he 56-kb ocfC gene cluster is required for occidiofungin production by Burkholderia contaminans strain MS14. However, the function of the ocfC gene remains unknown. Sequence analysis showed the putative protein encoded by ocfC shares significant identities to glycosyltransferase. Plate bioassays showed that occidiofungin production by the ocfC mutant (ocfC::nptII) was significantly reduced as compared with strain MS14. The occidiofungin produced by the ocfC mutant is xyloseree, which suggests the ocfC gene encodes a xylosyltransferase to add a xylose to the peptide backbone. Single variant optimization of culture condition and medium compositions was also performed in this study. The results indicated that occidiofungin production was promoted with higher cell density inoculum, additional casamino acid, xylose, urea, zinc ions, and at pH 5. The findings have provided insights into development of pharmaceutical drug and agricultural biofungicide.

Candida albicans is a polymorphic fungus that can grow as yeast (Y) and hypha (H). The Y-H morphological switching is controlled through the MAPK, Cek1p. With the prior work showing that occidiofungin prevents C. albicans from forming hypha when added at the time of Y-H switching, we aim to identify the impact of occidiofungin on signaling events associated with morphological switching specifically looking at Cek1 MAPK cascade. Results from this work have demonstrated that Cek1 MAPK is not required for occidiofungin bioactivity. Further, we report that morphologically switching cells are more sensitive to occidiofungin than their non-switching counterparts. Moreover, later stages of hyphal formation exhibit increased sensitivity towards occidiofungin suggesting occidiofungin targets hyphal initiation and/or elongation process. This work also demonstrates that addition of occidiofungin beyond a discrete time window required for hyphal initiation has minimal effect on hyphae formation and elongation.

Occidiofungin is a novel glycolipopeptide, synthesized and secreted by Burkholderia contaminans MS14, demonstrating broad-spectrum antifungal activity and potential for successful clinical applications. Its mechanism of action has not yet been determined but is known to exhibit fungicidal activity via the induction of apoptosis in a manner unique from that of currently approved antifungals. As an early investigation into occidiofungin’s mechanism of action, we aimed to identify environmental and cellular factors that significantly alter the susceptibility of the model organism, Saccharomyces cerevisiae. To that end, we have demonstrated that occidiofungin’s bioactivity requires active cellular growth, that new protein synthesis is necessary to adequately respond to occidiofungin exposure, and that alterations in transcriptional regulation in response to glucose and phosphate deprivation have synergistic and antagonist consequences, respectively, on occidiofungin’s effectiveness. Together, this data provides a foundation on which occidiofungin’s mechanism of action can be illuminated.

Fungal infections have a significant impact on the world population, with estimates of over 1.6 million deaths a year. One contributing factor is the increasing number of fungi resistant to the current clinical treatments, including the last approved family of antifungal compounds introduced into the market over a decade ago. This is driving the search for new antifungals with different biological targets. A new potential antifungal occidiofungin, is a cyclic glycolipopeptide isolated from the soil bacterium Burkholderia contaminans MS14 with a broad spectrum of activity against both human and plant pathogens. Kill kinetics demonstrated that this compound is fungicidal and activates the cell wall integrity pathway at suboptimal dosing as determined by Mkc1 MAPK phosphorylation studies. As three of the four classes of currently available antifungals target ergosterol or ergosterol biosynthesis, the bioactivity of occidiofungin was assayed in the presence of ergosterol containing DOPC vesicles and was shown to retain antifungal properties. Occidiofungin was also found to have a similar activity profile against the S. cerevisiae -1,3-glucan synthesis mutant, indicating that it does not share a target with the fourth class of antifungals. Stability testing showed occidiofungin retained in vitro potency in the presence of human serum, across a broad range of pH and temperature conditions, and was resistant to gastric proteases. Based on cell morphology, occidiofungin did not target a specific stage of the yeast cell cycle, however cells were smaller in size and acquired ‘dancing bodies’, both properties of apoptosis. This was confirmed with data showing concentration dependent increases in DNA fragmentation, reactive oxygen species, and extracellular localization of phosphatidylserine. In addition to these findings, cells deleted for the yeast caspase gene exhibit a 2old resistance to occidiofungin. While SEM showed no morphological differences between treated and untreated cells, TEM did identify a thinning of the cell wall and inclusion bodies in cells treated with occidiofungin. As a stable fungicidal compound that induces apoptosis in yeast, occidiofungin has a great potential to become a new candidate drug for clinical use in treating fungal infections, including those resistant to current antifungals.

Invasive fungal infections are a significant clinical challenge especially for hospitalized patients as traditional antifungal therapy often fails to resolve these infections. The ability of Candida to undergo yeast-to-hyphae morphological transition is central to this invasive behavior. Morphogenesis is also important for the formation of biofilms which are highly structured communities of microorganisms attached to one another or substratum and embedded within a protective extracellular matrix material. The refractory nature of cells within a biofilm to current antifungal therapies has created a need for alternative antifungal agents for the management of Candida biofilm-related infections. The novel antifungal occidiofungin is a natural product produced by the soil bacteria Burkholderia contaminans shown to be effective against a broad range of fungi including Candida spp. Prior studies have demonstrated that occidiofungin inhibits yeast-to-hyphae morphogenesis in the dimorphic yeast, C. albicans, likely through its impact on disrupting F-actin organization. To extend these findings, the efficacy of occidiofungin on morphogenesis of C. albicans and C. tropicalis strains under different inducing conditions was evaluated. Further, given the role of biofilm on pathogenicity, the anti-biofilm properties of occidiofungin against Candida species was examined using an in vitro static biofilm model developed on a silicon elastomer disk. The accumulated data indicate that occidiofungin inhibits hyphal transformation regardless of the inducing conditions used and prevents hyphal extension when added to cells post switching. Moreover, morphologically switching cells were more sensitive to occidiofungin than their yeast counterpart. In addition, occidiofungin effectively blocks biofilm formation at all stages of development and reduces dispersed cells from the biofilm for both C. albicans and C. tropicalis. Confocal data revealed alterations in actin organization with occidiofungin treatment for both morphologically switching and biofilm cells. These findings correlate with prior observations for occidiofungin activity on yeast form cells indicating the broad activity of occidiofungin against fungi at various stages of pathogenic growth and supports efforts to pursue occidiofungin as a potential therapeutic against Candida based infections.

Occidiofungin is an antifungal compound that targets the conserved cytoskeletal protein, actin. Despite >90% amino acid conservation between fungal actin proteins, sensitivity to occidiofungin has been shown to vary with C. albicans, F. oxysporum, and P. digitatum exhibiting a resistant profile relative to S. cerevisiae. To determine whether differences in the amino acid sequences of actin contribute to differences in occidiofungin susceptibility, we expressed the actin gene from these fungal organisms in the ACT1 S. cerevisiae shuffle strain. Functionality of actin gene products was determined by measuring growth kinetics, actin protein levels, nuclear position, and actin cable formation. Results demonstrated functional complementation for all actin orthologs. Analysis of occidiofungin susceptibility found that fungal actin ortholog expression resulted in a similar sensitivity profile as the wildtype S. cerevisiae. These findings suggest that amino acid differences in actin are not directly responsible for the resistance to occidiofungin identified for these fungal organisms.