The pathobiology of human fungal infections.

Human fungal infections are a historically neglected area of disease research, yet they cause more than 1.5 million deaths every year. Our understanding of the pathophysiology of these infections has increased considerably over the past decade, through major insights into both the host and pathogen factors that contribute to the phenotype and severity of these diseases. Recent studies are revealing multiple mechanisms by which fungi modify and manipulate the host, escape immune surveillance and generate complex comorbidities. Although the emergence of fungal strains that are less susceptible to antifungal drugs or that rapidly evolve drug resistance is posing new threats, greater understanding of immune mechanisms and host susceptibility factors is beginning to offer novel immunotherapeutic options for the future. In this Review, we provide a broad and comprehensive overview of the pathobiology of human fungal infections, focusing specifically on pathogens that can cause invasive life-threatening infections, highlighting recent discoveries from the pathogen, host and clinical perspectives. We conclude by discussing key future challenges including antifungal drug resistance, the emergence of new pathogens and new developments in modern medicine that are promoting susceptibility to infection.

Zinc prevents vaginal candidiasis by inhibiting expression of an inflammatory fungal protein.

Candida causes an estimated half-billion cases of vulvovaginal candidiasis (VVC) every year. VVC is most commonly caused by Candida albicans, which, in this setting, triggers nonprotective neutrophil infiltration, aggressive local inflammation, and symptomatic disease. Despite its prevalence, little is known about the molecular mechanisms underpinning the immunopathology of this fungal infection. In this study, we describe the molecular determinant of VVC immunopathology and a potentially straightforward way to prevent disease. In response to zinc limitation, C. albicans releases a trace mineral binding molecule called Pra1 (pH-regulated antigen). Here, we show that the PRA1 gene is strongly up-regulated during vaginal infections and that its expression positively correlated with proinflammatory cytokine concentrations in women. Genetic deletion of PRA1 prevented vaginal inflammation in mice, and application of a zinc solution down-regulated expression of the gene and also blocked immunopathology. We also show that treatment of women suffering from recurrent VVC with a zinc gel prevented reinfections. We have therefore identified a key mediator of symptomatic VVC, giving us an opportunity to develop a range of preventative measures for combatting this disease.

Is There a Relationship Between Mating and Pathogenesis in Two Human Fungal Pathogens, Candida albicans and Candida glabrata?

Purpose of Review: Human fungal pathogens are rapidly increasing in incidence and readily able to evade the host immune responses. Our ability to study the genetic behind this has been limited due to the apparent lack of a sexual cycle and forward genetic tools. In this review, we discuss the evolution of mating, meiosis, and pathogenesis and if these processes are advantageous to pathogens. Recent Findings: This review summarises what is currently known about the sexual cycles of two important human fungal pathogens, Candida albicans and Candida glabrata. This includes the identification of parasexual cycle in C. albicans and the observed low levels of recombination in C. glabrata populations. Summary: In this review, we present what is currently known about the mating types and mating/sexual cycles of two clinically important human fungal pathogens, Candida albicans and Candida glabrata. We discuss the evolution of meiosis using the knowledge that has been amassed from the decades of studying Saccharomyces cerevisiae and how this can be applied to fungal pathogens. We further discuss how the evolution of pathogenesis has played a role in influencing mating processes in human fungal pathogens and compare sexual cycles between C. albicans and C. glabrata, highlighting knowledge gaps and suggesting how these two fungi have evolved distinct mating niches to allow the development of disease in a human host.

The Candida glabrata Parent Strain Trap: How Phenotypic Diversity Affects Metabolic Fitness and Host Interactions.

