Gladiolin produced by pathogenic Burkholderia synergizes with amphotericin B through membrane lipid rearrangements.

Amphotericin B (AmpB) is an effective but toxic antifungal drug. Thus, improving its activity/toxicity relationship is of interest. AmpB disrupts fungal membranes by two proposed mechanisms: ergosterol sequestration from the membrane and pore formation. Whether these two mechanisms operate in conjunction and how they could be potentiated remains to be fully understood. Here, we report that gladiolin, a polyketide antibiotic produced by Burkholderia gladioli, is a strong potentiator of AmpB and acts synergistically against Cryptococcus and Candida species, including drug-resistant C. auris. Gladiolin also synergizes with AmpB against drug-resistant fungal biofilms, while exerting no mammalian cytotoxicity. To explain the mechanism of synergy, we show that gladiolin interacts with membranes via a previously unreported binding mode for polyketides. Moreover, gladiolin modulates lipid binding by AmpB and, in combination, causes faster and more pronounced lipid rearrangements relative to AmpB alone which include membrane thinning consistent with ergosterol extraction, areas of thickening, pore formation, and increased membrane destruction. These biophysical data provide evidence of a functional interaction between gladiolin and AmpB at the membrane interface. The data further indicate that the two proposed AmpB mechanisms (ergosterol sequestration and pore formation) act in conjunction to disrupt membranes, and that gladiolin synergizes by enhancing both mechanisms. Collectively, our findings shed light on AmpB's mechanism of action and characterize gladiolin as an AmpB potentiator, showing an antifungal mechanism distinct from its proposed antibiotic activity. We shed light on the synergistic mechanism at the membrane, and provide insights into potentiation strategies to improve AmpB's activity/toxicity relationship. IMPORTANCE: Amphotericin B (AmpB) is one of the oldest antifungal drugs in clinical use. It is an effective therapeutic, but it comes with toxicity issues due to the similarities between its fungal target (the membrane lipid ergosterol) and its mammalian counterpart (cholesterol). One strategy to improve its activity/toxicity relationship is by combinatorial therapy with potentiators, which would enable a lower therapeutic dose of AmpB. Here, we report on the discovery of the antibiotic gladiolin as a potentiator of AmpB against several priority human fungal pathogens and fungal biofilms, with no increased toxicity against mammalian cells. We show that gladiolin potentiates AmpB by increasing and accelerating membrane damage. Our findings also provide insights into the on-going debate about the mechanism of action of AmpB by indicating that both proposed mechanisms, extraction of ergosterol from membranes and pore formation, are potentiated by gladiolin.

Metabolic homeostasis in fungal infections from the perspective of pathogens, immune cells, and whole-body systems.

SUMMARYThe ability to overcome metabolic stress is a major determinant of outcomes during infections. Pathogens face nutrient and oxygen deprivation in host niches and during their encounter with immune cells. Immune cells require metabolic adaptations for producing antimicrobial compounds and mounting antifungal inflammation. Infection also triggers systemic changes in organ metabolism and energy expenditure that range from an enhanced metabolism to produce energy for a robust immune response to reduced metabolism as infection progresses, which coincides with immune and organ dysfunction. Competition for energy and nutrients between hosts and pathogens means that successful survival and recovery from an infection require a balance between elimination of the pathogen by the immune systems (resistance), and doing so with minimal damage to host tissues and organs (tolerance). Here, we discuss our current knowledge of pathogen, immune cell and systemic metabolism in fungal infections, and the impact of metabolic disorders, such as obesity and diabetes. We put forward the idea that, while our knowledge of the use of metabolic regulation for fungal proliferation and antifungal immune responses (i.e., resistance) has been growing over the years, we also need to study the metabolic mechanisms that control tolerance of fungal pathogens. A comprehensive understanding of how to balance resistance and tolerance by metabolic interventions may provide insights into therapeutic strategies that could be used adjunctly with antifungal drugs to improve patient outcomes.

