Conserved fungal genes as potential targets for broad-spectrum antifungal drug discovery

The discovery of novel classes of antifungal drugs depends to a certain extent on the identification of new, unexplored targets that are essential for growth of fungal pathogens. Likewise, the broad-spectrum capacity of future antifungals requires the target gene(s) to be conserved among key fungal pathogens. Using a genome comparison (or concordance) tool, we identified 240 conserved genes as candidates for potential antifungal targets in 10 fungal genomes. To facilitate the identification of essential genes in Candida albicans, we developed a repressible C. albicans MET3 (CaMET3) promoter system capable of evaluating gene essentiality on a genome-wide scale. The CaMET3 promoter was found to be highly amenable to controlled gene expression, a prerequisite for use in target-based whole-cell screening. When the expression of the known antifungal target C. albicans ERG1 was reduced via down-regulation of the CaMET3 promoter, the CaERG1 conditional mutant strain became hypersensitive, specifically to its inhibitor, terbinafine. Furthermore, parallel screening against a small compound library using the CaERG1 conditional mutant under normal and repressed conditions uncovered several hypersensitive compound hits. This work therefore demonstrates a streamlined process for proceeding from selection and validation of candidate antifungal targets to screening for specific inhibitors.     

Tissue Diagnosis of Invasive Fungal Infections: Current Limitations and the Emerging Use of Molecular Techniques

Invasive fungal diseases (IFD) due to opportunistic fungi are commonly treated using empirical antifungal therapy. Therefore, a comprehensive study of organisms associated with IFD is essential to define successful empiric therapies in each setting. Current diagnostic tests, such as culture, histology and serology are suboptimal, leading to delays in the initiation of antifungal therapies and resulting in high mortality rates despite the availability of several new antifungal agents. Using molecular methods to identify fungal pathogens directly from formalin-fixed, paraffin-embedded tissues is emerging as a diagnostic approach. The goal of this molecular approach is to complement conventional diagnostic tests through the reliable detection and identification of fungal nucleic acids or antigens in tissues so as to direct antiinfective therapies and improve patient outcomes. Here we review challenges and recent advances in the identification of fungal pathogens from tissue samples by conventional and molecular methods.     

Can plant defensins be used to engineer durable commercially useful fungal resistance in crop plants?

Plant defensins are cysteine-rich proteins that play an important role in defense against fungal pathogens. Because of their potent antifungal activity, they have a strong potential to be used for engineering disease resistance in crops. Significant advances have been made in elucidating their structure–activity relationships and modes of antifungal action. Their expression in transgenic plants provides resistance to fungal pathogens in crop plants. In this article, we review recent advances and offer future perspectives on the use of these proteins for engineering durable commercially useful disease resistance in transgenic crop plants.     

Dendritic cells in antifungal immunity and vaccine design

Life-threatening fungal infections have increased in recent years while treatment options remain limited. The development of vaccines against fungal pathogens represents a key advance sorely needed to combat the increasing fungal disease threat. Dendritic cells (DC) are uniquely able to shape antifungal immunity by initiating and modulating naive T?cell responses. Targeting DC may allow for the generation of potent vaccines against fungal pathogens. In the context of antifungal vaccine design, we describe the characteristics of the varied DC subsets, how DC recognize fungi, their function in immunity against fungal pathogens, and how DC can be targeted in order to create new antifungal vaccines. Ongoing studies continue to highlight the critical role of DC in antifungal immunity and will help guide DC-based vaccine strategies.     

Copper at the Fungal Pathogen-Host Axis

Fungal infections are responsible for millions of human deaths annually. Copper, an essential but toxic trace element, plays an important role at the host-pathogen axis during infection. In this review, we describe how the host uses either Cu compartmentalization within innate immune cells or Cu sequestration in other infected host niches such as in the brain to combat fungal infections. We explore Cu toxicity mechanisms and the Cu homeostasis machinery that fungal pathogens bring into play to succeed in establishing an infection. Finally, we address recent approaches that manipulate Cu-dependent processes at the host-pathogen axis for antifungal drug development.     

Plant β-1,3-glucanases: their biological functions and transgenic expression against phytopathogenic fungi

β-1,3-Glucanases are abundant in plants and have been characterized from a wide range of species. They play key roles in cell division, trafficking of materials through plasmodesmata, in withstanding abiotic stresses and are involved in flower formation through to seed maturation. They also defend plants against fungal pathogens either alone or in association with chitinases and other antifungal proteins. They are grouped in the PR-2 family of pathogenesis-related (PR) proteins. Use of β-1,3-glucanase genes as transgenes in combination with other antifungal genes is a plausible strategy to develop durable resistance in crop plants against fungal pathogens. These genes, sourced from alfalfa, barley, soybean, tobacco, and wheat have been co-expressed along with other antifungal proteins, such as chitinases, peroxidases, thaumatin-like proteins and α-1-purothionin, in various crop plants with promising results that are discussed in this review.     

