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Trehalose-6-phosphate synthase (TPS1) was identified as a virulence factor for Cryptococcus neoformans and a promising therapeutic target. This study reveals previously unknown roles of TPS1 in evasion of host defenses during pulmonary and disseminated phases of infection. In the pulmonary infection model, TPS1-deleted ( tps1Δ ) Cryptococci are rapidly cleared by mouse lungs whereas TPS1-sufficent WT (H99) and revertant ( tps1Δ : TPS1 ) strains expand in the lungs and disseminate, causing 100% mortality. Rapid pulmonary clearance of tps1Δ mutant is T-cell independent and relies on its susceptibility to lung resident factors and innate immune factors, exemplified by tps1Δ but not H99 inhibition in a coculture with dispersed lung cells and its rapid clearance coinciding with innate leukocyte infiltration. In the disseminated model of infection, which bypasses initial lung-fungus interactions, tps1Δ strain remains highly attenuated. Specifically, tps1Δ mutant is unable to colonize the lungs from the bloodstream or expand in spleens but is capable of crossing into the brain, where it remains controlled even in the absence of T cells. In contrast, strains H99 and tps1Δ:TPS1 rapidly expand in all studied organs, leading to rapid death of the infected mice. Since the rapid pulmonary clearance of tps1Δ mutant resembles a response to acapsular strains, the effect of tps1 deletion on capsule formation in vitro and in vivo was examined. Tps1Δ cryptococci form capsules but with a substantially reduced size. In conclusion, TPS1 is an important virulence factor, allowing C. neoformans evasion of resident pulmonary and innate defense mechanisms, most likely via its role in cryptococcal capsule formation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Goughenour, Creech, Xu, He, Hissong, Giamberardino, Tenor, Toffaletti, Perfect and Olszewski.)

Quorum sensing (QS) is a bacterial communication mechanism in which secreted signaling molecules impact population function and gene expression. QS-like phenomena have been reported in eukaryotes with largely unknown contributing molecules, functions, and mechanisms. We identify Qsp1, a secreted peptide, as a central signaling molecule that regulates virulence in the fungal pathogen Cryptococcus neoformans. QSP1 is a direct target of three transcription factors required for virulence, and qsp1Δ mutants exhibit attenuated infection, slowed tissue accumulation, and greater control by primary macrophages. Qsp1 mediates autoregulatory signaling that modulates secreted protease activity and promotes cell wall function at high cell densities. Peptide production requires release from a secreted precursor, proQsp1, by a cell-associated protease, Pqp1. Qsp1 sensing requires an oligopeptide transporter, Opt1, and remarkably, cytoplasmic expression of mature Qsp1 complements multiple phenotypes of qsp1Δ. Thus, C. neoformans produces an autoregulatory peptide that matures extracellularly but functions intracellularly to regulate virulence., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The trehalose pathway is essential for stress tolerance and virulence in fungi. We investigated the importance of this pathway for virulence of the pathogenic yeast Cryptococcus gattii using the highly virulent Vancouver Island, Canada, outbreak strain R265. Three genes putatively involved in trehalose biosynthesis, TPS1 (trehalose-6-phosphate [T6P] synthase) and TPS2 (T6P phosphatase), and degradation, NTH1 (neutral trehalose), were deleted in this strain, creating the R265tps1 Delta, R265tps2 Delta, and R265nth1 Delta mutants. As in Cryptococcus neoformans, cellular trehalose was reduced in the R265tps1 Delta and R265tps2 Delta mutants, which could not grow and died, respectively, at 37 degrees C on yeast extract-peptone-dextrose agar, suggesting that T6P accumulation in R265tps2 Delta is directly toxic. Characterizations of the cryptococcal hexokinases and trehalose mutants support their linkage to the control of glycolysis in this species. However, unlike C. neoformans, the C. gattii R265tps1 Delta mutant demonstrated, in addition, defects in melanin and capsule production, supporting an influence of T6P on these virulence pathways. Attenuated virulence of the R265tps1 Delta mutant was not due solely to its 37 degrees C growth defect, as shown in worm studies and confirmed by suppressor mutants. Furthermore, an intact trehalose pathway controls protein secretion, mating, and cell wall integrity in C. gattii. Thus, the trehalose synthesis pathway plays a central role in the virulence composites of C. gattii through multiple mechanisms. Deletion of NTH1 had no effect on virulence, but inactivation of the synthesis genes, TPS1 and TPS2, has profound effects on survival of C. gattii in the invertebrate and mammalian hosts. These results highlight the central importance of this pathway in the virulence composites of both pathogenic cryptococcal species.

