Biofilm-associated metabolism via ERG251 in Candida albicans.

Biofilm formation by the fungal pathogen Candida albicans is the basis for its ability to infect medical devices. The metabolic gene ERG251 has been identified as a target of biofilm transcriptional regulator Efg1, and here we report that ERG251 is required for biofilm formation but not conventional free-living planktonic growth. An erg251Δ/Δ mutation impairs biofilm formation in vitro and in an in vivo catheter infection model. In both in vitro and in vivo biofilm contexts, cell number is reduced and hyphal length is limited. To determine whether the mutant defect is in growth or some other aspect of biofilm development, we examined planktonic cell features in a biofilm-like environment, which was approximated with sealed unshaken cultures. Under those conditions, the erg251Δ/Δ mutation causes defects in growth and hyphal extension. Overexpression in the erg251Δ/Δ mutant of the paralog ERG25, which is normally expressed more weakly than ERG251, partially improves biofilm formation and biofilm hyphal content, as well as growth and hyphal extension in a biofilm-like environment. GC-MS analysis shows that the erg251Δ/Δ mutation causes a defect in ergosterol accumulation when cells are cultivated under biofilm-like conditions, but not under conventional planktonic conditions. Overexpression of ERG25 in the erg251Δ/Δ mutant causes some increase in ergosterol levels. Finally, the hypersensitivity of efg1Δ/Δ mutants to the ergosterol inhibitor fluconazole is reversed by ERG251 overexpression, arguing that reduced ERG251 expression contributes to this efg1Δ/Δ phenotype. Our results indicate that ERG251 is required for biofilm formation because its high expression levels are necessary for ergosterol synthesis in a biofilm-like environment.

Host populations, challenges, and commercialization of cryptococcal vaccines.

Vaccines are one of the most effective public health tools to prevent and manage infectious diseases. Since the first clinical use of vaccines in the late 18th century, many vaccines have been successfully developed to combat bacterial and viral infections, including the most recent Coronavirus Disease 2019 (COVID-19) pandemic. However, there remains no vaccine that is clinically available to treat or prevent invasive fungal diseases, including cryptococcal meningoencephalitis. This fungal disease is uniformly fatal without treatment and has a global mortality rate of over 70%. Despite a dire need for an effective cryptococcal vaccine, there are many scientific and economic challenges to overcome prior to making it a reality. Here, we discuss some of these challenges as well as steps that the community is taking for commercialization of effective cryptococcal vaccines.

The biological functions of sphingolipids in plant pathogenic fungi.

Sphingolipids are critically significant in a range of biological processes in animals, plants, and fungi. In mammalian cells, they serve as vital components of the plasma membrane (PM) in maintaining its structure, tension, and fluidity. They also play a key role in a wide variety of biological processes, such as intracellular signal transduction, cell polarization, differentiation, and migration. In plants, sphingolipids are important for cell development and for cell response to environmental stresses. In pathogenic fungi, sphingolipids are crucial for the initiation and the development of infection processes afflicting humans. However, our knowledge on the metabolism and function of the sphingolipid metabolic pathway of pathogenic fungi affecting plants is still very limited. In this review, we discuss recent developments on sphingolipid pathways of plant pathogenic fungi, highlighting their uniqueness and similarity with plants and animals. In addition, we discuss recent advances in the research and development of fungal-targeted inhibitors of the sphingolipid pathway, to gain insights on how we can better control the infection process occurring in plants to prevent or/and to treat fungal infections in crops.

SAR study of N'-(Salicylidene)heteroarenecarbohydrazides as promising antifungal agents.

