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A modified screen for the detection of cell wall-acting antifungal compounds.

One would logically expect that antifungal agents which act via the inhibition of cell wall biosynthesis should show fungicidal activity and low toxicity as the cell wall is essential for fungal cell growth and is not synthesized by mammalian cells. A variety of cell wall-acting antifungal agents have been discovered and some of these have become economically important1~3). However, new nontoxic antifungal agents are needed, especially for medical applications". While a variety of screens have been described for cell wall-acting antibacterial agents5~8), less work has been published on the identification of agents which inhibit fungal cell wall synthesis. A screen was recently described by SELITRENNIKOFF9) in which cell wall-acting antibiotics are detected by their ability to block the regeneration of Neurospora crassa protoplasts. This assay is based upon the characteristics of a N. crassa strain which carries a temperature sensitive allele of os-1 (allele NM233(t)). Although strains carrying this mutation produce a normal cell wall, mutations at the os-1 locus permit N. crassa cells to divide and grow as protoplasts on osmotically supported media, providing that the cell wall is first removed either by enzymatic or antibiotic treatment10). Strains carrying a temperature sensitive os-1 allele are able to grow as protoplasts at the restrictive temperature (37°C) and will regenerate a cell wall if shifted to the permissive temperature (22°C). In the screen described by SELITRENNIKOFF, plates are inoculated with a preparation of protoplasts, shifted to the permissive temperature and antibiotic disks are placed on the media. Most antibiotics produce a clear zone of growth inhibition while cell wall-acting antibiotics produce a hazy zone containing protoplasts which are unable to regenerate but, lacking a wall, are resistant to the antibiotic. These zones are somewhat difficult to score because the protoplasts grow poorly at 22°C. In addition, we were discouraged from employing this assay for mass screening because of the need to prepare and maintain large scale cultures of protoplasted cells. We argued, however, that it might be possible to employ this idea for screening without handling protoplasts by exploiting an inversion of the original principle. SELITRENNIKOFF'S method was based on the preparation of protoplasts by antibiotic treatment (polyoxin to inhibit chitin synthesis and sorbose to inhibit glucan synthesis) and screening for antibiotics which maintained the protoplasted state. We reasoned that it should be possible to detect cell wall inhibitors by virtue of their ability to protoplast walled os-1 cells grown on osmotically supported media. It also seemed likely that the response might be improved for the following reason. In SELITRENNIKOFF's assay, good protoplast growth is necessary to distinguish general inhibitory antibiotics which kill protoplasts from cell wall inhibitory antibiotics which are inactive against protoplasts. However, this assay is performed at the permissive temperature (22°C), a temperature at which the Neurospora cells would be expected to grow poorly as protoplasts. In screening for antibiotics which induce protoplast formation, one would perform the assay at the restrictive temperature (37°C) which would encourage protoplast growth.