Microbiota-derived acetate activates intestinal innate immunity via the Tip60 histone acetyltransferase complex

Microbe-derived acetate activates the Drosophila immunodeficiency (IMD) pathway in a subset of enteroendocrine cells (EECs) of the anterior midgut. In these cells, the IMD pathway co-regulates expression of antimicrobial and enteroendocrine peptides including tachykinin, a repressor of intestinal lipid synthesis. To determine whether acetate acts on a cell surface pattern recognition receptor or an intracellular target, we asked whether acetate import was essential for IMD signaling. Mutagenesis and RNA interference revealed that the putative monocarboxylic acid transporter Tarag was essential for enhancement of IMD signaling by dietary acetate. Interference with histone deacetylation in EECs augmented transcription of genes regulated by the steroid hormone ecdysone including IMD targets. Reduced expression of the histone acetyltransferase Tip60 decreased IMD signaling and blocked rescue by dietary acetate and other sources of intracellular acetyl-CoA. Thus, microbe-derived acetate induces chromatin remodeling within enteroendocrine cells, co-regulating host metabolism and intestinal innate immunity via a Tip60-steroid hormone axis that is conserved in mammals

Methionine Availability in the Arthropod Intestine Is Elucidated through Identification of Vibrio cholerae Methionine Acquisition Systems

International audienceMethionine is an essential amino acid involved in both biosynthetic and regulatory processes in the bacterial cell. To ensure an adequate supply of methionine, bacteria have evolved multiple systems to synthesize, import, and recover this amino acid. To explore the importance of methionine synthesis, transport, and recovery in any environment, all of these systems must be identified and mutagenized. Here, we have mutagenized every high-affinity methionine uptake system and methionine sulfoxide reductase encoded in the genome of the diarrheal pathogen V. cholerae . We use this information to determine that high-affinity methionine uptake systems are sufficient to acquire methionine in the intestine of the model arthropod Drosophila melanogaster but are not involved in virulence and that the intestinal concentration of methionine must be between 0.05 mM and 0.5 mM

Production of a soluble hydrogenase from R. Eutropha H16

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2) and are generally classified by the structure of the catalytic site. From a biotechnology perspective, hydrogenases have been extensively studied with a view to their potential application in H2-based energy systems. Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni-Fe] uptake hydrogenase (SH) produced by Ralstonia eutropha H16.The research conducted in this project can be divided into two main parts. Firstly, bioprocess optimisation to produce and purify the SH from R. eutropha H16 was undertaken. Partially optimised batch-fermentations in controlled bioreactors were carried out using Fructose-Glycerol-Nitrogen (FGN) media to induce the production of SH in a diauxic growth process. Subsequently, further optimisations were performed on the cell ultrasonication and ammonium sulphate precipitation methods followed by two consecutive ion exchange steps and a final size exclusion chromatography step to recover functional SH. As a result of this purification scheme developed, a pure active SH preparation thatexhibits a specific activity of 2.86 U/mg (NAD+ reduction) was obtained with 18.7% yieldand 13.1 fold purification. Subsequent electrochemical studies using cyclic voltammetry confirmed that the immobilised active enzyme on modified EPG electrodes was capable of oxidising H2 at the electrode surface. Secondly, molecular characterisation of the SH operon was investigated, involving two approaches: the analysis of the transcriptional regulation of the SH genes by quantifying the expressions of these genes using qRT-PCR, and development of a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. The expression of hoxF, hypF2 and hoxA genes were observed to be up-regulated by 4.6 + 0.94, 2.5 + 1.13 and 4.4 + 0.57 fold, respectively, under the derepressing growth condition. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by PSH promoter under derepressing conditions in FGN media was demonstrated in the recombinant R. eutropha

Investigations on a chloroform reductive dehalogenase

Halogenated organic compounds (organohalides) are globally prevalent, recalcitrant toxic and carcinogenic environmental pollutants contaminating soil and groundwater. Organohalide respiring bacteria provide a potential solution to remediate contaminated sites, as they are capable of utilising organohalides as electron acceptors for the generation of cellular energy. At the heart of these processes are reductive dehalogenases (RDase; EC 1.97.1.8), which are membrane bound enzymes that catalyse reductive dehalogenation reactions resulting in the generation of lesser-halogenated compounds that may be less toxic and more biodegradable.Chloroform (CF), primarily used in the production of refrigerants, is very prevalent and recalcitrant organohalide contamination and its improper disposal also caused groundwater pollution at industrial sites in Sydney, Australia. Its hazardous effect on environment and carcinogenic and organo-toxic effects on human health prompts bioremediation research towards its detoxification. Recently, Dehalobacter (Dhb) sp. strain UNSWDHB, which is capable of respiring CF and converting it to dichloromethane has been identified. The UNSWDHB strain was revealed to produce CF-RDase, as termed TmrA, which has been the focus of the research carried out in this thesis.Firstly, the response of Dhb sp. UNSWDHB to the addition of CF was evaluated from a transcriptomic and proteomic perspective. The elevated expressions of TmrABC proteins, key bioenergetics related membrane-associated and cytoplasmic proteins, enzymes associated with functional Wood-Ljungdahl pathway and complete corrinoid de novo synthesis were revealed, providing a broader view on the bioenergetics and general physiology of the Dehalobacter cells actively respiring with CF. Furthermore, TmrA was produced and purified from the membrane fraction of the UNSWDHB cells to apparent homogeneity, using detergent-based membrane solubilisation and anion exchange chromatographic purification. This allowed further biochemical characterisation of the purified enzyme. Lastly, an extensive study to heterologously express TmrA was conducted under several expression conditions to address a reportedly challenging issue with soluble, functional expression and purification of a respiratory RDase. The xylose-inducible expression in a corrinoid-producing Bacillus megaterium as an expression host and two liquid chromatographic steps resulted in a generation of a soluble and functional recombinant TmrA

Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein)

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni–Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression

Reductive Dehalogenases Come of Age in Biological Destruction of Organohalides

Halogenated organic compounds (organohalides) are globally prevalent, recalcitrant toxic, and carcinogenic environmental pollutants. Select microorganisms encode enzymes known as reductive dehalogenases (EC 1.97.1.8) that catalyze reductive dehalogenation reactions resulting in the generation of lesser-halogenated compounds that may be less toxic and more biodegradable. Recent breakthroughs in enzyme structure determination, elucidation of the mechanisms of reductive dehalogenation, and in heterologous expression of functional reductive dehalogenase enzymes have substantially increased our understanding of this fascinating class of enzymes. This knowledge has created opportunities for more versatile (in situ and ex situ) biologically-mediated organohalide destruction strategies

Organohalide Respiring Bacteria and Reductive Dehalogenases: Key Tools in Organohalide Bioremediation

Organohalides are recalcitrant pollutants that have been responsible for substantial contamination of soils and groundwater. Organohalide-respiring bacteria (ORB) provide a potential solution to remediate contaminated sites, through their ability to use organohalides as terminal electron acceptors to yield energy for growth (i.a, organohalide respiration). Ideally, this process results in non- or lesser-halogenated compounds that are mostly less toxic to the environment or more easily degraded. At the heart of these processes are reductive dehalogenases (RDases), which are membrane bound enzymes coupled with other components that facilitate dehalogenation of organohalides to generate cellular energy. This review focuses on RDases, concentrating on those which have been purified (partially or wholly) and functionally characterized. Further, the paper reviews the major bacteria involved in organohalide breakdown and the evidence for microbial evolution of RDases. Finally, the capacity for using ORB in a bioremediation and bioaugmentation capacity are discussed

Molecular Targets of Tannic Acid in Alzheimer\u27s Disease

Tannic acid (TA) is a naturally occurring plant-derived polyphenol found in several herbaceous and woody plants, including legumes, sorghum, beans, bananas, persimmons, raspberries, wines and a broad selection of teas. Clinically, TA has strong antioxidant/free radical scavenging, anti inflammatory, anti-viral/bacterial, and anti-carcinogenic properties. While the aetiology of Alzheimer’s disease (AD) remains unclear, this complex multifactorial neurodegenerative disorder remains the most common form of dementia, and is a growing public health concern worldwide. The neuroprotective effects of TA against AD have been shown in several in vitro and in vivo models of AD. Apart from its potent antioxidant and anti-inflammatory roles, evidence suggests that TA is also a natural inhibitor of β-secretase (BACE1) activity and protein expression. BACE1 is the primary enzyme responsible for the production and deposition of Aβ peptide. TA also destabilises neurotoxic amyloid beta (Aβ) fibrils in vitro. Apart from its effects on the Aβ cascade, TA can also inhibit the in vitro aggregation of tau peptide, a core component of intracellular neurofibrillary tangles (NFTs). This review summarizes the relevance of TA and TA-related vegetable extracts (tannins) in the pathogenesis of AD and its enzymatic targets. It also highlights the significance of TA as an important lead compound against AD

Resveratrol as a Potential Therapeutic Candidate for the Treatment and Management of Alzheimer\u27s Disease

Resveratrol (3,4\u27,5-trihydroxystilbene) is a naturally occurring phytochemical present in red wine, grapes, berries, chocolate and peanuts. Clinically, resveratrol has exhibited significant antioxidant, anti-inflammatory, anti-viral, and anti-cancer properties. Although resveratrol was first isolated in 1940, it was not until the last decade that it was recognised for its potential therapeutic role in reducing the risk of neurodegeneration, and Alzheimer\u27s disease (AD) in particular. AD is the primary cause of progressive dementia. Resveratrol has demonstrated neuroprotective effects in several in vitro and in vivo models of AD. Apart from its potent antioxidant and anti-inflammatory roles, evidence suggests that resveratrol also facilitates non-amyloidogenic breakdown of the amyloid precursor protein (APP), and promotes removal of neurotoxic amyloid beta (Aβ) peptides, a critical step in preventing and slowing down AD pathology. Resveratrol also reduces damage to neuronal cells via a variety of additional mechanisms, most notably is the activation of NAD+-dependent histone deacetylases enzymes, termed sirtuins. However in spite of the considerable advances in clarifying the mechanism of action of resveratrol, it is unlikely to be effective as monotherapy in AD due to its poor bioavailability, biotransformation, and requisite synergism with other dietary factors. This review summarizes the relevance of resveratrol in the pathophysiology of AD. It also highlights why resveratrol alone may not be an effective single therapy, and how resveratrol coupled to other compounds might yet prove an effective therapy with multiple targets