Cristina Lagido

Development and application of bioluminescent Caenorhabditis elegans as multicellular eukaryotic biosensors

We describe a novel approach to assess toxicity to the free‐living nematode Caenorhabditis elegans that relies on the ability of firefly luciferase to report on endogenous ATP levels. We have constructed bioluminescent C. elegans with the luc gene under control of a constitutive promoter. Light reduction was observed in response to increasing temperature, concentrations of copper, lead and 3,5‐dichlorophenol. This was due to increased mortality coupled with decreased metabolic activity in the surviving animals. The light emitted by the transgenic nematodes gave a rapid, real‐time indication of metabolic status. This forms the basis of rapid and biologically relevant toxicity tests.

Bridging the phenotypic gap: Real-time assessment of mitochondrial function and metabolism of the nematode Caenorhabditis elegans

BackgroundThe ATP levels of an organism are an important physiological parameter that is affected by genetic make up, ageing, stress and disease.ResultsWe have generated luminescent C. elegans through ubiquitous, constitutive expression of firefly luciferase, widely used for in vitro ATP determination. We hypothesise that whole animal luminescence reflects its intracellular ATP levels in vivo. To test this, we characterised the bioluminescence response of C. elegans during sublethal exposure to, and recovery from azide, a treatment that inhibits mitochondrial respiration reversibly, and causes ATP depletion. Consistent with our expectations, in vivo luminescence decreased with increasing sublethal azide levels, and recovered fully when worms were removed from azide. Firefly luciferase expression levels, stability and activity did not influence the final luminescence. Bioluminescence also reflected the lowered activity of the electron transport chain achieved with RNA interference (RNAi) of genes encoding respiratory chain components.ConclusionResults indicated that C. elegans luminescence reports on ATP levels in real-time. For the first time, we are able to directly assess the metabolism of a whole, living, multicellular organism by determination of the relative ATP levels. This will enable genetic analysis based on a readily quantifiable metabolic phenotype and will provide novel insights into mechanisms of fitness and disease that are likely to be of relevance for other organisms, as well as the worm.

A model for bacterial conjugal gene transfer on solid surfaces.

Abstract Quantitative models of bacterial conjugation are useful tools in environmental risk assessment and in studies of the ecology and evolution of bacterial communities. We constructed a mathematical model for gene transfer between bacteria growing on a solid surface. The model considers that donor and recipient cells will form separate colonies, which grow exponentially until nutrient exhaustion. Conjugation occurs when donor and recipient colonies meet, all recipient cells becoming transconjugants instantly, after which they act as donors. The model was tested theoretically by computer simulations that followed the histories of individual bacterial colonies and was validated for initial surface coverage of 60% or less, where confluent growth does not occur. Model predictions of final number of donors, recipients and transconjugants were tested experimentally using a filter mating system with two isogenic strains of Pseudomonas fluorescens MON787 acting as donor and recipient of plasmid RP4. Experimental trends resulting from varying donor and recipient inoculum numbers and donor:recipient ratios were well described by the model, although it often overestimated conjugation by 0.5-2 orders of magnitude. Predictions were greatly improved, generally to within half a log unit of experimental values, by consideration of the time for conjugative transfer. The model demonstrates the relationship between spatial separation of cells and nutrient availability on numbers of transconjugants. By providing a mechanistic approach to the study conjugation on surfaces, the model may contribute to the study of gene transfer in natural environments.

Rapid Sublethal Toxicity Assessment Using Bioluminescent Caenorhabditis elegans, a Novel Whole-Animal Metabolic Biosensor

Sublethal metabolic effects are informative toxicological end points. We used a rapid quantitative metabolic end point, bioluminescence of firefly luciferase expressing Caenorhabditis elegans, to assess effects of sublethal chronic exposure (19 h) to the oxidative stress agent and environmental pollutant cadmium (provided as chloride salt). Bioluminescence declined in a concentration-dependent manner in the concentration range tested (0-30 microM Cd), with comparable sensitivity to reproduction and developmental assay end points (after 67 and 72 h, respectively). Cd concentrations that resulted in 20% reduction in bioluminescence (EC(20)) were 11.8-13.0 microM, whereas the reproduction EC(20) (67 h exposure) was 10.2 microM. At low concentrations of Cd (< or = 15 microM), the decline in bioluminescence reflected a drop in ATP levels. At Cd concentrations of 15-30 microM, decreased bioluminescence was attributable both to effects of Cd on ATP levels and decreased production of luciferase proteins, concomitant with a decline in protein levels. We show that whole-animal bioluminescence is a valid toxicological end point and a rapid and sensitive predictor of effects of Cd exposure on development and reproduction. This provides a platform for high-throughput sublethal screening and will potentially contribute to reduction of testing in higher animals.

Toxicity of the bacterial luciferase substrate, n-decyl aldehyde, to Saccharomyces cerevisiae and Caenorhabditis elegans

This study determined that the bacterial luciferase fusion gene (luxAB) was not a suitable in vivo gene reporter in the model eukaryotic organisms Saccharomyces cerevisiae and Caenorhabditis elegans. LuxAB expressing S. cerevisiae strains displayed distinctive rapid decays in luminescence upon addition of the bacterial luciferase substrate, n‐decyl aldehyde, suggesting a toxic response. Growth studies and toxicity bioassays have subsequently confirmed, that the aldehyde substrate was toxic to both organisms at concentrations well tolerated by Escherichia coli. As the addition of aldehyde is an integral part of the bacterial luciferase activity assay, our results do not support the use of lux reporter genes for in vivo analyses in these model eukaryotic organisms.

Impact of Sublethal Levels of Environmental Pollutants Found in Sewage Sludge on a Novel Caenorhabditis elegans Model Biosensor

A transgenic strain of the model nematode Caenorhabditis elegans in which bioluminescence reports on relative, whole-organism ATP levels was used to test an environmentally-relevant mixture of pollutants extracted from processed sewage sludge. Changes in bioluminescence, following exposure to sewage sludge extract, were used to assess relative ATP levels and overall metabolic health. Reproductive function and longevity were also monitored. A short (up to 8 h) sublethal exposure of L4 larval stage worms to sewage sludge extract had a concentration-dependent, detrimental effect on energy status, with bioluminescence decreasing to 50–60% of the solvent control (1% DMSO). Following longer exposure (22–24 h), the energy status of the nematodes showed recovery as assessed by bioluminescence. Continuous exposure to sewage sludge extract from the L4 stage resulted in a shorter median lifespan relative to that of solvent or medium control animals, but only in the presence of 400–600 µM 5-fluoro-2′-deoxyuridine (FUdR), which was incorporated to inhibit reproduction. This indicated that FUdR increased lifespan, and that the effect was counteracted by SSE. Exposure to sewage sludge extract from the L1 stage led to slower growth and a delayed onset of egg laying. When L1 exposed nematodes reached the reproductive stage, no effect on egg laying rate or egg number in the uterus was observed. DMSO itself (1%) had a significant inhibitory effect on growth and development of C. elegans exposed from the L1 stage and on reproduction when exposed from the L4 stage. Results demonstrate subtle adverse effects on C. elegans of a complex mixture of environmental pollutants that are present, individually, in very low concentrations and indicate that our biosensor of energy status is a novel, sensitive, rapid, quantitative, whole-organism test system which is suitable for high throughput risk assessment of complex pollutant mixtures.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode’s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.