scholarly journals Laccase Affects the Rate of Cryptococcus neoformans Nonlytic Exocytosis from Macrophages

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Stefânia de Oliveira Frazão ◽  
Herdson Renney de Sousa ◽  
Lenise Gonçalves da Silva ◽  
Jéssica dos Santos Folha ◽  
Kaio César de Melo Gorgonha ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cryptococcus Neoformans ◽  
Laccase Activity ◽  
Severe Disease ◽  
Cell Types ◽  
Great Variability ◽  
Melanin Production ◽  
Content Type ◽  
Fungal Melanin ◽  
Laccase Enzyme

ABSTRACT Nonlytic exocytosis is a process in which previously ingested microbes are expelled from host phagocytes with the concomitant survival of both cell types. This process has been observed in the interaction of Cryptococcus spp. and other fungal cells with phagocytes as distant as mammalian, bird, and fish macrophages and ameboid predators. Despite a great amount of research dedicated to unraveling this process, there are still many questions about its regulation and its final benefits for host or fungal cells. During a study to characterize the virulence attributes of Brazilian clinical isolates of C. neoformans, we observed great variability in their rates of nonlytic exocytosis and noted a correlation between this process and fungal melanin production/laccase activity. Flow cytometry experiments using melanized cells, nonmelanized cells, and lac1Δ mutants revealed that laccase has a role in the process of nonlytic exocytosis that seems to be independent of melanin production. These results identify a role for laccase in virulence, independent of its role in pigment production, that represents a new variable in the regulation of nonlytic exocytosis. IMPORTANCE Cryptococcus neoformans is a yeast that causes severe disease, primarily in immunosuppressed people. It has many attributes that allow it to survive and cause disease, such as a polysaccharide capsule and the dark pigment melanin produced by the laccase enzyme. Upon infection, the yeast is ingested by cells called macrophages, whose function is to kill them. Instead, these fungal cells can exit from macrophages in a process called nonlytic exocytosis. We know that this process is controlled by both host and fungal factors, only some of which are known. As part of an ongoing study, we observed that C. neoformans isolates that produce melanin faster are more-frequent targets of nonlytic exocytosis. Further experiments showed that this is probably due to higher production of laccase, because fungi lacking this enzyme are nonlytically exocytosed less often. This shows that laccase is an important signal/regulator of nonlytic exocytosis of C. neoformans from macrophages.

scholarly journals Evaluation of laccases and melanization in clinical and environmental Cryptococcus neoformans samples by non-denaturing PAGE

2009 ◽  
Vol 58 (5) ◽  
pp. 563-566 ◽  
Author(s):  
Cristiane B. Pereira ◽  
Frank L. Bueno ◽  
Amanda L. T. Dias ◽  
Maísa R. P. L. Brigagão ◽  
Claudete R. Paula ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Enzyme Production ◽  
Laccase Activity ◽  
Opportunistic Pathogen ◽  
Clinical Strain ◽  
Melanin Production ◽  
Densitometry Analysis ◽  
Serotype B ◽  
Colour Quantification ◽  
Laccase Isoforms

The increased incidence of infections caused by the opportunistic pathogen Cryptococcus neoformans, which mainly affects immunocompromised patients but can also infect immunocompetent individuals, has needed additional studies on this micro-organism's pathogenicity and factors related to virulence, such as enzyme production, for a better understanding of the aetiology of cryptococcosis. The aim of this study was to verify the applicability of non-denaturing PAGE for analysis of laccases by quantification of the amount of melanin pigment produced by clinical and environmental strains of C. neoformans. After incubation of the gel with the substrate l-dopa, strains produced melanin spots of a bright brown to black colour. Quantification of these spots was performed by densitometry analysis and the amount of melanin produced was calculated and compared among the strains. All strains showed laccase activity. Serotype B strains showed a higher melanin intensity than serotype A strains. Over half of the clinical strains (56.2 %) showed the lowest melanin intensities, suggesting that melanin production may not be the main virulence factor against host defence. The clinical strain ICB 88 revealed two melanin spots on the gel, indicating the presence of two laccase isoforms. The environmental strains showed the highest values of melanin intensity, which may be related to previous exposure to environmental stress conditions.

scholarly journals Unraveling Melanin Biosynthesis and Signaling Networks in Cryptococcus neoformans

