The addition of occidiofungin had no effect on the polymerization or depolymerization properties of F-actin (see Fig. S3). Therefore, additional studies related to the perturbation of actin-based functions were performed to confirm that actin was the biological target of occidiofungin. As a dimorphic fungus,
C. albicans can grow as a yeast or hypha, and the ability to switch between these forms is linked to the pathogenicity of the organism (
29). Several reports have established that actin cables play an important role in the switch between yeast and hyphal forms (
30,
31). The impact of the antifungal on the morphological switching of
C. albicans cells was tested. The incubation of
C. albicans with a subinhibitory concentration of occidiofungin blocked hyphal formation in cells that were induced to undergo morphological switching (
Fig. 3 and Table S5). The morphogenesis of
C. albicans from yeast to filamentous forms involves actin dynamics, as treatments with cytochalasin A and latrunculin A or the elimination of myosin I function prevent hyphal formation (
32,
33). In
C. albicans, the maintenance of the actin scaffold is also necessary for endocytosis, DNA segregation, and cell division (
34,
35). To determine whether occidiofungin impacts other cellular activities linked to actin dynamics, we evaluated the effect of occidiofungin on endocytosis in fission yeast by staining cells with FM-464 (
Fig. 4). Cells exposed to 0.5× MIC and 1× MIC demonstrated concentration-dependent reductions in stained endocytic vesicles. Actin has also been linked to the proper positioning of the mitotic spindle during cell division, and mutants that lack actin cables exhibit an accumulation of multinucleated cells (
36,
37). Within 30 min of exposure, both
S. cerevisiae and
C. albicans cultures treated with a subinhibitory concentration of occidiofungin had more binucleated cells indicative of a disruption or a delay in nuclear transit through the mother-daughter neck (see Table S3). To further characterize the role of actin in the cellular response to occidiofungin, we analyzed haploid
S. cerevisiae mutants deleted for genes linked to actin polymerization and depolymerization. The deletant mutants were chosen for their role in actin organization and dynamics and screened for any deviation in observed activity (increase or decrease in occidiofungin susceptibility). Of the eighteen strains tested, only the Δ
tpm1 mutant showed altered sensitivity to occidiofungin, with the deletion mutant exhibiting a 4-fold resistance to occidiofungin (see Table S4). The observed increase in resistance to occidiofungin in the absence of the
tpm1 gene, which codes for the major isoform of tropomyosin, may be due to the mutant’s increased tolerance of cellular stressors (unpublished data) or a decrease in the cellular growth rate (
38). A decrease in cellular growth has previously been linked to occidiofungin resistance (
39).
To directly determine the effect of occidiofungin on actin organization
in vivo, fluorescence microscopy was carried out on diploid cells of
S. cerevisiae exposed to subinhibitory concentrations of occidiofungin. Within 30 min of exposure, an accumulation of actin patches and/or aggregates of F-actin were observed throughout the treated cells with a concomitant loss of actin cables (
Fig. 5A and
B; see also Table S6). Actin cables are formed by bundling F-actin. In
Fig. 5, the punctate structures in these cells are still likely filamentous actin, but occidiofungin disrupts the organization of F-actin to form cables at subinhibitory concentrations.