The C-terminal protein interaction domain of the chromatin reader Yaf9 is critical for pathogenesis of Candida albicans.

Fungal infections cause a large health burden but are treated by only a handful of antifungal drug classes. Chromatin factors have emerged as possible targets for new antifungals. These targets include the reader proteins, which interact with posttranslationally modified histones to influence DNA transcription and repair. The YEATS domain is one such reader recognizing both crotonylated and acetylated histones. Here, we performed a detailed structure/function analysis of the Candida albicans YEATS domain reader Yaf9, a subunit of the NuA4 histone acetyltransferase and the SWR1 chromatin remodeling complex. We have previously demonstrated that the homozygous deletion mutant yaf9Δ/Δ displays growth defects and is avirulent in mice. Here we show that a YEATS domain mutant expected to inactivate Yaf9's chromatin binding does not display strong phenotypes in vitro, nor during infection of immune cells or in a mouse systemic infection model, with only a minor virulence reduction in vivo. In contrast to the YEATS domain mutation, deletion of the C-terminal domain of Yaf9, a protein-protein interaction module necessary for its interactions with SWR1 and NuA4, phenocopies the null mutant. This shows that the C-terminal domain is essential for Yaf9 roles in vitro and in vivo, including C. albicans virulence. Our study informs on the strategies for therapeutic targeting of Yaf9, showing that approaches taken for the mammalian YEATS domains by disrupting their chromatin binding might not be effective in C. albicans, and provides a foundation for studying YEATS proteins in human fungal pathogens.IMPORTANCEThe scarcity of available antifungal drugs and rising resistance demand the development of therapies with new modes of action. In this context, chromatin regulation may be a target for novel antifungal therapeutics. To realize this potential, we must better understand the roles of chromatin regulators in fungal pathogens. Toward this goal, here, we studied the YEATS domain chromatin reader Yaf9 in Candida albicans. Yaf9 uses the YEATS domain for chromatin binding and a C-terminal domain to interact with chromatin remodeling complexes. By constructing mutants in these domains and characterizing their phenotypes, our data indicate that the Yaf9 YEATS domain might not be a suitable therapeutic drug target. Instead, the Yaf9 C-terminal domain is critical for C. albicans virulence. Collectively, our study informs how a class of chromatin regulators performs their cellular and pathogenesis roles in C. albicans and reveals strategies to inhibit them.

The short-chain fatty acid crotonate reduces invasive growth and immune escape of Candida albicans by regulating hyphal gene expression.

Microbes are exposed to nutritional and stress challenges in their environmental and host niches. To rise to these challenges, they regulate transcriptional programs that enable cellular adaptation. For instance, metabolite concentrations regulate post-translational modifications of chromatin, such as histone acetylation. In this way, metabolic signals are integrated with transcription. Over the last decade, several histone acylations have been discovered, including histone crotonylation. Their roles in microbial biology, environmental adaptation, and microbe-host interactions are incompletely defined. Here we show that the short-chain fatty acid crotonate, which is used to study histone crotonylation, changes cell morphology and immune interactions of Candida albicans. Crotonate reduces invasive hyphal morphogenesis of C. albicans within macrophages, thereby delaying macrophage killing and pathogen escape, as well as reducing inflammatory cytokine maturation. Crotonate's ability to reduce hyphal growth is environmentally contingent and pronounced within macrophages. Moreover, crotonate is a stronger hyphal inhibitor than butyrate under the conditions that we tested. Crotonate causes increased histone crotonylation in C. albicans under hyphal growth conditions and reduces transcription of hyphae-induced genes in a manner that involves the Nrg1 repressor pathway. Increasing histone acetylation by histone deacetylase inhibition partially rescues hyphal growth and gene transcription in the presence of crotonate. These results indicate that histone crotonylation might compete with acetylation in the regulation of hyphal morphogenesis. Based on our findings, we propose that diverse acylations of histones (and likely also non-histone proteins) enable C. albicans to respond to environmental signals, which in turn regulate its cell morphology and host-pathogen interactions.IMPORTANCEMacrophages curtail the proliferation of the pathogen Candida albicans within human body niches. Within macrophages, C. albicans adapts its metabolism and switches to invasive hyphal morphology. These adaptations enable fungal growth and immune escape by triggering macrophage lysis. Transcriptional programs regulate these metabolic and morphogenetic adaptations. Here we studied the roles of chromatin in these processes and implicate lysine crotonylation, a histone mark regulated by metabolism, in hyphal morphogenesis and macrophage interactions by C. albicans. We show that the short-chain fatty acid crotonate increases histone crotonylation, reduces hyphal formation within macrophages, and slows macrophage lysis and immune escape of C. albicans. Crotonate represses hyphal gene expression, and we propose that C. albicans uses diverse acylation marks to regulate its cell morphology in host environments. Hyphal formation is a virulence property of C. albicans. Therefore, a further importance of our study stems from identifying crotonate as a hyphal inhibitor.

