Methods: Using a broth dilution method, we determined the minimum fungicidal concentration (MFC) of a high molecular-weight (HMW) (150,000Da) water-soluble chitosan, a derivative shown in our previous study to be effective against common oral fungi, such as Candida albicans and the fluconazole-resistant species C.krusei. We employed Saccharomyces cerevisiae as a model for genome-wide analysis using the haploid yeast deletion library, a collection of ~5,000 yeast strains with individual deletions of virtually all non-essential Open Reading Frames in the yeast genome. The strains were spotted onto media containing a sublethal concentration of chitosan. Yeast strains that showed significantly less growth on chitosan-containing media than the wild-type were selected for further analysis.
Results: The MFC of the HMW water-soluble chitosan against S.cerevisiae was 2.5 mg/ml. A sublethal concentration of 0.7 mg/ml was chosen for the screen as it does not affect growth of the wild-type. From the library, 33 deletion strains are markedly hypersensitive to chitosan. Gene ontology analyses of this gene list indicated an enrichment of genes whose products localize in membranes and cytoskeleton, and those involved in cellular stress response, signal transduction, and regulation of metabolism and RNA synthesis.
Conclusions: Our genome-wide screen suggested that HMW water-soluble chitosan likely act on fungi by perturbing cell membranes and inducing cellular stress.