Antifungals discovery: an insight into new strategies to combat antifungal resistance

Undeniably, new antifungal treatments are necessary against pathogenic fungi. Fungal infections have significantly increased in recent decades, being highlighted as important causes of morbidity and mortality, particularly in immunocompromised patients. Five main antifungal classes are used: (i) azoles, (ii) echinocandins, (iii) polyenes, (iv) allylamines and (v) pyrimidine analogues. Moreover, the treatment of mycoses has several limitations, such as undesirable side effects, narrow activity spectrum, a small number of targets and fungal resistance, which are still of major concern in clinical practice. The discovery of new antifungals is mostly achieved by the screening of natural or synthetic/semisynthetic chemical compounds. The most recent discoveries in drug resistance mechanism and their avoidance were explored in a review, focusing on different antifungal targets, as well as new agents or strategies, such as combination therapy, that could improve antifungal therapy.

Significance and Impact of the Study

The failure to respond to antifungal therapy is complex and is associated with microbiological resistance and increased expression of virulence in fungal pathogens. Thus, this review offers an overview of current challenges in the treatment of fungal infections associated with increased antifungal drug resistance and the formation of biofilms in these opportunistic pathogens. Furthermore, the most recent and potential strategies to combat fungal pathogens are explored here, focusing on new agents as well as innovative approaches, such as combination therapy between antifungal drugs or with natural compounds.     

Inhibition of Candida albicans growth by brominated furanones

Candida albicans is the most virulent Candida species of medical importance, which presents a great threat to immunocompromised individuals such as HIV patients. Currently, there are only four classes of antifungal agents available for treating fungal infections: azoles, polyenes, pyrimidines, and echinocandins. The fast spread of multidrug resistant C. albicans strains has increased the demand for new antifungal drugs. In this study, we demonstrate the antifungal activity of brominated furanones on C. albicans. Studying the structure and activity of this class of furanones reveals that the exocyclic vinyl bromide conjugated with the carbonyl group is the most important structural element for fungal inhibition. Furthermore, gene expression analysis using DNA microarrays showed that 3 μg/mL of 4-bromo-5Z-(bromomethylene)-3-butylfuran-2-one (BF1) upregulated 32 C. albicans genes with functions of stress response, NADPH dehydrogenation, and small-molecule transport, and repressed 21 genes involved mainly in cell-wall maintenance. Interestingly, only a small overlap is observed between the gene expression changes caused by the representative brominated furanone (BF1) in this study and other antifungal drugs reported in literature. This result suggests that brominated furanones and other antifungal drugs may target different fungal proteins or genes. The existence of such new targets provides an opportunity for developing new agents to control fungal pathogens which are resistant to currently available drugs.     

Biotransformation of monoterpenoids and their antimicrobial activities

Biotransformation is an economically and ecologically viable technology which has been used extensively to modify the structures of many classes of biologically active products. The discovery of novel antimicrobial metabolites from biotransformation is an important alternative to overcome the increasing levels of drug resistance by plant and human pathogens. Monoterpenes, the main constituents of essential oils, are known for their antimicrobial activities. In 2004, Farooq, Atta-Ur-Rahman and Choudhary published a review on fungal transformation of monoterpenes which covers papers published up to 2002. The present review not only updates the previous one but also discusses the antimicrobial activities (antibacterial, antifungal and antiviral) of biotransformed compounds.     

An update on the pharmacoeconomics of antifungal pharmacotherapy

The incidence and severity of invasive fungal infections are on the rise and they pose a risk of significant morbidity and mortality. The cost burden of fungal infections in the United States is high. There are many newer, less toxic antifungal agents to manage these challenging infections; however, these agents also carry a high cost of their own. When considering an antifungal agent for a specific patient, it is important to consider safety, efficacy, and cost, thus making it essential to continually evaluate the antifungal pharmacoeconomic literature to assist in the therapeutic decision-making process for patients with invasive fungal infections. Unfortunately, there is a lack of pharmacoeconomic studies addressing the costs associated with the treatment and prevention of fungal infections. Future large-scale clinical studies should include pharmacoeconomic analyses and end points that encompass all costs associated with antifungal drug use, not solely drug acquisition costs.     

Resistance of human fungal pathogens to antifungal drugs

Resistance mechanisms can be engaged in clinically relevant fungal pathogens under different conditions when exposed to antifungal drugs. Over past years, active research was undertaken in the understanding of the molecular basis of antifungal drug resistance in these pathogens, and especially against the class of azole antifungals. The isolation of various alleles of the gene encoding the target of azoles has enabled correlation of the appearance of resistance with distinct mutations. Resistance mechanisms to azoles also converge to the upregulation of multidrug transporter genes, whose products have the capacity to extrude from cells several chemically unrelated antifungal agents and toxic compounds. Genome-wide studies of azole-resistant isolates are now permitting a more comprehensive analysis of the impact of resistance on gene expression, and may deliver new clues to their mechanisms. Several laboratories are also exploring, as well as possible alternative resistance pathways, the role of biofilm formation by several fungal species in the development of resistance to various antifungals, including azoles.     