The disaccharide trehalose has been found to play diverse roles, from energy source to stress protectant, and this sugar is found in organisms as diverse as bacteria, fungi, plants, and invertebrates but not in mammals. Recent studies in the pathobiology of Cryptococcus neoformans identified the presence of a functioning trehalose pathway during infection and suggested its importance for C. neoformans survival in the host. Therefore, in C. neoformans we created null mutants of the trehalose-6-phosphate (T6P) synthase (TPS1), trehalose-6-phophate phosphatase (TPS2), and neutral trehalase (NTH1) genes. We found that both TPS1 and TPS2 are required for high-temperature (37 degrees C) growth and glycolysis but that the block at TPS2 results in the apparent toxic accumulation of T6P, which makes this enzyme a fungicidal target. Sorbitol suppresses the growth defect in the tps1 and tps2 mutants at 37 degrees C, which supports the hypothesis that these sugars (trehalose and sorbitol) act primarily as stress protectants for proteins and membranes during exposure to high temperatures in C. neoformans. The essential nature of this pathway for disease was confirmed when a tps1 mutant strain was found to be avirulent in both rabbits and mice. Furthermore, in the system of the invertebrate C. elegans, in which high in vivo temperature is no longer an environmental factor, attenuation in virulence was still noted with the tps1 mutant, and this supports the hypothesis that the trehalose pathway in C. neoformans is involved in more host survival mechanisms than simply high-temperature stresses and glycolysis. These studies in C. neoformans and previous studies in other pathogenic fungi support the view of the trehalose pathway as a selective fungicidal target for use in antifungal development.

Topisomerase I is the target of several toxins and chemotherapy agents, and the enzyme is essential for viability in some organisms, including mice and drosophila. We have cloned the TOP1 gene encoding topoisomerase I from the opportunistic fungal pathogen Cryptococcus neoformans. The C. neoformans topoisomerase I contains a fungal insert also found in topoisomerase I from Candida albicans and Saccharomyces cerevisiae that is not present in the mammalian enzyme. We were unable to disrupt the topoisomerase I gene in this haploid organism by homologous recombination in over 8000 transformants analyzed. When a second functional copy of the TOP1 gene was introduced into the genome, the topoisomerase I gene could be readily disrupted by homologous recombination (at 7% efficiency). Thus, topoisomerase I is essential in C. neoformans. This new molecular strategy with C. neoformans may also be useful in identifying essential genes in other pathogenic fungi. To address the physiological and pathobiological functions of the enzyme, the TOP1 gene was fused to the GAL7 gene promoter. The resulting GAL7::TOP1 fusion gene was modestly regulated by carbon source in a serotype A strain of C. neoformans. Modest overexpression of topoisomerase I conferred sensitivity to heat shock, gamma-rays, and camptothecin. In contrast, alterations in topoisomerase I levels had no effect on the toxicity of a novel class of antifungal agents, the dicationic aromatic compounds (DACs), indicating that topoisomerase I is not the target of DACs. In an animal model of cryptococcal meningitis, topoisomerase I regulation was not critically important to established infection, but may impact on the initial stress response to infection. In summary, our studies reveal that topoisomerase I is essential in the human pathogen C. neoformans and represents a novel target for antifungal agents.

