Deming Xu

Pathway analysis of Candida albicans survival and virulence determinants in a murine infection model.

One potentially rich source of possible targets for antifungal therapy are those Candida albicans genes deemed essential for growth under the standard culture (i.e., in vitro) conditions; however, these genes are largely unexplored as drug targets because essential genes are not experimentally amenable to conventional gene deletion and virulence studies. Using tetracycline-regulatable promoter-based conditional mutants, we investigated a murine model of candidiasis in which repressing essential genes in the host was achieved. By adding doxycycline to the drinking water starting 3 days prior to (dox - 3D) or 2 days post (dox + 2D) infection, the phenotypic consequences of temporal gene inactivation were assessed by monitoring animal survival and fungal burden in prophylaxis and acute infection settings. Of 177 selected conditional shut-off strains tested, the virulence of 102 was blocked under both repressing conditions, suggesting that the corresponding genes are essential for growth and survival in a murine host across early and established infection periods. Among these genes were those previously identified as antifungal drug targets (i.e., FKS1, ERG1, and ERG11), verifying that this methodology can be used to validate potential new targets. We also identify genes either conditionally essential or dispensable for in vitro growth but required for survival and virulence, including those in late stage ergosterol synthesis, or early steps in fatty acid or riboflavin biosynthesis. This study evaluates the role of essential genes with respect to pathogen virulence in a large-scale, systems biology context, and provides a general method for gene target validation and for uncovering unexpected antimicrobial targets.

The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans.

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2‐hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast‐to‐hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24‐ceramides in membranes of RsAFP2‐treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.

PAP Inhibitor with In Vivo Efficacy Identified by Candida albicans Genetic Profiling of Natural Products

Natural products provide an unparalleled source of chemical scaffolds with diverse biological activities and have profoundly impacted antimicrobial drug discovery. To further explore the full potential of their chemical diversity, we survey natural products for antifungal, target-specific inhibitors by using a chemical-genetic approach adapted to the human fungal pathogen Candida albicans and demonstrate that natural-product fermentation extracts can be mechanistically annotated according to heterozygote strain responses. Applying this approach, we report the discovery and characterization of a natural product, parnafungin, which we demonstrate, by both biochemical and genetic means, to inhibit poly(A) polymerase. Parnafungin displays potent and broad spectrum activity against diverse, clinically relevant fungal pathogens and reduces fungal burden in a murine model of disseminated candidiasis. Thus, mechanism-of-action determination of crude fermentation extracts by chemical-genetic profiling brings a powerful strategy to natural-product-based drug discovery.

Chemical genetic profiling and characterization of small-molecule compounds that affect the biosynthesis of unsaturated fatty acids in Candida albicans.

The balance between saturated and unsaturated fatty acids plays a crucial role in determining the membrane fluidity. In the diploid fungal pathogen Candida albicans, the gene for fatty acid Δ9 desaturase, OLE1, is essential for viability. Using a reverse genetic approach, termed the fitness test, we identified a group of structurally related synthetic compounds that induce specific hypersensitivity of the OLE1+/− strain. Genetic repression of OLE1 and chemical inhibition by two selected compounds, ECC145 and ECC188, resulted in a marked decrease in the total unsaturated fatty acids and impaired hyphal development. The resulting auxotroph of both was suppressed by the exogenous monounsaturated fatty acids (16:1Δ9 and 18:1Δ9). These correlations suggest that both compounds affect the level of unsaturated fatty acids, likely by impairing Ole1p directly or indirectly. However, the residual levels of monounsaturated fatty acids (MUFAs) resulted from chemical inhibition were significantly higher than OLE1 repression, indicating even partial inhibition of MUFAs is sufficient to stop cellular proliferation. Although the essentiality of OLE1 was suppressed by MUFAs in vitro, we demonstrated that it was required for virulence in a murine model of systemic candidiasis even when the animals were supplemented with a high fat diet. Thus, the fungal fatty acid desaturase is an attractive antifungal drug target. Taking advantage of the inhibitors and the relevant conditional shut-off strains, we validated several chemical genetic interactions observed in the fitness test profiles that reveal novel genetic interactions between OLE1/unsaturated fatty acids and other cellular processes.

