Target For Antifungal Development Research Articles

High-resolution crystal structure and chemical screening reveal pantothenate kinase as a new target for antifungal development

Fungal infections are the leading cause of mortality by eukaryotic pathogens, with an estimated 150 million severe life-threatening cases and 1.7 million deaths reported annually. The rapid emergence of multidrug-resistant fungal isolates highlights the urgent need for new drugs with new mechanisms of action. In fungi, pantothenate phosphorylation, catalyzed by PanK enzyme, is the first step in the utilization of pantothenic acid and coenzyme A biosynthesis. In all fungi sequenced so far, this enzyme is encoded by a single PanK gene. Here, we report the crystal structure of a fungal PanK alone as well as with high-affinity inhibitors from a single chemotype identified through a high-throughput chemical screen. Structural, biochemical, and functional analyses revealed mechanisms governing substrate and ligand binding, dimerization, and catalysis and helped identify new compounds that inhibit the growth of several Candida species. The data validate PanK as a promising target for antifungal drug development.

Identification of a Pseudosubstrate Stimulatory Motif Uncovers Novel Roles of Cdc14 Phosphatase in Fungal Cell Wall Integrity

Fungal pathogens are a growing threat to human health and global food security as they are becoming increasingly resistant to the few existing antifungal treatments. Cdc14 is a protein phosphatase primarily known for its essential role in controlling mitotic exit in the model yeast Saccharomyces cerevisiae. However, in other fungal species Cdc14 is non‐essential and the reasons for its strict conservation across Dikarya remain unclear. Recently, Cdc14 was shown to be required for host infection by several plant pathogenic fungi. Here, we show that Cdc14 is also required for virulence in the human pathogen, Candida albicans.These observations, coupled with our structural and biochemical knowledge of Cdc14 phosphatases, suggest it could be a useful target for antifungal development. In our search for differences between fungal and animal Cdc14 enzymes that could be exploited for antifungal design, we discovered an invariant motif in the otherwise disordered C‐terminal tail of fungal Cdc14 orthologs. The motif resembles the recognition sequence of optimal Cdc14 substrates. We provide evidence from molecular modeling and enzyme kinetic studies that this motif is a “pseudosubstrate” that binds the Cdc14 active site and functions to accelerate the rate‐limiting catalytic step, providing a plausible mechanism for dynamic control of intracellular Cdc14 activity. The turnover rate for Cdc14 lacking a functional pseudosubstrate motif is reduced by an order of magnitude compared to the wild‐type enzyme. Both S. cerevisiaeand C. albicanscells expressing Cdc14 variants with point mutations in this motif exhibit a specific and pronounced sensitivity to cell wall stresses, including treatment with echinocandin antifungal drugs that inhibit cell wall synthesis. Moreover, signaling through the cell wall integrity pathway is chronically elevated in these cells, indicative of a cell wall structural defect. Preliminary evidence in synchronized S. cerevisiaecultures with reduced Cdc14 activity suggests that cell wall integrity is compromised around the time of cytokinesis and septation, consistent with its known functions in these processes. We are currently testing if the pseudosubstrate motif is required for virulence of C. albicans.Our findings reveal 1) a novel contribution to catalysis of a structural element outside the conserved catalytic core of Cdc14 enzymes and 2) novel roles of Cdc14 in promoting cell wall integrity and pathogenesis. More detailed characterization of the mechanisms by which Cdc14 contributes to cell wall integrity and host infection in human pathogenic fungi should help assess its future value for antifungal development.

DeORFanizing Candida albicans Genes using Coexpression.