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Dongpil Lee ◽  
Eun-Ha Jang ◽  
Minjae Lee ◽  
Sun-Woo Kim ◽  
Yeonseon Lee ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogens ◽  
Signaling Networks ◽  
Melanin Production ◽  
Nitrogen Base ◽  
Content Type ◽  
High Osmolarity ◽  
High Osmolarity Glycerol ◽  
The Core

ABSTRACT Melanin is an antioxidant polyphenol pigment required for the pathogenicity of many fungal pathogens, but comprehensive regulatory mechanisms remain unidentified. In this study, we systematically analyzed melanin-regulating signaling pathways in Cryptococcus neoformans and identified four melanin-regulating core transcription factors (TFs), Bzp4, Usv101, Mbs1, and Hob1, required for induction of the laccase gene (LAC1). Bzp4, Usv101, and Mbs1 independently regulate LAC1 induction, whereas Hob1 controls Bzp4 and Usv101 expression. Both Bzp4 and Usv101 are localized in the cytoplasm under nutrient-rich conditions (i.e., in the presence of yeast extract-peptone-dextrose [YPD] medium) but translocate into the nucleus upon nutrient starvation (i.e., in the presence of yeast nitrogen base [YNB] medium without glucose), and Mbs1 is constitutively localized in the nucleus. Notably, the cAMP pathway is not involved in regulation of the four TFs, but the high-osmolarity glycerol response (HOG) pathway negatively regulates induction of BZP4 and LAC1. Next, we searched for potential kinases upstream of the core TFs and identified nine core kinases; their deletion led to defective melanin production and LAC1 induction. Deletion of GSK3 or KIC1 abolished induction of LAC1 and BZP4 and perturbed nuclear translocation of Bzp4. Notably, Gsk3 also regulated expression of HOB1, USV101, and MBS1, indicating that it is a critical melanin-regulating kinase. Finally, an RNA sequencing-based transcriptome analysis of the wild-type strain and of bzp4Δ, usv101Δ, hob1Δ, and mbs1Δ strains under nutrient-rich and nutrient-starved conditions revealed that the melanin-regulating core TFs govern redundant and distinct classes of genes involved in a variety of biological processes. IMPORTANCE Melanins are dark green, brown, or black pigments that serve as antioxidant, reactive oxygen species (ROS) scavengers that protect fungal pathogens from radiation and host immune responses. Cryptococcus neoformans, the major etiological agent of fungal meningoencephalitis, also utilizes melanin as a key virulence factor. In this basidiomycete pathogen, melanin production is regulated by the cAMP and high-osmolarity glycerol response (HOG) pathways, and yet its complex signaling networks remain poorly described. In this study, we uncovered novel melanin synthesis regulatory networks consisting of core transcription factors (TFs), including Bzp4, Usv101, Hob1, and Mbs1, and core kinases Gsk3 and Kic1. These networks were identified through coupling systematic analyses of the expression and epistatic relationships of TF and kinase mutant libraries in the presence of diverse melanin substrates with transcriptome profiling of the core TF mutants. Thus, this report provides comprehensive insight into the melanin-regulating pathways in C. neoformans and other fungal pathogens.

scholarly journals Transcriptional Network of Multiple Capsule and Melanin Genes Governed by the Cryptococcus neoformans Cyclic AMP Cascade

2005 ◽  
Vol 4 (1) ◽  
pp. 190-201 ◽  
Author(s):  
Read Pukkila-Worley ◽  
Quincy D. Gerrald ◽  
Peter R. Kraus ◽  
Marie-Josée Boily ◽  
Matthew J. Davis ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Cryptococcus Neoformans ◽  
Glucose Deprivation ◽  
Melanin Production ◽  
Gene Encoding ◽  
Human Fungal Pathogen ◽  
Camp Pathway ◽  
Capsule Production ◽  
Capsule Assembly ◽  
Laccase Enzyme

