Strife concerning the accessibility of essential trace elements, such as transition metals, represents an important aspect of the dynamic interaction between a pathogenic fungus and its mammalian host. The host defends itself against infection by sequestering these essential micronutrients away from the invading pathogen via a phenomenon termed “nutritional immunity”. In turn, the fungus employs an array of tactics (scavenging and storage) to hoard micronutrients and support growth when these resources are scarce. In addition, micronutrient limitation triggers the expression of virulence determinants that can aggravate disease

Nutritional immunity is a process whereby an infected host manipulates essential micronutrients to defend against an invading pathogen. We reveal a dynamic aspect of nutritionalimmunity during infection that involves copper assimilation. Using a combination of laser ablation inductively coupled mass spectrometry (LA-ICP MS) and metal mapping, immunohistochemistry, and gene expression profiling from infected tissues, we show that readjustments in hepatic, splenic and renal copper homeostasis accompany disseminated Candidaalbicans infections in the mouse model. Localized host-imposed copper poisoning manifests itself as a transient increase in copper early in the kidney infection. Changes in renal copper are detected by the fungus, as revealed by gene expression profiling and fungal virulence studies. The fungus responds by differentially regulating the Crp1 copper efflux pump (higher expression during early infection and down-regulation late in infection) and the Ctr1 copper importer (lower expression during early infection, and subsequent up-regulation late in infection) to maintain copper homeostasis during disease progression. Both Crp1 and Ctr1 are required for full fungal virulence. Importantly, copper homeostasis influences other virulence traits—metabolic flexibility and oxidative stress resistance. Our study highlights the importance of copper homeostasis for host defence and fungal virulence during systemic disease.

Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype.

Opportunistic infections by environmental fungi are a growing clinical problem, driven by an increasing population of people with immunocompromising conditions. Spores of the Mucorales order are ubiquitous in the environment but can also cause acute invasive infections in humans through germination and evasion of the mammalian host immune system. How they achieve this and the evolutionary drivers underlying the acquisition of virulence mechanisms are poorly understood. Here, we show that a clinical isolate of Rhizopus microsporus contains a Ralstonia pickettii bacterial endosymbiont required for virulence in both zebrafish and mice and that this endosymbiosis enables the secretion of factors that potently suppress growth of the soil amoeba Dictyostelium discoideum, as well as their ability to engulf and kill other microbes. As amoebas are natural environmental predators of both bacteria and fungi, we propose that this tri-kingdom interaction contributes to establishing endosymbiosis and the acquisition of anti-phagocyte activity. Importantly, we show that this activity also protects fungal spores from phagocytosis and clearance by human macrophages, and endosymbiont removal renders the fungal spores avirulent in vivo. Together, these findings describe a new role for a bacterial endosymbiont in Rhizopus microsporus pathogenesis in animals and suggest a mechanism of virulence acquisition through environmental interactions with amoebas.

Pathogenic fungi have the remarkable ability to undergo morphological changes that can determine their virulence potential. In this issue of Cell Host & Microbe, Denham et al. identify a fungal morphotype that is uniquely adapted for extrapulmonary dissemination, contributing toward invasive infection and escaping host immune responses.

Fungal morphology significantly impacts the host response. Filamentation and tissue penetration by Candida and Aspergillus species are essential for virulence, while growth as a yeast allows the thermal dimorphic fungi Coccidiodes, Histoplasma, and Talaromyces to reside inside phagocytes and disseminate. The basidiomycete Cryptococcus neoformans exhibits an unusual yeast-to-titan transition thought to enhance pathogenicity by increasing fungal survival in the host lung and dissemination to the central nervous system. In a common laboratory strain (H99), in vitro and in vivo titan induction yields a heterogenous population including >10 μm titan cells, 5-7 μm yeast cells and 2-4 μm titanides. Previous reports have shown that titan cells are associated with enhanced virulence and the generation of aneuploid cells that facilitate stress adaptation and drug resistance, while small (>10 μm) cells are associated with increased dissemination. However, the relationship between titan cells, small cells, and titanides remains unclear. Here, we characterize titanides and small cells in H99 and three clinical isolates and show that titanides share the lipid membrane order of their titan mothers and the G 0 quiescent-like DNA staining of mating spores. In addition, we show that both titanizing and non-titanizing isolates exhibit altered capsule structure and PAMP exposure over time during in vitro culture, and generate aneuploidy in vivo. Author summary the human fungal pathogen Cryptococcus neoformans causes 200,000 HIV-associated deaths each year. In the lung, Cryptococcus makes an unusual yeast-to-titan morphological switch that contributes to disease development by altering immune polarization and introducing aneuploidy underlying host stress and drug resistance. Specifically, a proportion of 5 um haploid yeast endoreduplicate and swell, converting to large (> 10 um) polyploid titan cells that can then produce genetically distinct daughter cells. We recently developed an in vitro protocol for inducing large titan cells and additionally observed a novel small “titanide” cell type. Here we investigate the nature and origin of these small cells, demonstrating that they emerge during both in vitro and in vivo mouse-passaged titan induction in the well characterised lab strain H99 and are also apparent in a titanizing clinical isolate, Zc8. We show that these titanide cells share features with titan mothers (lipid order) and with spores produced during heterothalic mating. Finally, we show that the capacity of clinical isolates to produce both titan and titanide cells impacts aneuploidy and the emergence of drug resistance in vivo.

