Antifungal activity of microbial secondary metabolites

Abstract

Secondary metabolites are well known for their ability to impede other microorganisms. Reanalysis of a screen of natural products using the Caenorhabditis elegans-Candida albicans infection model identified twelve microbial secondary metabolites capable of conferring an increase in survival to infected nematodes. In this screen, the two compound treatments conferring the highest survival rates were members of the epipolythiodioxopiperazine (ETP) family of fungal secondary metabolites, acetylgliotoxin and a derivative of hyalodendrin. The abundance of fungal secondary metabolites indentified in this screen prompted further studies investigating the interaction between opportunistic pathogenic fungi and Aspergillus fumigatus, because of the ability of the fungus to produce a plethora of secondary metabolites, including the well studied ETP gliotoxin. We found that cell-free supernatant of A. fumigatus was able to inhibit the growth of Candida albicans through the production of a secreted product. Comparative studies between a wild-type and an A. fumigatus ΔgliP strain unable to synthesize gliotoxin demonstrate that this secondary metabolite is the major factor responsible for the inhibition. Although toxic to organisms, gliotoxin conferred an increase in survival to C. albicans-infected C. elegans in a dose dependent manner. As A. fumigatus produces gliotoxin in vivo, we propose that in addition to being a virulence factor, gliotoxin may also provide an advantage to A. fumigatus when infecting a host that harbors other opportunistic fungi.

Figure 1. Compound structures of secondary metabolites able to confer an increase in survival to Candida-infected nematodes.

The maximum nematode survival (%) and molecular weights are indicated for each of the compounds. Structures were provided by Analyticon Discovery.

Figure 2. Dose response of two ETP compounds from the C. albicans-C. elegans antifungal discovery assay.

A22 and A3 were as effective as amphotericin B in increasing nematode survival, however the decrease observed for compound A3 suggests there maybe toxicity associated with the compound at higher concentrations. The dose response experiment was conducted a single time as previously reported in Okoli et al., 2009.

Figure 3. Inhibition of C. albicans by A.f. supernatents and gliotoxin.

C. albicans strains DAY185 (A) and 95–120 (B) grown on Spider medium at 37°C overnight in the presence of discs containing the indicated amounts of AF293 supernatent (A.f. sup); supernatant from the ΔgliP mutant unable to synthesize gliotoxin (gliP sup); or pure gliotoxin. The growth of C. albicans strain 98–145 in the presence of the treatments was similar to 95–120 (data not shown). C. C. neoformans wild type strain KN99α grown on YPD at 30°C overnight in the presence of discs containing indicated amounts of A.f. sup or gliotoxin.

Figure 4. Assessment of C. albicans viability after treatment with gliotoxin.

Confocal laser microscopy of C. albicans after staining with the Live/Dead staining system, whereby dead cells stain red and live cells stain green. C. albicans strain DAY185 was grown overnight in YPD at 30°C or in Spider medium at 37°C, treated with DMSO, A. fumigatus supernatent (AFS; 3.2 mg/ml), or gliotoxin (2.0 µg/ml). The yellowish color is reflective of co-localization of both red and green dyes suggesting the cells have increased permeability and maybe potentially dying or already dead. White arrows showing intact nucleus with green color in DMSO and AFS treated cells, and partially dead nucleus with yellowish color in gliotoxin treated cells. There was growth (++) in case of DMSO treated cells, partial growth (+) in A.f. sup treated cells, and no growth (-) in gliotoxin treated cells. Gliotoxin treated cells were centrifuged before microscopy.

Figure 5. C. elegans-C. albicans co-infection assay to assess the ability of gliotoxin to promote nematode survival.

Representative assay wells from the C. elegans-C. albicans infection assay. The wells were treated with DMSO (negative control) or the indicated concentrations of A.f. supernatant (A.f. sup), gliotoxin, or C. neoformans supernatant (C.n. sup). Sinusoidal shaped worms are alive (thick black arrows) and rod shaped nematodes are dead (thin arrows).