Clinically available antifungal drugs have therapeutic limitations due to toxicity, narrow spectrum of activity, and intrinsic or acquired drug resistance. Thus, there is an urgent need for new broad-spectrum antifungal agents with low toxicity and a novel mechanism of action. In this context, we have successfully identified several highly promising lead compounds, i.e., aromatic N'-(salicylidene)carbohydrazides, exhibiting excellent antifungal activities against Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus and several other fungi both in vitro and in vivo. Building upon these highly promising results, 71 novel N'-(salicylidene)heteroarenecarbohydrazides 5 were designed, synthesized and their antifungal activities examined against fungi. Based on the SAR study, four highly promising lead compounds, i.e., 5.6a, 5.6b, 5.7b and 5.13a were identified, which exhibited excellent potency against C. neoformans, C. albicans and A. fumigatus, and displayed impressive time-kill profiles against C. neoformans with exceptionally high selectivity indices (SI ≥ 500). These four lead compounds also showed synergy with clinical antifungal drugs, fluconazole, caspofungin (CS) and amphotericin B against C. neoformans. For the SAR study, we also employed quantitative structure-activity relationship (QSAR) analysis by taking advantage of the accumulated data on a large number of aromatic and heteroaromatic N'-(salicylidene)carbohydrazides, which successfully led to rational design and selection of promising compounds for chemical synthesis and biological evaluation.

Cholesterol and sphingomyelin are critical for Fcγ receptor-mediated phagocytosis of Cryptococcus neoformans by macrophages.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening meningoencephalitis in lymphopenic patients. Pulmonary macrophages comprise the first line of host defense upon inhalation of fungal spores by aiding in clearance but can also potentially serve as a niche for their dissemination. Given that macrophages play a key role in the outcome of a cryptococcal infection, it is crucial to understand factors that mediate phagocytosis of C. neoformans. Since lipid rafts (high-order plasma membrane domains enriched in cholesterol and sphingomyelin [SM]) have been implicated in facilitating phagocytosis, we evaluated whether these ordered domains govern macrophages' ability to phagocytose C. neoformans. We found that cholesterol or SM depletion resulted in significantly deficient immunoglobulin G (IgG)-mediated phagocytosis of fungus. Moreover, repletion of macrophage cells with a raft-promoting sterol (7-dehydrocholesterol) rescued this phagocytic deficiency, whereas a raft-inhibiting sterol (coprostanol) significantly decreased IgG-mediated phagocytosis of C. neoformans. Using a photoswitchable SM (AzoSM), we observed that the raft-promoting conformation (trans-AzoSM) resulted in efficient phagocytosis, whereas the raft-inhibiting conformation (cis-AzoSM) significantly but reversibly blunted phagocytosis. We observed that the effect on phagocytosis may be facilitated by Fcγ receptor (FcγR) function, whereby IgG immune complexes crosslink to FcγRIII, resulting in tyrosine phosphorylation of FcR γ-subunit (FcRγ), an important accessory protein in the FcγR signaling cascade. Correspondingly, cholesterol or SM depletion resulted in decreased FcRγ phosphorylation. Repletion with 7-dehydrocholesterol restored phosphorylation, whereas repletion with coprostanol showed FcRγ phosphorylation comparable to unstimulated cells. Together, these data suggest that lipid rafts are critical for facilitating FcγRIII-mediated phagocytosis of C. neoformans.

The Future of Antifungal Drug Therapy: Novel Compounds and Targets.

Fungal infections are a universal problem and are routinely associated with high morbidity and mortality rates in immunocompromised patients. Existing therapies comprise five different classes of antifungal agents, four of which target the synthesis of ergosterol and cell wall glucans. However, the currently available antifungals have many limitations, including poor oral bioavailability, narrow therapeutic indices, and emerging drug resistance resulting from their use, thus making it essential to investigate the development of novel drugs which can overcome these limitations and add to the antifungal armamentarium. Advances have been made in antifungal drug discovery research and development over the past few years as evidenced by the presence of several new compounds currently in various stages of development. In the following minireview, we provide a comprehensive summary of compounds aimed at one or more novel molecular targets. We also briefly describe potential pathways relevant for fungal pathogenesis that can be considered for drug development in the near future.

Identification of Antifungal Compounds against Multidrug-Resistant Candida auris Utilizing a High-Throughput Drug-Repurposing Screen.

