Eukaryotes have evolved highly conserved vesicle transport machinery to deliver proteins to the vacuole. In this study we show that the filamentous fungus Aspergillus parasiticus employs this delivery system to perform new cellular functions, the synthesis, compartmentalization, and export of aflatoxin; this secondary metabolite is one of the most potent naturally occurring carcinogens known. Here we show that a highly pure vesicle-vacuole fraction isolated from A. parasiticus under aflatoxin-inducing conditions converts sterigmatocystin, a late intermediate in aflatoxin synthesis, to aflatoxin B 1 ; these organelles also compartmentalize aflatoxin. The role of vesicles in aflatoxin biosynthesis and export was confirmed by blocking vesicle-vacuole fusion using 2 independent approaches. Disruption of A. parasiticus vb1 (encodes a protein homolog of AvaA, a small GTPase known to regulate vesicle fusion in A. nidulans) or treatment with Sortin3 (blocks Vps16 function, one protein in the class C tethering complex) increased aflatoxin synthesis and export but did not affect aflatoxin gene expression, demonstrating that vesicles and not vacuoles are primarily involved in toxin synthesis and export. We also observed that development of aflatoxigenic vesicles (aflatoxisomes) is strongly enhanced under aflatoxin-inducing growth conditions. Coordination of aflatoxisome development with aflatoxin gene expression is at least in part mediated by Velvet (VeA), a global regulator of Aspergillus secondary metabolism. We propose a unique 2-branch model to illustrate the proposed role for VeA in regulation of aflatoxisome development and aflatoxin gene expression.S econdary metabolites, natural products generated by filamentous fungi, plants, bacteria, algae, and animals, have an enormous impact on humans due to their application in health, medicine, and agriculture. Many secondary metabolites are beneficial (antibiotics, statins, morphine, etc.), though phytotoxins (e.g., ricin, crotin, amygdalin) and fungal poisons called mycotoxins (e.g., aflatoxin, sterigmatocystin, fumonisin) are detrimental to humans and animals. To control or customize biosynthesis of these natural products we must understand how and where secondary metabolism is orchestrated within the cell.Vacuoles and vesicles are known to sequester secondary metabolites to protect host cells from self-toxicity (1). Enzymes involved in secondary metabolism are often found in vesicles and vacuoles, including those for biosynthesis of alkaloids (e.g., berberine, sanguinarine, camptotecin, and morphine; reviewed in refs. 1 and 2) and flavonoids (e.g., aurone) (reviewed in refs. 1 and 3) in plants and the nonribosomal peptide cyclosporin (4), the -lactam antibiotic penicillin (5) (localization of ACVS is still controversial), and the polyketide aflatoxin (6-8) in fungi. However, the functional role of these compartments in secondary metabolism was unclear because these organelles potentially could be involved in synthesis, storage, protein turnover, transport, or export of...
Human illness due to Camplyobacter jejuni infection is closely associated with consumption of poultry products. We previously demonstrated a 50 % shift in allele frequency (phase variation) in contingency gene Cj1139 (wlaN) during passage of C. jejuni NCTC11168 populations through Ross 308 broiler chickens. We hypothesized that phase variation in contingency genes during chicken passage could promote subsequent colonization and disease in humans. To test this hypothesis, we passaged C. jejuni strains NCTC11168, 33292, 81-176, KanR4 and CamR2 through broiler chickens and analysed the ability of passaged and non-passaged populations to colonize C57BL6 IL-10-deficient mice, our model for human colonization and disease. We utilized fragment analysis and nucleotide sequence analysis to measure phase variation in contingency genes. Passage through the chicken reservoir promoted phase variation in five specific contingency genes, and these 'successful' populations colonized mice. When phase variation did not occur in these same five contingency genes during chicken passage, these 'unsuccessful' populations failed to colonize mice. Phase variation during chicken passage generated small insertions or deletions (indels) in the homopolymeric tract (HT) in contingency genes. Singlecolony isolates of C. jejuni strain KanR4 carrying an allele of contingency gene Cj0170 with a 3These authors contributed equally to this work. Two supplementary tables, listing PCR primer sequences for fragment and sequence analysis used in this study, and results of sequence and fragment analysis to confirm large shifts in allele frequency, with a supplementary reference, are available with the online version of this paper. 10G HT colonized mice at high frequency and caused disease symptoms, whereas single-colony isolates carrying the 9G allele failed to colonize mice. Supporting results were observed for the successful 9G allele of Cj0045 in strain 33292. These data suggest that phase variation in Cj0170 and Cj0045 is strongly associated with mouse colonization and disease, and that the chicken reservoir can play an active role in natural selection, phase variation and disease.
