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ABSTRACT Systemic candidiasis remains a significant public health concern worldwide, with high mortality rates despite available antifungal drugs. Drug-resistant strains add to the urgency for alternative therapies. In this context, vaccination has reemerged as a prominent immune-based strategy. Extracellular vesicles (EVs), nanosized lipid bilayer particles, carry a diverse array of native fungal antigens, including proteins, nucleic acids, lipids, and glycans. Previous studies from our laboratory demonstrated that Candida albicans EVs triggered the innate immune response, activating bone marrow-derived dendritic cells (BMDCs) and potentially acting as a bridge between innate and adaptive immunity. Vaccination with C. albicans EVs induced the production of specific antibodies, modulated cytokine production, and provided protection in immunosuppressed mice infected with lethal C. albicans inoculum. To elucidate the mechanisms underlying EV-induced immune activation, our study investigated pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) involved in EVs-phagocyte engagement. EVs from wild-type and mutant C. albicans strains with truncated mannoproteins were compared for their ability to stimulate BMDCs. Our findings revealed that EV decoration with O- and N-linked mannans and the presence of β-1,3-glucans and chitin oligomers may modulate the activation of specific PRRs, in particular Toll-like receptor 4 (TLR4) and dectin-1. The protective effect of vaccination with wild-type EVs was found to be dependent on TLR4. These results suggest that fungal EVs can be harnessed in vaccine formulations to selectively activate PRRs in phagocytes, offering potential avenues for combating or preventing candidiasis.IMPORTANCESystemic candidiasis is a serious global health concern with high mortality rates and growing drug resistance. Vaccination offers a promising solution. A unique approach involves using tiny lipid-coated particles called extracellular vesicles (EVs), which carry various fungal components. Previous studies found that Candida albicans EVs activate the immune response and may bridge the gap between innate and adaptive immunity. To understand this better, we investigated how these EVs activate immune cells. We demonstrated that specific components on EV surfaces, such as mannans and glucans, interact with receptors on immune cells, including Toll-like receptor 4 (TLR4) and dectin-1. Moreover, vaccinating with these EVs led to strong immune responses and full protection in mice infected with Candida. This work shows how harnessing fungal EVs might lead to effective vaccines against candidiasis.

ABSTRACT Microbial species capable of co-existing with healthy individuals, such as the commensal fungus Candida albicans, exploit multifarious strategies to evade our immune defenses. These strategies include the masking of immunoinflammatory pathogen-associated molecular patterns (PAMPs) at their cell surface. We reported previously that C. albicans actively reduces the exposure of the proinflammatory PAMP, β-1,3-glucan, at its cell surface in response to host-related signals such as lactate and hypoxia. Here, we show that clinical isolates of C. albicans display phenotypic variability with respect to their lactate- and hypoxia-induced β-1,3-glucan masking. We have exploited this variability to identify responsive and non-responsive clinical isolates. We then performed RNA sequencing on these isolates to reveal genes whose expression patterns suggested potential association with lactate- or hypoxia-induced β-1,3-glucan masking. The deletion of two such genes attenuated masking: PHO84 and NCE103. We examined NCE103-related signaling further because NCE103 has been shown previously to encode carbonic anhydrase, which promotes adenylyl cyclase-protein kinase A (PKA) signaling at low CO2 levels. We show that while CO2 does not trigger β-1,3-glucan masking in C. albicans, the Sch9-Rca1-Nce103 signaling module strongly influences β-1,3-glucan exposure in response to hypoxia and lactate. In addition to identifying a new regulatory module that controls PAMP exposure in C. albicans, our data imply that this module is important for PKA signaling in response to environmental inputs other than CO2.IMPORTANCEOur innate immune defenses have evolved to protect us against microbial infection in part via receptor-mediated detection of “pathogen-associated molecular patterns” (PAMPs) expressed by invading microbes, which then triggers their immune clearance. Despite this surveillance, many microbial species are able to colonize healthy, immune-competent individuals, without causing infection. To do so, these microbes must evade immunity. The commensal fungus Candida albicans exploits a variety of strategies to evade immunity, one of which involves reducing the exposure of a proinflammatory PAMP (β-1,3-glucan) at its cell surface. Most of the β-1,3-glucan is located in the inner layer of the C. albicans cell wall, hidden by an outer layer of mannan fibrils. Nevertheless, some β-1,3-glucan can become exposed at the fungal cell surface. However, in response to certain specific host signals, such as lactate or hypoxia, C. albicans activates an anticipatory protective response that decreases β-1,3-glucan exposure, thereby reducing the susceptibility of the fungus to impending innate immune attack. Here, we exploited the natural phenotypic variability of C. albicans clinical isolates to identify strains that do not display the response to β-1,3-glucan masking signals observed for the reference isolate, SC5314. Then, using genome-wide transcriptional profiling, we compared these non-responsive isolates with responsive controls to identify genes potentially involved in β-1,3-glucan masking. Mutational analysis of these genes revealed that a sensing module that was previously associated with CO2 sensing also modulates β-1,3-glucan exposure in response to hypoxia and lactate in this major fungal pathogen of humans.

The impact of fungal infections on human health has been exacerbated by the rise of antifungal drug resistance. In this Review, the authors outline the problem of antifungal resistance and suggest how this growing threat might be mitigated.