Reference strains improve reproducibility by standardizing observations and methodology, which has ultimately led to important insights into fungal pathogenesis. However, recent investigations have highlighted significant genotypic and phenotypic heterogeneity across isolates that influence genetic circuitry and virulence within a species. Candida glabrata is the second leading cause of candidiasis, a life-threatening infection, and undergoes extensive karyotype and phenotypic changes in response to stress. Much of the work conducted on this pathogen has focused on two sequenced strains, CBS138 (ATCC 2001) and BG2. Few studies have compared these strains in detail, but key differences include mating type and altered patterns of expression of EPA adhesins. In fact, most C. glabrata isolates and BG2 are MATa, while CBS138 is MATα. However, it is not known if other phenotypic differences between these strains play a role in our understanding of C. glabrata pathogenesis. Thus, we set out to characterize metabolic, cell wall, and host-interaction attributes for CBS138 and BG2. We found that BG2 utilized a broader range of nitrogen sources and had reduced cell wall size and carbohydrate exposure than CBS138, which we hypothesized results in differences in innate immune interactions and virulence. We observed that, although both strains were phagocytosed to a similar extent, BG2 replicated to higher numbers in macrophages and was more virulent during Galleria mellonella infection than CBS138 in a dose-dependent manner. Interestingly, deletion of SNF3, a major nutrient sensor, did not affect virulence in G. mellonella for BG2, but significantly enhanced larval killing in the CBS138 background compared to the parent strain. Understanding these fundamental differences in metabolism and host interactions will allow more robust conclusions to be drawn in future studies of C. glabrata pathogenesis. IMPORTANCE Reference strains provide essential insights into the mechanisms underlying virulence in fungal pathogens. However, recent studies in Candida albicans and other species have revealed significant genotypic and phenotypic diversity within clinical isolates that are challenging paradigms regarding key virulence factors and their regulation. Candida glabrata is the second leading cause of candidiasis, and many studies use BG2 or CBS138 for their investigations. Therefore, we aimed to characterize important virulence-related phenotypes for both strains that might alter conclusions about C. glabrata pathogenesis. Our study provides context for metabolic and cell wall changes and how these may influence host interaction phenotypes. Understanding these differences is necessary to support robust conclusions about how virulence factors may function in these and other very different strain backgrounds.

Using Synthetic Genetic Interactions in Candida glabrata as a Novel Method to Detect Genes with Roles in Antifungal Drug Resistance.

Synthetic genetic interaction analysis is a powerful genetic strategy that analyzes the fitness and phenotypes of single- and double-gene mutant cells in order to dissect the interactions between genes, categorize into biological pathways, and characterize genes of unknown function. It has been extensively employed in model organisms for fundamental, systems-level assessment of the interactions between genes. However, more recently, genetic interaction mapping has been applied to fungal pathogens and has been instrumental for the study of clinically important infectious organisms. This protocol herein explains in the detail the methodology and analysis that can be employed to develop interaction maps in microbial pathogens. Such techniques can aid in bridging our understanding of complex genetic networks, with applications to diverse microbial pathogens to further our understanding of virulence, the use of antimicrobial therapies, and host-pathogen interactions.

Fluconazole resistant Candida auris clinical isolates have increased levels of cell wall chitin and increased susceptibility to a glucosamine-6-phosphate synthase inhibitor.

In 2009 Candida auris was first isolated as fungal pathogen of human disease from ear canal of a patient in Japan. In less than a decade, this pathogen has rapidly spread around the world and has now become a major health challenge that is of particular concern because many strains are resistant to multiple class of antifungal drugs. The lack of available antifungals and rapid increase of this fungal pathogen provides an incentive for the development of new and more potent anticandidal drugs and drug combinatorial treatments. Here we have explored the growth inhibitory activity against C. auris of a synthetic dipeptide glutamine analogue, L-norvalyl-N 3-(4-methoxyfumaroyl)-L-2,3- diaminopropanoic acid (Nva-FMDP), that acts as an inhibitor of glucosamine-6-phosphate (GlcN-6-P) synthase - a key enzyme in the synthesis of cell wall chitin. We observed that in contrast to FLC susceptible isolates of C. auris, FLC resistant isolates had elevated cell wall chitin and were susceptible to inhibition by Nva-FMDP. The growth kinetics of C. auris in RPMI-1640 medium revealed that the growth of FLC resistant isolates were 50-60% more inhibited by Nva-FMDP (8 µ g/ml) compared to a FLC susceptible isolate. Fluconazole resistant strains displayed increased transcription of CHS1, CHS2 and CHS3, and the chitin content of the fluconazole resistant strains was reduced following the Nva-FMDP treatment. Therefore, the higher chitin content in FLC resistant C. auris isolates may make the strain more susceptible to inhibition of the antifungal activity of the Nva-FMDP peptide conjugate.

Antifungal Exposure and Resistance Development: Defining Minimal Selective Antifungal Concentrations and Testing Methodologies.

This scoping review aims to summarise the current understanding of selection for antifungal resistance (AFR) and to compare and contrast this with selection for antibacterial resistance, which has received more research attention. AFR is an emerging global threat to human health, associated with high mortality rates, absence of effective surveillance systems and with few alternative treatment options available. Clinical AFR is well documented, with additional settings increasingly being recognised to play a role in the evolution and spread of AFR. The environment, for example, harbours diverse fungal communities that are regularly exposed to antifungal micropollutants, potentially increasing AFR selection risk. The direct application of effect concentrations of azole fungicides to agricultural crops and the incomplete removal of pharmaceutical antifungals in wastewater treatment systems are of particular concern. Currently, environmental risk assessment (ERA) guidelines do not require assessment of antifungal agents in terms of their ability to drive AFR development, and there are no established experimental tools to determine antifungal selective concentrations. Without data to interpret the selective risk of antifungals, our ability to effectively inform safe environmental thresholds is severely limited. In this review, potential methods to generate antifungal selective concentration data are proposed, informed by approaches used to determine antibacterial minimal selective concentrations. Such data can be considered in the development of regulatory guidelines that aim to reduce selection for AFR.