Candida auris is an emerging fatal fungal infection that has resulted in several outbreaks in hospitals and care facilities. Current treatment options are limited by the development of drug resistance. Identification of new pharmaceuticals to combat these drug-resistant infections will thus be required to overcome this unmet medical need. We have established a bioluminescent ATP-based assay to identify new compounds and potential drug combinations showing effective growth inhibition against multiple strains of multidrug-resistant Candida auris The assay is robust and suitable for assessing large compound collections by high-throughput screening (HTS). Utilizing this assay, we conducted a screen of 4,314 approved drugs and pharmacologically active compounds that yielded 25 compounds, including 6 novel anti-Candida auris compounds and 13 sets of potential two-drug combinations. Among the drug combinations, the serine palmitoyltransferase inhibitor myriocin demonstrated a combinational effect with flucytosine against all tested isolates during screening. This combinational effect was confirmed in 13 clinical isolates of Candida auris.

Antifungal activity of Acylhydrazone derivatives against Sporothrix spp.

Sporotrichosis is an emerging mycosis caused by members of the genus Sporothrix The disease affects humans and animals, particularly cats, which plays an important role in the zoonotic transmission. Feline sporotrichosis treatment options include itraconazole (ITC), potassium iodide and amphotericin B, drugs usually associated with deleterious adverse reactions and refractoriness in cats, especially when using ITC. Thus, affordable, non-toxic and clinically effective anti-Sporothrix agents are needed. Recently, acylhydrazones (AH), molecules targeting vesicular transport and cell cycle progression, exhibited a potent antifungal activity against several fungal species and displayed low toxicity when compared to the current drugs. In this work, the AH derivatives D13 and SB-AF-1002 were tested against Sporothrix schenckii and Sporothrix brasiliensis Minimal inhibitory concentrations of 0.12 - 1 µg/mL were observed for both species in vitro D13 and SB-AF-1002 showed an additive effect with itraconazole. Treatment with D13 promoted yeast disruption with release of intracellular components, as confirmed by transmission electron microscopy of S. brasiliensis exposed to the AH derivatives. AH-treated cells displayed thickening of the cell wall, discontinuity of the cell membrane and an intense cytoplasmic degeneration. In a murine model of sporotrichosis, treatment with AH derivatives was more efficient than ITC, the drug of choice for sporotrichosis. The results of the preliminary clinical study in cats indicate that D13 is safe and has potential to become a therapeutic option for sporotrichosis when associated to ITC. Our results expand the antifungal broadness of AH derivatives and suggest that these drugs could be exploited to combat sporotrichosis.

A Mechanism-Based Targeted Screen To Identify Epstein-Barr Virus-Directed Antiviral Agents.

Epstein-Barr virus (EBV) is one of nine human herpesviruses that persist latently to establish permanent residence in their hosts. Periodic activation into the lytic/replicative phase allows such viruses to propagate and spread, but can also cause disease in the host. This lytic phase is also essential for EBV to cause infectious mononucleosis and cancers, including B lymphocyte-derived Burkitt lymphoma and immunocompromise-associated lymphoproliferative diseases/lymphomas as well as epithelial cell-derived nasopharyngeal cell carcinoma. In the absence of anti-EBV agents, however, therapeutic options for EBV-related diseases are limited. In earlier work, we discovered that through the activities of the viral protein kinase conserved across herpesviruses and two cellular proteins, ATM and KAP1, a lytic cycle amplification loop is established, and disruption of this loop disables the EBV lytic cascade. We therefore devised a high-throughput screening assay, screened a small-molecule-compound library, and identified 17 candidates that impair the release of lytically replicated EBV. The identified compounds will (i) serve as lead compounds or may be modified to inhibit EBV and potentially other herpesviruses, and (ii) be developed into anticancer agents, as functions of KAP1 and ATM are tightly linked to cancer. Importantly, our screening strategy may also be used to screen additional compound libraries for antiherpesviral and anticancer drugs.IMPORTANCE Epstein-Barr virus, which is nearly ubiquitous in humans, is causal to infectious mononucleosis, chronic active EBV infection, and lymphoid and epithelial cancers. However, EBV-specific antiviral agents are not yet available. To aid in the identification of compounds that may be developed as antivirals, we pursued a mechanism-based approach. Since many of these diseases rely on EBV's lytic phase, we developed a high-throughput assay that is able to measure a key step that is essential for successful completion of EBV's lytic cascade. We used this assay to screen a library of small-molecule compounds and identified inhibitors that may be pursued for their anti-EBV and possibly even antiherpesviral potential, as this key mechanism appears to be common to several human herpesviruses. Given the prominent role of this mechanism in both herpesvirus biology and cancer, our screening assay may be used as a platform to identify both antiherpesviral and anticancer drugs.

