Growth on Douglas fir media facilitates Cryptococcus virulence factor production and enhances fungal survival against environmental and immune stressors.

The yeasts Cryptococcus neoformans and Cryptococcus gattii are fungal pathogens that can be isolated from the environment, including the surfaces of many plants. Cryptococcus gattii caused an outbreak on Vancouver Island, British Columbia beginning in 1999 that has since spread to the Pacific Northwest of the United States. Coastal Douglas fir (Pseudotsuga menziesii) is an important lumber species and a major component of the ecosystems in this area. Previous research has explored Cryptococcus survival and mating on Douglas fir plants and plant-derived material, but no studies have been done on the production of cryptococcal virulence factors by cells grown on those media. Here, we investigated the effects of growth on Douglas fir-derived media on the production of the polysaccharide capsule and melanin, two of the most important cryptococcal virulence factors. We found that while the capsule was mostly unchanged by growth in Douglas fir media compared to cells grown in defined minimal media, Cryptococcus spp. can use substrates present in Douglas fir to synthesize functional and protective melanin. These results suggest mechanisms by which Cryptococcus species may survive in the environment and emphasize the need to explore how association with Douglas fir trees could affect its epidemiology for human cryptococcosis.

Cryptococcus gattii is a fungal pathogen that can be found in the environment. It is responsible for causing an outbreak in British Columbia, Canada, in the late 90s. In our study, we created media from Douglas fir, a tree commonly found in the affected areas. We examined the production of virulence factors by Cryptococcus cells grown in this media.

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.

Caenorhabditis elegans DAF-16 regulates lifespan and immune responses to Cryptococcus neoformans and Cryptococcus gattii infections.

BACKGROUND: Cryptococcosis is a life-threatening infection is primarily caused by two sibling species Cryptococcus neoformans and Cryptococcus gattii. Several virulence-related factors of these cryptococci have been widely investigated in Caenorhabditis elegans, representing a facile in vivo model of host-pathogen interaction. While recent studies elucidated cryptococcal virulence factors, intrinsic host factors that affect susceptibility to infections by cryptococci remain unclear and poorly investigated. RESULTS: Here, we showed that defects in C. elegans insulin/insulin-like growth factor-1 (IGF-1) signaling (IIS) pathway influenced animal lifespan and mechanisms of host resistance in cryptococcal infections, which required the activation of aging regulator DAF-16/Forkhead box O transcription factor. Moreover, accumulation of lipofuscin, DAF-16 nuclear localization, and expression of superoxide dismutase (SOD-3) were elevated in C. elegans due to host defenses during cryptococcal infections. CONCLUSION: The present study demonstrated the relationship between longevity and immunity, which may provide a possibility for novel therapeutic intervention to improve host resistance against cryptococcal infections.

Cryptococcus gattii Species Complex as an Opportunistic Pathogen: Underlying Medical Conditions Associated with the Infection.

The Cryptococcus gattii species complex has often been referred to as a primary pathogen due to its high infection frequency among apparently immunocompetent patients. In order to scrutinize the immune status of patients and the lineages of etiologic agents, we analyzed patient histories and the molecular types of etiologic agents from 135 global C. gattii cases. Eighty-six of 135 patients had been diagnosed as immunocompetent, although some of them had underlying medical issues, and 49 were diagnosed as immunocompromised with risk factors similar to those seen in Cryptococcus neoformans infection. We focused on the 86 apparently immunocompetent patients and were able to obtain plasma from 32 (37%) to analyze for the presence of autoantibodies against the granulocyte-macrophage colony-stimulating factor (GM-CSF) since these antibodies have been reported as a hidden risk factor for C. gattii infection. Among the 32 patients, 25 were free from any known other health issues, and 7 had various medical conditions at the time of diagnosis for cryptococcosis. Importantly, plasma from 19 (76%) of 25 patients with no recognized underlying medical condition showed the presence of GM-CSF autoantibodies, supporting this antibody as a major hidden risk factor for C. gattii infection. These data indicate that seemingly immunocompetent people with C. gattii infection warrant detailed evaluation for unrecognized immunologic risks. There was no relationship between molecular type and underlying conditions of patients. Frequency of each molecular type was related to its geographic origin exemplified by the overrepresentation of VGIV in HIV-positive (HIV+) patients due to its prevalence in Africa. IMPORTANCE The C. neoformans and C. gattii species complex causes cryptococcosis. The C. neoformans species complex is known as an opportunistic pathogen since it primarily infects immunocompromised patients. C. gattii species complex has been referred to as a primary pathogen due to its high infection frequency in apparently immunocompetent people. We analyzed 135 global cases of C. gattii infection with documented patient history. Eighty-six of 135 patients were originally diagnosed as immunocompetent and 49 as immunosuppressed with similar underlying conditions reported for C. neoformans infection. A significant number of C. gattii patients without known underlying conditions possessed autoantibodies against granulocytes-macrophage colony-stimulating factor (GM-CSF) in their plasma, supporting the presence of GM-CSF antibodies as a hidden risk factor for C. gattii infection. No relationship was found between C. gattii lineages and the underlying conditions except for overrepresentation of the molecular type VGIV among HIV+ patients due to the prevalence of VGIV in Africa.

