Transcriptional and apoptotic responses of THP-1 cells to challenge with toxigenic, and non-toxigenic Bacillus anthracis

<p>Abstract</p> <p>Background</p> <p><it>Bacillus anthracis </it>secretes several virulence factors targeting different host organs and cell types during inhalational anthrax infection. The bacterial expression of a key virulence factor, lethal toxin (LeTx) is closely tied to another factor, edema toxin (EdTx). Both are transcribed on the same virulence plasmid (pXO1) and both have been the subject of much individual study. Their combined effect during virulent anthrax likely modulates both the global transcriptional and the phenotypic response of macrophages and phagocytes. In fact, responses brought about by the toxins may be different than each of their individual effects.</p> <p>Results</p> <p>Here we report the transcriptional and apoptotic responses of the macrophage-like phagocytic cell line THP-1 exposed to <it>B. anthracis </it>Sterne (pXO1⁺) spores, and <it>B. anthracis </it>Δ Sterne (pXO1^-) spores. These cells are resistant to LeTx-induced cytolysis, a phenotype seen in macrophages from several mouse strains which are sensitive to toxigenic anthrax infection. Our results indicate that the pXO1-containing strain induces higher pro-inflammatory transcriptional responses during the first 4 hours of interaction with bacterium, evident in the upregulation of several genes relevant to Nf-κB, phosphatases, prostaglandins, and TNF-α, along with decreases in expression levels of genes for mitochondrial components. Both bacterial strains induce apoptosis, but in the toxigenic strain-challenged cells, apoptosis is delayed.</p> <p>Conclusion</p> <p>This delay in apoptosis occurs despite the much higher level of TNF-α secretion induced by the toxigenic-strain challenge. Interestingly, CFLAR, an important apoptotic inhibitor which blocks apoptosis induced by large amounts of extracellular TNF-α, is upregulated significantly during toxigenic-strain infection, but not at all during non-toxigenic-strain infection, indicating that it may play a role in blocking or delaying TNF-α-mediated apoptosis. The suppression of apoptosis by the toxigenic anthrax strain is consistent with the notion that apoptosis itself may represent a protective host cell response.</p

The META tool optimizes metagenomic analyses across sequencing platforms and classifiers

A major challenge in the field of metagenomics is the selection of the correct combination of sequencing platform and downstream metagenomic analysis algorithm, or “classifier”. Here, we present the Metagenomic Evaluation Tool Analyzer (META), which produces simulated data and facilitates platform and algorithm selection for any given metagenomic use case. META-generated in silico read data are modular, scalable, and reflect user-defined community profiles, while the downstream analysis is done using a variety of metagenomic classifiers. Reported results include information on resource utilization, time-to-answer, and performance. Real-world data can also be analyzed using selected classifiers and results benchmarked against simulations. To test the utility of the META software, simulated data was compared to real-world viral and bacterial metagenomic samples run on four different sequencers and analyzed using 12 metagenomic classifiers. Lastly, we introduce “META Score”: a unified, quantitative value which rates an analytic classifier’s ability to both identify and count taxa in a representative sample

Longitudinal Metagenomic Analysis of the Water and Soil from Gulf of Mexico Beaches Affected by the Deep Water Horizon Oil Spill

Estimates of 7x105 cubic meters of crude oil were released into the Gulf of Mexico as a consequence of the April 20th, 2010 Deep Water Horizon drilling rig explosion, leaving thousands of square miles of earth&#x27;s surface covered in crude oil. Dispersants were used on large slicks and injected at the well head, resulting in oil being suspended throughout the water column. Starting in June 2010, oil reached hundreds of miles of Louisiana, Alabama, Mississippi, and Florida shoreline disturbing the ecological balance and economic stability of the region. While visible damages are evident in the wildlife populations and marine estuaries, the most significant affect may be on the most basic level of the ecosystems: the bacterial and plankton populations.We present results from high throughput DNA sequencing of close-to-shore water and beach soil samples before and during the appearance of oil in Louisiana and Mississippi. Sixteen samples were taken over a two month period at approximately two week intervals from Grand Isle, LA and Gulfport, MS and were sequenced using the Illumina GAIIx platform. Significant genomic-based population fluctuations were observed in the soil and water samples. These included large spikes in the human pathogen Vibrio cholera, a sharp increase in Rickettsiales sp., and decrease of Synechococus sp. in water samples. Analysis of the contiguous de-novo assembled DNAs (contigs) from the samples also suggested the loss of biodiversity in water samples by the time oil appeared at the shores in both locations. Our observations lead us to the conclusion that oil strongly influenced microbial population dynamics, had a striking impact on the phytoplankton and other flora present prior to the appearance of oil, and that the microbial community had not recovered to pre-spill conditions by the end of our observational period

