Botulinum neurotoxin A induced expression profiles in Raw264.7 cells
ABSTRACT: Botulinum neurotoxin type A (BoNT/A) is one of the most potent protein toxins, which makes it a possible biological weapon and therapeutic. Using microarray analysis we performed global transcriptional profiling of RAW264.7 cells, a murine alveolar macrophage cell line. Time dependent expression profiles after treatment of 1nM or 5nM Botulinum neurotoxin A
Project description:The purpose of this study was to determine the level of genomic content similarity among selected strains of Clostridium botuinum type F strains. In this study, strains were selected that had already been chacaterized by botulinum neurotoxin gene sequencing. Strains harboring bont/F1, bont/F4 and bont/F5 were compared.
Project description:Clostridium botulinum is a heterogeneous Gram-positive species that comprises four genetically and physiologically distinct groups of bacteria that share the ability to produce botulinum neurotoxin, the most poisonous toxin known to man, and the causative agent of botulism, a severe disease of humans and animals. We report here the complete genome sequence of a representative of Group I (proteolytic) C. botulinum (strain Hall A, ATCC 3502). The genome consists of a chromosome (3,886,916 bp) and a plasmid (16,344 bp) which carry 3,650 and 19 predicted genes, respectively. Consistent with the proteolytic phenotype of this strain, the genome harbours a large number of genes encoding secreted proteases and enzymes involved in uptake and metabolism of amino acids. The genome also reveals a hitherto unknown ability of C. botulinum to degrade chitin. There is a significant lack of recently acquired DNA, indicating a stable genomic content, in strong contrast to the fluid genome of C. difficile, which can form longer-term relationships with its host. Overall, the genome indicates that C. botulinum is adapted to a saprophytic lifestyle both in soil and aquatic environments. This pathogen relies on its toxin to rapidly kill a wide range of prey species, and to gain access to nutrient sources, it releases a large number of extracellular enzymes to soften and destroy rotting or decayed tissues.
Project description:The PFGRC has developed a cost effective alternative to complete genome sequencing in order to study the genetic differences between closely related species and/or strains. The comparative genomics approach combines Gene Discovery (GD) and Comparative Genomic Hybridization (CGH) techniques, resulting in the design and production of species microarrays that represent the diversity of a species beyond just the sequenced reference strain(s) used in the initial microarray design. These species arrays may then be used to interrogate hundreds of closely related strains in order to further unravel their evolutionary relationships. Clostridium botulinum produces botulinum neurotoxin (BoNT)and is classified as a “Category A” select agent. BoNT can be classified into seven serotypes designated A-G. There is considerable genetic variation within these serotypes, as demonstrated by the recognition of at least 47 subtypes. The most studied serotype, BoNT/A, has been found in a large and diverse group of clostridia, most of which express the subtype BoNT/A1. The BoNT/A1 producing C. botulinum strain ATCC 3502, used to obtain an initial annotated genome sequence, is not representative of the diverse clostridia group producing BoNT. Nearly 50% of C. botulinum strains producing BoNT/A1 have been shown to also encode unexpressed variants of BoNT/B with a distinct cluster arrangement. This nucleotide cluster is completely absent from the published genome sequence. In addition, a recently identified novel BoNT/A1 strain lacks the gene cluster seen in the genome sequence of ATCC 3502. Furthermore, a strain designated Hall A Hyper differs greatly from the sequenced strain as indicated by its ability to produce higher quantities of BoNT/A1. The genetic and phenotypic basis for this difference in BoNT expression is currently unknown, and the sequences of the BoNT gene and the cluster are identical in both strains. This observation supports the hypothesis that genes outside the toxin cluster are involved in the regulation and maturation of BoNT. The flow of genetic information within this group motivated us to identify novel genes for the purpose of creating a “species” DNA microarray to better understand the ancestral relationships among its members. Based on preliminary genotyping (MLST, and CGH using a single-genome-based array), 20 diverse C. botulinum strains were selected for sequencing. Sequence information obtained from this project, and from other publicly available sources, led to the development of a comprehensive species microarray for C. botulinum group members. The availability of the C. botulinum species DNA microarray has allowed us to carry out a collaborative CGH genotyping project to validate this microarray as well as understand the phylogenomic relationships among members of C. botulinum group. Overall design: One hundred fifty six query strains were investigated in this study, with each query strain hybridized against the reference strain, ATCC3502. Each strain has a single dye experiment. Each oligo is spotted on the C. botulinum species microarray once. Positive controls on the array consist of oligos designed from the sequenced reference genome, ATCC3502, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana.The microarrays also had Agilent internal controls.