Genetic interaction analysis in microbial pathogens: unravelling networks of pathogenesis, antimicrobial susceptibility and host interactions.

Genetic interaction (GI) analysis is a powerful genetic strategy that analyzes the fitness and phenotypes of single- and double-gene mutant cells in order to dissect the epistatic interactions between genes, categorize genes into biological pathways, and characterize genes of unknown function. GI analysis has been extensively employed in model organisms for foundational, systems-level assessment of the epistatic interactions between genes. More recently, GI analysis has been applied to microbial pathogens and has been instrumental for the study of clinically important infectious organisms. Here, we review recent advances in systems-level GI analysis of diverse microbial pathogens, including bacterial and fungal species. We focus on important applications of GI analysis across pathogens, including GI analysis as a means to decipher complex genetic networks regulating microbial virulence, antimicrobial drug resistance and host-pathogen dynamics, and GI analysis as an approach to uncover novel targets for combination antimicrobial therapeutics. Together, this review bridges our understanding of GI analysis and complex genetic networks, with applications to diverse microbial pathogens, to further our understanding of virulence, the use of antimicrobial therapeutics and host-pathogen interactions. .

Functional Characterization of a Novel Oxidative Stress Protection Protein in the Pathogenic Yeast Candida glabrata.

Candida species are important pathogens of humans and the fourth most commonly isolated pathogen from nosocomial blood stream infections. Although Candida albicans is the principle causative agent of invasive candidosis, the incidence of Candida glabrata infections has rapidly grown. The reason for this increase is not fully understood, but it is clear that the species has a higher innate tolerance to commonly administered azole antifungals, in addition to being highly tolerant to stresses especially oxidative stress. Taking the approach that using the model organism, Saccharomyces cerevisiae, with its intrinsic sensitivity to oxidative stress, we hypothesized that by expressing mediators of stress resistance from C. glabrata in S. cerevisiae, it would result in induced resistance. To test this we transformed, en-masse, the C. glabrata ORFeome library into S. cerevisiae. This resulted in 1,500 stress resistant colonies and the recovered plasmids of 118 ORFs. Sequencing of these plasmids revealed a total of 16 different C. glabrata ORFs. The recovery of genes encoding known stress protectant proteins such as GPD1, GPD2 and TRX3 was predicted and validated the integrity of the screen. Through this screen we identified a C. glabrata unique ORF that confers oxidative stress resistance. We set to characterise this gene herein, examining expression in oxidative stress sensitive strains, comet assays to measure DNA damage and synthetic genetic array analysis to identify genetic interaction maps in the presence and absence of oxidative stress.

Advances in Molecular Tools and In Vivo Models for the Study of Human Fungal Pathogenesis.

Pathogenic fungi represent an increasing infectious disease threat to humans, especially with an increasing challenge of antifungal drug resistance. Over the decades, numerous tools have been developed to expedite the study of pathogenicity, initiation of disease, drug resistance and host-pathogen interactions. In this review, we highlight advances that have been made in the use of molecular tools using CRISPR technologies, RNA interference and transposon targeted mutagenesis. We also discuss the use of animal models in modelling disease of human fungal pathogens, focusing on zebrafish, the silkworm, Galleria mellonella and the murine model.

Publisher Correction: Functional genomic characterization of metallothioneins in brown trout (Salmo trutta L.). using synthetic genetic analysis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Mechanisms of Mating in Pathogenic Fungi-A Plastic Trait.

The impact of fungi on human and plant health is an ever-increasing issue. Recent studies have estimated that human fungal infections result in an excess of one million deaths per year and plant fungal infections resulting in the loss of crop yields worth approximately 200 million per annum. Sexual reproduction in these economically important fungi has evolved in response to the environmental stresses encountered by the pathogens as a method to target DNA damage. Meiosis is integral to this process, through increasing diversity through recombination. Mating and meiosis have been extensively studied in the model yeast Saccharomyces cerevisiae, highlighting that these mechanisms have diverged even between apparently closely related species. To further examine this, this review will inspect these mechanisms in emerging important fungal pathogens, such as Candida, Aspergillus, and Cryptococcus. It shows that both sexual and asexual reproduction in these fungi demonstrate a high degree of plasticity.

