Mouse Model Of Cryptococcosis Research Articles

Models for Inducing Experimental Cryptococcosis in Mice.

Cryptococcus neoformans and Cryptococcus gattii are the predominant etiological agents of cryptococcosis, a particularly problematic disease in immunocompromised individuals. The increased clinical use of immunosuppressive drugs, the inherent ability of Cryptococcus species to suppress and evade host immune responses, and the emergence of drug-resistant yeast support the need for model systems that facilitate the design of novel immunotherapies and antifungals to combat disease progression. The mouse model of cryptococcosis is a widely used system to study Cryptococcus pathogenesis and the efficacy of antifungal drugs in vivo. In this chapter, we describe three commonly used strategies to establish cryptococcosis in mice: intranasal, intratracheal, and intravenous inoculations. Also, we discuss the methodology for delivering drugs to mice via intraperitoneal injection.

Inbred SJL mice recapitulate human resistance to Cryptococcus infection due to differential immune activation.

Cryptococcosis studies often utilize the common C57BL/6J mouse model. Unfortunately, infection in these mice fails to replicate the basic course of human disease, particularly hampering immunological studies. This work demonstrates that SJL/J mice can recapitulate human infection better than other mouse strains. The immunological response to Cryptococcus infection in SJL/J mice was markedly different from C57BL/6J and much more productive in combating this infection. Characterization of infected mice demonstrated strain-specific genetic linkage and differential regulation of multiple important immune-relevant genes in response to Cryptococcus infection. While our results validate many of the previously identified immunological features of cryptococcosis, we also demonstrate limitations from previous mouse models as they may be less translatable to human disease. We concluded that SJL/J mice more faithfully recapitulate human cryptococcosis serving as an exciting new animal model for immunological and genetic studies.

Extension of O-Linked Mannosylation in the Golgi Apparatus Is Critical for Cell Wall Integrity Signaling and Interaction with Host Cells in Cryptococcus neoformans Pathogenesis.

The human-pathogenic yeast Cryptococcus neoformans assembles two types of O-linked glycans on its proteins. In this study, we identified and functionally characterized the C. neoformans CAP6 gene, encoding an α1,3-mannosyltransferase responsible for the second mannose addition to minor O-glycans containing xylose in the Golgi apparatus. Two cell surface sensor proteins, Wml1 (WSC/Mid2-like) and Wml2, were found to be independent substrates of Cap6-mediated minor or Ktr3-mediated major O-mannosylation, respectively. The double deletion of KTR3 and CAP6 (ktr3Δ cap6Δ) completely blocked the mannose addition at the second position of O-glycans, resulting in the accumulation of proteins with O-glycans carrying only a single mannose. Tunicamycin (TM)-induced phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) was greatly decreased in both ktr3Δ cap6Δ and wml1Δ wml2Δ strains. Transcriptome profiling of the ktr3Δ cap6Δ strain upon TM treatment revealed decreased expression of genes involved in the Mpk1-dependent cell wall integrity (CWI) pathway. Consistent with its defective growth under several stress conditions, the ktr3Δ cap6Δ strain was avirulent in a mouse model of cryptococcosis. Associated with this virulence defect, the ktr3Δ cap6Δ strain showed decreased adhesion to lung epithelial cells, decreased proliferation within macrophages, and reduced transcytosis of the blood-brain barrier (BBB). Notably, the ktr3Δ cap6Δ strain showed reduced induction of the host immune response and defective trafficking of ergosterol, an immunoreactive fungal molecule. In conclusion, O-glycan extension in the Golgi apparatus plays critical roles in various pathobiological processes, such as CWI signaling and stress resistance and interaction with host cells in C. neoformans. IMPORTANCE Cryptococcus neoformans assembles two types of O-linked glycans on its surface proteins, the more abundant major O-glycans that do not contain xylose residues and minor O-glycans containing xylose. Here, we demonstrate the role of the Cap6 α1,3-mannosyltransferase in the synthesis of minor O-glycans. Previously proposed to be involved in capsule biosynthesis, Cap6 works with the related Ktr3 α1,2-mannosyltransferase to synthesize O-glycans on their target proteins. We also identified two novel C. neoformans stress sensors that require Ktr3- and Cap6-mediated posttranslational modification for full function. Accordingly, the ktr3Δ cap6Δ double O-glycan mutant strain displays defects in stress signaling pathways, CWI, and ergosterol trafficking. Furthermore, the ktr3Δ cap6Δ strain is completely avirulent in a mouse infection model. Together, these results demonstrate critical roles for O-glycosylation in fungal pathogenesis. As there are no human homologs for Cap6 or Ktr3, these fungus-specific mannosyltransferases are novel targets for antifungal therapy.