Functional characterization of open reading frames in nonmodel organisms, such as the common opportunistic fungal pathogen Candida albicans, can be labor-intensive. To meet this challenge, we built a comprehensive and unbiased coexpression network for C. albicans, which we call CalCEN, from data collected from 853 RNA sequencing runs from 18 large-scale studies deposited in the NCBI Sequence Read Archive. Retrospectively, CalCEN is highly predictive of known gene function annotations and can be synergistically combined with sequence similarity and interaction networks in Saccharomyces cerevisiae through orthology for additional accuracy in gene function prediction. To prospectively demonstrate the utility of the coexpression network in C. albicans, we predicted the function of underannotated open reading frames (ORFs) and identified CCJ1 as a novel cell cycle regulator in C. albicans This study provides a tool for future systems biology analyses of gene function in C. albicans We provide a computational pipeline for building and analyzing the coexpression network and CalCEN itself at http://github.com/momeara/CalCENIMPORTANCECandida albicans is a common and deadly fungal pathogen of humans, yet the genome of this organism contains many genes of unknown function. By determining gene function, we can help identify essential genes, new virulence factors, or new regulators of drug resistance, and thereby give new targets for antifungal development. Here, we use information from large-scale RNA sequencing (RNAseq) studies and generate a C. albicans coexpression network (CalCEN) that is robust and able to predict gene function. We demonstrate the utility of this network in both retrospective and prospective testing and use CalCEN to predict a role for C4_06590W/CCJ1 in cell cycle. This tool will allow for a better characterization of underannotated genes in pathogenic yeasts.

How Does Fusarium oxysporum Sense and Respond to Nicotinaldehyde, an Inhibitor of the NAD+ Salvage Biosynthesis Pathway?

Plant pathogenic fungi are a major threat to food security and impose a severe economic burden, thus there is a continuous need to develop new strategies to manage them. NAD+ is a co-factor in numerous enzymatic activities and determines the metabolic fate of the cell. Therefore, maintenance of NAD+ concentration is important for cellular viability. Consequently, the NAD+ biosynthetic pathway and redox homeostasis was suggested as a target for antifungal development. We aimed to study how Fusarium oxysporum senses and responds to nicotinaldehyde (NA), an inhibitor of Pnc1, a key enzyme in the salvage pathway of NAD+ biosynthesis. We were able to show that NA was inhibitory in high concentrations to several fungal plant pathogens, with much milder effects on tomato growth. Under low nutrient conditions NA reduced the total amounts of NAD+ in the fungal cell, a trend that was also observed in rich media, although without statistical significance. In low and high nutrient availability NA dramatically reduced the NAD+/NADH ratio. After exposure to NA, NADH levels were increased and NAD+ levels and the biomass were greatly reduced. Cells responded to NA by up-regulation of oxidoreductases, with hardly any up-regulation of the classic response to oxidative stress. Direct measurement of oxidative stress response showed that unlike formaldehyde and hydrogen peroxide, NA caused reductive rather than oxidative stress. Surprisingly, alcohol dehydrogenases were significantly up-regulated more than any other dehydrogenases, including aldehyde dehydrogenases. We propose that conidia of F. oxysporum efficiently detoxified the aldehyde group of NA by reducing NAD+ to NADH; the high concentrations of the latter provoked the expression of alcohol dehydrogenases that in yeast can act to reduce NADH and increase NAD+ amounts, respectively. Overall, the results suggest that targeting NAD+ biosynthesis pathway and redox homeostasis can be a potential approach to manage fungal plant pathogens. Many of the natural antifungal compounds produced by bio-control agents or even the natural biome are aldehydes, and thus the results presented here predict the possible response of Fusarium to wide sources of toxicity in the environment.

Analysis of the regulation, expression, and localisation of the isocitrate lyase from Aspergillus fumigatus, a potential target for antifungal drug development

Invasive aspergillosis, caused by Aspergillus fumigatus, is a severe systemic infection in immunocompromised patients. New drug targets are required, since therapeutic treatment often fails and is hampered by severe side effects of antifungals. Enzymes of the glyoxylate bypass are potential targets, since they are absent in humans, but required for growth of Aspergillus on C2-generating carbon sources. The key enzyme isocitrate lyase (ICL) can be inhibited by 3-nitropropionate, both as a purified enzyme and within intact cells, whereas the latter inhibition upregulates ICL promoter activity. ICL was found in distinct subcellular structures within growing hyphae, but only under conditions requiring ICL activity. In contrast, ICL was constitutively found in conidia, suggesting a specific role during germination. Lipids, as potential substrates, were detected in conidia and macrophages. Additionally, germinating conidia within macrophages contain ICL, suggesting that the glyoxylate shunt might be a relevant target for development of antifungals.