Functional genomic characterization of metallothioneins in brown trout (Salmo trutta L.). using synthetic genetic analysis.

Metal pollution has made a significant impact on the earth's ecosystems and tolerance to metals in a wide variety of species has evolved. Metallothioneins, a group of cysteine-rich metal-ion binding proteins, are known to be a key physiological mechanism in regulating protection against metal toxicity. Many rivers across the southwest of England are detrimentally affected by metal pollution, but brown trout (Salmo trutta L.) populations are known to reside within them. In this body of work, two isoforms of metallothionein (MetA and MetB) isolated from trout occupying a polluted and a control river are examined. Using synthetic genetic array (SGA) analyses in the model yeast, Saccharomyces cerevisiae, functional genomics is used to explore the role of metallothionein isoforms in driving metal tolerance. By harnessing this experimental system, S. cerevisiae is used to (i) determine the genetic interaction maps of MetA and MetB isoforms; (ii) identify differences between the genetic interactions in both isoforms and (iii) demonstrate that pre-exposure to metals in metal-tolerant trout influences these interactions. By using a functional genomics approach leveraged from the model yeast Saccharomyces cerevisiae, we demonstrate how such approaches could be used in understanding the ecology and evolution of a non-model species.

Attenuating the emergence of anti-fungal drug resistance by harnessing synthetic lethal interactions in a model organism.

Drug resistance is a rapidly emerging concern, thus prompting the development of novel therapeutics or combinatorial therapy. Currently, combinatorial therapy targets are based on knowledge of drug mode of action and/or resistance mechanisms, constraining the number of target proteins. Unbiased genome-wide screens could reveal novel genetic components within interaction networks as potential targets in combination therapies. Testing this, in the context of antimicrobial resistance, we implemented an unbiased genome-wide screen, performed in Saccharomyces cerevisiae expressing a Candida glabrata PDR1+ gain-of-function allele. Gain-of-function mutations in this gene are the principal mediators of fluconazole resistance in this human fungal pathogen. Eighteen synthetically lethal S. cerevisiae genetic mutants were identified in cells expressing C. glabrata PDR1+. One mutant, lacking the histone acetyltransferase Gcn5, was investigated further. Deletion or drug-mediated inhibition of Gcn5 caused a lethal phenotype in C. glabrata cells expressing PDR1+ alleles. Moreover, deletion or drug-mediated inactivation of Gcn5, inhibited the emergence of fluconazole-resistant C. glabrata isolates in evolution experiments. Thus, taken together, the data generated in this study provides proof of concept that synthetically lethal genetic screens can identify novel candidate proteins that when therapeutically targeted could allow effective treatment of drug-resistant infections.

Using DNA Barcoding to Investigate Patterns of Species Utilisation in UK Shark Products Reveals Threatened Species on Sale.

Many shark populations are in decline, primarily due to overexploitation. In response, conservation measures have been applied at differing scales, often severely restricting sales of declining species. Therefore, DNA barcoding was used to investigate sales of shark products in fishmongers and fish and chip takeaways in England. The majority of samples were identified as Spiny Dogfish (Squalus acanthias), which is critically endangered in the Northeast Atlantic and landings have been prohibited (although there is evidence of importation of this species). Significant differences in the species sold between retailer types were also identified, suggesting differing supply chains. The results underline issues surrounding the use of 'umbrella' sales terms where many species are labelled with the same designation. This denies consumer choice as purchasers cannot easily avoid declining species or those associated with high levels of toxicants. For the first time in Europe, minibarcodes are also used to identify species from dried shark fins. Despite a small sample size, analysis of UK wholesaler fins identified threatened sharks, including the endangered and CITES listed Scalloped Hammerhead (Sphyrna lewini). This highlights the global nature of the damaging trade in endangered shark species, in which Europe and the UK have a continuing role.

Utilising established SDL-screening methods as a tool for the functional genomic characterisation of model and non-model organisms.