ABSTRACT Cryptococcus neoformans is an opportunistic human fungal pathogen that elaborates several virulence attributes, including a polysaccharide capsule and melanin pigments. A conserved Gα protein/cyclic AMP (cAMP) pathway controls melanin and capsule production. To identify targets of this pathway, we used an expression profiling approach to define genes that are transcriptionally regulated by the Gα protein Gpa1. This approach revealed that Gpa1 transcriptionally regulates multiple genes involved in capsule assembly and identified two additional genes with a marked dependence on Gpa1 for transcription. The first is the LAC1 gene, encoding the laccase enzyme that catalyzes a rate-limiting step in diphenol oxidation and melanin production. The second gene identified (LAC2) is adjacent to the LAC1 gene and encodes a second laccase that shares 75% nucleotide identity with LAC1. Similar to the LAC1 gene, LAC2 is induced in response to glucose deprivation. However, LAC2 basal transcript levels are much lower than those for LAC1. Accordingly, a lac2 mutation results in only a modest delay in melanin formation. LAC2 overexpression suppresses the melanin defects of gpa1 and lac1 mutants and partially restores virulence of these strains. These studies provide mechanistic insights into the regulation of capsule and melanin production by the C. neoformans cAMP pathway and demonstrate that multiple laccases contribute to C. neoformans melanin production and pathogenesis.

scholarly journals Cryptococcus neoformans Host Adaptation: Toward Biological Evidence of Dormancy

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Alexandre Alanio ◽  
Frédérique Vernel-Pauillac ◽  
Aude Sturny-Leclère ◽  
Françoise Dromer
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Cryptococcus Neoformans ◽  
Immune Cells ◽  
Gene Expression Analysis ◽  
Yeast Cells ◽  
Content Type ◽  
Dormant Cells

ABSTRACTCryptococcosis is an opportunistic infection due to the ubiquitous yeastCryptococcus neoformans. This yeast interacts closely with innate immune cells, leading to various fates, including fungal persistence within cells, making possible the dissemination of the yeast cells with monocytes via a Trojan horse strategy. In humans, the natural history of the infection begins with primoinfection during childhood, which is followed by dormancy and, in some individuals, reactivation upon immunosuppression. To address the question of dormancy, we studiedC. neoformansinfection at the macrophage level (in vitroH99-macrophage interaction) and at the organ level in a murine model of cryptococcosis. We analyzed the diversity of yeast adaptation to the host by characterizing severalC. neoformanspopulations with new assays based on flow cytometry (quantitative flow cytometry, multispectral imaging flow cytometry, sorting), microscopy (dynamic imaging), and gene expression analysis. On the basis of parameters of multiplication and stress response, various populations of yeast cells were observed over timein vivoandin vitro. Cell sorting allowed the identification of a subpopulation that was less prone to grow under standard conditions than the other populations, with growth enhanced by the addition of serum. Gene expression analysis revealed that this population had specific metabolic characteristics that could reflect dormancy. Our data suggest that dormant yeast cells could existin vitroandin vivo.C. neoformansexhibits a huge plasticity and adaptation to hosts that deserves further study.In vitrogeneration of dormant cells is now the main challenge to overcome the limited number of yeast cells recovered in our models.IMPORTANCECryptococcus neoformansis a sugar-coated unicellular fungus that interacts closely with various cells and organisms, including amoebas, nematodes, and immune cells of mammals. This yeast is able to proliferate and survive in the intracellular environment.C. neoformanscauses cryptococcosis, and yeast dormancy in humans has been suggested on the basis of epidemiological evidence obtained years ago. By studying anin vitromodel of yeast-macrophage interaction and murine models of cryptococcosis, we observed that yeast cells evolve in heterogeneous populations during infection on the basis of global metabolic activity. We compared the growth ability and gene expression of yeast cells belonging to various populations in those two models. We eventually found a population of yeast cells with low metabolism that fit some of the criteria for dormant cells. This paves the way for further characterization of dormancy inC. neoformans.

scholarly journals Role of the ESCRT Pathway in Laccase Trafficking and Virulence of Cryptococcus neoformans

2020 ◽  
Vol 88 (7) ◽  
Author(s):  
Yoon-Dong Park ◽  
Shu Hui Chen ◽  
Emma Camacho ◽  
Arturo Casadevall ◽  
Peter R. Williamson
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Fluorescent Protein ◽  
Extracellular Vesicle ◽  
Multivesicular Bodies ◽  
Melanin Production ◽  
Content Type ◽  
Endosomal Sorting ◽  
Green Fluorescent