Cryptococcus gattii evades CD11b-mediated fungal recognition by coating itself with capsular polysaccharides.

Cryptococcus gattii is a capsular pathogenic fungus causing life-threatening cryptococcosis. Although the capsular polysaccharides (CPs) of C. gattii are considered as virulence factors, the physiological significance of CP biosynthesis and of CPs themselves is not fully understood, with many conflicting data reported. First, we demonstrated that CAP gene deletant of C. gattii completely lacked capsule layer and its virulence, and that the strain was susceptible to host-related factors including oxidizing, hypoxic, and hypotrophic conditions in vitro. Extracellular CPs recovered from culture supernatant bound specifically to C. gattii acapsular strains, not to other fungi and immune cells, and rendered them the immune escape effects. In fact, dendritic cells (DCs) did not efficiently uptake the CP-treated acapsular strains, which possessed no visible capsule layer, and a decreased amount of phosphorylated proteins and cytokine levels after the stimulation. DCs recognized C. gattii acapuslar cells via an immune receptor CD11b- and Syk-related pathway; however, CD11b did not bind to CP-treated acapsular cells. These results suggested that CPs support immune evasion by coating antigens on C. gattii and blocking the interaction between CD11b and C. gattii cells. Here, we describe the importance of CPs in pathogenicity and immune evasion mechanisms of C. gattii.

Zrg1, a cryptococcal protein associated with regulation of growth in nutrient deprivation conditions.

Cryptococcus gattii is one of the causes of cryptococcosis, a life-threatening disease generally characterized by pneumonia and/or meningitis. Zinc is an essential element for life, being required for the activity of many proteins with catalytic and structural roles. Here, we characterize ZRG1 (zinc-related gene 1), which codes a product involved in zinc metabolism. Transcriptional profiling revealed that zinc availability regulated the expression of ZRG1, and its null mutants demonstrated impaired growth in zinc- and nitrogen-limiting conditions. Moreover, zrg1 strains displayed alterations in the expression of the zinc homeostasis-related genes ZAP1 and ZIP1. Notably, cryptococcal cells lacking Zrg1 displayed upregulation of autophagy-like phenotypes. Despite no differences were detected in the classical virulence-associated traits; cryptococcal cells lacking ZRG1 displayed decreased capacity for survival inside macrophages and attenuated virulence in an invertebrate model. Together, these results indicate that ZRG1 plays an important role in proper zinc metabolism, and is necessary for cryptococcal fitness and virulence.

First report of cryptococcosis due to Cryptococcus gattii sensu stricto VGI in an Ivorian HIV negative patient.

