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$_2$, CO$_2$, CF$_4$ and C$_6$F$_6$ are presented. Emitting species observed were N$_2^+$ B$^2\Sigma_u^+$ - X$^2\Sigma_g^+$, CO$_2^+$ A$^2\Pi_u$ - X$^2\Pi_g$ and B$^2\Sigma_u^+$ - X$^2\Pi_g$, CF$_4$$^+$ C$^2$T$_2$ - X$^2$T$_1$ and C$^2$T$_2$ - A$^2$T$_2$, CF$_3$* $^2$A$^’_2$ - $^2$A$^”_2$, and C$_6$F$_6^+$ B$^2$A$_{2u}$ - X$^2$E$_{1g}$. 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 λ\lambda6614 Diffuse Interstellar Band Carrier

Analysis of high spectral resolution observations of the $\lambda$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

The T3SS effector EspT defines a new category of invasive enteropathogenic E. coli (EPEC) which form intracellular actin pedestals.

Enteropathogenic Escherichia coli (EPEC) strains are defined as extracellular pathogens which nucleate actin rich pedestal-like membrane extensions on intestinal enterocytes to which they intimately adhere. EPEC infection is mediated by type III secretion system effectors, which modulate host cell signaling. Recently we have shown that the WxxxE effector EspT activates Rac1 and Cdc42 leading to formation of membrane ruffles and lamellipodia. Here we report that EspT-induced membrane ruffles facilitate EPEC invasion into non-phagocytic cells in a process involving Rac1 and Wave2. Internalized EPEC resides within a vacuole and Tir is localized to the vacuolar membrane, resulting in actin polymerization and formation of intracellular pedestals. To the best of our knowledge this is the first time a pathogen has been shown to induce formation of actin comets across a vacuole membrane. Moreover, our data breaks the dogma of EPEC as an extracellular pathogen and defines a new category of invasive EPEC

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