ABSTRACT The endosomal sorting complex required for transport (ESCRT) plays a crucial role in the transportation and degradation of proteins. We determined that Vps27, a key protein of the ESCRT-0 complex, is required for the transport of the virulence factor laccase to the cell wall in Cryptococcus neoformans. Laccase activity was perturbed, as was melanin production, in vps27Δ strains. In the absence of VPS27, there was an accumulation of multivesicular bodies with vacuolar fragmentation and mistargeting of the vacuolar carboxypeptidase CPY/Prc1, resulting in an extracellular localization. In addition, deletion of VPS27 resulted in a defect in laccase targeting of a Lac1-green fluorescent protein (GFP) fusion to the cell wall with trapping within intracellular puncta; this deletion was accompanied by reduced virulence in a mouse model. However, the actin cytoskeleton remained intact, suggesting that the trafficking defect is not due to defects in actin-related localization. Extracellular vesicle maturation was also defective in the vps27Δ mutant, which had a larger vesicle size as measured by dynamic light scattering. Our data identify cryptococcal VPS27 as a required gene for laccase trafficking and attenuates virulence of C. neoformans in a mouse intravenous (i.v.) meningitis model.

scholarly journals Berberine Antifungal Activity in Fluconazole-Resistant Pathogenic Yeasts: Action Mechanism Evaluated by Flow Cytometry and Biofilm Growth Inhibition in Candida spp.

2016 ◽  
Vol 60 (6) ◽  
pp. 3551-3557 ◽  
Author(s):  
Anderson Ramos da Silva ◽  
João Batista de Andrade Neto ◽  
Cecília Rocha da Silva ◽  
Rosana de Sousa Campos ◽  
Rose Anny Costa Silva ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Antifungal Activity ◽  
Cryptococcus Neoformans ◽  
Fungal Infections ◽  
Cell Activity ◽  
Candida Spp ◽  
Antifungal Effect ◽  
Content Type ◽  
Broth Microdilution Method ◽  
Fluconazole Resistant

The incidence of fungal infections and, in particular, the incidence of fungal antibiotic resistance, which is associated with biofilm formation, have significantly increased, contributing to morbidity and mortality. Thus, new therapeutic strategies need to be developed. In this context, natural products have emerged as a major source of possible antifungal agents. Berberine is a protoberberine-type isoquinoline alkaloid isolated from the roots, rhizomes, and stem bark of natural herbs, such asBerberis aquifolium,Berberis vulgaris,Berberis aristata, andHydrastis canadensis, and ofPhellodendron amurense. Berberine has been proven to have broad antibacterial and antifungal activity. In the present study, the potential antifungal effect of berberine against fluconazole-resistantCandidaandCryptococcus neoformansstrains, as well as against the biofilm form ofCandidaspp., was assessed. The antifungal effect of berberine was determined by a broth microdilution method (the M27-A3 method of the Clinical and Laboratory Standards Institute) and flow cytometry techniques, in which the probable mechanism of action of the compound was also assessed. For biofilm assessment, a colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine the susceptibility of sessile cells. The isolates used in the study belonged to the Laboratory of Bioprospection and Experiments in Yeast (LABEL) of the Federal University of Ceará. After 24 and 72 h, fluconazole-resistantCandidaandCryptococcus neoformansstrains showed berberine MICs equal to 8 μg/ml and 16 μg/ml, respectively. Cytometric analysis showed that treatment with berberine caused alterations to the integrity of the plasma and mitochondrial membranes and DNA damage, which led to cell death, probably by apoptosis. Assessment of biofilm-forming isolates after treatment showed statistically significant reductions in biofilm cell activity (P< 0.001).

scholarly journals Developmental Cell Fate and Virulence Are Linked to Trehalose Homeostasis in Cryptococcus neoformans

2014 ◽  
Vol 13 (9) ◽  
pp. 1158-1168 ◽  
Author(s):  
Michael R. Botts ◽  
Mingwei Huang ◽  
Regen K. Borchardt ◽  
Christina M. Hull
Keyword(s):  
Cryptococcus Neoformans ◽  
Cell Fate ◽  
Spore Germination ◽  
Molecular Genetic ◽  
Cell Types ◽  
Mammalian Host ◽  
Spore Viability ◽  
Content Type ◽  
Cell Fate Decisions ◽  
Metabolic Adaptations

