Bailey DC et al. (2017) Environ Toxicol Pharmacol "Chronic exposure to a glyphosate-containing pesticide leads to mitochondrial dysfunction and increased reactive oxygen species production in Caenorhabditis elegans."

Pressley AS, Denney RD, Snapp IB, Negga R, Burchfield SL, Traynor WL, Bailey DC, Fitsanakis VA, Todt CE

[Environ Toxicol Pharmacol, 2017]

Glyphosate-containing herbicides are among the most widely-used in the world. Although glyphosate itself is relatively non-toxic, growing evidence suggests that commercial herbicide formulations may lead to increased oxidative stress and mitochondrial inhibition. In order to assess these mechanisms in vivo, we chronically (24h) exposed Caenorhabditis elegans to various concentrations of the glyphosate-containing herbicide TouchDown (TD). Following TD exposure, we evaluated the function of specific mitochondrial electron transport chain complexes. Initial oxygen consumption studies demonstrated inhibition in mid- and high-TD concentration treatment groups compared to controls. Results from tetramethylrhodamine ethyl ester and ATP assays indicated reductions in the proton gradient and ATP levels, respectively. Additional studies were designed to determine whether TD exposure resulted in increased reactive oxygen species (ROS) production. Data from hydrogen peroxide, but not superoxide or hydroxyl radical, assays showed statistically significant increases in this specific ROS. Taken together, these data indicate that exposure of Caenorhabditis elegans to TD leads to mitochondrial inhibition and hydrogen peroxide production.

Negga R et al. (2011) Neurotoxicology "Exposure to Mn/Zn ethylene-bis-dithiocarbamate and glyphosate pesticides leads to neurodegeneration in Caenorhabditis elegans."

Fitsanakis VA, Negga R, Fields AS, Davis NS, Rudd DA, Hatfield HE, Valente AL, Justice AN

[Neurotoxicology, 2011]

Epidemiological evidence suggests positive correlations between pesticide usage and the incidence of Parkinson's disease (PD). To further explore this relationship, we used wild type (N2) Caenorhabditis elegans (C. elegans) to test the following hypothesis: Exposure to a glyphosate-containing herbicide (TD) and/or a manganese/zinc ethylene-bis-dithiocarbamate-containing fungicide (MZ) may lead to neurotoxicity. We exposed N2 worms to varying concentrations of TD or MZ for 30 min (acute) or 24h (chronic). To replicate agricultural usage, a third population was exposed to TD (acute) followed by MZ (acute). For acute TD exposure, the LC(50)=8.0% (r(2)=0.6890), while the chronic LC(50)=5.7% (r(2)=0.9433). Acute MZ exposure led to an LC(50)=0.22% (r(2)=0.5093), and chronic LC(50)=0.50% (r(2)=0.9733). The combined treatment for TD+MZ yielded an LC(50)=12.5% (r(2)=0.6367). Further studies in NW1229 worms, a pan-neuronally green fluorescent protein (GFP) tagged strain, indicated a statistically significant (p<0.05) and dose-dependent reduction in green pixel number in neurons of treated worms following each paradigm. This reduction of pixel number was accompanied by visual neurodegeneration in photomicrographs. For the dual treatment, Bliss analysis suggested synergistic interactions. Taken together, these data suggest neuronal degeneration occurs in C. elegans following treatment with environmentally relevant concentrations of TD or MZ.

McVey KA et al. (2016) Neurotoxicol Teratol "Exposure of C. elegans eggs to a glyphosate-containing herbicide leads to abnormal neuronal morphology."