Cross-reactivity between vaccine antigens from the chitin deacetylase protein family improves survival in a mouse model of cryptococcosis.

Meningitis due to the fungal pathogen Cryptococcus neoformans is estimated to cause nearly 200,000 deaths annually, mostly in resource-limited regions. We previously identified cryptococcal protein antigens which, when delivered in glucan particles, afford vaccine-mediated protection against an otherwise lethal infection. Many of these proteins exhibit significant homology to other similar cryptococcal proteins leading us to hypothesize that protection may be augmented by immunologic cross-reactivity to multiple members of a protein family. To examine the significance of protein cross-reactivity in vaccination, we utilized strains of Cryptococcus that are genetically deficient in select antigens, yet are still lethal in mice. Vaccination with a protein without homologs (e.g., Mep1 and Lhc1) protected against challenge with wild-type Cryptococcus, but not against a deletion strain lacking that protein. Contrastingly, vaccination with a single chitin deacetylase (Cda) protein protected against the corresponding deletion strain, presumably due to host recognition of one or more other family members still expressed in this strain. Vaccination with a single Cda protein induced cross-reactive antibody and interferon-gamma (IFNγ) immune responses to other Cda protein family members. Paradoxically, we saw no evidence of cross-protection within the carboxypeptidase family of proteins. Factors such as in vivo protein expression and the degree of homology across the family could inform the extent to which vaccine-mediated immunity is amplified. Together, these data suggest a role for prioritizing protein families in fungal vaccine design: increasing the number of immune targets generated by a single antigen may improve efficacy while diminishing the risk of vaccine-resistant strains arising from gene mutations.

Vam6/Vps39/ TRAP1 ‐domain proteins influence vacuolar morphology, iron acquisition and virulence in Cryptococcus neoformans

The pathogenic fungus Cryptococcus neoformans must overcome iron limitation to cause disease in mammalian hosts. Previously, we reported a screen for insertion mutants with poor growth on haem as the sole iron source. In this study, we characterised one such mutant and found that the defective gene encoded a Vam6/Vps39/TRAP1 domain-containing protein required for robust growth on haem, an important iron source in host tissue. We designated this protein Vps3 based on reciprocal best matches with the corresponding protein in Saccharomyces cerevisiae. C. neoformans encodes a second Vam6/Vps39/TRAP1 domain-containing protein designated Vam6/Vlp1, and we found that this protein is also required for robust growth on haem as well as on inorganic iron sources. This protein is predicted to be a component of the homotypic fusion and vacuole protein sorting complex involved in endocytosis. Further characterisation of the vam6Δ and vps3Δ mutants revealed perturbed trafficking of iron acquisition functions (e.g., the high affinity iron permease Cft1) and impaired processing of the transcription factor Rim101, a regulator of haem and iron acquisition. The vps3Δ and vam6Δ mutants also had pleiotropic phenotypes including loss of virulence in a mouse model of cryptococcosis, reduced virulence factor elaboration and increased susceptibility to stress, indicating pleiotropic roles for Vps3 and Vam6 beyond haem use in C. neoformans. TAKE AWAYS: Two Vam6/Vps39/TRAP1-domain proteins, Vps3 and Vam6, support the growth of Cryptococcus neoformans on haem. Loss of Vps3 and Vam6 influences the trafficking and expression of iron uptake proteins. Loss of Vps3 or Vam6 eliminates the ability of C. neoformans to cause disease in a mouse model of cryptococcosis.

Oxidative Stress Causes Vacuolar Fragmentation in the Human Fungal Pathogen Cryptococcus neoformans.