ABSTRACT Candida albicans exists as a commensal of mucosal surfaces and the gastrointestinal tract without causing pathology. However, this fungus is also a common cause of mucosal and systemic infections when antifungal immune defenses become compromised. The activation of antifungal host defenses depends on the recognition of fungal pathogen-associated molecular patterns (PAMPs), such as β-1,3-glucan. In C. albicans, most β-1,3-glucan is present in the inner cell wall, concealed by the outer mannan layer, but some β-1,3-glucan becomes exposed at the cell surface. In response to host signals, such as lactate, C. albicans induces the Xog1 exoglucanase, which shaves exposed β-1,3-glucan from the cell surface, thereby reducing phagocytic recognition. We show here that β-1,3-glucan is exposed at bud scars and punctate foci on the lateral wall of yeast cells, that this exposed β-1,3-glucan is targeted during phagocytic attack, and that lactate-induced masking reduces β-1,3-glucan exposure at bud scars and at punctate foci. β-1,3-Glucan masking depends upon protein kinase A (PKA) signaling. We reveal that inactivating PKA, or its conserved downstream effectors, Sin3 and Mig1/Mig2, affects the amounts of the Xog1 and Eng1 glucanases in the C. albicans secretome and modulates β-1,3-glucan exposure. Furthermore, perturbing PKA, Sin3, or Mig1/Mig2 attenuates the virulence of lactate-exposed C. albicans cells in Galleria. Taken together, the data are consistent with the idea that β-1,3-glucan masking contributes to Candida pathogenicity. IMPORTANCE Microbes that coexist with humans have evolved ways of avoiding or evading our immunological defenses. These include the masking by these microbes of their “pathogen-associated molecular patterns” (PAMPs), which are recognized as “foreign” and used to activate protective immunity. The commensal fungus Candida albicans masks the proinflammatory PAMP β-1,3-glucan, which is an essential component of its cell wall. Most of this β-1,3-glucan is hidden beneath an outer layer of the cell wall on these microbes, but some can become exposed at the fungal cell surface. Using high-resolution confocal microscopy, we examine the nature of the exposed β-1,3-glucan at C. albicans bud scars and at punctate foci on the lateral cell wall, and we show that these features are targeted by innate immune cells. We also reveal that downstream effectors of protein kinase A (Mig1/Mig2, Sin3) regulate the secretion of major glucanases, modulate the levels of β-1,3-glucan exposure, and influence the virulence of C. albicans in an invertebrate model of systemic infection. Our data support the view that β-1,3-glucan masking contributes to immune evasion and the virulence of a major fungal pathogen of humans.

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.

In this study, the human immune response mechanisms against Sporothrix brasiliensis and Sporothrix schenckii, two causative agents of human and animal sporotrichosis, were investigated. The interaction of S. brasiliensis and S. schenckii with human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of Sporothrix yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two Sporothrix yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the Sporothrix yeast surfaces. PRM also showed direct interaction with CD11b, the specific component of the complement receptor-3 (CR3). Furthermore, the blockade of CR3 specifically impacted the interleukin (IL)-1β secretion by hMDM in response to both S. brasiliensis and S. schenckii, suggesting that the host complement system plays an essential role in the inflammatory immune response against these Sporothrix species. Nevertheless, the structural differences in the PRMs of the two Sporothrix species, as revealed by NMR, were related to the differences observed in the host complement activation pathways. Together, this work reports a new PAMP of the cell surface of pathogenic fungi playing a role through the activation of complement system and via CR3 receptor mediating an inflammatory response to Sporothrix species.

The authors show that the fungal pathogen Candida albicans exploits diverse host-associated signals, including specific nutrients and stresses, to promote immune evasion by masking cell wall β-glucan, a major pathogen-associated molecular pattern.

Late diagnosis and ineffective treatment of fungal infections lead to high mortality. Here, Rudkin et al. generate anti-Candida human monoclonal antibodies with diagnostic and therapeutic potential, by expressing recombinant antibodies from genes cloned from B cells of patients suffering candidiasis.

More than 95% of invasive Candida infections are caused by four Candida spp. (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis). C-type lectin-like receptors (CLRs), such as Dectin-1, Dectin-2, and Mincle mediate immune responses to C. albicans. Dectin-1 promotes clearance of C. albicans, C. glabrata, C. tropicalis, and C. parapsilosis, however, dependence on Dectin-1 for specific immune responses varies with the different Candida spp. Dectin-2 is important for host immunity to C. albicans and C. glabrata, and Mincle is important for the immune response to C. albicans. However, whether Dectin-2 drives host immunity to C. tropicalis or C. parapsilosis, and whether Mincle mediates host immunity to C. glabrata, C. tropicalis or C. parapsilosis is unknown. Therefore, we compared the roles of Dectin-2 and Mincle in response to these four Candida spp. We demonstrate that these four Candida spp. cell walls have differential mannan contents. Mincle and Dectin-2 play a key role in regulating cytokine production in response to these four Candida spp. and Dectin-2 is also important for clearance of all four Candida spp. during systemic infection. However, Mincle was only important for clearance of C. tropicalis during systemic infection. Our data indicate that multiple Candida spp. have different mannan contents, and dependence on the mannan-detecting CLRs, Mincle, and Dectin-2 varies between different Candida spp. during systemic infection.

ABSTRACT Amino acid metabolism is crucial for fungal growth and development. Ureohydrolases produce amines when acting on l-arginine, agmatine, and guanidinobutyrate (GB), and these enzymes generate ornithine (by arginase), putrescine (by agmatinase), or GABA (by 4-guanidinobutyrase or GBase). Candida albicans can metabolize and grow on arginine, agmatine, or guanidinobutyrate as the sole nitrogen source. Three related C. albicans genes whose sequences suggested that they were putative arginase or arginase-like genes were examined for their role in these metabolic pathways. Of these, Car1 encoded the only bona fide arginase, whereas we provide evidence that the other two open reading frames, orf19.5862 and orf19.3418, encode agmatinase and guanidinobutyrase (Gbase), respectively. Analysis of strains with single and multiple mutations suggested the presence of arginase-dependent and arginase-independent routes for polyamine production. CAR1 played a role in hyphal morphogenesis in response to arginine, and the virulence of a triple mutant was reduced in both Galleria mellonella and Mus musculus infection models. In the bloodstream, arginine is an essential amino acid that is required by phagocytes to synthesize nitric oxide (NO). However, none of the single or multiple mutants affected host NO production, suggesting that they did not influence the oxidative burst of phagocytes. IMPORTANCE We show that the C. albicans ureohydrolases arginase (Car1), agmatinase (Agt1), and guanidinobutyrase (Gbu1) can orchestrate an arginase-independent route for polyamine production and that this is important for C. albicans growth and survival in microenvironments of the mammalian host.