抗真菌植物基因工程的策略和进展

所有高等植物都受多种真菌的侵害,水稻的240多种病害中真菌性痫害占90％。,可见真菌病害是世界范围内危害作物产蘑的主要因素之一,是长期以来作物育种学家一直在努力攻克的难题。目前国     

Inhibiting GPI anchor biosynthesis in fungi stresses the endoplasmic reticulum and enhances immunogenicity

In fungi, the anchoring of proteins to the plasma membrane via their covalent attachment to glycosylphosphatidylinositol (GPI) is essential and thus provides a valuable point of attack for the development of antifungal therapeutics. Unfortunately, studying the underlying biology of GPI-anchor synthesis is difficult, especially in medically relevant fungal pathogens because they are not genetically tractable. Compounding difficulties, many of the genes in this pathway are essential in Saccharomyces cerevisiae. Here, we report the discovery of a new small molecule christened gepinacin (for GPI acylation inhibitor) which selectively inhibits Gwt1, a critical acyltransferase required for the biosynthesis of fungal GPI anchors. After delineating the target specificity of gepinacin using genetic and biochemical techniques, we used it to probe key, therapeutically relevant consequences of disrupting GPI anchor metabolism in fungi. We found that, unlike all three major classes of antifungals in current use, the direct antimicrobial activity of this compound results predominantly from its ability to induce overwhelming stress to the endoplasmic reticulum. Gepinacin did not affect the viability of mammalian cells nor did it inhibit their orthologous acyltransferase. This enabled its use in co-culture experiments to examine Gwt1's effects on host-pathogen interactions. In isolates of Candida albicans, the most common fungal pathogen in humans, exposure to gepinacin at sublethal concentrations impaired filamentation and unmasked cell wall β-glucan to stimulate a pro-inflammatory cytokine response in macrophages. Gwt1 is a promising antifungal drug target, and gepanacin is a useful probe for studying how disrupting GPI-anchor synthesis impairs viability and alters host-pathogen interactions in genetically intractable fungi.     

Engineering of an industrial polyoxin producer for the rational production of hybrid peptidyl nucleoside antibiotics

Polyoxins and nikkomycins are potent antifungal peptidyl nucleoside antibiotics, which inhibit fungal cell wall biosynthesis. They consist of a nucleoside core and one or two independent peptidyl moieties attached to the core at different sites. Making mutations and introducing heterologous genes into an industrial Streptomyces aureochromogenes polyoxin producer, resulted in the production of four polyoxin-nikkomycin hybrid antibiotics designated as polyoxin N and nikkoxin B-D, whose structures were confirmed using high resolution MS and NMR. Two of the hybrid antibiotics, polyoxin N and nikkoxin D, were significantly more potent against some human or plant fungal pathogens than their parents. The data provides an example for rational generation of novel peptidyl nucleoside antibiotics in an industrial producer.     

Current Status in Diagnosis of Scedosporium Infections: What Is the Impact of New Molecular Methods?

Scedosporium species are increasingly encountered as fungal pathogens. Species identification is important due to species-specific differences in epidemiology, antifungal susceptibility and virulence. Histology and culture-based identification are hampered by their low sensitivity and specificity. The use of new selective media has improved the recovery rate from clinical samples. Molecular methods, including multiplex PCR, PCR-RFLP analysis, DNA sequencing, oligonucleotide arrays, real-time PCR, rolling circle amplification, are increasingly used for species identification. Most recently, Matrix-Assisted Laser Desorption-Time of Flight Mass Spectrometry has been successfully applied as a tool for rapid identification of clinically relevant Scedosporium species. This review aims to summarize the methods currently used to guide the clinical microbiology laboratory in the selection of the most appropriate identification techniques. This will aid the laboratory in making a fast and reliable diagnosis that enables the clinician to make correct treatment choices.     

Identification and characterization of candidate Rlm4 blackleg resistance genes in Brassica napus using next-generation sequencing

A thorough understanding of the relationships between plants and pathogens is essential if we are to continue to meet the agricultural needs of the world's growing population. The identification of genes underlying important quantitative trait loci is extremely challenging in complex genomes such as Brassica napus (canola, oilseed rape or rapeseed). However, recent advances in next-generation sequencing (NGS) enable much quicker identification of candidate genes for traits of interest. Here, we demonstrate this with the identification of candidate disease resistance genes from B.?napus for its most devastating fungal pathogen, Leptosphaeria maculans (blackleg fungus). These two species are locked in an evolutionary arms race whereby a gene-for-gene interaction confers either resistance or susceptibility in the plant depending on the genotype of the plant and pathogen. Preliminary analysis of the complete genome sequence of Brassica rapa, the diploid progenitor of B.?napus, identified numerous candidate genes with disease resistance characteristics, several of which were clustered around a region syntenic with a major locus (Rlm4) for blackleg resistance on A7 of B.?napus. Molecular analyses of the candidate genes using B.?napus NGS data are presented, and the difficulties associated with identifying functional gene copies within the highly duplicated Brassica genome are discussed.