Vacuoles are dynamic cellular organelles, and their morphology is altered by various stimuli or stresses. Vacuoles play an important role in the physiology and virulence of many fungal pathogens. For example, a Cryptococcus neoformans mutant deficient in vacuolar functions showed significantly reduced expression of virulence factors such as capsule and melanin synthesis and was avirulent in a mouse model of cryptococcosis. In the current study, we found significantly increased vacuolar fragmentation in the C. neoformans mutants lacking SOD1 or SOD2, which respectively encode Zn, Cu-superoxide dismutase and Mn-superoxide dismutase. The sod2 mutant showed a greater level of vacuole fragmentation than the sod1 mutant. We also observed that the vacuoles were highly fragmented when wild-type cells were grown in a medium containing high concentrations of iron, copper, or zinc. Moreover, elevated temperature and treatment with the antifungal drug fluconazole caused increased vacuolar fragmentation. These conditions also commonly cause an increase in the levels of intracellular reactive oxygen species in the fungus, suggesting that vacuoles are fragmented in response to oxidative stress. Furthermore, we observed that Sod2 is not only localized in mitochondria but also in the cytoplasm within phagocytosed C. neoformans cells, possibly due to copper or iron limitation.

Sensitive bioluminescence imaging of fungal dissemination to the brain in mouse models of cryptococcosis.

ABSTRACTCryptococcus neoformans is a leading cause of fungal brain infection, but the mechanism of dissemination and dynamics of cerebral infection following pulmonary disease are poorly understood. To address these questions, non-invasive techniques that can study the dynamic processes of disease development and progression in living animal models or patients are required. As such, bioluminescence imaging (BLI) has emerged as a powerful tool to evaluate the spatial and temporal distribution of infection in living animals. We aimed to study the time profile of the dissemination of cryptococcosis from the lung to the brain in murine models by engineering the first bioluminescent C. neoformans KN99α strain, expressing a sequence-optimized red-shifted luciferase. The high pathogen specificity and sensitivity of BLI was complemented by the three-dimensional anatomical information from micro-computed tomography (μCT) of the lung and magnetic resonance imaging (MRI) of the brain. These non-invasive imaging techniques provided longitudinal readouts on the spatial and temporal distribution of infection following intravenous, intranasal or endotracheal routes of inoculation. Furthermore, the imaging results correlated strongly with the fungal load in the respective organs. By obtaining dynamic and quantitative information about the extent and timing of brain infections for individual animals, we found that dissemination to the brain after primary infection of the lung is likely a late-stage event with a timeframe that is variable between animals. This novel tool in Cryptococcus research can aid the identification of host and pathogen factors involved in this process, and supports development of novel preventive or therapeutic approaches.

The Monothiol Glutaredoxin Grx4 Regulates Iron Homeostasis and Virulence in Cryptococcus neoformans.

The acquisition of iron and the maintenance of iron homeostasis are important aspects of virulence for the pathogenic fungus Cryptococcus neoformans In this study, we characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis and virulence in C. neoformans Monothiol glutaredoxins are important regulators of iron homeostasis because of their conserved roles in [2Fe-2S] cluster sensing and trafficking. We initially identified Grx4 as a binding partner of Cir1, a master regulator of iron-responsive genes and virulence factor elaboration in C. neoformans We confirmed that Grx4 binds Cir1 and demonstrated that iron repletion promotes the relocalization of Grx4 from the nucleus to the cytoplasm. We also found that a grx4 mutant lacking the GRX domain displayed iron-related phenotypes similar to those of a cir1Δ mutant, including poor growth upon iron deprivation. Importantly, the grx4 mutant was avirulent in mice, a phenotype consistent with observed defects in the key virulence determinants, capsule and melanin, and poor growth at 37°C. A comparative transcriptome analysis of the grx4 mutant and the WT strain under low-iron and iron-replete conditions confirmed a central role for Grx4 in iron homeostasis. Dysregulation of iron-related metabolism was consistent with grx4 mutant phenotypes related to oxidative stress, mitochondrial function, and DNA repair. Overall, the phenotypes of the grx4 mutant lacking the GRX domain and the transcriptome sequencing (RNA-Seq) analysis of the mutant support the hypothesis that Grx4 functions as an iron sensor, in part through an interaction with Cir1, to extensively regulate iron homeostasis.IMPORTANCE Fungal pathogens cause life-threatening diseases in humans, particularly in immunocompromised people, and there is a tremendous need for a greater understanding of pathogenesis to support new therapies. One prominent fungal pathogen, Cryptococcus neoformans, causes meningitis in people suffering from HIV/AIDS. In the present study, we focused on characterizing mechanisms by which C. neoformans senses iron availability because iron is both a signal and a key nutrient for proliferation of the pathogen in vertebrate hosts. Specifically, we characterized a monothiol glutaredoxin protein, Grx4, that functions as a sensor of iron availability and interacts with regulatory factors to control the ability of C. neoformans to cause disease. Grx4 regulates key virulence factors, and a mutant is unable to cause disease in a mouse model of cryptococcosis. Overall, our study provides new insights into nutrient sensing and the role of iron in the pathogenesis of fungal diseases.