ABSTRACT The cell wall provides a major physical interface between fungal pathogens and their mammalian host. This extracellular armor is critical for fungal cell homeostasis and survival. Fungus-specific cell wall moieties, such as β-1,3-glucan, are recognized as pathogen-associated molecular patterns (PAMPs) that activate immune-mediated clearance mechanisms. We have reported that the opportunistic human fungal pathogen Candida albicans masks β-1,3-glucan following exposure to lactate, hypoxia, or iron depletion. However, the precise mechanism(s) by which C. albicans masks β-1,3-glucan has remained obscure. Here, we identify a secreted exoglucanase, Xog1, that is induced in response to lactate or hypoxia. Xog1 functions downstream of the lactate-induced β-glucan “masking” pathway to promote β-1,3-glucan “shaving.” Inactivation of XOG1 blocks most but not all β-1,3-glucan masking in response to lactate, suggesting that other activities contribute to this phenomenon. Nevertheless, XOG1 deletion attenuates the lactate-induced reductions in phagocytosis and cytokine stimulation normally observed for wild-type cells. We also demonstrate that the pharmacological inhibition of exoglucanases undermines β-glucan shaving, enhances the immune visibility of the fungus, and attenuates its virulence. Our study establishes a new mechanism underlying environmentally induced PAMP remodeling that can be manipulated pharmacologically to influence immune recognition and infection outcomes. IMPORTANCE The immune system plays a critical role in protecting us against potentially fatal fungal infections. However, some fungal pathogens have evolved evasion strategies that reduce the efficacy of our immune defenses. Previously, we reported that the fungal pathogen Candida albicans exploits specific host-derived signals (such as lactate and hypoxia) to trigger an immune evasion strategy that involves reducing the exposure of β-glucan at its cell surface. Here, we show that this phenomenon is mediated by the induction of a major secreted exoglucanase (Xog1) by the fungus in response to these host signals. Inactivating XOG1-mediated “shaving” of cell surface-exposed β-glucan enhances immune responses against the fungus. Furthermore, inhibiting exoglucanase activity pharmacologically attenuates C. albicans virulence. In addition to revealing the mechanism underlying a key immune evasion strategy in a major fungal pathogen of humans, our work highlights the potential therapeutic value of drugs that block fungal immune evasion.

ABSTRACT The fungal kingdom includes at least 6 million eukaryotic species and is remarkable with respect to its profound impact on global health, biodiversity, ecology, agriculture, manufacturing, and biomedical research. Approximately 625 fungal species have been reported to infect vertebrates, 200 of which can be human associated, either as commensals and members of our microbiome or as pathogens that cause infectious diseases. These organisms pose a growing threat to human health with the global increase in the incidence of invasive fungal infections, prevalence of fungal allergy, and the evolution of fungal pathogens resistant to some or all current classes of antifungals. More broadly, there has been an unprecedented and worldwide emergence of fungal pathogens affecting animal and plant biodiversity. Approximately 8,000 species of fungi and Oomycetes are associated with plant disease. Indeed, across agriculture, such fungal diseases of plants include new devastating epidemics of trees and jeopardize food security worldwide by causing epidemics in staple and commodity crops that feed billions. Further, ingestion of mycotoxins contributes to ill health and causes cancer. Coordinated international research efforts, enhanced technology translation, and greater policy outreach by scientists are needed to more fully understand the biology and drivers that underlie the emergence of fungal diseases and to mitigate against their impacts. Here, we focus on poignant examples of emerging fungal threats in each of three areas: human health, wildlife biodiversity, and food security.

ABSTRACT The morphogenetic switching between yeast cells and filaments (true hyphae and pseudohyphae) is a key cellular feature required for full virulence in many polymorphic fungal pathogens, such as Candida albicans. In the recently emerged yeast pathogen Candida auris, occasional elongation of cells has been reported. However, environmental conditions and genetic triggers for filament formation have remained elusive. Here, we report that induction of DNA damage and perturbation of replication forks by treatment with genotoxins, such as hydroxyurea, methyl methanesulfonate, and the clinically relevant fungistatic 5-fluorocytosine, cause filamentation in C. auris. The filaments formed were characteristic of pseudohyphae and not parallel-sided true hyphae. Pseudohyphal growth is apparently signaled through the S phase checkpoint and, interestingly, is Tup1 independent in C. auris. Intriguingly, the morphogenetic switching capability is strain specific in C. auris, highlighting the heterogenous nature of the species as a whole. IMPORTANCE Candida auris is a newly emerged fungal pathogen of humans. This species was first reported in 2009 when it was identified in an ear infection of a patient in Japan. However, despite intense interest in this organism as an often multidrug-resistant fungus, there is little knowledge about its cellular biology. During infection of human patients, fungi are able to change cell shape from ellipsoidal yeast cells to elongated filaments to adapt to various conditions within the host organism. There are different types of filaments, which are triggered by reactions to different cues. Candida auris fails to form filaments when exposed to triggers that stimulate yeast filament morphogenesis in other fungi. Here, we show that it does form filaments when its DNA is damaged. These conditions might arise when Candida auris cells interact with host immune cells or during growth in certain host tissues (kidney or bladder) or during treatment with antifungal drugs.

ABSTRACT The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin (TOR) signaling, oxidative stress resistance, and virulence of this fungal pathogen. It also contributes to C. albicans’ tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild-type cells recovering from phosphate starvation. Nonphosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar, GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. We posit that low substrate concentrations of beta-d-glucan- and chitin synthases, together with pharmacologic inhibition of their activity, diminish enzymatic reaction rates as well as the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-d-glucans or chitin. Hence, inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans. IMPORTANCE Candida species cause hundreds of thousands of invasive infections with high mortality each year. Developing novel antifungal agents is challenging due to the many similarities between fungal and human cells. Maintaining phosphate balance is essential for all organisms but is achieved completely differently by fungi and humans. A protein that imports phosphate into fungal cells, Pho84, is not present in humans and is required for normal cell wall stress resistance and cell wall integrity signaling in C. albicans. Nucleotide sugars, which are phosphate-containing building block molecules for construction of the cell wall, are diminished in cells lacking Pho84. Cell wall-constructing enzymes may be slowed by lack of these building blocks, in addition to being inhibited by drugs. Combined targeting of Pho84 and cell wall-constructing enzymes may provide a strategy for antifungal therapy by which two sequential steps of cell wall maintenance are blocked for greater potency.

Four Candida spp. (albicans, glabrata, tropicalis, parapsilosis) cause >95% of invasive Candida infections. C. albicans elicits immune responses via pathogen recognition receptors including C-type lectin-like receptors (CLRs). The CLR, Dectin-1 is important for host immunity to C. albicans and C. glabrata, however, whether Dectin-1 is important for host defense against C. tropicalis or C. parapsilosis is unknown. Therefore, we compared the involvement of Dectin-1 in response to these four diverse Candida spp. We found that Dectin-1 mediates innate cytokine responses to these Candida spp. in a species- and cell-dependent manner. Dectin-1 KO mice succumbed to infection with highly virulent C. albicans while they mostly survived infection with less virulent Candida spp. However, Dectin-1 KO mice displayed increased fungal burden following infection with each Candida spp. Additionally, T cells from Dectin-1 KO mice displayed enhanced effector functions likely due to the inability of Dectin-1 KO mice to clear the infections. Together, these data indicate that Dectin-1 is important for host defense to multiple Candida spp., although the specific roles for Dectin-1 varies with different Candida spp.

