Sha Zheng

Trapping toxins within lipid droplets is a resistance mechanism in fungi.

Lipid droplets (LDs) act as intracellular storage organelles in most types of cells and are principally involved in energy homeostasis and lipid metabolism. However, the role of LDs in resistance to toxins in fungi remains largely unknown. Here, we show that the trapping of endogenous toxins by LDs is a self-resistance mechanism in the toxin producer, while absorbing external lipophilic toxins is a resistance mechanism in the toxin recipient that acts to quench the production of reactive oxygen species. We found that an endolichenic fungus that generates phototoxic perylenequinones (PQs) trapped the PQs inside LDs. Using a model that incorporates the fungicidal action of hypocrellin A (HA), a PQ derivative, we showed that yeast cells escaped killing by trapping toxins inside LDs. Furthermore, LD-deficient mutants were hypersusceptible to HA-mediated phototoxins and other fungicides. Our study identified a previously unrecognised function of LDs in fungi that has implications for our understanding of environmental adaptation strategies for fungi and antifungal drug discovery.

Biatriosporin D displays anti-virulence activity through decreasing the intracellular cAMP levels

&NA; Candidiasis has long been a serious human health problem, and novel antifungal approaches are greatly needed. During both superficial and systemic infection, C. albicans relies on a battery of virulence factors, such as adherence, filamentation, and biofilm formation. In this study, we found that a small phenolic compound, Biatriosporin D (BD), isolated from an endolichenic fungus, Biatriospora sp., displayed anti‐virulence activity by inhibiting adhesion, hyphal morphogenesis and biofilm formation of C. albicans. Of note is the high efficacy of BD in preventing filamentation with a much lower dose than its MIC value. Furthermore, BD prolonged the survival of worms infected by C. albicans in vivo. Quantitative real‐time PCR analysis, exogenous cAMP rescue experiments and intracellular cAMP measurements revealed that BD regulates the Ras1‐cAMP‐Efg1 pathway by reducing cAMP levels to inhibit the hyphal formation. Further investigation showed that BD could upregulate Dpp3 to synthesize much more farnesol, which could inhibit the activity of Cdc35 and reduce the generation of cAMP. Taken together, these findings indicate that BD stimulates the expression of Dpp3 to synthesize more farnesol that directly inhibits the Cdc35 activity, reducing intracellular cAMP and thereby disrupting the morphologic transition and attenuating the virulence of C. albicans. Our study uncovers the underlying mechanism of BD as a prodrug in fighting against pathogenic C. albicans and provides a potential application of BD in fighting clinically relevant fungal infections by targeting fungal virulence. HighlightsBD inhibits the filamentation of C. albicans in multiple hypha‐inducing conditions.BD can prolong the survival of nematodes infected by C. albicans.BD stimulates the expression of Dpp3 to synthesize more farnesol.BD reduces intracellular cAMP and regulates Ras1‐cAMP‐PKA pathway.

Quinone derivatives isolated from the endolichenic fungus Phialocephala fortinii are Mdr1 modulators that combat azole resistance in Candida albicans.

One of the main azole-resistance mechanisms in Candida pathogens is the upregulation of drug efflux pumps, which compromises the efficacy of azoles and results in treatment failure. The combination of azole-antifungal agents with efflux pump inhibitors represents a promising strategy to combat fungal infection. High-throughput screening of 150 extracts obtained from endolichenic fungal cultures led to the discovery that the extract of Phialocephala fortinii exhibits potent activity for the reversal of azole resistance. From P. fortinii cultures, a total of 15 quinone derivatives, comprising 11 new derivatives and 4 known compounds, were obtained. Among these compounds, palmarumycin P3 (3) and phialocephalarin B (8) specifically modulate the expression of MDR1 to inhibit the activity of drug efflux pumps and therefore reverse azole resistance. The present study revealed Mdr1 targeting as an alternative mechanism for the discovery of new agents to fight antifungal drug resistance.

