Anthrax Lethal Toxin-Mediated Killing of Human and Murine Dendritic Cells Impairs the Adaptive Immune Response

Many pathogens have acquired strategies to combat the immune response. Bacillus anthracis interferes with host defenses by releasing anthrax lethal toxin (LT), which inactivates mitogen-activated protein kinase pathways, rendering dendritic cells (DCs) and T lymphocytes nonresponsive to immune stimulation. However, these cell types are considered resistant to killing by LT. Here we show that LT kills primary human DCs in vitro, and murine DCs in vitro and in vivo. Kinetics of LT-mediated killing of murine DCs, as well as cell death pathways induced, were dependent upon genetic background: LT triggered rapid necrosis in BALB/c-derived DCs, and slow apoptosis in C57BL/6-derived DCs. This is consistent with rapid and slow killing of LT-injected BALB/c and C57BL/6 mice, respectively. We present evidence that anthrax LT impairs adaptive immunity by specifically targeting DCs. This may represent an immune-evasion strategy of the bacterium, and contribute to anthrax disease progression. We also established that genetic background determines whether apoptosis or necrosis is induced by LT. Finally, killing of C57BL/6-derived DCs by LT mirrors that of human DCs, suggesting that C57BL/6 DCs represent a better model system for human anthrax than the prototypical BALB/c macrophages

Distribution, functional impact, and origin mechanisms of copy number variation in the barley genome

BACKGROUND There is growing evidence for the prevalence of copy number variation (CNV) and its role in phenotypic variation in many eukaryotic species. Here we use array comparative genomic hybridization to explore the extent of this type of structural variation in domesticated barley cultivars and wild barleys. RESULTS A collection of 14 barley genotypes including eight cultivars and six wild barleys were used for comparative genomic hybridization. CNV affects 14.9% of all the sequences that were assessed. Higher levels of CNV diversity are present in the wild accessions relative to cultivated barley. CNVs are enriched near the ends of all chromosomes except 4H, which exhibits the lowest frequency of CNVs. CNV affects 9.5% of the coding sequences represented on the array and the genes affected by CNV are enriched for sequences annotated as disease-resistance proteins and protein kinases. Sequence-based comparisons of CNV between cultivars Barke and Morex provided evidence that DNA repair mechanisms of double-strand breaks via single-stranded annealing and synthesis-dependent strand annealing play an important role in the origin of CNV in barley. CONCLUSIONS We present the first catalog of CNVs in a diploid Triticeae species, which opens the door for future genome diversity research in a tribe that comprises the economically important cereal species wheat, barley, and rye. Our findings constitute a valuable resource for the identification of CNV affecting genes of agronomic importance. We also identify potential mechanisms that can generate variation in copy number in plant genomes.This work was financially supported by the following grants: project GABI-BARLEX, German Federal Ministry of Education and Research (BMBF), #0314000 to MP, US, KFXM and NS; Triticeae Coordinated Agricultural Project, USDA-NIFA #2011-68002-30029 to GJM; and Agriculture and Food Research Initiative Plant Genome, Genetics and Breeding Program of USDA’s Cooperative State Research and Extension Service, #2009-65300- 05645 to GJM

Distribution, functional impact, and origin mechanisms of copy number variation in the barley genome

BACKGROUND: There is growing evidence for the prevalence of copy number variation (CNV) and its role in phenotypic variation in many eukaryotic species. Here we use array comparative genomic hybridization to explore the extent of this type of structural variation in domesticated barley cultivars and wild barleys. RESULTS: A collection of 14 barley genotypes including eight cultivars and six wild barleys were used for comparative genomic hybridization. CNV affects 14.9% of all the sequences that were assessed. Higher levels of CNV diversity are present in the wild accessions relative to cultivated barley. CNVs are enriched near the ends of all chromosomes except 4H, which exhibits the lowest frequency of CNVs. CNV affects 9.5% of the coding sequences represented on the array and the genes affected by CNV are enriched for sequences annotated as disease-resistance proteins and protein kinases. Sequence-based comparisons of CNV between cultivars Barke and Morex provided evidence that DNA repair mechanisms of double-strand breaks via single-stranded annealing and synthesis-dependent strand annealing play an important role in the origin of CNV in barley. CONCLUSIONS: We present the first catalog of CNVs in a diploid Triticeae species, which opens the door for future genome diversity research in a tribe that comprises the economically important cereal species wheat, barley, and rye. Our findings constitute a valuable resource for the identification of CNV affecting genes of agronomic importance. We also identify potential mechanisms that can generate variation in copy number in plant genomes

