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Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli

ABSTRACT: Isopentenyl pyrophosphate (IPP) is the universal C5 precursor for isoprenoids, the largest class of natural products and frequent targets for metabolic engineering. In engineered microbial systems, IPP toxicity presents a challenge since its formation is unavoidable in the production of high-value, long-chain terpenes. In this work, we develop an experimental platform to study IPP toxicity in E. coli engineered to produce isoprenol, an IPP-derived C5 alcohol. We first characterize the physiological response to IPP accumulation, demonstrating that elevated levels of IPP are linked to growth inhibition, altered morphology, and reduced cell viability. We show that IPP toxicity selects for pathway “breakage”, using proteomics to identify a reduction in phosphomevalonate kinase (PMK) as a probable recovery mechanism. Next, we demonstrate that endogenous E. coli metabolism is globally impacted by IPP accumulation, which results in inhibited nutrient uptake, reduced ATP levels, and an apparent “pause” in metabolism. Finally, we suggest that IPP toxicity is mediated by the formation of an isoprenyl-ATP analog (ApppI). The complementary data presented here represent the most comprehensive assessment of IPP stress to date and suggest potential strategies for the alleviation of IPP and prenyl diphosphate toxicity.

ORGANISM(S): Escherichia coli DH1

PROVIDER: GSE102672 | GEO | 2018/03/14

REPOSITORIES: GEO

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Project description:Isoprenoids are a class of ubiquitous organic molecules synthesized from the five-carbon starter unit isopentenyl pyrophosphate (IPP). Comprising more than 30,000 known natural products, isoprenoids serve various important biological functions in many organisms. In bacteria, undecaprenyl pyrophosphate is absolutely required for the formation of cell wall peptidoglycan and other cell surface structures, while ubiquinones and menaquinones, both containing an essential prenyl moiety, are key electron carriers in respiratory energy generation. There is scant knowledge on the nature and regulation of bacterial isoprenoid pathways. In order to explore the cellular responses to perturbations in the mevalonate pathway, responsible for producing the isoprenoid precursor IPP in many Gram-positive bacteria and eukaryotes, we constructed three strains of Staphylococcus aureus in which each of the mevalonate pathway genes is regulated by an IPTG inducible promoter. We used DNA microarrays to profile the transcriptional effects of downregulating the components of the mevalonate pathway in S. aureus and demonstrate that decreased expression of the mevalonate pathway leads to widespread downregulation of primary metabolism genes, an upregulation in virulence factors and cell wall biosynthetic determinants, and surprisingly little compensatory expression in other isoprenoid biosynthetic genes. We subsequently correlate these transcriptional changes with downstream metabolic consequences. We used microarrays to profile the transcriptional changes incurred when downregulating mvaS, mvaA, or mvaK in S. aureus. Keywords: stress response S. aureus mutants were generated in which mvaS, mvaA, or mvaK were placed under control of the IPTG-inducible Pspac promoter. These mutants, as well as wildtype RN4220 S. aureus, were grown with or without 1 mM IPTG, harvested in exponential phase, and their total RNA was extracted and hybridized to Affymetrix GeneChips. Experiments were done in triplicate.

Project description:Isopropylated phenyl phosphates (IPP) are an additive organophosphate flame retardant (OPFR) which has been extensively used in furniture, electronics, automobiles, plastics, and children’s products to slow down the spread of fire. The processing and distribution of IPP- containing products have been prohibited but its continuous leaching from end use products has retained the concern of its toxicity. The present study was designed to evaluate IPP-induced developmental toxicity using zebrafish embryos. We first performed range finding experiments with embryonic zebrafish, exposed to 0 - 200 M IPP from 6 to 120 h post fertilization and found significant morphological impacts like pericardial edema, yolk sac edema and spinal curvature at higher concentrations. Following this, relying on secondary analyses of our whole embryo mRNA-seq data, we quantified neurotransmitters and found significant increase in the levels of dopamine and its metabolite, 3-methoxytyramine. We then conducted, in vitro retinoic acid receptor (RAR) signaling assay and noticed significant inhibition of RARα, but not RAR and RAR. For behavioral readouts, we performed larval photomotor response (LPR) assay at sublethal concentrations and observed hypoactive locomotory behavior in exposed larvae. Whole-mount immunohistochemistry for 5-methylcytosine and global DNA methylation assay showed significant IPP-induced hypermethylation in situ. Finally, based on whole embryo RNA-seq data, we hypothesized that IPP affects the development of brain and eyes. Firstly, we performed global DNA methylation in brain and eyes, but did not find significant effects. Then, we conducted mRNA sequencing on dissected brains and eyes, and found 2 and 135 differentially expressed genes, respectively. Gene ontology revealed that IPP affect voltage gated ion channel activity, synaptic transmission and neurotransmitter signaling. Collectively, our data shows that IPP induces morphological abnormalities and disrupts larval photo motor response, potentially through RA inhibition and methylomic regulation. Finally, we observed that IPP affects gene expression within the developing eye, establishing synaptic transmission, vision and muscle contraction as a potential causative factor for LPR responses.

Project description:Systemic inflammation like in sepsis is still lacking specific diagnostic markers and effective therapeutics. The first line of defense against intruding pathogens and endogenous damage signals is pattern recognition by e.g., complement and Toll-like receptors (TLR). Combined inhibition of a key complement component (C3 and C5) and TLR-co-receptor CD14 has been shown to attenuate certain systemic inflammatory responses. Using DNA microarray and gene annotation analyses, we aimed to decipher the effect of combined inhibition of C3 and CD14 on the transcriptional response to bacterial challenge in human whole blood. Importantly, combined inhibition reversed the transcriptional changes of 70% of the 2335 genes which significantly responded to heat-inactivated Escherichia coli by on average 80%. Single inhibition was less efficient (p<0.001) but revealed a suppressive effect of C3 on 21% of the responding genes which was partially counteracted by CD14. Furthermore, CD14 dependency of the Escherichia coli-induced response was increased in C5-deficient compared to C5-sufficient blood. The observed crucial distinct and synergistic roles for complement and CD14 on the transcriptional level correspond to their broad impact on the inflammatory response in human blood, and their combined inhibition may become inevitable in the early treatment of acute systemic inflammation. A microarray study was performed on whole blood from two healthy donors, a C5-deficient patient (C5D) (with and without C5-reconstitution) incubated for 120 min at 37oC with (i) PBS, (ii) E. coli or (iii) E. coli and inhibitors of complement (C3 or C5aR) and/or CD14 or inhibitor controls. The experiments were performed as biological replicates on two different days (healthy donor and C5D). Due to lack of a second C5-deficient patient, microarray analyses were prerformed twice (technical replicate) for C5D and day 1. Expression data in presence of PBS were used as reference for those in pressence of E. coli. Expression data in presence of E. coli were used as reference for those in pressence of E. coli and inhibitors. Expression data from 0 min incubation with PBS represent the initial state (T0). Whole blood was lysed in Applied Biosystems AB6100 total RNA chemistry lysis buffer and stored at -80oC prior to RNA extraction.

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Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli

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