Preclinical Evaluation of Acylhydrazone SB-AF-1002 as a Novel Broad-Spectrum Antifungal Agent.

The incidence of invasive fungal infections is rising due to the increase in susceptible populations. Current clinically available drugs have therapeutic limitations due to toxicity, a narrow spectrum of activity, and, more importantly, the consistent rise of fungal species that are intrinsically resistant or that develop resistance due to prolonged therapy. Thus, there is an urgent need for new broad-spectrum antifungal agents with low toxicity and a novel mechanism of action. We previously reported a new class of potent antifungal compounds, acylhydrazones, that target the fungal sphingolipid pathway. Based upon our initial lead molecules, (E)-N'-(5-bromo-2-hydroxybenzylidene)-2-methylbenzohydrazide and D13, we performed a structure-activity relationship study, synthesizing ca. 300 new compounds. Of these, 5 compounds were identified to be the most promising for further studies, based on their broad-spectrum activity and low toxicity in mammalian cells lines. Among these top 5 lead compounds, we report here the impressive in vivo activity of 2,4-dibromo-N'-(5-bromo-2-hydroxybenzylidene)benzohydrazide (SB-AF-1002) in several models of systemic fungal infection. Our data show that SB-AF-1002 is efficacious and outperforms current standard-of-care drugs in models of invasive fungal infections, such as cryptococcosis, candidiasis, and aspergillosis. Specifically, animals treated with SB-AF-1002 not only survived the infection but also showed a clearing of fungal cells from key organs. Moreover, SB-AF-1002 was very effective in an aspergillosis model as a prophylactic therapy. SB-AF-1002 also displayed acceptable pharmacokinetic properties in mice, similar to those of the parent compound, D13. These results clearly indicate that our novel acylhydrazones constitute a new class of highly potent and efficacious antifungal agents which warrant further development for the treatment of invasive fungal infections.

Fenbendazole Controls In Vitro Growth, Virulence Potential, and Animal Infection in the Cryptococcus Model.

The human diseases caused by the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are associated with high indices of mortality and toxic and/or cost-prohibitive therapeutic protocols. The need for affordable antifungals to combat cryptococcal disease is unquestionable. Previous studies suggested benzimidazoles as promising anticryptococcal agents combining low cost and high antifungal efficacy, but their therapeutic potential has not been demonstrated so far. In this study, we investigated the antifungal potential of fenbendazole, the most effective anticryptococcal benzimidazole. Fenbendazole was inhibitory against 17 different isolates of C. neoformans and C. gattii at a low concentration. The mechanism of anticryptococcal activity of fenbendazole involved microtubule disorganization, as previously described for human parasites. In combination with fenbendazole, the concentrations of the standard antifungal amphotericin B required to control cryptococcal growth were lower than those required when this antifungal was used alone. Fenbendazole was not toxic to mammalian cells. During macrophage infection, the anticryptococcal effects of fenbendazole included inhibition of intracellular proliferation rates and reduced phagocytic escape through vomocytosis. Fenbendazole deeply affected the cryptococcal capsule. In a mouse model of cryptococcosis, the efficacy of fenbendazole to control animal mortality was similar to that observed for amphotericin B. These results indicate that fenbendazole is a promising candidate for the future development of an efficient and affordable therapeutic tool to combat cryptococcosis.