ABSTRACTAmong pathogenic environmental fungi, spores are thought to be infectious particles that germinate in the host to cause disease. The meningoencephalitis-causing yeastCryptococcus neoformansis found ubiquitously in the environment and sporulates in response to nutrient limitation. While the yeast form has been studied extensively, relatively little is known about spore biogenesis, and spore germination has never been evaluated at the molecular level. Using genome transcript analysis of spores and molecular genetic approaches, we discovered that trehalose homeostasis plays a key role in regulating sporulation ofC. neoformans, is required for full spore viability, and influences virulence. Specifically, we found that genes involved in trehalose metabolism, including a previously uncharacterized secreted trehalase (NTH2), are highly overrepresented in dormant spores. Deletion of the two predicted trehalases in theC. neoformansgenome,NTH1andNTH2, resulted in severe defects in spore production, a decrease in spore germination, and an increase in the production of alternative developmental structures. This shift in cell types suggests that trehalose levels modulate cell fate decisions during sexual development. We also discovered that deletion of theNTH2trehalase results in hypervirulence in a murine model of infection. Taken together, these data show that the metabolic adaptations that allow this fungus to proliferate ubiquitously in the environment play unexpected roles in virulence in the mammalian host and highlight the complex interplay among the processes of metabolism, development, and pathogenesis.

scholarly journals Effect of Allium cepa on LAC1 gene expression and physiological activities in Cryptococcus neoformans

2021 ◽  
Author(s):  
Seyed Afzal Musavinasab-Mobarakeh ◽  
Masoomeh Shams-Ghahfarokhi ◽  
Mehdi Razzaghi-Abyaneh
Keyword(s):  
Gene Expression ◽  
Cell Membrane ◽  
Cryptococcus Neoformans ◽  
Allium Cepa ◽  
Laccase Activity ◽  
Inhibitory Concentration ◽  
Biological Activities ◽  
Antifungal Drugs ◽  
Melanin Production ◽  
Ergosterol Content

Background and Purpose: This study aimed to investigate the effects of Allium cepa ethanolic extract (EAC) on Cryptococcus neoformans biological activities and LAC1 gene expression. Materials and Methods: The minimum inhibitory concentration (MIC) of EAC was determined based on the Clinical and Laboratory Standards Institute M27-A4 method at a concentration range of 125- 4000 µg/ml. The EAC synergism activity was determined in combination with fluconazole (FCZ) as an antifungal azole. Laccase activity, melanin production, and cell membrane ergosterol content of C. neoformans were assessed at the 0.5× MIC concentration of EAC (1000 μg/ml) and FCZ (64μg/ml) by approved methods. The expression of the LAC1 gene was studied in the fungus exposed to 0.5× MIC concentration of EAC and FCZ using the real-time polymerase chain reaction. Results: Based on obtained results, MIC of EAC and FCZ were 2000 and 128 μg/ml,respectively. A combinatory effect was reported for FCZ and EAC by a fractional inhibitory concentration index of 0.25. The cell membrane ergosterol content was inhibited in EAC- and FCZ-treated C. neoformans by 58.25% and 49.85%, respectively.The laccase activity and melanin production were reduced in EAC-treated C. neoformans by 45.37% and 51.57%, and in FCZ-treated fungus by 54.64% and 53.68%, respectively.The expression of fungal LAC1 at messenger RNA (mRNA) level was measured 0.46 and 0.58 folds and significantly decreased in both EAC- and FCZ-treated C. neoformans at the 0.5×MIC concentration, respectively (p <0.05). Conclusion: The findings revealed that EAC contains inhibitory compounds which interact with biological activities in C. neoformans and thereby, it could be considered as a potential source for the development of novel antifungal drugs.

scholarly journals Cryptococcosis Serotypes Impact Outcome and Provide Evidence of Cryptococcus neoformans Speciation

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Marie Desnos-Ollivier ◽  
Sweta Patel ◽  
Dorothée Raoux-Barbot ◽  
Joseph Heitman ◽  
Françoise Dromer ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Clinical Data ◽  
Mating Type ◽  
Severe Disease ◽  
Nationwide Survey ◽  
High Serum ◽  
Content Type ◽  
Serotype A ◽  
Population Structures ◽  
Ad Hybrids