ATP-binding cassette (ABC) superfamily members have a key role as nutrient importers and exporters in bacteria. However, their role as drug exporters in eukaryotes brought this superfamily member to even greater prominence. The capacity of ABC transporters to efflux a broad spectrum of xenobiotics represents one of the major mechanisms of clinical multidrug resistance in pathogenic fungi including Candida species. Candida auris, a newly emerged multidrug-resistant fungal pathogen of humans, has been responsible for multiple outbreaks of drug-resistant infections in hospitals around the globe. Our study has analyzed the entire complement of ABC superfamily transporters to assess whether these play a major role in drug resistance mechanisms of C. auris. Our bioinformatics analyses identified 28 putative ABC proteins encoded in the genome of the C. auris type-strain CBS 10913T; 20 of which contain transmembrane domains (TMDs). Quantitative real-time PCR confirmed the expression of all 20 TMD transporters, underlining their potential in contributing to the C. auris drug-resistant phenotype. Changes in transcript levels after short-term exposure of drugs and in drug-resistant C. auris isolates suggested their importance in the drug resistance phenotype of this pathogen. CAUR_02725 orthologous to CDR1, a major multidrug exporter in other yeasts, showed consistently higher expression in multidrug-resistant strains of C. auris. Homologs of other ABC transporter genes, such as CDR4, CDR6, and SNQ2, also displayed raised expression in a sub-set of clinical isolates. Together, our analysis supports the involvement of these transporters in multidrug resistance in C. auris.

Complement is a tightly controlled arm of the innate immune system, facilitating phagocytosis and killing of invading pathogens. Factor H (FH) is the main fluid-phase inhibitor of the alternative pathway. Many pathogens can hijack FH from the host and protect themselves from complement-dependent killing. Candida albicans is a clinically important opportunistic yeast, expressing different FH binding molecules on its cell surface, which allow complement evasion. One such FH binding molecule is the transmembrane protein “High affinity glucose transporter 1” (Hgt1p), involved in glucose metabolism. This study demonstrated that Hgt1p transcription and expression is induced and highest at the low, but physiological glucose concentration of 0.1%. Thus, this concentration was used throughout the study. We also demonstrated the transport of Hgt1p to the fungal cell wall surface by vesicle trafficking and its release by exosomes containing Hgt1p integrated in the vesicular membrane. We corroborated Hgt1p as FH binding molecule. A polyclonal anti-Hgt1p antibody was created which interfered with the binding of FH, present in normal human serum to the fungal cell wall. A chimeric molecule consisting of FH domains 6 and 7 fused to human IgG1 Fc (FH6.7/Fc) even more comprehensively blocked FH binding, likely because FH6.7/Fc diverted FH away from fungal FH ligands other than Hgt1p. Reduced FH binding to the yeast was associated with a concomitant increase in C3b/iC3b deposition and resulted in significantly increased in vitro phagocytosis and killing by human neutrophils. In conclusion, Hgt1p also exhibits non-canonical functions such as binding FH after its export to the cell wall. Blocking Hgt1p-FH interactions may represent a tool to enhance complement activation on the fungal surface to promote phagocytosis and killing of C. albicans.

ABSTRACT Organisms must adapt to changes in oxygen tension if they are to exploit the energetic benefits of reducing oxygen while minimizing the potentially damaging effects of oxidation. Consequently, organisms in all eukaryotic kingdoms display robust adaptation to hypoxia (low oxygen levels). This is particularly important for fungal pathogens that colonize hypoxic niches in the host. We show that adaptation to hypoxia in the major fungal pathogen of humans Candida albicans includes changes in cell wall structure and reduced exposure, at the cell surface, of β-glucan, a key pathogen-associated molecular pattern (PAMP). This leads to reduced phagocytosis by murine bone marrow-derived macrophages and decreased production of IL-10, RANTES, and TNF-α by peripheral blood mononuclear cells, suggesting that hypoxia-induced β-glucan masking has a significant effect upon C. albicans-host interactions. We show that hypoxia-induced β-glucan masking is dependent upon both mitochondrial and cAMP-protein kinase A (PKA) signaling. The decrease in β-glucan exposure is blocked by mutations that affect mitochondrial functionality (goa1Δ and upc2Δ) or that decrease production of hydrogen peroxide in the inner membrane space (sod1Δ). Furthermore, β-glucan masking is enhanced by mutations that elevate mitochondrial reactive oxygen species (aox1Δ). The β-glucan masking defects displayed by goa1Δ and upc2Δ cells are suppressed by exogenous dibutyryl-cAMP. Also, mutations that inactivate cAMP synthesis (cyr1Δ) or PKA (tpk1Δ tpk2Δ) block the masking phenotype. Our data suggest that C. albicans responds to hypoxic niches by inducing β-glucan masking via a mitochondrial cAMP-PKA signaling pathway, thereby modulating local immune responses and promoting fungal colonization. IMPORTANCE Animal, plant, and fungal cells occupy environments that impose changes in oxygen tension. Consequently, many species have evolved mechanisms that permit robust adaptation to these changes. The fungal pathogen Candida albicans can colonize hypoxic (low oxygen) niches in its human host, such as the lower gastrointestinal tract and inflamed tissues, but to colonize its host, the fungus must also evade local immune defenses. We reveal, for the first time, a defined link between hypoxic adaptation and immune evasion in C. albicans. As this pathogen adapts to hypoxia, it undergoes changes in cell wall structure that include masking of β-glucan at its cell surface, and it becomes better able to evade phagocytosis by innate immune cells. We also define the signaling mechanisms that mediate hypoxia-induced β-glucan masking, showing that they are dependent on mitochondrial signaling and the cAMP-protein kinase pathway. Therefore, hypoxia appears to trigger immune evasion in this fungal pathogen.

Early detection is critical to the successful treatment of life-threatening infections caused by fungal pathogens, as late diagnosis of systemic infection almost always equates with a poor prognosis. The field of fungal diagnostics has some tests that are relatively simple, rapid to perform and are potentially suitable at the point of care. However, there are also more complex high-technology methodologies that offer new opportunities regarding the scale and precision of fungal diagnosis, but may be more limited in their portability and affordability. Future developments in this field are increasingly incorporating new technologies provided by the use of new format biosensors. This overview provides a critical review of current fungal diagnostics and the development of new biophysical technologies that are being applied for selective new sensitive fungal biosensors to augment traditional diagnostic methodologies.

