fibers and filopodia, suggesting that nanoscopic bundles are a generic feature of actin filament assemblies involved in motility; presumably, they provide the necessary stiffness. We propose a mechanism for the initiation of comet tail assembly and two scenarios that occur either independently or in concert for the ensuing actin-based motility, both emphasizing the role of filament bundling
Recruitment of the Major Vault Protein by InlK: A Listeria monocytogenes Strategy to Avoid Autophagy
L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK− bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles
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