Leveraging the MMV Pathogen Box to Engineer an Antifungal Compound with Improved Efficacy and Selectivity against Candida auris.

Fungal infections pose a significant and increasing threat to human health, but the current arsenal of antifungal drugs is inadequate. We screened the Medicines for Malaria Venture (MMV) Pathogen Box for new antifungal agents against three of the most critical Candida species (Candida albicans, Candida auris, and Candida glabrata). Of the 14 identified hit compounds, most were active against C. albicans and C. auris. We selected the pyrazolo-pyrimidine MMV022478 for chemical modifications to build structure-activity relationships and study their antifungal properties. Two analogues, 7a and 8g, with distinct fluorine substitutions, greatly improved the efficacy against C. auris and inhibited fungal replication inside immune cells. Additionally, analogue 7a had improved selectivity toward fungal killing compared to mammalian cytotoxicity. Evolution experiments generating MMV022478-resistant isolates revealed a change in morphology from oblong to round cells. Most notably, the resistant isolates blocked the uptake of the fluorescent dye rhodamine 6G and showed reduced susceptibility toward fluconazole, indicative of structural changes in the yeast cell surface. In summary, our study identified a promising antifungal compound with activity against high-priority fungal pathogens. Additionally, we demonstrated how structure-activity relationship studies of known and publicly available compounds can expand the repertoire of molecules with antifungal efficacy and reduced cytotoxicity to drive the development of novel therapeutics.

Candida auris uses metabolic strategies to escape and kill macrophages while avoiding robust activation of the NLRP3 inflammasome response.

Metabolic adaptations regulate the response of macrophages to infection. The contributions of metabolism to macrophage interactions with the emerging fungal pathogen Candida auris are poorly understood. Here, we show that C. auris-infected macrophages undergo immunometabolic reprogramming and increase glycolysis but fail to activate a strong interleukin (IL)-1ß cytokine response or curb C. auris growth. Further analysis shows that C. auris relies on its own metabolic capacity to escape from macrophages and proliferate in vivo. Furthermore, C. auris kills macrophages by triggering host metabolic stress through glucose starvation. However, despite causing macrophage cell death, C. auris does not trigger robust activation of the NLRP3 inflammasome. Consequently, inflammasome-dependent responses remain low throughout infection. Collectively, our findings show that C. auris uses metabolic regulation to eliminate macrophages while remaining immunologically silent to ensure its own survival. Thus, our data suggest that host and pathogen metabolism could represent therapeutic targets for C. auris infections.

The proteasome regulator Rpn4 controls antifungal drug tolerance by coupling protein homeostasis with metabolic responses to drug stress.