Chiloscyphenol A derived from Chinese liverworts exerts fungicidal action by eliciting both mitochondrial dysfunction and plasma membrane destruction

This study aimed to characterize the antifungal effects of chiloscyphenol A (CA), a natural small molecule isolated from Chinese liverworts, and investigate its mode of action. CA was effective against five tested Candida species with a minimal inhibitory concentration (MIC) of 8–32 μg/ml and exhibited fungicidal activity against Candida albicans in both the planktonic state and mature biofilms. The in vivo study using Caenorhabditis elegans showed that CA prolonged the survival of C. albicans infected worms. Further investigations revealed that CA resulted in mitochondrial dysfunction as indicated by mtΔψ hyperpolarization, increased ATP production and intracellular ROS accumulation, and aggregated distribution of Tom70. In addition, CA caused perturbation of the cell membrane and increased membrane permeability, as demonstrated by specific staining and confocal microscopic and transmission electron microscopy (TEM) observations and by calcein-leakage measurements. This conclusion was further confirmed by the decreased cell size of CA-treated cells via three-dimensional contour-plot analysis using flow cytometry. Taken together, these results suggest that CA exerts fungicidal activity by eliciting both mitochondrial dysfunction and plasma membrane destruction in C. albicans. The elucidated mechanism supports the potential application of CA against clinical fungal infections.

Als1 and Als3 regulate the intracellular uptake of copper ions when Candida albicans biofilms are exposed to metallic copper surfaces.

Copper surfaces possess efficient antimicrobial effect. Here, we reported that copper surfaces could inactivate Candida albicans biofilms within 40 min. The intracellular reactive oxygen species in C. albicans biofilms were immediately stimulated during the contact of copper surfaces, which might be an important factor for killing the mature biofilms. Copper release assay demonstrated that the copper ions automatically released from the surface of 1 mm thick copper coupons with over 99.9% purity are not the key determinant for the copper-mediated killing action. The susceptibility test to copper surfaces by using C. albicans mutant strains, which were involved in efflux pumps, adhesins, biofilms formation or osmotic stress response showed that als1/als1 and als3/als3 displayed higher resistance to the copper surface contact than other mutants did. The intracellular concentration of copper ions was lower in als1/als1 and als3/als3 than that in wild-type strain. Transcriptional analysis revealed that the expression of copper transporter-related gene, CRP1, was significantly increased in als1/als1, als3/als3, suggesting a potential role of ALS1 and ALS3 in absorbing ions by regulating the expression of CRP1 This study provides a potential application in treating pathogenic fungi by using copper surfaces and uncovers the roles of ALS1 and ALS3 in absorbing copper ions for C. albicans.

The cleavage of perylenequinones through photochemical oxidation acts as a detoxification mechanism for the producer

Perylenequinones (PQs) belong to a class of photosensitizers, generated by some fungi for parasitization or combating invaders. However, PQs generate reactive oxygen when exposed to light irradiation and cause nonselective damage to the producer host. The mechanism underlying the self-resistance of the producer is less understood. By using high-performance liquid chromatography and UV-visible absorption spectroscopic analysis, we found that PQs from an endolichenic fungus Phaeosphaeria sp. were transformed into new derivatives when the culture was exposed to visible or ultraviolet light. This transformation was accompanied by the reduction of its antimicrobial activity. In order to unveil the underlying mechanism, the purified hypocrellin A and calphostin D were employed in the photochemical analysis. The obtained light cleaved products were found to be nontoxic to the tested microbes and this photo-driven detoxification could be taken as a self-resistant strategy for the producer.

Floricolin C elicits intracellular reactive oxygen species accumulation and disrupts mitochondria to exert fungicidal action

Candida albicans, one of the most prevalent fungal pathogens, causes severe mucosal and invasive infections in predisposed individuals. The rise of fungal infection and drug resistance demands the development of novel antifungal agents. In this study, we observed that floricolin C (FC), a p-terphenyl pigment from an endolichenic fungus, killed C. albicans cells in the planktonic state or within biofilms through reactive oxygen species (ROS) accumulation. Further tests revealed that FC could directly damage the mitochondria to cause ROS accumulation. In addition, FC can quench thiol-based agents through a Michael reaction involving the α,β-unsaturated carbonyl group, whose effect may chelate intracellular thiol-based molecules or proteins in C. albicans, resulting in an imbalance in redox homeostasis. Increased ROS generation led to mitochondrial dysfunction, nuclear dispersion and consequently cell death. We further demonstrated that FC could prevent biofilm formation of other Candida species and eradicate their pre-formed biofilms. An in vivo study demonstrated that FC prolonged the survival of C. albicans-infected Caenorhabditis elegans. Taken together, our study provides the basis for the application of FC to combat Candida infections.