Johnson MB, McVey KA, Negga R, Snapp IB, Fitsanakis VA, Pressley AS

[Neurotoxicol Teratol, 2016]

Recent data demonstrate that chronic exposure of Caenorhabditis elegans (C. elegans) to a high-use glyphosate-containing herbicide, Touchdown (TD), potentially damages the adult nervous system. It is unknown, however, whether unhatched worms exposed to TD during the egg stage show abnormal neurodevelopment post-hatching. Therefore, we investigated whether early treatment with TD leads to aberrant neuronal or neurite development in C. elegans. Studies were completed in three different worm strains with green fluorescent protein (GFP)-tagged neurons to facilitate visual neuronal assessment. Initially, eggs from C. elegans with all neurons tagged with GFP were chronically exposed to TD. Visual inspection suggested decreased neurite projections associated with ventral nerve cord neurons. Data analysis showed a statistically significant decrease in overall green pixel numbers at the fourth larval (L4) stage (*p<0.05). We further investigated whether specific neuronal populations were preferentially vulnerable to TD by treating eggs from worms that had all dopaminergic (DAergic) or -aminobutyric acid (GABAergic) neurons tagged with GFP. As before, green pixel number associated with these discrete neuronal populations was analyzed at multiple larval stages. Data analysis indicated statistically significant decreases in pixel number associated with DAergic, but not GABAergic, neurons (***p<0.001) at all larval stages. Finally, statistically significant decreases (at the first larval stage, L1) or increases (at the fourth larval stage, L4) in superoxide levels, a developmental signaling molecule, were detected (*p<0.05). These data suggest that early exposure to TD may impair neuronal development, perhaps through superoxide perturbation. Since toxic insults during development may late render individuals more vulnerable to neurodegenerative diseases in adulthood, these studies provide some of the first evidence in this model organism that early exposure to TD may adversely affect the developing nervous system.

Burchfield SL et al. (2018) Environ Toxicol Pharmacol "Acute exposure to a glyphosate-containing herbicide formulation inhibits Complex II and increases hydrogen peroxide in the model organism Caenorhabditis elegans."

Fitsanakis VA, Todt CE, Burchfield SL, Denney RD, Negga R, Bailey DC

[Environ Toxicol Pharmacol, 2018]

Glyphosate-based herbicides, such as Touchdown (TD) and Roundup, are among the most heavily-used herbicides in the world. While the active ingredient is generally considered non-toxic, the toxicity resulting from exposure to commercially-sold formulations is less clear. In many cases, cell cultures or various model organisms exposed to glyphosate formulations show toxicity and, in some cases, lethality. Using Caenorhabditis elegans, we assessed potential toxic mechanisms through which a highly-concentrated commercial formulation of TD promotes neurodegeneration. Following a 30-min treatment, we assayed mitochondrial electron transport chain function and reactive oxygen species (ROS) production. Initial oxygen consumption studies indicated general mitochondrial inhibition compared to controls (^*p<0.05). When Complex II activity was further assessed, inhibition was observed in all TD-treated groups (^*p<0.05). Complex IV activity, however, was not adversely affected by TD. This electron transport chain inhibition also resulted in reduced ATP levels (^*p<0.05). Furthermore, hydrogen peroxide levels, but not other ROS, were increased (^*p<0.05). Taken together, these data indicate that commercially-available formulations of TD may exert neurotoxicity through Complex II (succinate dehydrogenase) inhibition, decreased ATP levels, and increased hydrogen peroxide production.

Kriegeskorte A et al. (2014) MBio "Inactivation of thyA in Staphylococcus aureus attenuates virulence and has a strong impact on metabolism and virulence gene expression."

Kahl BC, Becker K, Lore NI, Baum C, Seggewiss J, Peters G, Neumann C, Hertel P, Liebau E, Drescher M, Block D, Bragonzi A, Proctor RA, Windmuller N, Kriegeskorte A, Mellmann A

[MBio, 2014]