Fungal pathogens overcome antifungal drug therapy by classic resistance mechanisms, such as increased efflux or changes to the drug target. However, even when a fungal strain is susceptible, trailing or persistent microbial growth in the presence of an antifungal drug can contribute to therapeutic failure. This trailing growth is caused by adaptive physiological changes that enable the growth of a subpopulation of fungal cells in high drug concentrations, in what is described as drug tolerance. Mechanistically, antifungal drug tolerance is incompletely understood. Here we report that the transcriptional activator Rpn4 is important for drug tolerance in the human fungal pathogen Candida albicans. Deletion of RPN4 eliminates tolerance to the commonly used antifungal drug fluconazole. We defined the mechanism and show that Rpn4 controls fluconazole tolerance via two target pathways. First, Rpn4 activates proteasome gene expression, which enables sufficient proteasome capacity to overcome fluconazole-induced proteotoxicity and the accumulation of ubiquitinated proteins targeted for degradation. Consistently, inhibition of the proteasome with MG132 eliminates fluconazole tolerance and resistance, and phenocopies the rpn4Δ/Δ mutant for loss of tolerance. Second, Rpn4 is required for wild type expression of the genes required for the synthesis of the membrane lipid ergosterol. Our data indicates that this function of Rpn4 is required for mitigating the inhibition of ergosterol biosynthesis by fluconazole. Based on our findings, we propose that Rpn4 is a central hub for fluconazole tolerance in C. albicans by coupling the regulation of protein homeostasis (proteostasis) and lipid metabolism to overcome drug-induced proteotoxicity and membrane stress.

The escape of Candida albicans from macrophages is enabled by the fungal toxin candidalysin and two host cell death pathways.

The egress of Candida hyphae from macrophages facilitates immune evasion, but it also alerts macrophages to infection and triggers inflammation. To better define the mechanisms, here we develop an imaging assay to directly and dynamically quantify hyphal escape and correlate it to macrophage responses. The assay reveals that Candida escapes by using two pore-forming proteins to permeabilize macrophage membranes: the fungal toxin candidalysin and Nlrp3 inflammasome-activated Gasdermin D. Candidalysin plays a major role in escape, with Nlrp3 and Gasdermin D-dependent and -independent contributions. The inactivation of Nlrp3 does not reduce hyphal escape, and we identify ETosis via macrophage extracellular trap formation as an additional pathway facilitating hyphal escape. Suppressing hyphal escape does not reduce fungal loads, but it does reduce inflammatory activation. Our findings explain how Candida escapes from macrophages by using three strategies: permeabilizing macrophage membranes via candidalysin and engaging two host cell death pathways, Gasdermin D-mediated pyroptosis and ETosis.

Disruption of Iron Homeostasis and Mitochondrial Metabolism Are Promising Targets to Inhibit Candida auris.