Project description:The advances in chemical proteomics have significantly expanded our understanding of the diversity and abundance of fatty-acylated proteins in eukaryotes, and reveal novel functions for these lipid protein modifications. Nonetheless, quantitative comparative proteomic analysis of fatty-acylated proteins in different cellular states is still challenging. To address these limitations, we systematically evaluated different proteomic methods (alk-16 chemical reporter and acyl-RAC) and established robust conditions to selectively and quantitatively profile fatty-acylated proteins in mammalian cells. Using a combination of metabolic labeling with fatty acid chemical reporters, selective chemical enrichment and label-free proteomics, we performed a quantitative analysis of fatty-acylated proteins in naïve and activated macrophages. These studies revealed novel fatty-acylated proteins associated with host immunity that are differently expressed and lipid-modified in different cellular states.
Project description:Transcriptome analysis of RNA extracted from human induced pluripotent stem cell (hipsc)-derived neurons exposed to botulinum neurotoxin type A1 (BoNT/A1) and an atoxic derivative, BoNT/A ad. Botulinum neurotoxin type A1 (BoNT/A1) is a potent protein toxin responsible for the potentially fatal human illness botulism. Despite this, the long-lasting flaccid muscle paralysis caused by BoNT/A has led to its rise as a powerful and versatile bio-pharmaceutical. The flaccid paralysis is due to specific cleavage of SNAREs by BoNTs inside neurons. However, potential effects of BoNTs on intoxicated neurons besides the cleavage of SNAREs have not been studied in detail. In this study we investigated by microarray analysis the effects of BoNT/A and a catalytically inactive derivative (BoNT/A ad) on the transcriptome of human induced pluripotent stem cell (hiPSC)-derived neurons at 2 days and 2 weeks after exposure. While there were only minor changes in expression levels at 2 days post exposure, at 2 weeks post exposure 492 genes were differentially expressed more than 2-fold in BoNT/A1-exposed cells when compared to non-exposed populations, and 682 genes were differentially expressed in BoNT/A ad-exposed cells. The vast majority of genes were similarly regulated in BoNT/A1 and BoNT/A ad-exposed neurons, and the few genes differentially regulated between BoNT/A1 and BoNT/A ad-exposed neurons were regulated less than 3.5 fold. These data indicate a similar response of neurons to BoNT/A1 and BoNT/A ad exposure. The most highly regulated genes in cells exposed to either BoNT/A1 or BoNT/A ad are involved in neurite outgrowth and calcium channel sensitization. 18 samples were used for this project: 6 populations were exposed to BoNT/A1, 6 to BoNT/A ad, and 6 were non-exposed. All exposures lasted for 48h, at which point 3 samples of each exposure group were harvested (2d condition), and the remaining 3 samples were grown for an additional 12d (2wk condition). Analysis was performed using the Affymetrix HG U133 Plus 2.0 array, and data was extracted using the Affymetrix Expression Console v 18.104.22.168 software
Project description:RAW264.7 macrophages infected with MNV-1 and mock infected gene expression measured by microarray. To be published in Waugh, E. Chen, A. Baird, M. Fleming, S. Brown, C.M. and V K. Ward (2014) Characterization of the chemokine response of RAW264.7 cells to infection by murine norovirus. Virus Genes Four Samples, two mock and two MNV-1 infected.
Project description:Profound understanding of the mechanisms foodborne pathogenic bacteria utilize in adaptation to the environmental stress they encounter during food processing and storage is of paramount importance in design of control measures. Chill temperature is a central control measure applied in minimally processed foods; however, data on the mechanisms the foodborne pathogen Clostridium botulinum activates upon cold stress are scarce. Global gene expression analysis on the C. botulinum ATCC 3502 strain upon temperature downshift from 37 °C to 15 °C was performed to identify the cold-responsive gene set of this organism. Significant up- or down-regulation of 16 and 11 genes, respectively, was observed already 1 h after the cold shock. At 5 h after the temperature downshift, 199 and 210 genes were up- and down-regulated, respectively. Thus, the cold shock rapidly affected the expression of a gene set of a relatively small size, indicating a targeted acute response to cold shock, whereas extensive metabolic remodeling took place after prolonged exposure to cold. Induction of genes related to fatty acid biosynthesis, oxidative stress response, and iron uptake and storage was observed, in addition to mechanisms previously characterized as cold-tolerance related in bacteria. Furthermore, induction of several uncharacterized DNA-binding transcriptional regulator-encoding genes was observed, suggesting involvement of novel regulatory mechanisms in the cold shock response of C. botulinum. The role of such regulatory proteins, CBO0477 and CBO0558A, in cold tolerance of C. botulinum ATCC 3502 was demonstrated by the deteriorated growth of mutants of the respective genes at 17 °C. C. botulinum ATCC 3502 wild type cold-shocked vs. pre-cold-shock; 3 replicates; growth at 37C in TPGY broth batch culture and subjected to cold shock to 15C; sampling at mid-log growth phase before cold shock, and 1 h and 5 h after temperature downshift to 15C (= 3 time points). Dye-swapped hybridization.