Staphylococcus aureus thymidine-dependent small-colony variants (TD-SCVs) are frequently isolated from patients with chronic S. aureus infections after long-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX). While it has been shown that TD-SCVs were associated with mutations in thymidylate synthase (TS; thyA), the impact of such mutations on protein function is lacking. In this study, we showed that mutations in thyA were leading to inactivity of TS proteins, and TS inactivity led to tremendous impact on S. aureus physiology and virulence. Whole DNA microarray analysis of the constructed thyA mutant identified severe alterations compared to the wild type. Important virulence regulators (agr, arlRS, sarA) and major virulence determinants (hla, hlb, sspAB, and geh) were downregulated, while genes important for colonization (fnbA, fnbB, spa, clfB, sdrC, and sdrD) were upregulated. The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly. NupC was identified as a major nucleoside transporter, which supported growth of the mutant during TMP-SMX exposure by uptake of extracellular thymidine. The thyA mutant was strongly attenuated in virulence models, including a Caenorhabditis elegans killing model and an acute pneumonia mouse model. This study identified inactivation of TS as the molecular basis of clinical TD-SCV and showed that thyA activity has a major role for S. aureus virulence and physiology. Importance: Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus carry mutations in the thymidylate synthase (TS) gene (thyA) responsible for de novo synthesis of thymidylate, which is essential for DNA synthesis. TD-SCVs have been isolated from patients treated for long periods with trimethoprim-sulfamethoxazole (TMP-SMX) and are associated with chronic and recurrent infections. In the era of community-associated methicillin-resistant S. aureus, the therapeutic use of TMP-SMX is increasing. Today, the emergence of TD-SCVs is still underestimated due to misidentification in the diagnostic laboratory. This study showed for the first time that mutational inactivation of TS is the molecular basis for the TD-SCV phenotype and that TS inactivation has a strong impact on S. aureus virulence and physiology. Our study helps to understand the clinical nature of TD-SCVs, which emerge frequently once patients are treated with TMP-SMX.

Zuryn S et al. (2014) Science "Transdifferentiation. Sequential histone-modifying activities determine the robustness of transdifferentiation."

White ER, Margueron R, Zuryn S, Morin MC, Jarriault S, Ahier A, Portoso M

[Science, 2014]

Natural interconversions between distinct somatic cell types have been reported in species as diverse as jellyfish and mice. The efficiency and reproducibility of some reprogramming events represent unexploited avenues in which to probe mechanisms that ensure robust cell conversion. We report that a conserved H3K27me3/me2 demethylase, JMJD-3.1, and the H3K4 methyltransferase Set1 complex cooperate to ensure invariant transdifferentiation (Td) of postmitotic Caenorhabditis elegans hindgut cells into motor neurons. At single-cell resolution, robust conversion requires stepwise histone-modifying activities, functionally partitioned into discrete phases of Td through nuclear degradation of JMJD-3.1 and phase-specific interactions with transcription factors that have conserved roles in cell plasticity and terminal fate selection. Our results draw parallels between epigenetic mechanisms underlying robust Td in nature and efficient cell reprogramming in vitro.

Biswas SN et al. (1996) Res Commun Mol Pathol Pharmacol "A phenomenological model describing pharyngeal pulsing in the nematode Caenorhabditis elegans anesthetized by alcohol."

Okada H, Shingai R, Biswas SN, Ebina Y

[Res Commun Mol Pathol Pharmacol, 1996]

The recovery process of the suppressed pharyngeal pulsation in the nematode has been investigated for several concentrations of a homologous primary alcohol series (CnH2n-1OH, n = 1,2,3). A mathematical model describing the time course of the recovery process is phenomenologically constructed by using two time constants of delay time tD and recovery time tau. The values of tD and tau are obtained by fitting the equation to experimental data. The obtained values increase with increasing alcohol concentration. To observe the characteristics of tD and tau against the alcohol of order n, the inverse of these time constants are computed at 25 v/v% concentration and plotted on a semi-logarithmic scale. The plot curves decrease non-linearly and are dissimilar to the well-known curves illustrating the importance of lipid solubility in the cell membrane in anesthetic phenomena.

Bonaterra A et al. (2014) Int Microbiol "Phenotypic comparison of clinical and plant-beneficial strains of Pantoea agglomerans."

