Assays for Toxicity Studies in &lt;i&gt;C. elegans&lt;/i&gt; With Bt Crystal Proteins

Bischof, Larry J.; Huffman, Danielle L.; Aroian, Raffi V.

doi:10.1385/1-59745-151-7:139

Cited by 77 publications

(134 citation statements)

References 30 publications

Supporting

Mentioning

132

Contrasting

Order By: Relevance

“…With this in mind, we wanted to find out whether the Bmp1 protein could enhance the activity of Cry5Ba against C. elegans. To investigate this, we conducted quantitative growth analyses, mortality tests, and brood size assays to test whether the enhanced activity occurs (18). For the quantitative growth test, the bioassay data against C.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Bacillus thuringiensis Metalloproteinase Bmp1 Functions as a Nematicidal Virulence Factor

Luo

Chen

Huang

et al. 2013

Appl Environ Microbiol

View full text Add to dashboard Cite

Some Bacillus thuringiensis strains have high toxicity to nematodes. Nematicidal activity has been found in several families of crystal proteins, such as Cry5, Cry6, and Cry55. The B. thuringiensis strain YBT-1518 has three cry genes that have high nematicidal activity. The whole genome sequence of this strain contains multiple potential virulence factors. To evaluate the pathogenic potential of virulence factors, we focused on a metalloproteinase called Bmp1. It encompasses a consecutive N-terminal signal peptide, an FTP superfamily domain, an M4 neutral protease GluZincin superfamily, two Big-3 superfamily motifs, and a Grampositive anchor superfamily motif as a C-terminal domain. Here, we showed that purified Bmp1 protein showed metalloproteinase activity and toxicity against Caenorhabditis elegans (the 50% lethal concentration is 610 ؎ 9.37 g/ml). In addition, mixing Cry5Ba with Bmp1 protein enhanced the toxicity 7.9-fold (the expected toxicity of the two proteins calculated from their separate toxicities) against C. elegans. Confocal microscopic observation revealed that Bmp1 protein was detected from around the mouth and esophagus to the intestine. Striking microscopic images revealed that Bmp1 degrades intestine tissues, and the Cry5Ba causes intestinal shrinkage from the body wall. Thus, the B. thuringiensis Bmp1 metalloproteinase is a nematicidal virulence factor. These findings give a new insight into the relationship between B. thuringiensis and its host nematodes. Bacillus thuringiensis is a rod-shaped, Gram-positive, sporeforming bacterium. It is the most successful insect pathogen used for insect control (1). The action to the insect pest relies on insecticidal toxin and array of virulence factors (2). Upon sporulation, it produces insecticidal crystal inclusion that is formed by a variety of insecticidal proteins called Cry or Cyt proteins. These insecticidal crystal proteins are toxic to insects in the orders Lepidoptera, Dipteran, Coleoptera, Hymenoptera, Homoptera, Orthoptera, and Mallophage (2), and they are also toxic to nematodes. Until now, there are several families of Cry proteins (Cry5, Cry6, Cry12, Cry13, Cry14, Cry21, and Cry55) known to be toxic to the larvae of a number of free-living or parasitic nematodes (3). Besides having crystal proteins that are toxic to insects, B. thuringiensis has many virulence factors that contribute to its pathogenic effects. These virulence factors contain exotoxins and extracellular proteases (2). During the stationary growth phase, some B. thuringiensis strains secrete exotoxins, which are heat-stable, watersoluble, and low-molecular-mass compounds (701 Da). These compounds are highly toxic to a wide range of insect species by the oral route (4, 5). In addition, extracellular proteases, e.g., serine protease, chitinase, and collagenase (6), have been reported to be insect pests virulence factors (2).In the past few years, some insecticidal virulence factors have been isolated from B. thuringiensis. The action mode of virulence factors against insect c...

show abstract

Section: Resultsmentioning

confidence: 99%

“…The entire bioassays were repeated a minimum of three times to calculate the activity for each dose-response of proteins. The bioassay procedure and 50% lethal concentration (LC 50 ) evaluation were undertaken according to the method of Bischof et al (18).…”

mentioning

confidence: 99%

Bacillus thuringiensis Metalloproteinase Bmp1 Functions as a Nematicidal Virulence Factor