The putative flippase Apt1 is required for intracellular membrane architecture and biosynthesis of polysaccharide and lipids in Cryptococcus neoformans.

Flippases are responsible for the asymmetric distribution of phospholipids in biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the putative flippase Apt1 is an important regulator of polysaccharide secretion and pathogenesis in mice by unknown mechanisms. In this study, we analyzed the role of C. neoformans Apt1 in intracellular membrane architecture and synthesis of polysaccharide and lipids. Analysis of wild type (WT), apt1Δ (mutant) and apt1Δ::APT1 (complemented) strains by transmission electron microscopy revealed that deletion of APT1 resulted in the formation of irregular vacuoles. Disorganization of vacuolar membranes in apt1Δ cells was accompanied by a significant increase in the amounts of intra-vacuolar and pigment-containing vesicles. Quantitative immunogold labeling of C. neoformans cells with a monoclonal antibody raised to a major capsular component suggested impaired polysaccharide synthesis. APT1 deletion also affected synthesis of phosphatidylserine, phosphatidylethanolamine, inositolphosphoryl ceramide, glucosylceramide and ergosterylglycoside. These results reveal novel functions of Apt1 and are in agreement with the notion that this putative flippase plays an important role in the physiology of C. neoformans.

The effect of sterol structure upon clathrin-mediated and clathrin-independent endocytosis.

Ordered lipid domains (rafts) in plasma membranes have been hypothesized to participate in endocytosis based on inhibition of endocytosis by removal or sequestration of cholesterol. To more carefully investigate the role of the sterol in endocytosis, we used a substitution strategy to replace cholesterol with sterols that show various raft-forming abilities and chemical structures. Both clathrin-mediated endocytosis of transferrin and clathrin-independent endocytosis of clustered placental alkaline phosphatase were measured. A subset of sterols reversibly inhibited both clathrin-dependent and clathrin-independent endocytosis. The ability of a sterol to support lipid raft formation was necessary for endocytosis. However, it was not sufficient, because a sterol lacking a 3ß-OH group did not support endocytosis even though it had the ability to support ordered domain formation. Double bonds in the sterol rings and an aliphatic tail structure identical to that of cholesterol were neither necessary nor sufficient to support endocytosis. This study shows that substitution using a large number of sterols can define the role of sterol structure in cellular functions. Hypotheses for how sterol structure can similarly alter clathrin-dependent and clathrin-independent endocytosis are discussed.

Generational distribution of a Candida glabrata population: Resilient old cells prevail, while younger cells dominate in the vulnerable host.

Similar to other yeasts, the human pathogen Candida glabrata ages when it undergoes asymmetric, finite cell divisions, which determines its replicative lifespan. We sought to investigate if and how aging changes resilience of C. glabrata populations in the host environment. Our data demonstrate that old C. glabrata are more resistant to hydrogen peroxide and neutrophil killing, whereas young cells adhere better to epithelial cell layers. Consequently, virulence of old compared to younger C. glabrata cells is enhanced in the Galleria mellonella infection model. Electron microscopy images of old C. glabrata cells indicate a marked increase in cell wall thickness. Comparison of transcriptomes of old and young C. glabrata cells reveals differential regulation of ergosterol and Hog pathway associated genes as well as adhesion proteins, and suggests that aging is accompanied by remodeling of the fungal cell wall. Biochemical analysis supports this conclusion as older cells exhibit a qualitatively different lipid composition, leading to the observed increased emergence of fluconazole resistance when grown in the presence of fluconazole selection pressure. Older C. glabrata cells accumulate during murine and human infection, which is statistically unlikely without very strong selection. Therefore, we tested the hypothesis that neutrophils constitute the predominant selection pressure in vivo. When we altered experimentally the selection pressure by antibody-mediated removal of neutrophils, we observed a significantly younger pathogen population in mice. Mathematical modeling confirmed that differential selection of older cells is sufficient to cause the observed demographic shift in the fungal population. Hence our data support the concept that pathogenesis is affected by the generational age distribution of the infecting C. glabrata population in a host. We conclude that replicative aging constitutes an emerging trait, which is selected by the host and may even play an unanticipated role in the transition from a commensal to a pathogen state.