INTRODUCTION: Cryptococcus gattii species complex is endemic to tropical and subtropical regions and is described as a causative agent of cryptococcosis in immunocompetent individuals. CASE PRESENTATION: We describe the first case of cryptococcosis in a HIV-negative patient from Ivory Coast infected by Cryptococcus gattii sensu stricto VGI. Isolates were recovered from cerebrospinal fluid (CSF) prior to systemic antifungal treatment up to 42 days after detection of the presence of yeasts in the CSF. Eighteen isolates were recovered, genetic diversity and antifungal susceptibility analyses were performed. All the isolates belonged to the Cryptococcus gattii sensu stricto (B;VGI) and were identified as a new sequence type (ST) 553 by Multilocus Sequence Typing (MLST) analyses. Susceptibility testing showed that all the strains had a wild-type phenotype for fluconazole, amphotericin B and flucytosine. Treatment with fluconazole (1200mg/day) was initiated with success. CONCLUSION: This is the first case report of the presence of C. gattii sensu stricto VGI in a HIV-negative ivorian patient and the second report of the presence of species from the C. gattii complex species in this country.

Cryptococcal meningitis in non-HIV patients in the State of Amazonas, Northern Brazil.

Cryptococcosis is a life-threatening fungal infection caused by the Cryptococcus neoformans/Cryptococcus gattii species complex. Most cases are recorded in patients suffering from HIV/AIDS (human immunodeficiency virus/acquired immunodeficiency syndrome). However, this infection also occurs in non-HIV patients with a proportion of 10-30% of all cases. The study aimed at the clinical and molecular characterization of non-HIV patients diagnosed with cryptococcosis at the Tropical Medicine Foundation (FMT-HVD) from July 2016 to June 2019. Medical records of respective patients were analyzed to describe the course of cryptococcosis in non-HIV patients. In addition, multi-locus sequence typing (MLST) was applied to identify the sequence types of the isolated Cryptococcus strains, to perform phylogenetic analysis, and to evaluate the isolates' genetic relationship to global reference strains. Antifungal susceptibility profiles to amphotericin B, fluconazole, and itraconazole were assessed by broth microdilution. From a total of 7 patients, 4 were female, the age range varied between 10 and 53 years (median of 36.3 years). Cryptococcal meningitis was the common clinical manifestation (100%). The period between onset of symptoms and confirmed diagnosis ranged from 15 to 730 days (mean value of 172.9 days), and the observed mortality was 57.1%. Of note, comorbidities of the assessed cryptococcosis patients comprised hypertension, diabetes mellitus, and intestinal tuberculosis. Genotyping applying PCR-RFLP of the URA5 gene identified all clinical isolates as C. gattii genotype VGII. Using MLST, it was possible to discriminate the sequence types ST20 (n = 4), ST5 (n = 3), and the newly identified sequence type ST560 (n = 1). The antifungals amphotericin B, fluconazole, and itraconazole showed satisfactory inhibitory activity (microdilution test) against all C. gattii VGII strains.

Population diversity and virulence characteristics of Cryptococcus neoformans/C. gattii species complexes isolated during the pre-HIV-pandemic era.

Cryptococcosis has become a major global health problem since the advent of the HIV pandemic in 1980s. Although its molecular epidemiology is well-defined, using isolates recovered since then, no pre-HIV-pandemic era epidemiological data exist. We conducted a molecular epidemiological study using 228 isolates of the C. neoformans/C. gattii species complexes isolated before 1975. Genotypes were determined by URA5 restriction fragment length polymorphism analysis and multi-locus sequence typing. Population genetics were defined by nucleotide diversity measurements, neutrality tests, and recombination analysis. Growth at 37°C, melanin synthesis, capsule production, and urease activity as virulence factors were quantified. The pre-HIV-pandemic isolates consisted of 186 (81.5%) clinical, 35 (15.4%) environmental, and 7 (3.1%) veterinary isolates. Of those, 204 (89.5%) belonged to C. neoformans VNI (64.0%), VNII (14.9%) and VNIV (10.5%) while 24 (10.5%) belonged to C. gattii VGIII (7.5%), VGI (2.6%) and VGII (0.5%). Among the 47 sequence types (STs) identified, one of VNII and 8 of VNIV were novel. ST5/VNI (23.0%) in C. neoformans and ST75/VGIII (25.0%) in C. gattii were the most common STs in both species complexes. Among C. neoformans, VNIV had the highest genetic diversity (Hd = 0.926) and the minimum recombination events (Rm = 10), and clinical isolates had less genetic diversity (Hd = 0.866) than environmental (Hd = 0.889) and veterinary isolates (Hd = 0.900). Among C. gattii, VGI had a higher nucleotide diversity (π = 0.01436) than in VGIII (π = 0.00328). The high-virulence genotypes (ST5/VNI and VGIIIa/serotype B) did not produce higher virulence factors levels than other genotypes. Overall, high genetic variability and recombination rates were found for the pre-HIV-pandemic era among strains of the C. neoformans/C. gattii species complexes. Whole genome analysis and in vivo virulence studies would clarify the evolution of the genetic diversity and/or virulence of isolates of the C. neoformans/C. gattii species complexes during the pre- and post-HIV-pandemic eras.