Isolation and structure elucidation of parnafungins, antifungal natural products that inhibit mRNA polyadenylation.

The Candida albicans Fitness Test, a whole-cell screening platform, was used to profile crude fermentation extracts for novel antifungal natural products with interesting mechanisms of action. An extract with intrinsic antifungal activity from the fungus Fusarium larvarum displayed a Fitness Test profile that strongly implicated mRNA processing as the molecular target responsible for inhibition of fungal growth. Isolation of the active components from this sample identified a novel class of isoxazolidinone-containing natural products, which we have named parnafungins. These natural products were isolated as an interconverting mixture of four structural- and stereoisomers. The isomerization of the parnafungins was due to a retro-Michael ring-opening and subsequent reformation of a xanthone ring system. This interconversion was blocked by methylation of an enol moiety. Structure elucidation of purified parnafungin derivatives was accomplished by X-ray crystallography and NMR analysis. The biochemical target of these natural products has been identified as the fungal polyadenosine polymerase. Parnafungins demonstrated broad spectrum antifungal activity with no observed activity against gram-positive or gram-negative bacteria. The intact isoxazolidinone ring was required for antifungal activity. In addition, the natural products were efficacious in a mouse model of disseminated candidiasis.

Isolation, structure elucidation, and biological activity of virgineone from Lachnum virgineum using the genome-wide Candida albicans fitness test

A glycosylated tetramic acid, virgineone (1), was isolated from saprotrophic Lachnum virgineum. The antifungal activity of the fermentation extract of L. virgineum was characterized in the Candida albicans fitness test as distinguishable from other natural products tested. Bioassay-guided fractionation yielded 1, a tyrosine-derived tetramic acid with a C-22 oxygenated chain and a beta-mannose. It displayed broad-spectrum antifungal activity against Candida spp. and Aspergillus fumigatus with a MIC of 4 and 16 microg/mL, respectively. Virgineone was also identified in a number of Lachnum strains collected from diverse geographies and habitats.

Discovery of the parnafungins, antifungal metabolites that inhibit mRNA polyadenylation, from the Fusarium larvarum complex and other Hypocrealean fungi.

Evaluation of fungal fermentation extracts with whole cell Candida albicans activity resulted in the identification of a novel class of isoxazolidinone-containing metabolites named parnafungins. Chemical-genetic profiling with the C. albicans fitness test identified the biochemical target as inhibition of poly-adenosine polymerase, a component of the mRNA cleavage and polyadenylation complex. Parnafungins were discovered from fermentation extracts of fungi resembling F. larvarum isolated from plants, plant litter and lichens. Furthermore authentic strains of F. larvarum var. larvarum and F. larvarum var. rubrum could be induced to produce parnafungins and their degradation products in low titers. Relationships among strains of the F. larvarum complex (FLC), including parnafungin-producing strains, were examined by cladistic analyses of rDNA, mitochondrial rDNA, and two protein-coding genes, comparisons of antifungal activity and antifungal metabolite profiles, and morphological phenotypes. Integrated analyses of these data led to the conclusion that the diversity within the FLC exceeded the one-to-one correspondence between F. larvarum and its teleomorph Cosmospora aurantiicola. Based on multiple gene sequence analyses, strains of the FLC formed a monophyletic clade inclusive of the parnafungin-producing strains. The FLC, including newly discovered parnafungin-producing strains, could be resolved into at least six different lineages, possibly representing cryptic species, of which one was not fully resolved from F. larvarum var. rubrum. Fusarium larvarum var. rubrum represents a species distinct from var. larvarum. Finally we report that two other species from the Hypocreales, Trichonectria rectipila and Cladobotryum pinarense, are able to produce parnafungins and their open-ring forms.