The trend for large-scale genetic and phenotypic screens has revealed a wealth of information on biological systems. A major challenge is understanding how genes function and putative roles in networks. The majority of current gene knowledge is garnered from studies utilising the model yeast Saccharomyces cerevisiae. We demonstrate that synthetic dosage lethal genetic array methodologies can be used to study genetic networks in other yeasts, namely the fungal pathogen Candida glabrata, which has limited forward genetic tools, due to the lack of 'natural' mating. We performed two SDL screens in S. cerevisiae, overexpressing the transcriptional regulator UME6 as bait in the first screen and its C. glabrata ortholog CAGL0F05357g in the second. Analysis revealed that SDL maps share 204 common interactors, with 10 genetic interactions unique to C. glabrata indicating a level of genetic rewiring, indicative of linking genotype to phenotype in fungal pathogens. This was further validated by incorporating our results into the global genetic landscape map of the cell from Costanzo et al. to identify common and novel gene attributes. This data demonstrated the utility large data sets and more robust analysis made possible by interrogating exogenous genes in the context of the eukaryotic global genetic landscape.

False interpretation of diagnostic serology tests for patients treated with pooled human immunoglobulin G infusions: a trap for the unwary.

Therapeutic immunoglobulin G (IgG) products are produced from numerous plasma donations, and are infused in many medical conditions. The serological testing of patients who have received IgG infusions may well produce falsely positive and misleading results from this infused IgG, rather than endogenously produced IgG. We present two example cases of clinical situations where this could cause concern. We tested multiple IgG products with a range of serological tests performed in infective or autoimmune conditions, including hepatitis B, syphilis, human immunodeficiency virus, human T-lymphotropic virus, anti-neutrophil cytoplasmic antibody (ANCA), anti-nuclear antibody (ANA), anti-cardiolipin antibodies and anti-double stranded DNA (dsDNA) antibody. We found positivity within these products for hepatitis B surface and core antibody, syphilis, ANCA, ANA, anti-cardiolipin IgG and dsDNA antibody, which may result from specific or non-specific reactivity. The serological testing of patients who have received IgG treatment detects the administered IgG in addition to IgG produced by the patient.

Combinatorial stresses kill pathogenic Candida species.

Pathogenic microbes exist in dynamic niches and have evolved robust adaptive responses to promote survival in their hosts. The major fungal pathogens of humans, Candida albicans and Candida glabrata, are exposed to a range of environmental stresses in their hosts including osmotic, oxidative and nitrosative stresses. Significant efforts have been devoted to the characterization of the adaptive responses to each of these stresses. In the wild, cells are frequently exposed simultaneously to combinations of these stresses and yet the effects of such combinatorial stresses have not been explored. We have developed a common experimental platform to facilitate the comparison of combinatorial stress responses in C. glabrata and C. albicans. This platform is based on the growth of cells in buffered rich medium at 30°C, and was used to define relatively low, medium and high doses of osmotic (NaCl), oxidative (H(2)O(2)) and nitrosative stresses (e.g., dipropylenetriamine (DPTA)-NONOate). The effects of combinatorial stresses were compared with the corresponding individual stresses under these growth conditions. We show for the first time that certain combinations of combinatorial stress are especially potent in terms of their ability to kill C. albicans and C. glabrata and/or inhibit their growth. This was the case for combinations of osmotic plus oxidative stress and for oxidative plus nitrosative stress. We predict that combinatorial stresses may be highly significant in host defences against these pathogenic yeasts.

Chemical and Synthetic Genetic Array Analysis Identifies Genes that Suppress Xylose Utilization and Fermentation in Saccharomyces cerevisiae.

Though highly efficient at fermenting hexose sugars, Saccharomyces cerevisiae has limited ability to ferment five-carbon sugars. As a significant portion of sugars found in cellulosic biomass is the five-carbon sugar xylose, S. cerevisiae must be engineered to metabolize pentose sugars, commonly by the addition of exogenous genes from xylose fermenting fungi. However, these recombinant strains grow poorly on xylose and require further improvement through rational engineering or evolutionary adaptation. To identify unknown genes that contribute to improved xylose fermentation in these recombinant S. cerevisiae, we performed genome-wide synthetic interaction screens to identify deletion mutants that impact xylose utilization of strains expressing the xylose isomerase gene XYLA from Piromyces sp. E2 alone or with an additional copy of the endogenous xylulokinase gene XKS1. We also screened the deletion mutant array to identify mutants whose growth is affected by xylose. Our genetic network reveals that more than 80 nonessential genes from a diverse range of cellular processes impact xylose utilization. Surprisingly, we identified four genes, ALP1, ISC1, RPL20B, and BUD21, that when individually deleted improved xylose utilization of both S. cerevisiae S288C and CEN.PK strains. We further characterized BUD21 deletion mutant cells in batch fermentations and found that they produce ethanol even the absence of exogenous XYLA. We have demonstrated that the ability of laboratory strains of S. cerevisiae to utilize xylose as a sole carbon source is suppressed, which implies that S. cerevisiae may not require the addition of exogenous genes for efficient xylose fermentation.