Sphingosine-1-phosphate receptors and innate immunity.

Sphingosine-1-phosphate (S1P) is a signalling lipid that regulates many cellular processes in mammals. One well-studied role of S1P signalling is to modulate T-cell trafficking, which has a major impact on adaptive immunity. Compounds that target S1P signalling pathways are of interest for immune system modulation. Recent studies suggest that S1P signalling regulates many more cell types and processes than previously appreciated. This review will summarise current understanding of S1P signalling, focusing on recent novel findings in the roles of S1P receptors in innate immunity.

Acylhydrazones as Antifungal Agents Targeting the Synthesis of Fungal Sphingolipids.

The incidence of invasive fungal infections has risen dramatically in recent decades. Current antifungal drugs are either toxic, likely to interact with other drugs, have a narrow spectrum of activity, or induce fungal resistance. Hence, there is a great need for new antifungals, possibly with novel mechanisms of action. Previously our group reported an acylhydrazone called BHBM that targeted the sphingolipid pathway and showed strong antifungal activity against several fungi. In this study, we screened 19 derivatives of BHBM. Three out of 19 derivatives were highly active against Cryptococcus neoformansin vitro and had low toxicity in mammalian cells. In particular, one of them, called D13, had a high selectivity index and showed better activity in an animal model of cryptococcosis, candidiasis, and pulmonary aspergillosis. D13 also displayed suitable pharmacokinetic properties and was able to pass through the blood-brain barrier. These results suggest that acylhydrazones are promising molecules for the research and development of new antifungal agents.

Analysis of sphingolipids, sterols, and phospholipids in human pathogenic Cryptococcus strains.

Cryptococcus species cause invasive infections in humans. Lipids play an important role in the progression of these infections. Independent studies done by our group and others provide some detail about the functions of these lipids in Cryptococcus infections. However, the pathways of biosynthesis and the metabolism of these lipids are not completely understood. To thoroughly understand the physiological role of these Cryptococcus lipids, a proper structure and composition analysis of Cryptococcus lipids is demanded. In this study, a detailed spectroscopic analysis of lipid extracts from Cryptococcus gattii and Cryptococcus grubii strains is presented. Sphingolipid profiling by LC-ESI-MS/MS was used to analyze sphingosine, dihydrosphingosine, sphingosine-1-phosphate, dihydrosphingosine-1-phosphate, ceramide, dihydroceramide, glucosylceramide, phytosphingosine, phytosphingosine-1-phosphate, phytoceramide, α-hydroxy phytoceramide, and inositolphosphorylceramide species. A total of 13 sterol species were identified using GC-MS, where ergosterol is the most abundant species. The 31P-NMR-based phospholipid analysis identified phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidyl-N,N-dimethylethanolamine, phosphatidyl-N-monomethylethanolamine, phosphatidylglycerol, phosphatidic acid, and lysophosphatidylethanolamine. A comparison of lipid profiles among different Cryptococcus strains illustrates a marked change in the metabolic flux of these organisms, especially sphingolipid metabolism. These data improve our understanding of the structure, biosynthesis, and metabolism of common lipid groups of Cryptococcus and should be useful while studying their functional significance and designing therapeutic interventions.

Changes in glucosylceramide structure affect virulence and membrane biophysical properties of Cryptococcus neoformans.