The discovery of liquid water at several locations in the solar system raises the possibility that microbial life may have evolved outside Earth and as such could be accidently introduced into the Earth’s ecosystem. Unusual sugars or amino acids, like non-proteinogenic isovaline and α-aminoisobutyric acid that are vanishingly rare or absent from life forms on Earth, have been found in high abundance on non-terrestrial carbonaceous meteorites. It is therefore conceivable that exo-microorganisms might contain proteins that include these rare amino acids. We therefore asked whether the mammalian immune system would be able to recognize and induce appropriate immune responses to putative proteinaceous antigens that include these rare amino acids. To address this, we synthesised peptide antigens based on a backbone of ovalbumin and introduced isovaline and α-aminoisobutyric acid residues and demonstrated that these peptides can promote naïve OT-I cell activation and proliferation, but did so less efficiently than the canonical peptides. This is relevant to the biosecurity of missions that may retrieve samples from exoplanets and moons that have conditions that may be permissive for life, suggesting that accidental contamination and exposure to exo-microorganisms with such distinct proteomes might pose an immunological challenge.

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.

ABSTRACT Knowing the full set of essential genes for a given organism provides important information about ways to promote, and to limit, its growth and survival. For many non-model organisms, the lack of a stable haploid state and low transformation efficiencies impede the use of conventional approaches to generate a genome-wide comprehensive set of mutant strains and the identification of the genes essential for growth. Here we report on the isolation and utilization of a highly stable haploid derivative of the human pathogenic fungus Candida albicans, together with a modified heterologous transposon and machine learning (ML) analysis method, to predict the degree to which all of the open reading frames are required for growth under standard laboratory conditions. We identified 1,610 C. albicans essential genes, including 1,195 with high “essentiality confidence” scores, thereby increasing the number of essential genes (currently 66 in the Candida Genome Database) by >20-fold and providing an unbiased approach to determine the degree of confidence in the determination of essentiality. Among the genes essential in C. albicans were 602 genes also essential in the model budding and fission yeasts analyzed by both deletion and transposon mutagenesis. We also identified essential genes conserved among the four major human pathogens C. albicans, Aspergillus fumigatus, Cryptococcus neoformans, and Histoplasma capsulatum and highlight those that lack homologs in humans and that thus could serve as potential targets for the design of antifungal therapies. IMPORTANCE Comprehensive understanding of an organism requires that we understand the contributions of most, if not all, of its genes. Classical genetic approaches to this issue have involved systematic deletion of each gene in the genome, with comprehensive sets of mutants available only for very-well-studied model organisms. We took a different approach, harnessing the power of in vivo transposition coupled with deep sequencing to identify >500,000 different mutations, one per cell, in the prevalent human fungal pathogen Candida albicans and to map their positions across the genome. The transposition approach is efficient and less labor-intensive than classic approaches. Here, we describe the production and analysis (aided by machine learning) of a large collection of mutants and the comprehensive identification of 1,610 C. albicans genes that are essential for growth under standard laboratory conditions. Among these C. albicans essential genes, we identify those that are also essential in two distantly related model yeasts as well as those that are conserved in all four major human fungal pathogens and that are not conserved in the human genome. This list of genes with functions important for the survival of the pathogen provides a good starting point for the development of new antifungal drugs, which are greatly needed because of the emergence of fungal pathogens with elevated resistance and/or tolerance of the currently limited set of available antifungal drugs.

ABSTRACT Candida auris has recently emerged as an important, multidrug-resistant fungal pathogen of humans. Comparative studies indicate that despite high levels of genetic divergence, C. auris is as virulent as the most pathogenic member of the genus, Candida albicans. However, key virulence attributes of C. albicans, such as morphogenetic switching, are not utilized by C. auris, indicating that this emerging pathogen employs alternative strategies to infect and colonize the host. An important trait required for the pathogenicity of many fungal pathogens is the ability to adapt to host-imposed stresses encountered during infection. Here, we investigated the relative resistance of C. auris and other pathogenic Candida species to physiologically relevant stresses and explored the role of the evolutionarily conserved Hog1 stress-activated protein kinase (SAPK) in promoting stress resistance and virulence. In comparison to C. albicans, C. auris is relatively resistant to hydrogen peroxide, cationic stress, and cell-wall-damaging agents. However, in contrast to other Candida species examined, C. auris was unable to grow in an anaerobic environment and was acutely sensitive to organic oxidative-stress-inducing agents. An analysis of C. auris hog1Δ cells revealed multiple roles for this SAPK in stress resistance, cell morphology, aggregation, and virulence. These data demonstrate that C. auris has a unique stress resistance profile compared to those of other pathogenic Candida species and that the Hog1 SAPK has pleiotropic roles that promote the virulence of this emerging pathogen. IMPORTANCE The rapid global emergence and resistance of Candida auris to current antifungal drugs highlight the importance of understanding the virulence traits exploited by this human fungal pathogen to cause disease. Here, we characterize the stress resistance profile of C. auris and the role of the Hog1 stress-activated protein kinase (SAPK) in stress resistance and virulence. Our findings that C. auris is acutely sensitive to certain stresses may facilitate control measures to prevent persistent colonization in hospital settings. Furthermore, our observation that the Hog1 SAPK promotes C. auris virulence akin to that reported for many other pathogenic fungi indicates that antifungals targeting Hog1 signaling would be broad acting and effective, even on emerging drug-resistant pathogens.

ABSTRACT The fungal cell wall is a critically important structure that represents a permeability barrier and protective shield. We probed Candida albicans and Cryptococcus neoformans with liposomes containing amphotericin B (AmBisome), with or without 15-nm colloidal gold particles. The liposomes have a diameter of 60 to 80 nm, and yet their mode of action requires them to penetrate the fungal cell wall to deliver amphotericin B to the cell membrane, where it binds to ergosterol. Surprisingly, using cryofixation techniques with electron microscopy, we observed that the liposomes remained intact during transit through the cell wall of both yeast species, even though the predicted porosity of the cell wall (pore size, ~5.8 nm) is theoretically too small to allow these liposomes to pass through intact. C. albicans mutants with altered cell wall thickness and composition were similar in both their in vitro AmBisome susceptibility and the ability of liposomes to penetrate the cell wall. AmBisome exposed to ergosterol-deficient C. albicans failed to penetrate beyond the mannoprotein-rich outer cell wall layer. Melanization of C. neoformans and the absence of amphotericin B in the liposomes were also associated with a significant reduction in liposome penetration. Therefore, AmBisome can reach cell membranes intact, implying that fungal cell wall viscoelastic properties are permissive to vesicular structures. The fact that AmBisome can transit through chemically diverse cell wall matrices when these liposomes are larger than the theoretical cell wall porosity suggests that the wall is capable of rapid remodeling, which may also be the mechanism for release of extracellular vesicles. IMPORTANCE AmBisome is a broad-spectrum fungicidal antifungal agent in which the hydrophobic polyene antibiotic amphotericin B is packaged within a 60- to 80-nm liposome. The mode of action involves perturbation of the fungal cell membrane by selectively binding to ergosterol, thereby disrupting membrane function. We report that the AmBisome liposome transits through the cell walls of both Candida albicans and Cryptococcus neoformans intact, despite the fact that the liposome is larger than the theoretical cell wall porosity. This implies that the cell wall has deformable, viscoelastic properties that are permissive to transwall vesicular traffic. These observations help explain the low toxicity of AmBisome, which can deliver its payload directly to the cell membrane without unloading the polyene in the cell wall. In addition, these findings suggest that extracellular vesicles may also be able to pass through the cell wall to deliver soluble and membrane-bound effectors and other molecules to the extracellular space.