The RNase II family of 3'-5' exoribonucleases are present in all domains of life, and eukaryotic family members Dis3 and Dis3L2 play essential roles in RNA degradation. Ascomycete yeasts contain both Dis3 and inactive RNase II-like "pseudonucleases". These function as RNA-binding proteins that affect cell growth, cytokinesis, and fungal pathogenicity. Here, we show how these pseudonuclease homologs, including Saccharomyces cerevisiae Ssd1, are descended from active Dis3L2 enzymes. During fungal evolution, active site mutations in Dis3L2 homologs have arisen at least four times, in some cases following gene duplication. The N-terminal cold-shock domains and regulatory features are conserved across diverse dikarya and mucoromycota, suggesting that the non-nuclease function require this region. In the basidiomycete pathogenic yeast Cryptococcus neoformans, the single Ssd1/Dis3L2 homolog is required for cytokinesis from polyploid "titan" growth stages and yet retains an active site sequence signature. We propose that that a nuclease-independent function for Dis3L2 arose in an ancestral hyphae-forming fungus. This second function has been conserved across hundreds of millions of years, while the RNase activity was lost repeatedly in independent lineages.

Elizabeth Ballou works in the field of medical mycology. In this mSphere of Influence article, she reflects on how two papers by Okagaki et al. (PLoS Pathog 6:e1000953, 2010, https://doi.org/10.1371/journal.ppat.1000953) and Zaragoza et al. (PLoS Pathog 6:e1000945, 2010, https://doi.org/10.1371/journal.ppat.1000945) made an impact on her career by demonstrating an alternative to destructive publication practices.

Traditional in vivo investigation of fungal infection and new antifungal therapies in mouse models is usually carried out using post mortem methodologies. However, biomedical imaging techniques focusing on non-invasive techniques using bioluminescent and fluorescent proteins have become valuable tools. These new techniques address ethical concerns as they allow reduction in the number of animals required to evaluate new antifungal therapies. They also allow better understanding of the growth and spread of the pathogen during infection. In this review, we concentrate on imaging technologies using different fungal reporter proteins. We discuss the advantages and limitations of these different reporters and compare the efficacy of bioluminescent and fluorescent proteins for fungal research.

Increasing resistance to frontline antifungals is a growing threat to global health. In the face of high rates of relapse for patients with cryptococcal meningitis and frequent drug resistance in clinical isolates, recent insights into Cryptococcus neoformans morphogenesis and genome plasticity take on new and urgent meaning. Here we review the state of the understanding of mechanisms of drug resistance in the context of host-relevant changes in Cryptococcus morphology and cell ploidy.

Mucorales spores, the causative agents of mucormycosis, interact with the innate immune system to cause acute, chronic, or resolving infection. Understanding the factors that influence disease initiation and progression is key to understanding mucormycosis and developing new treatments. Complicating this, mucormycosis can be caused by a number of species that span the Mucorales order and may be host to bacterial endosymbionts. This study sets out to examine the differences between two species in the Mucorales order by characterising their differential interactions with the innate immune system, and their interactions with environmental bacterial endosymbionts. Through a holistic approach, this study examines the transcriptional responses of Rhizopus delemar and Rhizopus microsporus , two of the most commonly diagnosed species, to innate immune cells. This study also examines the immune cell response and assesses the variation in these responses, given the presence or absence of bacterial endosymbionts within the fungi. We see that the fungal response is driven by interaction with innate immune cells. Moreover, the effect of the bacterial endosymbiont on the fungus is species-specific and strongly influences fungal transcription during phagocyte stress. The macrophage response varies depending on the infecting fungal species, and depending on endosymbiont status. Macrophages are better able to survive when germination is inhibited, or upon a pro-inflammatory response. This work reveals species-specific host responses to related Mucorales spores and shows that bacterial endosymbionts have an important role to play by impacting both innate immune cell response, and fungal response when under stress.