Role of clathrin-mediated endocytosis in the use of heme and hemoglobin by the fungal pathogen Cryptococcus neoformans.

Heme is a major source of iron for pathogens of humans, and its use is critical in determining the outcome of infection and disease. Cryptococcus neoformans is an encapsulated fungal pathogen that causes life-threatening infections in immunocompromised individuals. C.neoformans effectively uses heme as an iron source, but the underlying mechanisms are poorly defined. Non-iron metalloporphyrins (MPPs) are toxic analogues of heme and are thought to enter microbial cells via endogenous heme acquisition systems. We therefore carried out a mutant screen for susceptibility against manganese MPP (MnMPP) to identify new components for heme uptake in C.neoformans. We identified several genes involved in signalling, DNA repair, sugar metabolism, and trafficking that play important roles in susceptibility to MnMPP and in the use of heme as an iron source. We focused on investigating the role of clathrin-mediated endocytosis (CME) and found that several components of CME including Chc1, Las17, Rvs161, and Rvs167 are required for growth on heme and hemoglobin and for endocytosis and intracellular trafficking of these molecules. We show that the hemoglobin uptake process in C.neoformans involves clathrin heavy chain, Chc1, which appears to colocalise with hemoglobin-containing vesicles and to potentially assist in proper delivery of hemoglobin to the vacuole. Additionally, C.neoformans strains lacking Chc1, Las17, Rvs161, or Rvs167 were defective in the elaboration of several key virulence factors, and a las17 mutant was avirulent in a mouse model of cryptococcosis. Overall, this study unveils crucial functions of CME in the use of heme iron by C.neoformans and reveals a role for CME in fungal pathogenesis.

The Sec1/Munc18 (SM) protein Vps45 is involved in iron uptake, mitochondrial function and virulence in the pathogenic fungus Cryptococcus neoformans.

The battle for iron between invading microorganisms and mammalian hosts is a pivotal determinant of the outcome of infection. The pathogenic fungus, Cryptococcus neoformans, employs multiple mechanisms to compete for iron during cryptococcosis, a disease primarily of immunocompromised hosts. In this study, we examined the role of endocytic trafficking in iron uptake by characterizing a mutant defective in the Sec1/Munc18 (SM) protein Vps45. This protein is known to regulate the machinery for vesicle trafficking and fusion via interactions with SNARE proteins. As expected, a vps45 deletion mutant was impaired in endocytosis and showed sensitivity to trafficking inhibitors. The mutant also showed poor growth on iron-limited media and a defect in transporting the Cfo1 ferroxidase of the high-affinity iron uptake system from the plasma membrane to the vacuole. Remarkably, we made the novel observation that Vps45 also contributes to mitochondrial function in that a Vps45-Gfp fusion protein associated with mitotracker, and a vps45 mutant showed enhanced sensitivity to inhibitors of electron transport complexes as well as changes in mitochondrial membrane potential. Consistent with mitochondrial function, the vps45 mutant was impaired in calcium homeostasis. To assess the relevance of these defects for virulence, we examined cell surface properties of the vps45 mutant and found increased sensitivity to agents that challenge cell wall integrity and to antifungal drugs. A change in cell wall properties was consistent with our observation of altered capsule polysaccharide attachment, and with attenuated virulence in a mouse model of cryptococcosis. Overall, our studies reveal a novel role for Vps45-mediated trafficking for iron uptake, mitochondrial function and virulence.

Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis.

Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus.

Role of dendritic cell-pathogen interactions in the immune response to pulmonary cryptococcal infection.

This review discusses the unique contributions of dendritic cells (DCs) to T-cell priming and the generation of effective host defenses against Cryptococcus neoformans (C.neo) infection. We highlight DC subsets involved in the early and later stages of anticryptococcal immune responses, interactions between C.neo pathogen-associated molecular patterns and pattern recognition receptors expressed by DC, and the influence of DC on adaptive immunity. We emphasize recent studies in mouse models of cryptococcosis that illustrate the importance of DC-derived cytokines and costimulatory molecules and the potential role of DC epigenetic modifications that support maintenance of these signals throughout the immune response to C.neo. Lastly, we stipulate where these advances can be developed into new, immune-based therapeutics for treatment of this global pathogen.

Role of Sterylglucosidase 1 (Sgl1) on the pathogenicity of Cryptococcus neoformans: potential applications for vaccine development.

Cryptococcosis caused by Cryptococcus neoformans and Cryptococcus gattii affects a large population and is a cause of significant morbidity and mortality. Despite its public health burden, there are currently no vaccines against cryptococcosis and new strategies against such infections are needed. In this study, we demonstrate that C. neoformans has the biochemical ability to metabolize sterylglucosides (SGs), a class of immunomodulatory glycolipids. Genetic manipulations that eliminate cryptococccal sterylglucosidase lead to the accumulation of SGs and generate a mutant strain (Δsgl1) that is non-pathogenic in the mouse models of cryptococcosis. Interestingly, this mutant strain acts as a vaccine strain and protects mice against cryptococcosis following infection with C. neoformans or C. gattii. The immunity induced by the Δsgl1 strain is not CD4+ T-cells dependent. Immunocompromised mice, which lack CD4+ T-cells, are able to control the infection by Δsgl1 and acquire immunity against the challenge by wild-type C. neoformans following vaccination with the Δsgl1 strain. These findings are particularly important in the context of HIV/AIDS immune deficiency and suggest that the Δsgl1 strain might provide a potential vaccination strategy against cryptococcosis.

Unraveling the Novel Structure and Biosynthetic Pathway of O-Linked Glycans in the Golgi Apparatus of the Human Pathogenic Yeast Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated basidiomycete causing cryptococcosis in immunocompromised humans. The cell surface mannoproteins of C. neoformans were reported to stimulate the host T-cell response and to be involved in fungal pathogenicity; however, their O-glycan structure is uncharacterized. In this study, we performed a detailed structural analysis of the O-glycans attached to cryptococcal mannoproteins using HPLC combined with exoglycosidase treatment and showed that the major C. neoformans O-glycans were short manno-oligosaccharides that were connected mostly by α1,2-linkages but connected by an α1,6-linkage at the third mannose residue. Comparison of the O-glycan profiles from wild-type and uxs1Δ mutant strains strongly supports the presence of minor O-glycans carrying a xylose residue. Further analyses of C. neoformans mutant strains identified three mannosyltransferase genes involved in O-glycan extensions in the Golgi. C. neoformans KTR3, the only homolog of the Saccharomyces cerevisiae KRE2/MNT1 family genes, was shown to encode an α1,2-mannosyltransferase responsible for the addition of the second mannose residue via an α1,2-linkage to the major O-glycans. C. neoformans HOC1 and HOC3, homologs of the Saccharomyces cerevisiae OCH1 family genes, were shown to encode α1,6-mannosyltransferases that can transfer the third mannose residue, via an α1,6-linkage, to minor O-glycans containing xylose and to major O-glycans without xylose, respectively. Moreover, the C. neoformans ktr3Δ mutant strain, which displayed increased sensitivity to SDS, high salt, and high temperature, showed attenuated virulence in a mouse model of cryptococcosis, suggesting that the extended structure of O-glycans is required for cell integrity and full pathogenicity of C. neoformans.

