ABSTRACT
BACKGROUND: Heterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products. METHODOLOGY: We have developed a new Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ(54) directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ(54) promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways. CONCLUSIONS: We have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ(54) may be a viable method for the production of additional polyketide products.
Subject(s)
Biosynthetic Pathways , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Polyketides/metabolism , RNA Polymerase Sigma 54/metabolism , Anti-Bacterial Agents/pharmacology , Base Sequence , Benzothiazoles , Diamines , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial/drug effects , Molecular Sequence Data , Organic Chemicals/metabolism , Oxytetracycline/biosynthesis , Oxytetracycline/chemistry , Oxytetracycline/pharmacology , Peptides/metabolism , Polyketides/chemistry , Promoter Regions, Genetic/genetics , Quinolines , RNA Polymerase Sigma 54/genetics , Transcription, Genetic/drug effectsABSTRACT
To identify therapeutic opportunities for oncolytic viral therapy, we conducted genome-wide RNAi screens to search for host factors that modulate rhabdoviral oncolysis. Our screens uncovered the endoplasmic reticulum (ER) stress response pathways as important modulators of rhabdovirus-mediated cytotoxicity. Further investigation revealed an unconventional mechanism whereby ER stress response inhibition preconditioned cancer cells, which sensitized them to caspase-2-dependent apoptosis induced by a subsequent rhabdovirus infection. Importantly, this mechanism was tumor cell specific, selectively increasing potency of the oncolytic virus by up to 10,000-fold. In vivo studies using a small molecule inhibitor of IRE1α showed dramatically improved oncolytic efficacy in resistant tumor models. Our study demonstrates proof of concept for using functional genomics to improve biotherapeutic agents for cancer.
Subject(s)
Endoplasmic Reticulum Stress , Endoplasmic Reticulum/physiology , Oncolytic Viruses/physiology , Animals , Apoptosis/physiology , Caspase 2/metabolism , Caspase 2/physiology , Cell Line, Tumor , Cysteine Endopeptidases/metabolism , Cysteine Endopeptidases/physiology , Endoribonucleases/antagonists & inhibitors , Female , Genomics/methods , Glioblastoma/drug therapy , Glioblastoma/pathology , Glioblastoma/virology , Humans , Mice , Mice, Nude , Oncolytic Virotherapy/methods , Oncolytic Viruses/immunology , Oncolytic Viruses/metabolism , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Ovarian Neoplasms/virology , Protein Serine-Threonine Kinases/antagonists & inhibitors , RNA Interference , Rhabdoviridae/physiologyABSTRACT
BACKGROUND AIMS: The ability to expand and maintain bone marrow (BM)-derived mesenchymal stem cells (MSC) in vitro is an important aspect of their therapeutic potential. Despite this, the exact composition of stromal cell types within these cultures and the potential effects of non-stem cells on the maintenance of MSC are poorly understood. METHODS: C57BL/6J BM stroma was investigated as a model to determine the relationship between MSC and non-multipotent cells in vitro. Whole BM and single-cell derived cultures were characterized using flow cytometry and cell sorting combined with multipotent differentiation. Proliferation of individual stromal populations was evaluated using BrdU. RESULTS: At a single-cell level, MSC were distinguished from committed progenitors, and cells lacking differentiation ability, by the expression of CD105 (CD105+). A 3-fold reduction in the percentage of CD105+ cells was detected after prolonged culture and correlated with loss of MSC. Depletion of CD105+ cells coincided with a 10-20% increase in the frequency of proliferating CD105(-) cells. Removal of CD105(-) stroma caused increased proliferation in CD105+ cells, which could be diminished by conditioned media from parent cultures. Comparison of the multipotent differentiation potential in purified and non-purified CD105+ cells determined that MSC were detectable for at least 3 weeks longer when cultured in the absence of CD105(-) cells. CONCLUSIONS: This work identifies a simple model for characterizing the different cellular components present in BM stromal cultures and demonstrates that stromal cells lacking multipotent differentiating capacity greatly reduce the longevity of MSC.