Luo

Chen

Huang

et al. 2013

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…19. Images were collected after treating adult worms with ascribed compound in M9 media for 1 h and were then washing with M9 media to clear each probe from the digestive track, unless noted otherwise.…”

Section: Supplementary Materialsmentioning

confidence: 99%

Metabolite Induction of Caenorhabditis elegans Dauer Larvae Arises via Transport in the Pharynx

et al. 2008

View full text Add to dashboard Cite

Caenorhabditis elegans sense natural chemicals in their environment and use them as cues to regulate their development. This investigation probes the mechanism of sensory trafficking by evaluating the processing of fluorescent derivatives of natural products in C. elegans. Fluorescent analogs of daumone, an ascaroside, and apigenin were prepared by total synthesis and evaluated for their ability to induce entry into a nonaging dauer state. Fluorescent imaging detailed the uptake and localization of every labeled compound at each stage of the C. elegans life cycle.Comparative analyses against natural products that did not induce dauer indicated that dauertriggering natural products accumulated in the cuticle of the pharnyx. Subsequent transport of these molecules to amphid neurons signaled entry into the dauer state. These studies provide cogent evidence supporting the roles of the glycosylated fatty acid daumone and related ascarosides and the ubiquitous plant flavone apigenin as chemical cues regulating C. elegans development.In nature, the multifaceted lifecycle of nematodes typically involves passage through multiple host-guest relations (1). While genomic and proteomic experiments provide a blueprint of an organism, they do not provide a complete explanation of the molecular communication between organisms and their environment. Environmental signals often trigger responses that are far more complex than those elicited by a single genetic or proteomic event (2). Within recent years, the discovery and elucidation of small molecule signals that induce C. elegans into a dauer or resting state has opened a new avenue for understanding how environmental cues regulate development and aging (3). In this investigation, we focus attention on the physiological mechanisms by which C. elegans sense and process fluorescent analogs of natural products. Our studies compare endogenous and xenobiotic signals with an attempt to identify the molecular plasticity of the C. elegans HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptC. elegans sense environmental signals in part through ciliated amphids at the tips of their heads and phasmids in their tails (4-6). Early tracking experiments established that fluorescent dyes such as fluorescein isothiocyanate (FITC) move from solution through the cilia at the top of their heads to sensory neurons within the amphid (7). While genetic approaches have uncovered aspects of the signal transduction pathways involved in this reception (8-10), the underlying mechanisms by which the chemical signals are transported and processed within C. elegans remain unresolved.Small molecules, including damone (1), trigger the entry of L1 larvae into a nonaging dauer state. Recently, the structure of 1 was determined by isolation from large scale extracts of C. elegans followed by a combination of NMR studies (11) and total syntheses (11,12). Using this molecule as a starting point, we investigated the transport of 1 and other non-dauerinducing natural products in C. elegans....

show abstract

“…An L1 larvae growth assay and a lethality assay were performed by the methods of Bischof et al and Wang et al 4,32) When the lethality of the L4 worms was determined, two different methods were applied. The assays were set up with snodprot solution of 50 mmol/L of sodium citrate buffer (pH 6.0) and 50-100 L4 worms, which was incubated for 24 h, or with E. coli OP50 0 in S medium, chloromycetin, 5-fluorodeoxyuridine, the protein, and approximately 20 L4 worms, and this was incubated for 5 d at 25…”

Section: Methodsmentioning

confidence: 99%

The Function of Snodprot in the Cerato-Platanin Family fromDactylellina cionopagain Nematophagous Fungi

Duan²,

Wang

et al. 2012

Bioscience, Biotechnology, and Biochemistry

View full text Add to dashboard Cite

Assays for Toxicity Studies in <i>C. elegans</i> With Bt Crystal Proteins

Cited by 77 publications

References 30 publications

Bacillus thuringiensis Metalloproteinase Bmp1 Functions as a Nematicidal Virulence Factor

Bacillus thuringiensis Metalloproteinase Bmp1 Functions as a Nematicidal Virulence Factor

Metabolite Induction of Caenorhabditis elegans Dauer Larvae Arises via Transport in the Pharynx

The Function of Snodprot in the Cerato-Platanin Family fromDactylellina cionopagain Nematophagous Fungi

Contact Info

Product

Resources

About