Fungal glucosylceramide (GlcCer) is a plasma membrane sphingolipid in which the sphingosine backbone is unsaturated in carbon position 8 (C8) and methylated in carbon position 9 (C9). Studies in the fungal pathogen, Cryptococcus neoformans, have shown that loss of GlcCer synthase activity results in complete loss of virulence in the mouse model. However, whether the loss of virulence is due to the lack of the enzyme or to the loss of the sphingolipid is not known. In this study, we used genetic engineering to alter the chemical structure of fungal GlcCer and studied its effect on fungal growth and pathogenicity. Here we show that unsaturation in C8 and methylation in C9 is required for virulence in the mouse model without affecting fungal growth in vitro or common virulence factors. However, changes in GlcCer structure led to a dramatic susceptibility to membrane stressors resulting in increased cell membrane permeability and rendering the fungal mutant unable to grow within host macrophages. Biophysical studies using synthetic vesicles containing GlcCer revealed that the saturated and unmethylated sphingolipid formed vesicles with higher lipid order that were more likely to phase separate into ordered domains. Taken together, these studies show for the first time that a specific structure of GlcCer is a major regulator of membrane permeability required for fungal pathogenicity.

The Aspergillus fumigatus SchASCH9 kinase modulates SakAHOG1 MAP kinase activity and it is essential for virulence.

The serine-threonine kinase TOR, the Target of Rapamycin, is an important regulator of nutrient, energy and stress signaling in eukaryotes. Sch9, a Ser/Thr kinase of AGC family (the cAMP-dependent PKA, cGMP- dependent protein kinase G and phospholipid-dependent protein kinase C family), is a substrate of TOR. Here, we characterized the fungal opportunistic pathogen Aspergillus fumigatus Sch9 homologue (SchA). The schA null mutant was sensitive to rapamycin, high concentrations of calcium, hyperosmotic stress and SchA was involved in iron metabolism. The ΔschA null mutant showed increased phosphorylation of SakA, the A. fumigatus Hog1 homologue. The schA null mutant has increased and decreased trehalose and glycerol accumulation, respectively, suggesting SchA performs different roles for glycerol and trehalose accumulation during osmotic stress. The schA was transcriptionally regulated by osmotic stress and this response was dependent on SakA and MpkC. The double ΔschA ΔsakA and ΔschA ΔmpkC mutants were more sensitive to osmotic stress than the corresponding parental strains. Transcriptomics and proteomics identified direct and indirect targets of SchA post-exposure to hyperosmotic stress. Finally, ΔschA was avirulent in a low dose murine infection model. Our results suggest there is a complex network of interactions amongst the A. fumigatus TOR, SakA and SchA pathways.

The Granuloma Response Controlling Cryptococcosis in Mice Depends on the Sphingosine Kinase 1-Sphingosine 1-Phosphate Pathway.

Cryptococcus neoformans is a fungal pathogen that causes pulmonary infections, which may progress into life-threatening meningitis. In commonly used mouse models of C. neoformans infections, fungal cells are not contained in the lungs, resulting in dissemination to the brain. We have previously reported the generation of an engineered C. neoformans strain (C. neoformans Δgcs1) which can be contained in lung granulomas in the mouse model and have shown that granuloma formation is dependent upon the enzyme sphingosine kinase 1 (SK1) and its product, sphingosine 1-phosphate (S1P). In this study, we have used four mouse models, CBA/J and C57BL6/J (both immunocompetent), Tgε26 (an isogenic strain of strain CBA/J lacking T and NK cells), and SK(-/-) (an isogenic strain of strain C57BL6/J lacking SK1), to investigate how the granulomatous response and SK1-S1P pathway are interrelated during C. neoformans infections. S1P and monocyte chemotactic protein-1 (MCP-1) levels were significantly elevated in the bronchoalveolar lavage fluid of all mice infected with C. neoformans Δgcs1 but not in mice infected with the C. neoformans wild type. SK1(-/-) mice did not show elevated levels of S1P or MCP-1. Primary neutrophils isolated from SK1(-/-) mice showed impaired antifungal activity that could be restored by the addition of extracellular S1P. In addition, high levels of tumor necrosis factor alpha were found in the mice infected with C. neoformans Δgcs1 in comparison to the levels found in mice infected with the C. neoformans wild type, and their levels were also dependent on the SK1-S1P pathway. Taken together, these results suggest that the SK1-S1P pathway promotes host defense against C. neoformans infections by regulating cytokine levels, promoting extracellular killing by phagocytes, and generating a granulomatous response.