ABSTRACTCryptococcus neoformansis a human opportunistic fungal pathogen causing severe disseminated meningoencephalitis, mostly in patients with cellular immune defects. This species is divided into three serotypes: A, D, and the AD hybrid. Our objectives were to compare population structures of serotype A and D clinical isolates and to assess whether infections with AD hybrids differ from infections with the other serotypes. For this purpose, we analyzed 483 isolates and the corresponding clinical data from 234 patients enrolled during the CryptoA/D study or the nationwide survey on cryptococcosis in France. Isolates were characterized in terms of ploidy, serotype, mating type, and genotype, utilizing flow cytometry, serotype- and mating type-specific PCR amplifications, and multilocus sequence typing (MLST) methods. Our results suggest thatC. neoformansserotypes A and D have different routes of multiplication (primarily clonal expansion versus recombination events for serotype A and serotype D, respectively) and important genomic differences. Cryptococcosis includes a high proportion of proven or probable infections (21.5%) due to a mixture of genotypes, serotypes, and/or ploidies. Multivariate analysis showed that parameters independently associated with failure to achieve cerebrospinal fluid (CSF) sterilization by week 2 were a high serum antigen titer, the lack of flucytosine during induction therapy, and the occurrence of mixed infection, while infections caused by AD hybrids were more likely to be associated with CSF sterilization. Our study provides additional evidence for the possible speciation ofC. neoformansvar.neoformansandgrubiiand highlights the importance of careful characterization of causative isolates.IMPORTANCECryptococcus neoformansis an environmental fungus causing severe disease, estimated to be responsible for 600,000 deaths per year worldwide. This species is divided into serotypes A and D and an AD hybrid, and these could be considered two different species and an interspecies hybrid. The objectives of our study were to compare population structures of serotype A and serotype D and to assess whether infections with AD hybrids differ from infections with serotype A or D isolates in terms of clinical presentation and outcome. For this purpose, we used clinical data and strains from patients diagnosed with cryptococcosis in France. Our results suggest that, according to the serotype, isolates have different routes of multiplication and high genomic differences, confirming the possible speciation of serotypes A and D. Furthermore, we observed a better prognosis for infections caused by AD hybrid than those caused by serotype A or D, at least for those diagnosed in France.

scholarly journals The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in Cryptococcus neoformans

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Brian T. Maybruck ◽  
Woei C. Lam ◽  
Charles A. Specht ◽  
Ma. Xenia G. Ilagan ◽  
Maureen J. Donlin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Wall ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Cryptococcus Neoformans ◽  
Cannabinoid Receptors ◽  
Biological Activities ◽  
Screening Method ◽  
Mammalian Host ◽  
Content Type

ABSTRACT Cryptococcus neoformans can cause fatal meningoencephalitis in patients with AIDS or other immunocompromising conditions. Current antifungals are suboptimal to treat this disease; therefore, novel targets and new therapies are needed. Previously, we have shown that chitosan is a critical component of the cryptococcal cell wall and is required for survival in the mammalian host and that chitosan deficiency results in rapid clearance from the mammalian host. We had also identified several specific proteins that were required for chitosan biosynthesis, and we hypothesize that screening for compounds that inhibit chitosan biosynthesis would identify additional genes/proteins that influence chitosan biosynthesis. To identify these compounds, we developed a robust and novel cell-based flow cytometry screening method to identify small-molecule inhibitors of chitosan production. We screened the ICCB Known Bioactives library and identified 8 compounds that reduced chitosan in C. neoformans. We used flow cytometry-based counterscreens and confirmatory screens, followed by a biochemical secondary screen to refine our primary screening hits to 2 confirmed hits. One of the confirmed hits that reduced chitosan content was the aminoalkylindole BML-190, a known inverse agonist of mammalian cannabinoid receptors. We demonstrated that BML-190 likely targets the C. neoformans G-protein-coupled receptor Gpr4 and, via the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, contributes to an intracellular accumulation of cAMP that results in decreased chitosan. Our discovery suggests that this approach could be used to identify additional compounds and pathways that reduce chitosan biosynthesis and could lead to potential novel therapeutics against C. neoformans. IMPORTANCE Cryptococcus neoformans is a fungal pathogen that kills ∼200,000 people every year. The cell wall is an essential organelle that protects fungi from the environment. Chitosan, the deacetylated form of chitin, has been shown to be an essential component of the cryptococcal cell wall during infection of a mammalian host. In this study, we screened a set of 480 compounds, which are known to have defined biological activities, for activity that reduced chitosan production in C. neoformans. Two of these compounds were confirmed using an alternative method of measuring chitosan, and one of these was demonstrated to impact the cAMP signal transduction pathway. This work demonstrates that the cAMP pathway regulates chitosan biosynthesis in C. neoformans and validates that this screening approach could be used to find potential antifungal agents.

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