Molecular typing, in vitro susceptibility and virulence of Cryptococcus neoformans/Cryptococcus gattii species complex clinical isolates from south-eastern Brazil.

BACKGROUND: Cryptococcus neoformans/ Cryptococcus gattii species complex is composed of encapsulated yeast species that are causative agents of cryptococcosis. The characterisation of pathogenic Cryptococcus species provides useful data for epidemiological studies as well as the clinical diagnosis and treatment of patients. OBJECTIVES: This study aimed to characterise the epidemiology, antifungal susceptibility and virulence of 72 clinical strains isolated from cryptococcosis cases between 2012 and 2017 in a tertiary reference hospital in south-eastern Brazil. METHODS: Species and molecular types were molecularly assessed by PCR and PCR-restriction fragment length polymorphism (RFLP) of the URA5 gene. Antifungal susceptibility testing was performed according to the CLSI protocols. The virulence was studied in a Galleria mellonella infection model. RESULTS: The most frequently isolated strain was C. neoformans molecular type VNI (61/72; 84.7%), although C. neoformans molecular type VNII (3/72; 4.2%) was also isolated. Additionally, C. deuterogattii molecular type VGII (8/72; 11.1%) was present, but most frequently from non-HIV-infected patients. Non-wild-type phenotype to the antifungals was observed in 26.4% (19/72) of the C. neoformans and C. deuterogattii clinical isolates, and the latter demonstrated higher MIC to fluconazole and itraconazole than C. neoformans clinical isolates. Finally, the virulence of C. neoformans and C. deuterogattii clinical isolates was diverse in G mellonella larvae and uncorrelated with the virulence factors of melanin and capsule. CONCLUSIONS: The assessment of the spread of cryptococcal species and molecular types as well as the pattern of corresponding antifungal susceptibility and virulence aids in surveil the emergence of resistant strains, ensuring more accurate management of the cryptococcal infection.

Cryptococcal infection in haematologic malignancies and haematopoietic stem cell transplantation.

This review summarises both the recent and relevant studies about cryptococcal infections in haematologic malignancies and haematopoietic stem cell transplantation. Although uncommon in this patient population, this infection carries a high mortality, especially if left untreated. Given the limited data, we draw some conclusions with respect to management from the solid organ transplantation and HIV-infected literature. Herein, we discuss cryptococcosis with a particular attention to its background, epidemiology, risk factors, clinical presentation, diagnosis, treatment and prevention in this group.

Participation of Zip3, a ZIP domain-containing protein, in stress response and virulence in Cryptococcus gattii.

Cryptococcus gattii is an etiologic agent of cryptococcosis, a potentially fatal disease that affects humans and animals. The successful infection of mammalian hosts by cryptococcal cells relies on their ability to infect and survive in macrophages. Such phagocytic cells present a hostile environment to intracellular pathogens via the production of reactive nitrogen and oxygen species, as well as low pH and reduced nutrient bioavailability. To overcome the low-metal environment found during infection, fungal pathogens express high-affinity transporters, including members of the ZIP family. Previously, we determined that functional zinc uptake driven by Zip1 and Zip2 is necessary for full C.gattiivirulence. Here, we characterized the ZIP3 gene of C. gattii, an ortholog of the Saccharomyces cerevisiae ATX2, which codes a manganese transporter localized to the membrane of the Golgi apparatus. Cryptococcal cells lacking Zip3 were tolerant to toxic concentrations of manganese and had imbalanced expression of intracellular metal transporters, such as the vacuolar Pmc1 and Vcx1, as well as the Golgi Pmr1. Moreover, null mutants of the ZIP3 gene displayed higher sensitivity to reactive oxygen species (ROS) and substantial alteration in the expression of ROS-detoxifying enzyme-coding genes. In line with these phenotypes, cryptococcal cells displayed decreased virulence in a non-vertebrate model of cryptococcosis. Furthermore, we found that the ZIP3 null mutant strain displayed decreased melanization and secretion of the major capsular component glucuronoxylomannan, as well as an altered extracellular vesicle dimensions profile. Collectively, our data suggest that Zip3 activity impacts the physiology, and consequently, several virulence traits of C. gattii.