Assessment of methods and analysis of outcomes for comprehensive optimization of nucleofection

BACKGROUND: Nucleofection is an emerging technology for delivery of nucleic acids into both the cytoplasm and nucleus of eukaryotic cells with high efficiency. This makes it an ideal technology for gene delivery and siRNA applications. A 96-well format has recently been made available for high-throughput nucleofection, however conditions must be optimized for delivery into each specific cell type. Screening each 96-well plate can be expensive, and descriptions of methods and outcomes to determine the best conditions are lacking in the literature. Here we employ simple methods, including cell counting, microscopy, viability and cytotoxicity assays to describe the minimal experimental methods required to optimize nucleofection conditions for a given cell line. METHODS: We comprehensively measured and analyzed the outcomes of the 96-well nucleofection of pmaxGFP plasmids encoding green fluorescent protein (GFP) into the A-549 human lung epithelial cell line. Fluorescent microscopy and a plate reader were used to respectively observe and quantify green fluorescence in both whole and lysed cells. Cell viability was determined by direct counting/permeability assays, and by both absorbance and fluorescence-based plate reader cytotoxicity assays. Finally, an optimal nucleofection condition was used to deliver siRNA and gene specific knock-down was demonstrated. RESULTS: GFP fluorescence among conditions ranged from non-existent to bright, based upon the fluorescent microscopy and plate reader results. Correlation between direct counting of cells and plate-based cytotoxicity assays were from R = .81 to R = .88, depending on the assay. Correlation between the GFP fluorescence of lysed and unlysed cells was high, ranging from R = .91 to R = .97. Finally, delivery of a pooled sample of siRNAs targeting the gene relA using an optimized nucleofection condition resulted in a 70–95% knock down of the gene over 48 h with 90–97% cell viability. CONCLUSION: Our results show the optimal 96-well nucleofection conditions for the widely-used human cell line, A-549. We describe simple, effective methods for determining optimal conditions with high confidence, providing a useful road map for other laboratories planning optimization of specific cell lines or primary cells. Our analysis of outcomes suggests the need to only measure unlysed, whole-cell fluorescence and cell metabolic activity using a plate reader cytotoxicity assay to determine the best conditions for 96-well nucleofection

Carboxysome Genomics: A Status Report

Carboxysomes, microcompartments that enhance the fixation of carbon dioxide by Rubisco, are found in several chemoautotrophs and in all cyanobacteria thus far examined. The genes for Rubisco large (cbbL) and small (cbbS) subunits (cbb for Calvin-Benson-Bassham), along with the genes (csoS) for the carboxysome shell peptides, are organized in a putative operon in Halothiobacillus neapolitanus in the following order: cbbL,cbbS, csoS2, csoS3, orfA, orfB, csoS1C, csoS1A, and csoS1B. DNA sequencing has revealed essentially the same operon in three other thiobacilli, Acidithiobacillus ferrooxidans, Thiomonas intermedia, and Thiobacillus denitrificans. The carboxysome genes are also clustered inSynechococcus sp. and Synechocystis sp., although in some cases certain genes lie outside the cluster. The genes, labelled ccm for CO2 concentrating mechanism, exist in Synechococcus PCC7942 in the order ccmK, ccmL, ccmM, ccmN, and ccmO, and are located upstream of the Rubisco genes. ccmO is absent, and multiple copies of ccmK exist in some species. The ccmK/ccmO and ccmL genes are homologues of csoS1CAB andorfAB, respectively. The ccmM and ccmN genes have no apparent counterpart in the thiobacilli. More recently, the genome sequence of four additional cyanobacteria has become available. The carboxysome genes in Nostoc punctiforme are clustered like, and are similar to, the genes of the earlier mentioned cyanobacteria. However, the three marine organisms Prochlorococcus marinus MIT9313, P. marinus MED4, and Synechococcus WH8102, possess an operon nearly identical to that found in thiobacilli. Furthermore, the genes exhibit surprising sequence identity to the carboxysome genes of the thiobacilli

Organization of Carboxysome Genes in the Thiobacilli

The order of genes in the carboxysome gene clusters of four thiobacilli was examined and the possibility of the cluster forming an operon evaluated. Furthermore, carboxysome peptide homologs were compared with respect to similarities in primary sequence, and the unique structural features of the shell protein CsoS2 were described

Organization of carboxysome genes in the thiobacilli

The order of genes in the carboxysome gene clusters of four thiobacilli was examined and the possibility of the cluster forming an operon evaluated. Furthermore, carboxysome peptide homologs were compared with respect to similarities in primary sequence, and the unique structural features of the shell protein CsoS2 were described.U.S. Department of Agriculture, under Agreement No. 92-37306-7663 and by NSF Grant MCB-951348