Morbidity and mortality associated with fungal infections have increased in recent decades, but current drugs are unable to meet the growing demand for antifungal therapeutics. We exploited the group III HHK-expressing S. cerevisiae reporter strain to establish a simple high-throughput bioassay to quickly screen microbial extracts, made from actinomycetes isolated from unexplored and underexplored ecological niches, to identify novel antifungal natural products. Our assay successfully detected antifungal microbial extracts with potent, broad-spectrum activity. Dereplication of one of the active extracts, made from Streptomyces sp. YIM56295, resulted in the identification of the macrotetrolides (nonactin, monactin, dinactin, and trinactin) as responsible for the observed antifungal activity. Whereas the macrotetrolides may not act directly upon group III HHKs, we obtained novel insights about the antifungal properties of this class of compounds, including broader and more potent activity than previously appreciated, and possible new insight about their mode of action.
The macrotetrolides are a group of closely related compounds produced by various Streptomyces
). They are broadly active antimicrobials which affect bacteria, fungi, and mites [32
]. Macrotetrolides were initially reported to have only limited activity against C. albicans and C. neoformans
. We investigated by disk diffusion whether they might have broader and more potent activity against fungal pathogens than previously reported because the macrotetrolides showed such potent activity in our high-throughput bioassay [32
We found here that monactin, dinactin, and trinactin had robust activity against clinical isolates of C. albicans, Cryptococcus
, and fluconazole resistant C. krusei
and C. albicans
. The purity of the macrotetrolides used and the different fungal isolates in our studies are two possible explanations for the difference between our results and those reported by Ando et al
]. However, the most likely cause for the disparity is the method that they employed to assess antifungal activity. These authors [32
] used the agar dilution method and noted that the macrotetrolides were insoluble in water, resulting in very small zones of growth inhibition. We utilized the microbroth dilution method to quantify the activities of the macrotetrolides and observed no solubility problems. We recognize that our studies represent a relatively small sampling of clinical isolates and that our findings need to be confirmed with a larger sample size of clinical isolates. Consistent with prior reports [14
], nonactin showed no activity in our study.
biofilms on implanted medical devices are a major source of life-threatening blood stream infections. Monactin, dinactin, and trinactin had robust in vitro
activity against C. albicans
in biofilms. Fluconazole, a frontline drug for the treatment of C. albicans
infections, was relatively ineffective against the yeast in such biofilms, while amphotericin B was active against them. Dinactin was active in a rat catheter model of C. albicans
infection but the animals did not tolerate intravenous administration at 100 µg/ml. However, the macrotetrolides are well tolerated by vertebrates via other routes of administration. The LD50
for mice is > 300 mg/kg after intraperitoneal delivery and > 1500 mg/kg following oral administration [32
]. Although our preliminary in vivo
results demonstrated activity of dinactin against C. albicans
biofilm, pharmacokinetic studies are needed to improve these attractive drug leads for treatment of C. albicans
Our analysis of the antifungal action of the macrotetrolides revealed that they did not act directly on group III HHKs. We observed that S. cerevisiae
grown on glucose enabled this yeast to resist the macrotetrolides, whereas growth on galactose significantly enhanced its sensitivity to them. Moreover, the parental and Drk1-expressing S. cerevisiae
strains were equally sensitive to the macrotetrodlides, suggesting that the sensitivity of S. cerevisiae
is dependent on the carbon source of the media, not Drk1 expression. Since the macrotetrolides are known to be ionophores capable of transporting potassium across lipid membranes [33
], a possible explanation for these findings is the effect of carbon source on potassium transport in S. cerevisiae
. Since glucose stimulates potassium uptake in S. cerevisiae
], we hypothesize that the macrotetrolides promote death of yeast by depleting their intracellular potassium stores. The increased potassium uptake of S. cerevisiae
grown on glucose may buffer the cells against potassium loss, whereas yeast grown on galactose would be unable to replenish their potassium stores.
The leakage of potassium is thought to be involved in the mechanism of action of amphotericin B in C. albicans
]. However, the loss of intracellular potassium is not likely involved in macrotetrolides activity against C. albicans
since the compounds exerted their activity against C. albicans
independent of the carbon source.
Macrotetrolides were inactive against five out of six of the fluconazole-resistant C. albicans
isolates tested. Only fluconazole-resistant isolate 2823 was sensitive to the macrotetrolides. This strain over-expresses Mdr1, a major facilitator superfamily protein, and Erg11, a cytochrome P-450 lanosterol 14-alpha-demethylase, the enzyme target of fluconazole [37
]. The fact that over-expression of Erg11 does not engender resistance to the macrotetrolides indicates that it is unlikely that the compounds target ergosterol biosynthesis in the same manner as fluconazole.
The mechanism of resistance to fluconazole is known for three of the five macrotetrolide-resistant strains of C. albicans
. C. albicans
strain C48 over-expresses Erg11 and Cdr1, an ATP-Binding Cassette (ABC) transporter drug efflux pump [38
]; isolate 12–99 over-expresses Cdr1, Mdr1 and Erg11; and strain FH5 over-expresses Cdr1 [37
]. The common phenotype of these macrotetrolide-resistant C. albicans
isolates is the over-expression of Cdr1. As a drug efflux pump, Cdr1 can shuttle a wide variety of compounds across the plasma membrane [39
]. In contrast, Mdr1 is thought to be capable of causing the efflux of only fluconazole [40
]. Therefore, the decreased activity of the macrotetrolides against C. albicans
isolates that over-express Cdr1 is likely due to the drug efflux pumps preventing accumulation of macrotetrolide within the cell.
In summary, we developed a simple, high-throughput, yeast bioassay and demonstrated its utility in discovering natural products as novel antifungal drug leads, as exemplified by the isolation of the macrotetrolides from Streptomyces
sp. YIM56295. Our results should be interpreted with caution due to the limited number of strains available for testing in our study. This point notwithstanding, the macrotetrolides have potent activity against human fungal pathogens C. albicans
and C. neoformans
. Some fluconazole-resistant strains of C. krusei
are also sensitive to the macrotetrolides, while fluconazole-resistant strains of C. albicans
are most often resistant. Importantly, monactin, dinactin, and trinactin exert potent activity against C. albicans
biofilms. Dinactin also showed in vivo
activity against C. albicans
biofilm. Despite their robust activity against medically important fungi, macrotetrolides were well tolerated by mammals in previous studies [32
]. Although our study was small, our results suggest that macrotetrolides have broader and more potent activity against human fungal pathogens than reported previously, and suggest that these compounds may represent promising antifungal drug leads.