Titan cell formation is unique to Cryptococcus species complex.

Members of the Cryptococcus species complex stand out by unique virulence factors that allowed evolutionary transition to pathogenesis. Among the factors contributing to cryptococcosis is a morphological transformation into giant (Titan) cells. It remains unclear whether species outside of the C. neoformans/C. gattii species complex are capable of titanization. We utilized two recently developed protocols that allow obtaining Titan cells in vitro to test if titanization occurs in non-C. neoformans/C. gattii species. We find that none of the tested strains, representing 10 species of basidiomycetous yeasts and the ascomycetous yeast Saccharomyces cerevisiae, undergo significant titanization under conditions that promote robust Titan cell formation in C. neoformans/C. gattii species complex. C. terreus formed occasional enlarged cells through a mechanism potentially similar to that of titanization. Our findings suggest that titanization is a rare phenomenon among basidiomycetous yeasts that occurs mostly in members of the C. neoformans/C. gattii species complex.

Landmark clinical observations and immunopathogenesis pathways linked to HIV and Cryptococcus fatal central nervous system co-infection.

Cryptococcal meningitis remains one of the leading causes of death among HIV-infected adults in the fourth decade of HIV era in sub-Saharan Africa, contributing to 10%-20% of global HIV-related deaths. Despite widespread use and early induction of ART among HIV-infected adults, incidence of cryptococcosis remains significant in those with advanced HIV disease. Cryptococcus species that causes fatal infection follows systemic spread from initial environmental acquired infection in lungs to antigenaemia and fungaemia in circulation prior to establishment of often fatal disease, cryptococcal meningitis in the CNS. Cryptococcus person-to-person transmission is uncommon, and deaths related to blood infection without CNS involvement are rare. Keen to the persistent high mortality associated with HIV-cryptococcal meningitis, seizures are common among a third of the patients, altered mental status is frequent, anaemia is prevalent with ensuing brain hypoxia and at autopsy, brain fibrosis and infarction are evident. In addition, fungal burden is 3-to-4-fold higher in those with seizures. And high immune activation together with exacerbated inflammation and elevated PD-1/PD-L immune checkpoint expression is immunomodulated phenotypes elevated in CSF relative to blood. Lastly, though multiple Cryptococcus species cause disease in this setting, observations are mostly generalised to cryptococcal infection/meningitis or regional dominant species (C neoformans or gattii complex) that may limit our understanding of interspecies differences in infection, progression, treatment or recovery outcome. Together, these factors and underlying mechanisms are hypotheses generating for research to find targets to prevent infection or adequate therapy to prevent persistent high mortality with current optimal therapy.

Fungal kinases and transcription factors regulating brain infection in Cryptococcus neoformans.

Cryptococcus neoformans causes fatal fungal meningoencephalitis. Here, we study the roles played by fungal kinases and transcription factors (TFs) in blood-brain barrier (BBB) crossing and brain infection in mice. We use a brain infectivity assay to screen signature-tagged mutagenesis (STM)-based libraries of mutants defective in kinases and TFs, generated in the C. neoformans H99 strain. We also monitor in vivo transcription profiles of kinases and TFs during host infection using NanoString technology. These analyses identify signalling components involved in BBB adhesion and crossing, or survival in the brain parenchyma. The TFs Pdr802, Hob1, and Sre1 are required for infection under all the conditions tested here. Hob1 controls the expression of several factors involved in brain infection, including inositol transporters, a metalloprotease, PDR802, and SRE1. However, Hob1 is dispensable for most cellular functions in Cryptococcus deuterogattii R265, a strain that does not target the brain during infection. Our results indicate that Hob1 is a master regulator of brain infectivity in C. neoformans.

