Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1996-11-27
2003-07-08
Richter, Johann (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
Reexamination Certificate
active
06589762
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates to the identification of potential fungicides and insecticides using a screening method that reverses the calcofluor inhibition of chitin biosynthesis in yeast.
BACKGROUND OF THE INVENTION
The polysaccharide chitin is a structural cell wall component of all fungi except some
Basidiomycetes fungi
and most Oomycetes, and is the most abundant organic skeletal component of invertebrates, making up, for example, from about 25 to 60% of the dry weight of insect cuticles. Chitin consists primarily of linear polymers of the amino sugar N-acetyl-D-glucosamine joined in 1,4-&bgr;-glucosidic linkage. Thus, chitin bears a close resemblance to cellulose, the major structural polysaccharide of plants; indeed, the only chemical difference between them is that in chitin the hydroxyl group on the 2-position is an acetoamido group instead of an hydroxyl. However, because of its widespread occurrence in fungi and arthropods, the total world-wide production of chitin vastly exceeds cellulose (Neville, A. C.,
The Biology of the Arthropod Cuticle,
Springer-Verlag, New York, 1975, pages 71 to 76).
Many fungi and arthropods having chitinous cell walls or exoskeletons are injurious to plants and animals, causing a legion number of diseases. To name but a few, fungal species containing chitin cause wheat eyespot, rice sheath blight, damping off, apple scab, pepper botrytis, rice blast, sugar beet cercospora, tomato early blight, wheat leaf rust, and wheat powdery mildew. Fungal species also cause myriad cutaneous and systemic mycoses in human beings and other animals, including candidiasis, histoplasmosis, blastomycosis, sporotrichosis, cryptococcosis, and the like. Insects are vectors of viruses causing arboviral encephalitides, yellow fever, and dengue, protozoa causing malarias, trypanosomiases, and leishmaniases, and various harmful helminths. Crustaceans carry some infectious helminths and trematodes.
Most fungicides and insecticides that are used to control or cure these diseases by killing or controlling their causative agents, intermediate hosts, or vectors employ various modes of action including physical poisons that suffocate or dessicate organisms; protoplasmic poisons such as arsenicals that kill by precipitating or deactivating proteins, enzymes or other cellular constituents; respiratory poisons that deactivate respiratory enzymes; and various poisons that affect different tissue systems such as tubules or nerves. Of course, preferred agents do not injure the host plant or animal, and most preferably have no effect whatsoever on the host. Because of the complexity and interdependence of life processes, however, this goal is not always achieved, so that many fungicides and insecticides exhibit some toxicity to the host. Others cause unexpected side effects.
Since chitin is not a usual constituent of most plants and vertebrates, chitin biosynthesis inhibitors can be employed as selective antifungal and/or insecticide agents. Applied to ornamental or edible plants or animals, these offer the advantage of targeting undesirable fungi or insects without harming significantly the host plant or vertebrate animal. 1-(2,6-Dichlorobenzoyl)-3-(3,4-dichlorophenyl)urea, for example, has been suggested as a chitin-inhibiting insecticide (Neville, cited above). Antifungals that inhibit chitin synthesis include nikkomycin and polyoxin D.
Calcofluor white is a fluorescent brightener used commercially to whiten textiles and paper. The fluorochrome has been used as a stain for cell wall materials in fungi, algae, and higher plants. It can exhibit antifungal properties, binding to nascent chitin microfibrils in fungal cell walls containing chitin. Exposure of yeast (
Saccharomyces cerevisiae
) to calcofluor white, for example, induces abnormally thick walls between mother and daughter cells during cell division as a result of the massive deposition of anomalous crystallized chitin. The dye interaction appears to enhance the rate of chitin polymerization, producing levels of chitin that are inhibitory to cell growth and viability (Roncero, C., et al.,
J. Bact.
170: 1945-1949 (1988)). Microscopic examination of calcofluor-inhibited cells reveals high levels of chitin deposition. This putative mechanism for calcofluor white action is supported by the observation that mutants selected for calcofluor white resistance show decreased levels of chitin synthesis (Roncero, C., et al.,
J. Bact.
170: 1950-1954 (1988)).
The interaction of calcofluor white with growing
S. cerevisiae
cells generally requires a pH of above 4 and close to 6 or 6.5 (Roncero, et al., cited above at 1946). This can perhaps be understood in view of the fact that a major chitinase in yeast is active only under acidic conditions (Correa, J., et al.,
J. Biol. Chem.
257: 1392-1397 (1982)). It is possible that this enzyme opposes the action of calcofluor, and thus the activity of calcofluor is augmented under conditions of reduced chitinase activity.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a screening test for the identification of agents exhibiting potential fungicidal and insecticidal activity for a wide variety of agricultural, medical, and veterinary uses.
It is a further and more specific object of the invention to identify agents that inhibit chitin biosynthesis.
These and other objects are accomplished by the present invention, which provides a method for the identification of agents which inhibit chitin synthesis, and thus possess fungicidal and insecticidal activity making them potentially suitable as selective fungicides or insecticides. The method is a screening test whereby test samples are incubated in a fungal culture with calcofluor white. Agents exhibiting potentially desirable fungicidal or insecticidal properties inhibit chitin synthesis, and, by doing so, reverse calcofluor white inhibition of the culture. Agents that are positive in the test produce enhanced fungus growth because they rescue the growing fungus from the adverse effects of calcofluor white.
In the practice of this inventive method for screening for the presence or absence of chitin synthesis inhibition by a test sample, the test sample is added to a chitin-producing fungus, e.g., a yeast, culture or culture area containing calcofluor white. The culture is incubated with the test sample for such time under such conditions sufficient to observe yeast cell growth inhibition in a corresponding culture containing calcofluor but no test sample. The extent of growth in the culture or culture area containing test sample is then compared with the extent of growth in the culture or culture area containing no test sample. The presence of chitin synthesis inhibition is determined by observation of whether culture growth in the presence of test sample exceeds growth in its absence.
In a preferred screening test, a
Saccharomyces cerevisiae
strain exhibiting little or no calcofluor white resistance is grown in culture at neutral pH in the presence of calcofluor white and test samples. Potentially active agents are identified by the observation of enhanced growth of the cultured yeast. In especially preferred embodiments, a positive control is employed to assist in the identification of potential agents. In these embodiments, a known chitin sythesis inhibitor such as nikkomycin Z is added to the culture or culture area, and this is compared to the culture with the test sample.
In a particularly preferred embodiment, the yeast is grown in a solidified media in the presence of calcofluor in a plate or dish, so that test samples and positive controls can be observed visually and simultaneously as regions of the same culture. Actives produce a turbid zone of growth around the test sample in the lawn of the culture.
DETAILED DESCRIPTION OF THE INVENTION
This invention is based upon the finding that chemical and biochemical agents of potential value as fungicides or insecticides are identified in
Saccharomyces cerevisiae
cultures containing calcofluor white, a fluorochrome that causes le
Hsien-Fen Kuh Lai Margaret
Kirsch Donald Richard
Silverman Sanford J.
BASF - Aktiengesellschaft
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Owens Howard V.
Richter Johann
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