BackgroundFilamentous fungi in the genus Aspergillus produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by Aspergillus parasiticus in an attempt to define the association of secondary metabolism with other metabolic and cellular processes.ResultsVolatile compounds were examined using solid phase microextraction - gas chromatography/mass spectrometry. In the wild type strain Aspergillus parasiticus SU-1, the largest group of volatiles included compounds derived from catabolism of branched chain amino acids (leucine, isoleucine, and valine); we also identified alcohols, esters, aldehydes, and lipid-derived volatiles. The number and quantity of the volatiles produced depended on media composition, time of incubation, and light-dark status. A block in aflatoxin biosynthesis or disruption of the global regulator veA affected the volatile profile. In addition to its multiple functions in secondary metabolism and development, VeA negatively regulated catabolism of branched chain amino acids and synthesis of ethanol at the transcriptional level thus playing a role in controlling carbon flow within the cell. Finally, we demonstrated that volatiles generated by a veA disruption mutant are part of the complex regulatory machinery that mediates the effects of VeA on asexual conidiation and sclerotia formation.Conclusions1) Volatile profiling provides a rapid, effective, and powerful approach to identify changes in intracellular metabolic networks in filamentous fungi. 2) VeA coordinates the biosynthesis of secondary metabolites with catabolism of branched chain amino acids, alcohol biosynthesis, and β-oxidation of fatty acids. 3) Intracellular chemical development in A. parasiticus is linked to morphological development. 4) Understanding carbon flow through secondary metabolic pathways and catabolism of branched chain amino acids is essential for controlling and customizing production of natural products.
In vitro cell proliferation and apoptosis assays are widely used to study cancer cell biology. Commonly used methodologies are however performed at a single, user-defined endpoint. We describe a kinetic multiplex assay incorporating the CellPlayer(TM) NucLight Red reagent to measure proliferation and the CellPlayer(TM) Caspase-3/7 reagent to measure apoptosis using the two-color, live-content imaging platform, IncuCyte(TM) ZOOM. High-definition phase-contrast images provide an additional qualitative validation of cell death based on morphological characteristics. The kinetic data generated using this strategy can be used to derive informed pharmacology measurements to screen potential cancer therapeutics.
Campylobacter jejuniis an important foodborne pathogen of humans and its primary reservoir is the gastrointestinal (GI) tract of chickens. Our previous studies demonstrated that phase variation to specific “successful alleles” at C. jejuni contingency loci Cj0045 (successful alleles carry 9G or 10G homopolymeric tracts) and Cj0170 (successful allele carries a 10G homopolymeric tract) in C. jejuni populations is strongly associated with colonization and enteritis in C57BL/6 IL-10 deficient mice. In the current study, we strengthened the association between locus Cj0170, Cj0045, and mouse colonization. We generated 8 independent strains derived from C. jejuni 11168 strain KanR4 that carried a Cj0170 gene disruption and these were all non motile. Two randomly chosen strains with the Cj0170 gene disruption (DM0170-2 and DM0170-6) were gavaged into mice. DM0170-2 and DM0170-6 failed to colonize mice while the control strain that carried a “successful” Cj0170 10G allele was motile and did colonize mice. In parallel studies, when we inoculated C. jejuni strain 33292 into mice, the “unsuccessful” Cj0045 11G allele experienced phase variation to “successful” 9G and 10G alleles in 2 independent experiments prior to d4 post inoculation in mice while the “successful” 9G allele in the control strain remained stable through d21 post inoculation or shifted to other successful alleles. These data confirm that locus Cj0170 regulates motility in C. jejuni strain KanR4 and is a virulence factor in the mouse model. The data also support a possible role of locus Cj0045 as a virulence factor in strain 33292 in infection of mice.