Pathogenic microorganisms often face acute micronutrient limitation during infection due to the action of host-mediated nutritional immunity. The human fungal pathogen Candida albicans is polymorphic and its morphological plasticity is one of its most widely recognized pathogenicity attributes. Here we investigated the effect of zinc, iron, manganese, and copper limitation on C. albicans morphology. Restriction of zinc specifically resulted in the formation of enlarged, spherical yeasts, a phenotype which we term Goliath cells. This cellular response to zinc restriction was conserved in C. albicans, C. dubliniensis and C. tropicalis, but not in C. parapsilosis, C. lusitaniae or Debaryomyces hansenii, suggesting that it may have emerged in the last common ancestor of these related pathogenic species. Cell wall analysis revealed proportionally more chitin exposure on the Goliath cell surface. Importantly, these cells were hyper-adherent, suggesting a possible role in pathogenicity. Interestingly, the zincophore-encoding gene PRA1 was expressed by Goliath cells in zinc limited media and lack of Pra1 inhibited both cellular enlargement and adhesion. Goliath cells represent a further layer of Candida phenotypic plasticity.

Phosphomannosylation is a modification of cell wall proteins that occurs in some species of yeast-like organisms, including the human pathogen Candida albicans. These modified mannans confer a negative charge to the wall, which is important for the interactions with phagocytic cells of the immune systems and cationic antimicrobial peptides. In Saccharomyces cerevisiae, the synthesis of phosphomannan relies on two enzymes, the phosphomannosyltransferase Ktr6 and its positive regulator Mnn4. However, in C. albicans, at least three phosphomannosyltransferases, Mnn4, Mnt3 and Mnt5, participate in the addition of phosphomannan. In addition to MNN4, C. albicans has a MNN4-like gene family composed of seven other homologous members that have no known function. Here, using the classical mini-Ura-blaster approach and the new gene knockout CRISPR-Cas9 system for gene disruption, we generated mutants lacking single and multiple genes of the MNN4 family; and demonstrate that, although Mnn4 has a major impact on the phosphomannan content, MNN42 was also required for full protein phosphomannosylation. The reintroduction of MNN41, MNN42, MNN46, or MNN47 in a genetic background lacking MNN4 partially restored the phenotype associated with the mnn4Δ null mutant, suggesting that there is partial redundancy of function between some family members and that the dominant effect of MNN4 over other genes could be due to its relative abundance within the cell. We observed that additional copies of alleles number of any of the other family members, with the exception of MNN46, restored the phosphomannan content in cells lacking both MNT3 and MNT5. We, therefore, suggest that phosphomannosylation is achieved by three groups of proteins: [i] enzymes solely activated by Mnn4, [ii] enzymes activated by the dual action of Mnn4 and any of the products of other MNN4-like genes, with exception of MNN46, and [iii] activation of Mnt3 and Mnt5 by Mnn4 and Mnn46. Therefore, although the MNN4-like genes have the potential to functionally redundant with Mnn4, they apparently do not play a major role in cell wall mannosylation under most in vitro growth conditions. In addition, our phenotypic analyses indicate that several members of this gene family influence the ability of macrophages to phagocytose C. albicans cells.

Candida albicans is a human opportunist pathogen that can grow as yeast, pseudohyphae, or true hyphae in vitro and in vivo, depending on environmental conditions. Reversible cellular morphogenesis is an important virulence factor that facilitates invasion of host tissues, escape from phagocytes, and dissemination in the blood stream. The innate immune system is the first line of defense against C. albicans infections and is influenced by recognition of wall components that vary in composition in different morphological forms. However, the relationship between cellular morphogenesis and immune recognition of this fungus is not fully understood. We therefore studied various vegetative cell types of C. albicans, singly and in combination, to assess the consequences of cellular morphogenesis on selected immune cytokine outputs from human monocytes. Hyphae stimulated proportionally lower levels of certain cytokines from monocytes per unit of cell surface area than yeast cells, but did not suppress cytokine response when copresented with yeast cells. Pseudohyphal cells induced intermediate cytokine responses. Yeast monomorphic mutants had elevated cytokine responses under conditions that otherwise supported filamentous growth and mutants of yeast and hyphal cells that were defective in cell wall mannosylation or lacking certain hypha-specific cell wall proteins could variably unmask or deplete the surface of immunostimulatory ligands. These observations underline the critical importance of C. albicans morphology and morphology-associated changes in the cell wall composition that affect both immune recognition and pathogenesis.