Badosa E, Duffy B, Montesinos E, Rezzonico F, Bonaterra A

[Int Microbiol, 2014]

Certain strains of Pantoea are used as biocontrol agents for the suppression of plant diseases. However, their commercial registration is hampered in some countries because of biosafety concerns. This study compares clinical and plant-beneficial strains of P. agglomerans and related species using a phenotypic analysis approach in which plant-beneficial effects, adverse effects in nematode models, and toxicity were evaluated. Plant-beneficial effects were determined as the inhibition of apple fruit infection by Penicillium expansum and apple flower infection by Erwinia amylovora. Clinical strains had no general inhibitory activity against infection by the fungal or bacterial plant pathogens, as only one clinical strain inhibited P. expansum and three inhibited E. amylovora. By contrast, all biocontrol strains showed activity against at least one of the phytopathogens, and three strains were active against both. The adverse effects in animals were evaluated in the plant-parasitic nematode Meloidogyne javanica and the bacterial-feeding nematode Caenorhabditis elegans. Both models indicated adverse effects of the two clinical strains but not of any of the plant-beneficial strains. Toxicity was evaluated by means of hemolytic activity in blood, and genotoxicity with the Ames test. None of the strains, whether clinical or plant-beneficial, showed any evidence of toxicity.

Zheng J et al. (2016) Proc Biol Sci "The Ditylenchus destructor genome provides new insights into the evolution of plant parasitic nematodes."

Ruan L, Zheng J, Xu M, Chen L, Chen F, Peng D, Liu H, Sun M, Ju S

[Proc Biol Sci, 2016]

Plant-parasitic nematodes were found in 4 of the 12 clades of phylum Nematoda. These nematodes in different clades may have originated independently from their free-living fungivorous ancestors. However, the exact evolutionary process of these parasites is unclear. Here, we sequenced the genome sequence of a migratory plant nematode, Ditylenchus destructor We performed comparative genomics among the free-living nematode, Caenorhabditis elegans and all the plant nematodes with genome sequences available. We found that, compared with C. elegans, the core developmental control processes underwent heavy reduction, though most signal transduction pathways were conserved. We also found D. destructor contained more homologies of the key genes in the above processes than the other plant nematodes. We suggest that Ditylenchus spp. may be an intermediate evolutionary history stage from free-living nematodes that feed on fungi to obligate plant-parasitic nematodes. Based on the facts that D. destructor can feed on fungi and has a relatively short life cycle, and that it has similar features to both C. elegans and sedentary plant-parasitic nematodes from clade 12, we propose it as a new model to study the biology, biocontrol of plant nematodes and the interaction between nematodes and plants.

Ganapathy M et al. (2014) Plant Cell Rep "Immunogenicity of Brugia malayi Abundant Larval Transcript-2, a potential filarial vaccine candidate expressed in tobacco."

Palaniswamy H, Perumal A, Ganapathy M, Mohan C, Perumal K

[Plant Cell Rep, 2014]

KEY MESSAGE: Transgenic tobacco plants with Bm ALT-2, a filarial vaccine candidate, were developed. The plant-produced antigen showed immunogenicity on par with the E.coli product. Transgenic tobacco plants were developed using Brugia malayi Abundant Larval Transcript-2 (Bm ALT-2), a major antigen produced from recombinant E.coli found to be experimentally successful as potential vaccine candidate against lymphatic filariasis. Results of experiments on the transformation and expression of the Bm ALT-2 in tobacco plant to produce plant-based vaccine are presented here. We have successfully transformed the tobacco plant with Bm ALT-2 and confirmed that the plants expressed the filarial protein by PCR analysis and Western blotting. The level of expression varied from 50 to 90ng/g of total soluble protein for ALT-2. Immunization of mice with plant-extracted protein indicated that the plant-produced protein had immunological characteristics similar to the E.coli-produced protein. Antibody titres produced by plant-produced recombinant ALT 2-immunized mice were on par with those immunized with recombinant protein produced by E.coli. Antibody isotype assay showed that plant-produced recombinant ALT-2 induced significant IgG1, whereas E.coli-produced recombinant ALT-2 induced IgG3. This result is a step forward towards the development of a model eukaryotic system for the production of recombinant filarial proteins, which can be utilized to produce therapeutic and diagnostic molecules against lymphatic filariasis, a neglected tropical infectious disease which has a negative impact on socioeconomic development. In addition, this is the first report of the immunogenicity of a plant-derived filarial antigen.