Fungal infections are a global threat, but treatments are limited due to a paucity in antifungal drug targets and the emergence of drug-resistant fungi such as Candida auris. Metabolic adaptations enable microbial growth in nutrient-scarce host niches, and they further control immune responses to pathogens, thereby offering opportunities for therapeutic targeting. Because it is a relatively new pathogen, little is known about the metabolic requirements for C. auris growth and its adaptations to counter host defenses. Here, we establish that triggering metabolic dysfunction is a promising strategy against C. auris. Treatment with pyrvinium pamoate (PP) induced metabolic reprogramming and mitochondrial dysfunction evident in disrupted mitochondrial morphology and reduced tricarboxylic acid (TCA) cycle enzyme activity. PP also induced changes consistent with disrupted iron homeostasis. Nutrient supplementation experiments support the proposition that PP-induced metabolic dysfunction is driven by disrupted iron homeostasis, which compromises carbon and lipid metabolism and mitochondria. PP inhibited C. auris replication in macrophages, which is a relevant host niche for this yeast pathogen. We propose that PP causes a multipronged metabolic hit to C. auris: it restricts the micronutrient iron to potentiate nutritional immunity imposed by immune cells, and it further causes metabolic dysfunction that compromises the utilization of macronutrients, thereby curbing the metabolic plasticity needed for growth in host environments. Our study offers a new avenue for therapeutic development against drug-resistant C. auris, shows how complex metabolic dysfunction can be caused by a single compound triggering antifungal inhibition, and provides insights into the metabolic needs of C. auris in immune cell environments. IMPORTANCE Over the last decade, Candida auris has emerged as a human pathogen around the world causing life-threatening infections with wide-spread antifungal drug resistance, including pandrug resistance in some cases. In this study, we addressed the mechanism of action of the antiparasitic drug pyrvinium pamoate against C. auris and show how metabolism could be inhibited to curb C. auris proliferation. We show that pyrvinium pamoate triggers sweeping metabolic and mitochondrial changes and disrupts iron homeostasis. PP-induced metabolic dysfunction compromises the utilization of both micro- and macronutrients by C. auris and reduces its growth in vitro and in immune phagocytes. Our findings provide insights into the metabolic requirements for C. auris growth and define the mechanisms of action of pyrvinium pamoate against C. auris, demonstrating how this compound works by inhibiting the metabolic flexibility of the pathogen. As such, our study characterizes credible avenues for new antifungal approaches against C. auris.

The novel Dbl homology/BAR domain protein, MsgA, of Talaromyces marneffei regulates yeast morphogenesis during growth inside host cells.

Microbial pathogens have evolved many strategies to evade recognition by the host immune system, including the use of phagocytic cells as a niche within which to proliferate. Dimorphic pathogenic fungi employ an induced morphogenetic transition, switching from multicellular hyphae to unicellular yeast that are more compatible with intracellular growth. A switch to mammalian host body temperature (37 °C) is a key trigger for the dimorphic switch. This study describes a novel gene, msgA, from the dimorphic fungal pathogen Talaromyces marneffei that controls cell morphology in response to host cues rather than temperature. The msgA gene is upregulated during murine macrophage infection, and deletion results in aberrant yeast morphology solely during growth inside macrophages. MsgA contains a Dbl homology domain, and a Bin, Amphiphysin, Rvs (BAR) domain instead of a Plekstrin homology domain typically associated with guanine nucleotide exchange factors (GEFs). The BAR domain is crucial in maintaining yeast morphology and cellular localisation during infection. The data suggests that MsgA does not act as a canonical GEF during macrophage infection and identifies a temperature independent pathway in T. marneffei that controls intracellular yeast morphogenesis.

Immunometabolism in fungal infections: the need to eat to compete.

Immune cells, including macrophages and monocytes, remodel their metabolism and have specific nutritional needs when dealing with microbial pathogens. While we are just beginning to understand immunometabolism in fungal infections, emerging themes include recognition of fungal cell surface molecule driving metabolic remodelling to increase glycolysis, the critical role of glycolysis in the production of antifungal cytokines and fungicidal effector molecules, and the need for maintaining host glucose homeostasis to defeat fungal infections. A crosstalk between host and pathogen metabolic pathways determines the fate of immune cells and fungi when they interact. Thus, immunometabolic interactions offer potential for innovation in antifungal treatments in the future. For this to become a reality, we must decipher the mechanisms by which diverse fungal pathogens activate and manipulate immunometabolism.

A unique aspartyl protease gene expansion in Talaromyces marneffei plays a role in growth inside host phagocytes.

Aspartyl proteases are a widely represented class of proteolytic enzymes found in eukaryotes and retroviruses. They have been associated with pathogenicity in a range of disease-causing microorganisms. The dimorphic human-pathogenic fungus Talaromyces marneffei has a large expansion of these proteases identified through genomic analyses. Here we characterize the expansion of these genes (pop - paralogue of pep) and their role in T. marneffei using computational and molecular approaches. Many of the genes in this monophyletic family show copy number variation and positive selection despite the preservation of functional regions and possible redundancy. We show that the expression profile of these genes differs and some are expressed during intracellular growth in the host. Several of these proteins have distinctive localization as well as both additive and epistatic effects on the formation of yeast cells during macrophage infections. The data suggest that this is a recently evolved aspartyl protease gene family which affects intracellular growth and contributes to the pathogenicity of T. marneffei.

Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo.

Neutrophils and macrophages provide the first line of cellular defence against pathogens once physical barriers are breached, but can play very different roles for each specific pathogen. This is particularly so for fungal pathogens, which can occupy several niches in the host. We developed an infection model of talaromycosis in zebrafish embryos with the thermally-dimorphic intracellular fungal pathogen Talaromyces marneffei and used it to define different roles of neutrophils and macrophages in infection establishment. This system models opportunistic human infection prevalent in HIV-infected patients, as zebrafish embryos have intact innate immunity but, like HIV-infected talaromycosis patients, lack a functional adaptive immune system. Importantly, this new talaromycosis model permits thermal shifts not possible in mammalian models, which we show does not significantly impact on leukocyte migration, phagocytosis and function in an established Aspergillus fumigatus model. Furthermore, the optical transparency of zebrafish embryos facilitates imaging of leukocyte/pathogen interactions in vivo. Following parenteral inoculation, T. marneffei conidia were phagocytosed by both neutrophils and macrophages. Within these different leukocytes, intracellular fungal form varied, indicating that triggers in the intracellular milieu can override thermal morphological determinants. As in human talaromycosis, conidia were predominantly phagocytosed by macrophages rather than neutrophils. Macrophages provided an intracellular niche that supported yeast morphology. Despite their minor role in T. marneffei conidial phagocytosis, neutrophil numbers increased during infection from a protective CSF3-dependent granulopoietic response. By perturbing the relative abundance of neutrophils and macrophages during conidial inoculation, we demonstrate that the macrophage intracellular niche favours infection establishment by protecting conidia from a myeloperoxidase-dependent neutrophil fungicidal activity. These studies provide a new in vivo model of talaromycosis with several advantages over previous models. Our findings demonstrate that limiting T. marneffei's opportunity for macrophage parasitism and thereby enhancing this pathogen's exposure to effective neutrophil fungicidal mechanisms may represent a novel host-directed therapeutic opportunity.

Organism-wide studies into pathogenicity and morphogenesis in Talaromyces marneffei.

Organism-wide approaches examining the genetic mechanisms controlling growth and proliferation have proven to be a powerful tool in the study of pathogenic fungi. For many fungal pathogens techniques to study transcription and protein expression are particularly useful, and offer insights into infection processes by these species. Here we discuss the use of approaches such as differential display, suppression subtractive hybridization, microarray, RNA-seq, proteomics, genetic manipulation and infection models for the AIDS-defining pathogen Talaromyces marneffei. Together these methods have broadened our understanding of the biological processes, and genes that underlie them, which are involved in switching between the saprophytic and pathogenic states of T. marneffei, the maintenance of these two specialized cell types and its ability to cause disease.

Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei.

Penicillium marneffei is an opportunistic pathogen of humans and displays a temperature dependent dimorphic transition. Like many fungi, exogenous DNA introduced by DNA mediated transformation is integrated randomly into the genome resulting in inefficient gene deletion and position-specific effects. To enhance successful gene targeting, the consequences of perturbing components of the non-homologous end joining recombination pathway have been examined. The deletion of the KU70 and LIG4 orthologs, pkuA and ligD, respectively, dramatically enhanced the observed homologous recombination frequency leading to efficient gene deletion. While ΔpkuA was associated with reduced genetic stability over-time, ΔligD represents a suitable recipient strain for downstream applications and combined with a modified Gateway™ system for the rapid generation of gene deletion constructs, this represents an efficient pipeline for characterizing gene function in P. marneffei.