Cryptococcus neoformans dual GDP-mannose transporters and their role in biology and virulence.

Cryptococcus neoformans is an opportunistic yeast responsible for lethal meningoencephalitis in humans. This pathogen elaborates a polysaccharide capsule, which is its major virulence factor. Mannose constitutes over one-half of the capsule mass and is also extensively utilized in cell wall synthesis and in glycosylation of proteins and lipids. The activated mannose donor for most biosynthetic reactions, GDP-mannose, is made in the cytosol, although it is primarily consumed in secretory organelles. This compartmentalization necessitates specific transmembrane transporters to make the donor available for glycan synthesis. We previously identified two cryptococcal GDP-mannose transporters, Gmt1 and Gmt2. Biochemical studies of each protein expressed in Saccharomyces cerevisiae showed that both are functional, with similar kinetics and substrate specificities in vitro. We have now examined these proteins in vivo and demonstrate that cells lacking Gmt1 show significant phenotypic differences from those lacking Gmt2 in terms of growth, colony morphology, protein glycosylation, and capsule phenotypes. Some of these observations may be explained by differential expression of the two genes, but others suggest that the two proteins play overlapping but nonidentical roles in cryptococcal biology. Furthermore, gmt1 gmt2 double mutant cells, which are unexpectedly viable, exhibit severe defects in capsule synthesis and protein glycosylation and are avirulent in mouse models of cryptococcosis.

Defects in Phosphate Acquisition and Storage Influence Virulence of Cryptococcus neoformans

Nutrient acquisition and sensing are critical aspects of microbial pathogenesis. Previous transcriptional profiling indicated that the fungal pathogen Cryptococcus neoformans, which causes meningoencephalitis in immunocompromised individuals, encounters phosphate limitation during proliferation in phagocytic cells. We therefore tested the hypothesis that phosphate acquisition and polyphosphate metabolism are important for cryptococcal virulence. Deletion of the high-affinity uptake system interfered with growth on low-phosphate medium, perturbed the formation of virulence factors (capsule and melanin), reduced survival in macrophages, and attenuated virulence in a mouse model of cryptococcosis. Additionally, analysis of nutrient sensing functions for C. neoformans revealed regulatory connections between phosphate acquisition and storage and the iron regulator Cir1, cyclic AMP (cAMP)-dependent protein kinase A (PKA), and the calcium-calmodulin-activated protein phosphatase calcineurin. Deletion of the VTC4 gene encoding a polyphosphate polymerase blocked the ability of C. neoformans to produce polyphosphate. The vtc4 mutant behaved like the wild-type strain in interactions with macrophages and in the mouse infection model. However, the fungal load in the lungs was significantly increased in mice infected with vtc4 deletion mutants. In addition, the mutant was impaired in the ability to trigger blood coagulation in vitro, a trait associated with polyphosphate. Overall, this study reveals that phosphate uptake in C. neoformans is critical for virulence and that its regulation is integrated with key signaling pathways for nutrient sensing.

Synthesis and antifungal activities of miltefosine analogs

Miltefosine is an alkylphosphocholine that shows broad-spectrum in vitro antifungal activities and limited in vivo efficacy in mouse models of cryptococcosis. To further explore the potential of this class of compounds for the treatment of systemic mycoses, nine analogs (3a−3i) were synthesized by modifying the choline structural moiety and the alkyl chain length of miltefosine. In vitro testing of these compounds against the opportunistic fungal pathogens Candida albicans, Candida glabrata, Candida krusei, Aspergillus fumigatus, and Cryptococcus neoformans revealed that N-benzyl-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3a), N,N-dimethyl-N-(4-nitrobenzyl)-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3d), and N-(4-methoxybenzyl)-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3e) exhibited minimum inhibitory concentrations (MIC) of 2.5–5.0μg/mL against all tested pathogens, when compared to miltefosine with MICs of 2.5–3.3μg/mL. Compound 3a showed low in vitro cytotoxicity against three mammalian cell lines similar to miltefosine. In vivo testing of 3a and miltefosine against C. albicans in a mouse model of systemic infection did not demonstrate efficacy. The results of this study indicate that further investigation will be required to determine the potential usefulness of the alkylphosphocholines in the treatment of invasive fungal infections.