Despite the high prevalence of women in graduate degree programs and equal or more women earning PhDs, MDs, and MD/PhDs, and despite efforts at individual and institutional levels to promote women in STEM fields, there remains a disparity in pay and academic advancement of women. Likewise, there is a paucity of women in top scientific and academic leadership positions. The causes of this gender disparity are complex and multi-factorial and to date no "magic bullet" approach has been successful in changing the landscape for women in academic and scientific fields. In this report we detail our experiences with a novel mechanism for promoting discussion and raising awareness of the challenges of gender disparity in the sciences. The Gordon Research Conferences (GRC) launched the Power Hour at its meetings in 2016: a dedicated, scheduled session held during the scientific meeting to facilitate discussion of challenges specific to women in science. Here we share our experience with hosting the second Power Hour at the 2019 GRC Immunology of Fungal Infections (IFI) meeting held in Galveston, TX. We will discuss the overall structure, key discussion points, and feedback from participants with the aim of supporting future efforts to empower women and underrepresented minority groups in science.

Opportunistic infections by environmental fungi are a growing clinical problem, driven by an increasing population of people with immunocompromising conditions. Spores of the Mucorales order are ubiquitious in the environment but can also cause acute invasive infections in humans through germination and evasion of the mammalian host immune system. How they achieve this, and the evolutionary drivers underlying the acquisition of virulence mechanisms, are poorly understood. Here we show that a clinical isolate of Rhizopus microsporus contains a Ralstonia pickettii bacterial endosymbiont required for virulence in both zebrafish and mice, and that this endosymbiosis enables secretion of factors that potently suppress growth of the soil amoeba Dictyostelium discoideum , as well as their ability to engulf and kill other microbes. As amoebae are natural environmental predators of both bacteria and fungi, we propose this tri-kingdom interaction contributes to establishing the endosymbiosis, and acquisition of anti-phagocyte activity. Importantly, we show this activity also protects fungal spores from phagocytosis and clearance by human macrophages, and endosymbiont removal renders the fungal spores avirulent in vivo. Together, these findings describe a novel role for a bacterial endosymbiont in Rhizopus microsporus pathogenesis in animals, and suggest a mechanism of virulence acquisition through environmental interactions with amoebae. In brief How environmental fungi evolved the mechanisms that enable them to cause opportunistic infections in humans is unclear. Here, we identify a novel tri-kingdom interaction, whereby a bacterial endosymbiont, living within a clinical isolate of the ubiquitous environmental fungus Rhizopus microsporus , causes the generation of a secreted activity that blocks the growth and predatory activity of amoebae. We suggest this provides a new evolutionary driver for the establishment of bacterial/fungal endosymbiosis and demonstrate this is critical for fungal pathogenicity in vivo.

The balance between reactive oxygen species and reactive nitrogen species production by the host and stress response by fungi is a key axis of the host-pathogen interaction. This review will describe emerging themes in fungal pathogenesis underpinning this axis.

Purpose of Review: During infection, the human fungal pathogen Cryptococcus neoformans undergoes an unusual change in size, from small haploid yeast to large polyploid Titan cells. This transition is now well recognized as a virulence factor, but significant questions remain about how Titanisation is regulated and how it influences disease progression. Progress has been impeded by the lack of an in vitro model for the yeast-to-Titan transition, a challenge that was recently overcome by three independent groups. Recent Findings: Here, we review Titanization in the context of patient samples and animal models and set the stage for three new reports describing in vitro Titan cell induction assays. We compare and contrast key findings, place them in the broader research context, and identify areas of further interest. Summary: New in vitro models will allow pressing questions about molecular mechanisms driving the yeast-to-Titan transition and their influence on drug resistance and pathogenesis to be addressed.

Candida albicans is able to proliferate in environments that vary dramatically in ambient pH, a trait required for colonising niches such as the stomach, vaginal mucosal and the GI tract. Here we show that growth in acidic environments involves cell wall remodelling which results in enhanced chitin and β-glucan exposure at the cell wall periphery. Unmasking of the underlying immuno-stimulatory β-glucan in acidic environments enhanced innate immune recognition of C. albicans by macrophages and neutrophils, and induced a stronger proinflammatory cytokine response, driven through the C-type lectin-like receptor, Dectin-1. This enhanced inflammatory response resulted in significant recruitment of neutrophils in an intraperitoneal model of infection, a hallmark of symptomatic vaginal colonisation. Enhanced chitin exposure resulted from reduced expression of the cell wall chitinase Cht2, via a Bcr1-Rim101 dependent signalling cascade, while increased β-glucan exposure was regulated via a non-canonical signalling pathway. We propose that this "unmasking" of the cell wall may induce non-protective hyper activation of the immune system during growth in acidic niches, and may attribute to symptomatic vaginal infection.