Apoptosis, the biological process by which cells undergo programmed cell death, is required for normal tissue maintenance and development. However, aberrations in apoptotic signaling networks are implicated in numerous human diseases including neurodegeneration and cancer. Stimulation of either the extrinsic or intrinsic apoptotic pathways triggers a signaling cascade that typically results in the activation of executioner caspase-3. Numerous in vitro assays have been designed to measure the activation of caspase-3. The majority of these assays utilize reagent substrates that incorporate the DEVD motif which is recognized by both activated caspases 3 and 7. Such assays result in single, user-defined time point measurements of caspase-3/7 activity and typically require multiple wash steps or cell lifting prior to data collection; potentially resulting in the loss of cells or critical data in experiments where cells undergo apoptosis at different rates according to treatment conditions. In this study, we optimized an assay system incorporating a bi-functional reagent containing the DEVD motif linked to a DNA dye for use on the IncuCyte FLR imaging system. When added to cell culture growth medium, this relatively inert, non-fluorescent molecule crosses the cell membrane where it is available to be a substrate for activated caspase-3/7. The reaction product is a green fluorescent DNA dye that labels the nuclei of cells undergoing apoptosis. Our data show that this strategy can quantitatively measure the kinetic activation of caspase-3/7 through extrinsic stimulation of human tumor derived MDA-MB-231 breast adenocarcinoma cells, HeLa cervical adenocarcinoma cells, A549 lung epithelial carcinoma cells, and HT 1080 fibrosarcoma cells following treatment with TNFα and cycloheximide. Moreover, we exemplify the use of this strategy in 96-well plate screening protocols using both HT 1080 and MDA-MB-231 cells to measure the induction of caspase-3/7 activity in the presence of staurosporine, a well-known general protein kinase inhibitor and inducer of caspase-3/7 mediated apoptosis with Z’ values α0.6. We provide evidence that this stable, kinetic, mix-and-read assay can provide both quantitative and qualitative assessments of caspase-3/7 activity using fluorescent images and high-definition phase contrast images, respectively. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4046. doi:1538-7445.AM2012-4046
The tumor microenvironment is fundamentally involved in the response of a tumor to anti-cancer therapies. In this study, we present a novel method for kinetically measuring the proliferation of one or more cell types in mixed, co-culture models. Specifically, lentiviral based reagents are used to label nuclei of multiple cell populations within a single culture. Real time cell counts are acquired using an automated, Live Content Imaging approach in which 96-well and 384-well microplates are imaged every 2-3 hours over the course of the full 2-3 day assay. Using this strategy, we confirm data illustrating that culturing HER-2 positive breast cancer cells (SK-BR-3) in the presence of either Human Mammary Fibroblasts (HMFs) or normal skin fibroblasts (CCD-1068Sk) are resistant to the anti-proliferative and cytotoxic effects of the HER-2 and EGFR tyrosine kinase inhibitor lapatinib. Extending this data, we also completed pharmacolgical analyses, using area under the curve of the full kinetic trace, revealing nearly a 2-log difference in lapatinib sensitivity between SK-BR-3 cells grown in the presence or absence lapatinib. The data presented here reveal a novel, automated, kinetic assay for measuring proliferation of one or more cell types in mixed culture models. More importantly, these data further clarify the pharmacolgical effect stromal cells can have on the resistance of breast cancer cells to lapatinib treatment. Citation Format: Katherine Artymovich, Clare Szybut, Kalpana Patel, Tim O'Callaghan, Tim Dale, Del Trezise, Daniel M. Appledorn. Stromal cells confer drug resistance to breast cancer cells in a kinetic co-culture model. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B03.
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