ABSTRACT The opportunistic human fungal pathogen Candida albicans can cause a variety of diseases, ranging from superficial mucosal infections to life-threatening systemic infections. Phagocytic cells of the innate immune response, such as neutrophils and macrophages, are important first-line responders to an infection and generate reactive oxygen and nitrogen species as part of their protective antimicrobial response. During an infection, host cells generate nitric oxide through the enzyme inducible nitric oxide synthase (iNOS) to kill the invading pathogen. Inside the phagocyte, iNOS competes with the enzyme arginase-1 for a common substrate, the amino acid l-arginine. Several pathogenic species, including bacteria and parasitic protozoans, actively modulate the production of nitric oxide by inducing their own arginases or the host’s arginase activity to prevent the conversion of l-arginine to nitric oxide. We report here that C. albicans blocks nitric oxide production in human-monocyte-derived macrophages by induction of host arginase activity. We further determined that purified chitin (a fungal cell wall polysaccharide) and increased chitin exposure at the fungal cell wall surface induces this host arginase activity. Blocking the C. albicans-induced arginase activity with the arginase-specific substrate inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) or the chitinase inhibitor bisdionin F restored nitric oxide production and increased the efficiency of fungal killing. Moreover, we determined that C. albicans influences macrophage polarization from a classically activated phenotype toward an alternatively activated phenotype, thereby reducing antimicrobial functions and mediating fungal survival. Therefore, C. albicans modulates l-arginine metabolism in macrophages during an infection, potentiating its own survival. IMPORTANCE The availability and metabolism of amino acids are increasingly recognized as crucial regulators of immune functions. In acute infections, the conversion of the “conditionally essential” amino acid l-arginine by the inducible nitric oxide synthase to nitric oxide is a resistance factor that is produced by the host to fight pathogens. Manipulation of these host defense mechanisms by the pathogen can be key to successful host invasion. We show here that the human opportunistic fungal pathogen Candida albicans influences l-arginine availability for nitric oxide production by induction of the substrate-competing host enzyme arginase-1. This led to a reduced production of nitric oxide and, moreover, reduced eradication of the fungus by human macrophages. We demonstrate that blocking of host arginase-1 activity restored nitric oxide production and increased the killing potential of macrophages. These results highlight the therapeutic potential of l-arginine metabolism in fungal diseases.

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.

In addition to the bio-guided investigation of the antifungal activity of Plinia cauliflora leaves against different Candida species, the major aim of the present study was the search for targets on the fungal cell. The most active antifungal fraction was purified by chromatography and characterized by NMR and mass spectrometry. The antifungal activity was evaluated against five Candida strains according to referenced guidelines. Cytotoxicity against fibroblast cells was determined. The likely targets of Candida albicans cells were assessed through interactions with ergosterol and cell wall composition, porosity and architecture. The chemical major component within the most active antifungal fraction of P. cauliflora leaves identified was the hydrolysable tannin casuarinin. The cytotoxic concentration was higher than the antifungal one. The first indication of plant target on cellular integrity was suggested by the antifungal activity ameliorated when using an osmotic support. The most important target for the tannin fraction studied was suggested by ultrastructural analysis of yeast cell walls revealing a denser mannan outer layer and wall porosity reduced. It is possible to imply that P. cauliflora targeted the C. albicans cell wall inducing some changes in the architecture, notably the outer glycoprotein layer, affecting the cell wall porosity without alteration of the polysaccharide or protein level.

SUMMARY Candida spp. are opportunistic pathogens in humans, and their systemic infections display upwards of 30% mortality in immunocompromised patients. Current mammalian model systems have certain disadvantages in that obtaining results is time consuming owing to the relatively long life spans and these results have low statistical resolution because sample sizes are usually small. We have therefore evaluated the potential of Drosophila melanogaster as an additional model system with which to dissect the host-pathogen interactions that occur during Candida albicans systemic infection. To do this, we monitored the survival of wild-type flies infected with various C. albicans clinical isolates that were previously ranked for murine virulence. From our lifetime data we computed two metrics of virulence for each isolate. These correlated significantly with murine survival, and were also used to group the isolates, and this grouping made relevant predictions regarding their murine virulence. Notably, differences in virulence were not predictably resolvable using immune-deficient spz−/− flies, suggesting that Toll signalling might actually be required to predictably differentiate virulence. Our analysis reveals wild-type D. melanogaster as a sensitive and relevant model system; one that offers immense genetic tractability (having an extensive RNA interference library that enables tissue-specific gene silencing), and that is easy to manipulate and culture. Undoubtedly, it will prove to be a valuable addition to the model systems currently used to study C. albicans infection.

ABSTRACT The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. IMPORTANCE The C. albicans cell wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antifungal therapy. Its tensile strength is conferred by a network of cell wall polysaccharides, which are remodeled in response to growth conditions and environmental stress. However, little is known about how cell wall elasticity is regulated and how it affects adaptation to stresses such as sudden changes in osmolarity. We show that elasticity is critical for survival under conditions of osmotic shock, before stress signaling pathways have time to induce gene expression and drive glycerol accumulation. Critical cell wall remodeling enzymes control cell wall flexibility, and its regulation is strongly dependent on host nutritional inputs. We also demonstrate an entirely new level of cell wall dynamism, where significant architectural changes and structural realignment occur within seconds of an osmotic shock.

ABSTRACT 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 host-pathogen 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. IMPORTANCE Innate cells engulf and destroy invading organisms by phagocytosis, which is essential for the elimination of fungal cells to protect against systemic life-threatening infections. Yet comparatively little is known about what controls the maturation of phagosomes following ingestion of fungal cells. We used live-cell microscopy and fluorescent protein reporter macrophages to understand how C. albicans viability, filamentous growth, and cell wall composition affect phagosome maturation and the survival of the pathogen within host macrophages. We have demonstrated that cell wall glycosylation and yeast-hypha morphogenesis are required for disruption of host processes that function to inactivate pathogens, leading to survival and escape of this fungal pathogen from within host phagocytes. The methods employed here are applicable to study interactions of other pathogens with phagocytic cells to dissect how specific microbial features impact different stages of phagosome maturation and the survival of the pathogen or host.

Candida albicans is an opportunistic fungal pathogen that infects immunocompromised patients. Infection control requires phagocytosis by innate immune cells, including macrophages. Migration towards, and subsequent recognition of, C. albicans fungal cell wall components by macrophages is critical for phagocytosis. Using live-cell imaging of phagocytosis, the macrophage cell line J774.1 showed enhanced movement in response to C. albicans cell wall mutants, particularly during the first 30 min, irrespective of the infection ratio. However, phagocyte migration was reduced up to 2-fold within a C. albicans biofilm compared to planktonic fungal cells. Biofilms formed from C. albicans glycosylation mutant cells also inhibited macrophage migration to a similar extent as wildtype Candida biofilms, suggesting that the physical structure of the biofilm, rather than polysaccharide matrix composition, may hamper phagocyte migration. These data illustrate differential macrophage migratory capacities, dependent upon the form of C. albicans encountered. Impaired migration of macrophages within a C. albicans biofilm may contribute to the recalcitrant nature of clinical infections in which biofilm formation occurs.