Upon Cryptococcus neoformans infection of the host lung, the fungus enters a nutrient poor environment and must adapt to a variety of host-specific stress conditions (temperature, nutrient limitation, pH, CO2). Fungal spores enter this milieu with limited nutritional reserves, germinate, and begin proliferating by budding as yeast. Although relatively little is known about the initial stages of infection, recent work has characterized changes that occur upon germination. This program and subsequent yeast-phase proliferation progress in a dynamic environment as host nutrient immunity responds to the infection via toxic accumulation or sequestration of essential micronutrients and innate immune cells are recruited to the site of infection. Adaptation to the host environment and evasion of the immune response through pathogenicity factor expression allows proliferation and dissemination to multiple sites throughout the body, including, most significantly for human disease, the central nervous system. Here we will discuss recent insights into mechanisms underlying C. neoformans interactions with the host during infection.

As they proliferate, fungi expose antigens at their cell surface that are potent stimulators of the innate immune response, and yet the commensal fungus Candida albicans is able to colonize immuno competent individuals. We show that C. albicans may evade immune detection by presenting a moving immunological target. We report that the exposure of β-glucan, a key pathogen-associated molecular pattern (PAMP) located at the cell surface of C. albicans and other pathogenic Candida species, is modulated in response to changes in the carbon source. Exposure to lactate induces β-glucan masking in C. albicans via a signalling pathway that has recruited an evolutionarily conserved receptor (Gpr1) and transcriptional factor (Crz1) from other well-characterized pathways. In response to lactate, these regulators control the expression of cell-wall-related genes that contribute to β-glucan masking. This represents the first description of active PAMP masking by a Candida species, a process that reduces the visibility of the fungus to the immune system.

Efficient carbon assimilation is critical for microbial growth and pathogenesis. The environmental yeast Saccharomyces cerevisiae is “Crabtree positive”, displaying a rapid metabolic switch from the assimilation of alternative carbon sources to sugars. Following exposure to sugars, this switch is mediated by the transcriptional repression of genes (carbon catabolite repression) and the turnover (catabolite inactivation) of enzymes involved in the assimilation of alternative carbon sources. The pathogenic yeast Candida albicans is Crabtree negative. It has retained carbon catabolite repression mechanisms, but has undergone posttranscriptional rewiring such that gluconeogenic and glyoxylate cycle enzymes are not subject to ubiquitin-mediated catabolite inactivation. Consequently, when glucose becomes available, C. albicans can continue to assimilate alternative carbon sources alongside the glucose. We show that this metabolic flexibility promotes host colonization and virulence. The glyoxylate cycle enzyme isocitrate lyase (CaIcl1) was rendered sensitive to ubiquitin-mediated catabolite inactivation in C. albicans by addition of a ubiquitination site. This mutation, which inhibits lactate assimilation in the presence of glucose, reduces the ability of C. albicans cells to withstand macrophage killing, colonize the gastrointestinal tract and cause systemic infections in mice. Interestingly, most S. cerevisiae clinical isolates we examined (67%) have acquired the ability to assimilate lactate in the presence of glucose (i.e. they have become Crabtree negative). These S. cerevisiae strains are more resistant to macrophage killing than Crabtree positive clinical isolates. Moreover, Crabtree negative S. cerevisiae mutants that lack Gid8, a key component of the Glucose-Induced Degradation complex, are more resistant to macrophage killing and display increased virulence in immunocompromised mice. Thus, while Crabtree positivity might impart a fitness advantage for yeasts in environmental niches, the more flexible carbon assimilation strategies offered by Crabtree negativity enhance the ability of yeasts to colonize and infect the mammalian host.

All organisms must secure essential trace nutrients, including iron, zinc, manganese and copper for survival and proliferation. However, these very nutrients are also highly toxic if present at elevated levels. Mammalian immunity has harnessed both the essentiality and toxicity of micronutrients to defend against microbial invasion-processes known collectively as 'nutritional immunity'. Therefore, pathogenic microbes must possess highly effective micronutrient assimilation and detoxification mechanisms to survive and proliferate within the infected host. In this review we compare and contrast the micronutrient homeostatic mechanisms of Cryptococcus and Candida-yeasts which, despite ancient evolutionary divergence, account for over a million life-threatening infections per year. We focus on two emerging arenas within the host-pathogen battle for essential trace metals: adaptive responses to zinc limitation and copper availability.