Melanin deposition in two Cryptococcus species depends on cell-wall composition and flexibility.

Cryptococcus neoformans and Cryptococcus gattii are two species complexes in the large fungal genus Cryptococcus and are responsible for potentially lethal disseminated infections. These two complexes share several phenotypic traits, such as production of the protective compound melanin. In C. neoformans, the pigment associates with key cellular constituents that are essential for melanin deposition within the cell wall. Consequently, melanization is modulated by changes in cell-wall composition or ultrastructure. However, whether similar factors influence melanization in C. gattii is unknown. Herein, we used transmission EM, biochemical assays, and solid-state NMR spectroscopy of representative isolates and "leaky melanin" mutant strains from each species complex to examine the compositional and structural factors governing cell-wall pigment deposition in C. neoformans and C. gattii. The principal findings were the following. 1) C. gattii R265 had an exceptionally high chitosan content compared with C. neoformans H99; a rich chitosan composition promoted homogeneous melanin distribution throughout the cell wall but did not increase the propensity of pigment deposition. 2) Strains from both species manifesting the leaky melanin phenotype had reduced chitosan content, which was compensated for by the production of lipids and other nonpolysaccharide constituents that depended on the species or mutation. 3) Changes in the relative rigidity of cell-wall chitin were associated with aberrant pigment retention, implicating cell-wall flexibility as an independent variable in cryptococcal melanin assembly. Overall, our results indicate that cell-wall composition and molecular architecture are critical factors for the anchoring and arrangement of melanin pigments in both C. neoformans and C. gattii species complexes.

Acute Fluctuant Neurological Symptoms in Stable Chronic Cryptococcus gattii Cryptococcomas: A Novel Disease Complication.

BACKGROUND: Cryptococcus, a yeast-like fungus, is the most common cause of fungal meningitis worldwide. The Cryptococcus gattii variety is concentrated in Australia has a greater propensity to infect immunocompetent hosts, cause meningitis and form crytococcomas. This case presents a novel disease complication, that is, acute neurological symptoms without seizures, disease progression or reactivation. CASE PRESENTATION: A 58-year-old immunocompetent male was brought to the emergency department with dysarthria and right arm paraesthesias. Computed tomography of the brain brain and magnetic resonance imaging revealed no stroke but found several previously identified crytococcomas that demonstrated no interval change. Blood tests and lumbar puncture found only a low cryptococcal antigen complex titer (CRAG) (1:10) and a negative cell culture. He had remained compliant on his maintenance fluconazole therapy and had no immunocompromise or seizure activity. He was initially treated as a relapse of cryptococcal disease and restarted on induction therapy but after the cell culture returned negative and the symptoms resolved over the following days he was reverted back to maintenance therapy. DISCUSSION AND CONCLUSIONS: Central nervous system cryptococcomas are difficult to treat, chronic infections, that in our patient had lasted over 10 years despite treatment compliance. A true cryptococcal meningitis relapse is indicated by positive cell cultures in previously sterile fluid but cryptococcoma progression is measured by serial magnetic resonance imaging or computed tomography scans. In the case of progression or relapse induction and consolidation therapy should be restarted. Our patient demonstrated neither relapse nor progression but presented with a novel disease complication of acute fluctuating neurology in chronic stable cryptococcomas.

Contribution of Laccase Expression to Immune Response against Cryptococcus gattii Infection.