The Mannoprotein Cig1 Supports Iron Acquisition From Heme and Virulence in the Pathogenic Fungus Cryptococcus neoformans

Iron acquisition is critical for virulence of the human pathogenic fungus Cryptococcus neoformans. The cryptococcal transcript for the extracellular mannoprotein Cig1 is highly regulated by iron and abundant in iron-starved cells, suggesting a role in iron acquisition. Indeed, loss of Cig1 resulted in delayed growth on heme at physiological pH. Expression of CIG1 is regulated by the pH-responsive transcription factor Rim101, and loss of Rim101 also impaired growth on heme. A cig1Δ mutant was less susceptible than the wild-type strain to noniron metalloporphyrins, further indicating a role for Cig1 in heme uptake. Recombinant Cig1 exhibited the absorbance spectrum of a heme-binding protein upon heme titration, and Cig1 may therefore function as a hemophore at the cell surface. Cig1 contributed to virulence in a mouse model of cryptococcosis but only in a mutant that also lacked the high-affinity iron uptake system. Overall, Cig1-mediated heme uptake is a potential therapeutic target in C. neoformans.

Cryptococcus neoformans Requires the ESCRT Protein Vps23 for Iron Acquisition from Heme, for Capsule Formation, and for Virulence

Iron availability is a key regulator of virulence factor elaboration in Cryptococcus neoformans, the causative agent of fungal meningoencephalitis in HIV/AIDS patients. In addition, iron is an essential nutrient for pathogen proliferation in mammalian hosts but little is known about the mechanisms of iron sensing and uptake in fungal pathogens that attack humans. In this study, we mutagenized C. neoformans by Agrobacterium-mediated T-DNA insertion and screened for mutants with reduced growth on heme as the sole iron source. Among 34 mutants, we identified a subset with insertions in the gene for the ESCRT-I (endosomal sorting complex required for transport) protein Vps23 that resulted in a growth defect on heme, presumably due to a defect in uptake via endocytosis or misregulation of iron acquisition from heme. Remarkably, vps23 mutants were also defective in the elaboration of the cell-associated capsular polysaccharide that is a major virulence factor, while overexpression of Vps23 resulted in cells with a slightly enlarged capsule. These phenotypes were mirrored by a virulence defect in the vps23 mutant in a mouse model of cryptococcosis and by hypervirulence of the overexpression strain. Overall, these results reveal an important role for trafficking via ESCRT functions in both heme uptake and capsule formation, and they further reinforce the connection between iron and virulence factor deployment in C. neoformans.

The GATA-type transcriptional activator Gat1 regulates nitrogen uptake and metabolism in the human pathogen Cryptococcus neoformans

Nitrogen uptake and metabolism are essential to microbial growth. Gat1 belongs to a conserved family of zinc finger containing transcriptional regulators known as GATA-factors. These factors activate the transcription of Nitrogen Catabolite Repression (NCR) sensitive genes when preferred nitrogen sources are absent or limiting. Cryptococcus neoformansGAT1 is an ortholog to the Aspergillus nidulansAreA and Candida albicans GAT1 genes. In an attempt to define the function of this transcriptional regulator in C. neoformans, we generated null mutants (gat1Δ) of this gene. The gat1 mutant exhibited impaired growth on all amino acids tested as sole nitrogen sources, with the exception of arginine and proline. Furthermore, the gat1 mutant did not display resistance to rapamycin, an immunosuppressant drug that transiently mimics a low-quality nitrogen source. Gat1 is not required for C. neoformans survival during macrophage infection or for virulence in a mouse model of cryptococcosis. Microarray analysis allowed the identification of target genes that are regulated by Gat1 in the presence of proline, a poor and non-repressing nitrogen source. Genes involved in ergosterol biosynthesis, iron uptake, cell wall organization and capsule biosynthesis, in addition to NCR-sensitive genes, are Gat1-regulated in C. neoformans.