ABSTRACT Immune cells exploit reactive oxygen species (ROS) and cationic fluxes to kill microbial pathogens, such as the fungus Candida albicans. Yet, C. albicans is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity of neutrophils? We show that simultaneous exposure to oxidative and cationic stresses is much more potent than the individual stresses themselves and that this combinatorial stress kills C. albicans synergistically in vitro. We also show that the high fungicidal activity of human neutrophils is dependent on the combinatorial effects of the oxidative burst and cationic fluxes, as their pharmacological attenuation with apocynin or glibenclamide reduced phagocytic potency to a similar extent. The mechanistic basis for the extreme potency of combinatorial cationic plus oxidative stress—a phenomenon we term stress pathway interference—lies with the inhibition of hydrogen peroxide detoxification by the cations. In C. albicans this causes the intracellular accumulation of ROS, the inhibition of Cap1 (a transcriptional activator that normally drives the transcriptional response to oxidative stress), and altered readouts of the stress-activated protein kinase Hog1. This leads to a loss of oxidative and cationic stress transcriptional outputs, a precipitous collapse in stress adaptation, and cell death. This stress pathway interference can be suppressed by ectopic catalase (Cat1) expression, which inhibits the intracellular accumulation of ROS and the synergistic killing of C. albicans cells by combinatorial cationic plus oxidative stress. Stress pathway interference represents a powerful fungicidal mechanism employed by the host that suggests novel approaches to potentiate antifungal therapy. IMPORTANCE The immune system combats infection via phagocytic cells that recognize and kill pathogenic microbes. Human neutrophils combat Candida infections by killing this fungus with a potent mix of chemicals that includes reactive oxygen species (ROS) and cations. Yet, Candida albicans is relatively resistant to these stresses in vitro. We show that it is the combination of oxidative plus cationic stresses that kills yeasts so effectively, and we define the molecular mechanisms that underlie this potency. Cations inhibit catalase. This leads to the accumulation of intracellular ROS and inhibits the transcription factor Cap1, which is critical for the oxidative stress response in C. albicans. This triggers a dramatic collapse in fungal stress adaptation and cell death. Blocking either the oxidative burst or cationic fluxes in human neutrophils significantly reduces their ability to kill this fungal pathogen, indicating that combinatorial stress is pivotal to immune surveillance.

ABSTRACT An important first line of defense against Candida albicans infections is the killing of fungal cells by professional phagocytes of the innate immune system, such as polymorphonuclear cells (PMNs) and macrophages. In this study, we employed live-cell video microscopy coupled with dynamic image analysis tools to provide insights into the complexity of C. albicans phagocytosis when macrophages and PMNs were incubated with C. albicans alone and when both phagocyte subsets were present. When C. albicans cells were incubated with only one phagocyte subtype, PMNs had a lower overall phagocytic capacity than macrophages, despite engulfing fungal cells at a higher rate once fungal cells were bound to the phagocyte surface. PMNs were more susceptible to C. albicans-mediated killing than macrophages, irrespective of the number of C. albicans cells ingested. In contrast, when both phagocyte subsets were studied in coculture, the two cell types phagocytosed and cleared C. albicans at equal rates and were equally susceptible to killing by the fungus. The increase in macrophage susceptibility to C. albicans-mediated killing was a consequence of macrophages taking up a higher proportion of hyphal cells under these conditions. In the presence of both PMNs and macrophages, C. albicans yeast cells were predominantly cleared by PMNs, which migrated at a greater speed toward fungal cells and engulfed bound cells more rapidly. These observations demonstrate that the phagocytosis of fungal pathogens depends on, and is modified by, the specific phagocyte subsets present at the site of infection. IMPORTANCE Extensive work investigating fungal cell phagocytosis by macrophages and PMNs of the innate immune system has been carried out. These studies have been informative but have examined this phenomenon only when one phagocyte subset is present. The current study employed live-cell video microscopy to break down C. albicans phagocytosis into its component parts and examine the effect of a single phagocyte subset, versus a mixed phagocyte population, on these individual stages. Through this approach, we identified that the rate of fungal cell engulfment and rate of phagocyte killing altered significantly when both macrophages and PMNs were incubated in coculture with C. albicans compared to the rate of either phagocyte subset incubated alone with the fungus. This research highlights the significance of studying pathogen-host cell interactions with a combination of phagocytes in order to gain a greater understanding of the interactions that occur between cells of the host immune system in response to fungal invasion.

ABSTRACT Microbes must assimilate carbon to grow and colonize their niches. Transcript profiling has suggested that Candida albicans, a major pathogen of humans, regulates its carbon assimilation in an analogous fashion to the model yeast Saccharomyces cerevisiae, repressing metabolic pathways required for the use of alterative nonpreferred carbon sources when sugars are available. However, we show that there is significant dislocation between the proteome and transcriptome in C. albicans. Glucose triggers the degradation of the ICL1 and PCK1 transcripts in C. albicans, yet isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are stable and are retained. Indeed, numerous enzymes required for the assimilation of carboxylic and fatty acids are not degraded in response to glucose. However, when expressed in C. albicans, S. cerevisiae Icl1 (ScIcl1) is subjected to glucose-accelerated degradation, indicating that like S. cerevisiae, this pathogen has the molecular apparatus required to execute ubiquitin-dependent catabolite inactivation. C. albicans Icl1 (CaIcl1) lacks analogous ubiquitination sites and is stable under these conditions, but the addition of a ubiquitination site programs glucose-accelerated degradation of CaIcl1. Also, catabolite inactivation is slowed in C. albicans ubi4 cells. Ubiquitination sites are present in gluconeogenic and glyoxylate cycle enzymes from S. cerevisiae but absent from their C. albicans homologues. We conclude that evolutionary rewiring of ubiquitination targets has meant that following glucose exposure, C. albicans retains key metabolic functions, allowing it to continue to assimilate alternative carbon sources. This metabolic flexibility may be critical during infection, facilitating the rapid colonization of dynamic host niches containing complex arrays of nutrients. IMPORTANCE Pathogenic microbes must assimilate a range of carbon sources to grow and colonize their hosts. Current views about carbon assimilation in the pathogenic yeast Candida albicans are strongly influenced by the Saccharomyces cerevisiae paradigm in which cells faced with choices of nutrients first use energetically favorable sugars, degrading enzymes required for the assimilation of less favorable alternative carbon sources. We show that this is not the case in C. albicans because there has been significant evolutionary rewiring of the molecular signals that promote enzyme degradation in response to glucose. As a result, this major pathogen of humans retains enzymes required for the utilization of physiologically relevant carbon sources such as lactic acid and fatty acids, allowing it to continue to use these host nutrients even when glucose is available. This phenomenon probably enhances efficient colonization of host niches where sugars are only transiently available.