Cryptococcosis is an infectious disease caused by two fungal species, Cryptococcus neoformans and Cryptococcus gattii While C. neoformans affects mainly immunocompromised patients, C. gattii infects both immunocompetent and immunocompromised individuals. Laccase is an important virulence factor that contributes to the virulence of C. neoformans by promoting pulmonary growth and dissemination to the brain. The presence of laccase in C. neoformans can shift the host immune response toward a nonprotective Th2-type response. However, the role of laccase in the immune response against C. gattii remains unclear. In this study, we characterized laccase activity in C. neoformans and C. gattii isolates from Thailand and investigated whether C. gattii that is deficient in laccase might modulate immune responses during infection. C. gattii was found to have higher laccase activity than C. neoformans, indicating the importance of laccase in the pathogenesis of C. gattii infection. The expression of laccase promoted intracellular proliferation in macrophages and inhibited in vitro fungal clearance. Mice infected with a lac1Δ mutant strain of C. gattii had reduced lung burdens at the early but not the late stage of infection. Without affecting type-1 and type-2 responses, the deficiency of laccase in C. gattii induced cryptococcus-specific interleukin-17 (IL-17) cytokine, neutrophil accumulation, and expression of the neutrophil-associated cytokine gene Csf3 and chemokine genes Cxcl1, Cxcl2, and Cxcl5 in vivo, as well as enhanced neutrophil-mediated phagocytosis and killing in vitro Thus, our data suggest that laccase constitutes an important virulence factor of C. gattii that plays roles in attenuating Th17-type immunity, neutrophil recruitment, and function during the early stage of infection.

Repurposing the thrombopoietin receptor agonist eltrombopag as an anticryptococcal agent.

In this study, a Food and Drug Administration (FDA)-approved drug with previously unreported antifungal activity was investigated for suitability for use as an anticryptococcal agent. First, we screened a compound library of 1018 FDA-approved drugs against Cryptococcus neoformans. Of 52 drugs possessing anti-Cryptococcus activity, eltrombopag was chosen due to its novel activity. The susceptibility of Cryptococcus against eltrombopag was then studied by determining the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC), while the synergy of eltrombopag with other drugs was tested by fractional inhibitory concentration index (FICI). Eltrombopag had a limited spectrum of antifungal activity against C. neoformans/C. gattii species complex (MICs of 0.125 mg/l), Candida glabrata (MIC, 0.25 mg/l), and Trichophyton rubrum (MIC, 0.5 mg/l). Eltrombopag affected cryptococcal virulence factors, including capsule and biofilm formation, melanin production, and growth ability at 37°C. Further, RNA sequencing and deletion mutant library screening experiments revealed that genes involved in the calcineurin pathway, lipid biosynthesis, membrane component, and transporter genes were associated with eltrombopag. In addition, eltrombopag showed synergism with the calcineurin inhibitor FK506 (FICI < 0.5) against Cryptococcus species. In conclusion, eltrombopag exhibited excellent antifungal activity against Cryptococcus species potentially via a mode of action which interferes with virulence factors and the calcineurin pathway, indicating that eltrombopag might be usefully repurposed as an antifungal agent for treating cryptococcosis.

Effect of non-antifungal agrochemicals on the pathogenic fungus Cryptococcus gattii.

The chemical control of pests and weeds is employed to improve crop production and the quality of agricultural products. The intensive use of pesticides, however, may cause environmental contamination, thus altering microbial communities. Cryptococcus gattii is an environmental yeast and the causative agent of cryptococcosis in both humans and animals. Up to this day, the effects of agrochemicals on human pathogens living in nature are still widely unknown. In this work, we analyzed the susceptibility of C. gattii to nonfungicide agrochemicals (herbicides and insecticides). Microdilution and drug-combination susceptibility tests were performed for the herbicides flumioxazin (FLX), glyphosate (GLY), isoxaflutole (ISO), pendimethalin (PEND), and also for the insecticide fipronil (FIP). Moreover, these compounds were combined with the clinical antifungals amphotericin B and fluconazole. The MIC values found for the agrochemicals were the following: < 16 µg/ml, for flumioxazin; 128 to 256 µg/ml, for FIP, ISO, and PEND; and >256 µg/ml, for GLY. Synergistic and antagonistic interactions, depending on the strain and concentration tested, were also observed. All strains had undergone adaptation to increasing levels of agrochemicals, in order to select the less susceptible subpopulations. During this process, one C. gattii strain (196 L/03) tolerated high concentrations (50 to 900 µg/ml) of all pesticides assessed. Subsequently, the strain adapted to flumioxazin, isoxaflutole and pendimethalin showed a reduction in the susceptibility to agrochemicals and clinical antifungals, suggesting the occurrence of cross-resistance. Our data point to the risk of exposing C. gattii to agrochemicals existing in the environment, once it might impact the susceptibility of clinical antifungals.