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

spores germinate in the presence of a single germinant, inosine, yet with a significant lag period. spores. spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination

A chromosome conformation capture ordered sequence of the barley genome

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BALB/c- and C57BL/6-Derived DCs Differ in Their Response to LT

<div><p>(A) BALB/c and C57BL/6-derived BMDCs were treated with LT (500 ng/ml PA and 250 ng/ml LF), and cell survival was determined by MTT assay.</p><p>(B) Caspase-3 activation of LT-treated BALB/c and C57BL/6 DCs as determined by a colorimetric caspase-3 cleavage assay. A representative experiment is shown.</p><p>(C) The caspase inhibitors Z-VAD-FMK (10 μg/ml) and BOC-D-FMK (40 μg/ml) prevent caspase-3 activation in LT-treated BALB/c BMDCs.</p><p>(D) The caspase inhibitors Z-VAD-FMK (10 μg/ml) and BOC-D-FMK (40 μg/ml) do not prevent LT killing of BALB/c BMDCs as determined by MTT assay.</p><p>(E) C57BL6, but not BALB/c DCs, were TUNEL-positive post-LT exposure. BALB/c and C57BL/6-derived BMDCs were treated with LT (500 ng/ml PA and 250 ng/ml LF), and were stained using a TUNEL reaction and a Hoechst counterstain.</p><p>(F) BALB/c and C57BL/6-derived BMDCs were analyzed by electron microscopy 4 and 48 h post-LT exposure, respectively. Bars, 1 μm.</p></div

FACS Profile of BMDCs

<div><p>(A) BMDCs were derived from a BALB/c mouse and analyzed on day 10. Expression of CD11b, CD11c, CD14, and CD80 was assessed by flow cytometry. The data are representative of four similar experiments in BALB/c and C57BL/6 mice.</p><p>(B) BALB/c and C57BL/6-derived BMDCs were stimulated by LPS for 18 h, and CD86 expression was measured by flow cytometry. Filled histograms represent isotype-matched controls.</p></div

Killing of Human DCs by LT

<div><p>(A) MoDCs from a representative human subject were treated with LT (500 ng/ml PA and 250 ng/ml LF) or 10 μM camptothecin (Campt.) as a positive control, and annexin V/PI staining was measured by flow cytometry 48 h post-LT exposure.</p><p>(B) Percentages of annexin V-positive untreated and LT-treated MoDCs from five different subjects were determined by flow cytometry 48 h post-LT exposure.</p><p>(C) MTT assay of LT or camptothecin-treated human MoDCs. Mean + standard deviation from three independent experiments are shown.</p><p>(D) LT-treated human MoDCs show signs of apoptotic cell death, as analyzed by electron microscopy 48 h post-LT exposure. Bars: 1 μm.</p><p>(E) Human MoDCs were TUNEL-positive 48 h post-LT exposure. Untreated and LT-treated MoDCs were subjected to TUNEL and Hoechst staining.</p></div

FACS Profile of Immature Human MoDCs

<p>MoDCs were derived from human peripheral blood monocytes. Expression of HLA-DR, CD11c, CD14, CD16, CD80, CD86, and DEC-205 was assessed by flow cytometry. The data were collected from two subjects and are representative of similar experiments. Filled histograms represent isotype-matched controls.</p