Isolation, structure, and biological activities of fellutamides C and D from an undescribed Metulocladosporiella (Chaetothyriales) using the genome-wide Candida albicans fitness test

In a whole-cell mechanism of action (MOA)-based screening strategy for discovery of antifungal agents, Candida albicans was used, followed by testing of active extracts in the C. albicans fitness test (CaFT), which provides insight into the mechanism of action. A fermentation extract of an undescribed species of Metulocladosporiella that inhibited proteasome activity in a C. albicans fitness test was identified. The chemical genomic profile of the extract contained hypersensitivity of heterozygous deletion strains (strains that had one of the genes of the diploid genes knocked down) of genes represented by multiple subunits of the 25S proteasome. Two structurally related peptide aldehydes, named fellutamides C and D, were isolated from the extract. Fellutamides were active against C. albicans and Aspergillus fumigatus with MICs ranging from 4 to 16 μg/mL and against fungal proteasome (IC₅₀ 0.2 μg/mL). Both compounds showed proteasome activity against human tumor cell lines, potently inhibiting the growth of PC-3 prostate carcinoma cells, but not A549 lung carcinoma cells. In PC-3 cells compound treatment produced a G2M cell cycle block and induced apoptosis. Preliminary SAR studies indicated that the aldehyde group is critical for the antifungal activity and that the two hydroxy groups are quantitatively important for potency.

Isolation, structure and biological activity of phomafungin, a cyclic lipodepsipeptide from a widespread tropical Phoma sp.

We isolated a cyclic lipodepsipeptide, phomafungin, from a Phoma sp. The distinct antifungal activity of phomafungin in the crude extract was initially discovered by mechanistic profiling in the Candida albicans fitness test. The purified compound contains a 28 member ring consisting of eight amino acids and a beta-hydroxy-gamma-methyl-hexadecanoic acid, and displays a broad spectrum of antifungal activity against Candida spp., Aspergillus fumigatus and Trichophyton mentagrophytes with MIC of 2-8 microg/ml, and toxicity to mice at 25 mg/kg. The linear peptide derived from opening of the lactone ring was devoid of antifungal activity as well as toxicity. Phomafungin has been identified in a number of Phoma spp. collected from Africa and the Indian and Pacific Ocean islands.

Isolation, structure, and biological activity of phaeofungin, a cyclic lipodepsipeptide from a Phaeosphaeria sp. Using the genome-wide Candida albicans fitness test

Phaeofungin (1), a new cyclic depsipeptide isolated from Phaeosphaeria sp., was discovered by application of reverse genetics technology, using the Candida albicans fitness test (CaFT). Phaeofungin is comprised of seven amino acids and a β,γ-dihydroxy-γ-methylhexadecanoic acid arranged in a 25-membered cyclic depsipeptide. Five of the amino acids were assigned with d-configurations. The structure was elucidated by 2D-NMR and HRMS-MS analysis of the natural product and its hydrolyzed linear peptide. The absolute configuration of the amino acids was determined by Marfeys method by complete and partial hydrolysis of 1. The CaFT profile of the phaeofungin-containing extract overlapped with that of phomafungin (3), another structurally different cyclic lipodepsipeptide isolated from a Phoma sp. using the same approach. Comparative biological characterization further demonstrated that these two fungal lipodepsipeptides are functionally distinct. While phomafungin was potentiated by cyclosporin A (an inhibitor of the calcineurin pathway), phaeofungin was synergized with aureobasidin A (2) (an inhibitor of the sphingolipid biosynthesis) and to some extent caspofungin (an inhibitor of glucan synthase). Furthermore, phaeofungin caused ATP release in wild-type C. albicans strains but phomafungin did not. It showed modest antifungal activity against C. albicans (MIC 16-32 μg/mL) and better activity against Aspergillus fumigatus (MIC 8-16 μg/mL) and Trichophyton mentagrophytes (MIC 4 μg/mL). The linear peptide was inactive, suggesting that the macrocyclic depsipeptide ring is essential for target engagement and antifungal activity.