Candida albicans is a major life-threatening human fungal pathogen in the immunocompromised host. Host defense against systemic Candida infection relies heavily on the capacity of professional phagocytes of the innate immune system to ingest and destroy fungal cells. A number of pathogens, including C. albicans, have evolved mechanisms that attenuate the efficiency of phagosome-mediated inactivation, promoting their survival and replication within the host. Here we visualize hostpathogen interactions using live-cell imaging and show that viable, but not heat- or UV-killed C. albicans cells profoundly delay phagosome maturation in macrophage cell lines and primary macrophages. The ability of C. albicans to delay phagosome maturation is dependent on cell wall composition and fungal morphology. Loss of cell wall O-mannan is associated with enhanced acquisition of phagosome maturation markers, distinct changes in Rab GTPase acquisition by the maturing phagosome, impaired hyphal growth within macrophage phagosomes, profound changes in macrophage actin dynamics, and ultimately a reduced ability of fungal cells to escape from macrophage phagosomes. The loss of cell wall O-mannan leads to exposure of β-glucan in the inner cell wall, facilitating recognition by Dectin-1, which is associated with enhanced phagosome maturation.

Precise genome modification is essential for the molecular dissection of Candida albicans, and is yielding invaluable information about the roles of specific gene functions in this major fungal pathogen of humans. C. albicans is naturally diploid, unable to undergo meiosis, and utilizes a non-canonical genetic code. Hence, specialized tools have had to be developed for gene disruption in C. albicans that permit the deletion of both target alleles, and in some cases, the recycling of the Candida-specific selectable markers. Previously, we developed a tool based on the Cre recombinase, which recycles markers in C. albicans with 90-100% efficiency via site-specific recombination between loxP sites. Ironically, the utility of this system was hampered by the extreme efficiency of Cre, which prevented the construction in Escherichia coli of stable disruption cassettes carrying a methionine-regulatable CaMET3p-cre gene flanked by loxP sites. Therefore, we have significantly enhanced this system by engineering new Clox cassettes that carry a synthetic, intron-containing cre gene. The Clox kit facilitates efficient transformation and marker recycling, thereby simplifying and accelerating the process of gene disruption in C. albicans. Indeed, homozygous mutants can be generated and their markers resolved within two weeks. The Clox kit facilitates strategies involving single marker recycling or multi-marker gene disruption. Furthermore, it includes the dominant NAT1 marker, as well as URA3, HIS1 and ARG4 cassettes, thereby permitting the manipulation of clinical isolates as well as genetically marked strains of C. albicans. The accelerated gene disruption strategies afforded by this new Clox system are likely to have a profound impact on the speed with which C. albicans pathobiology can be dissected. © 2014 Shahana et al.

Candida albicans is a major fungal pathogen of humans. This yeast is carried by many individuals as a harmless commensal, but when immune defences are perturbed it causes mucosal infections (thrush). Additionally, when the immune system becomes severely compromised, C. albicans often causes life-threatening systemic infections. A battery of virulence factors and fitness attributes promote the pathogenicity of C. albicans. Fitness attributes include robust responses to local environmental stresses, the inactivation of which attenuates virulence. Stress signalling pathways in C. albicans include evolutionarily conserved modules. However, there has been rewiring of some stress regulatory circuitry such that the roles of a number of regulators in C. albicans have diverged relative to the benign model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This reflects the specific evolution of C. albicans as an opportunistic pathogen obligately associated with warm-blooded animals, compared with other yeasts that are found across diverse environmental niches. Our understanding of C. albicans stress signalling is based primarily on the in vitro responses of glucose-grown cells to individual stresses. However, in vivo this pathogen occupies complex and dynamic host niches characterised by alternative carbon sources and simultaneous exposure to combinations of stresses (rather than individual stresses). It has become apparent that changes in carbon source strongly influence stress resistance, and that some combinatorial stresses exert non-additive effects upon C. albicans. These effects, which are relevant to fungus-host interactions during disease progression, are mediated by multiple mechanisms that include signalling and chemical crosstalk, stress pathway interference and a biological transistor.

Cryptococcus neoformans was first recognized as a human pathogen over 100 years ago when it was independently isolated from a patient with a tibial infection and from environmental sources (peach juice). This basidiomycete has subsequently been isolated from most regions of the world, causing a significant number of lethal infections each year, especially in AIDS patients. Originally described as a yeast-like fungus causing human and animal infections, C. neoformans is now known to undergo morphological transitions that are important for its survival and dissemination. Some of the signaling pathways that control yeast and hyphal morphogenesis in this organism are also central regulators of its pathogenesis. © 2012 Springer-Verlag Berlin Heidelberg.