Cryptococcal meningitis is a fungal infection, caused by Cryptococcus neoformans, which is prevalent in immunocompromised patient populations. Treatment failures of this disease are emerging in the clinic, usually associated with long-term treatment with existing antifungal agents. The fungal cell wall is an attractive target for drug therapy because the syntheses of cell wall glucan and chitin are processes that are absent in mammalian cells. Echinocandins comprise a class of lipopeptide compounds known to inhibit 1,3-beta-glucan synthesis, and at least two compounds belonging to this class are currently in clinical trials as therapy for life-threatening fungal infections. Studies of Saccharomyces cerevisiae and Candida albicans mutants identify the membrane-spanning subunit of glucan synthase, encoded by the FKS genes, as the molecular target of echinocandins. In vitro, the echinocandins show potent antifungal activity against Candida and Aspergillus species but are much less potent against C. neoformans. In order to examine why C. neoformans cells are less susceptible to echinocandin treatment, we have cloned a homolog of S. cerevisiae FKS1 from C. neoformans. We have developed a generalized method to evaluate the essentiality of genes in Cryptococcus and applied it to the FKS1 gene. The method relies on homologous integrative transformation with a plasmid that can integrate in two orientations, only one of which will disrupt the target gene function. The results of this analysis suggest that the C. neoformans FKS1 gene is essential for viability. The C. neoformans FKS1 sequence is closely related to the FKS1 sequences from other fungal species and appears to be single copy in C. neoformans. Furthermore, amino acid residues known to be critical for echinocandin susceptibility in Saccharomyces are conserved in the C. neoformans FKS1 sequence.

Comparative studies of 5-aminoimidazole ribonucleotide (AIR) carboxylases from Escherichia coli and Gallus gallus have identified this central step in de novo purine biosynthesis as a case for unusual divergence in primary metabolism. Recent discoveries establish the fungal AIR carboxylase, encoded by the ADE2 gene, as essential for virulence in certain pathogenic organisms. This investigation is a biochemical analysis that links the fungal ADE2 protein to the function of the E. coli AIR carboxylase system. A cDNA clone of ADE2 from Cryptococcus neoformans was isolated by genetic complementation of a purE-deficient strain of E. coli. High-level expression of the C. neoformans ADE2 was achieved, which enabled the production and purification of AIR carboxylase. Amino acid sequence alignments, C-terminal deletion mutants, and biochemical assays indicate that the ADE2 enzyme is a two-domain, bifunctional protein. The N-terminal domain is related to E. coli PurK and a series of kinetic experiments show that the ADE2-PurK activity uses AIR, ATP, and HCO3- as substrates. The biosynthetic product of the ADE2-PurK reaction was identified as N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) by 1H NMR, thus confirming that the C-terminal domain contains a catalytic activity similar to that of the E. coli PurE. By using an in situ system for substrate production, the steady-state kinetic constants for turnover of N5-CAIR by ADE2 were determined and together with stoichiometry measurements, these data indicate that ADE2 has a balance in the respective catalytic turnovers to ensure efficient flux. Distinctive features of the PurE active site were probed using 4-nitro-5-aminoimidazole ribonucleotide (NAIR), an analog of the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). NAIR was shown to be a selective inhibitor of the ADE2-PurE activity (K1 = 2.4 microM), whereas it is a slow-binding inhibitor of the G. gallus enzyme which further distinguishes the fungal ADE2 from the G. gallus AIR carboxylase. As such, this enzyme represents a novel intracellular target for the discovery of antifungal agents.

An expression plasmid carrying a heterologous gene fusion between the Cryptococcus neoformans actin promoter and the Escherichia coli reporter gene, LACZ, was constructed to study actin regulation in C. neoformans. Two randomly stable transformants, designated 20.6 and 20.9, were selected for further examination. Both ectopic and homologous recombination with vector insertion in tandem repeats occurred in these transformants. Transformant 20.9 carried more copies of ACTp::LACZ in its genome than 20.6 and this was reflected in expressing higher levels of beta-galactosidase activity. In vitro, these transformants showed higher levels of beta-galactosidase activity expressed when the transformants were propagated at higher temperatures (37 degrees C vs 30 degrees C). However, beta-galactosidase expression in the transformants was variable during logarithmic and stationary growth phases and this differential expression was temperature dependent. This report shows that the constitutive actin gene in C. neoformans is regulated by temperature and growth and this fact should be taken into consideration when actin expression is used as a standard to compare the expression of other regulated genes. Also, a more sensitive reporter construct will be needed for in vivo gene analysis of regulation.

Cyclosporin A (CsA) and FK506 are antimicrobial, immunosuppressive natural products that inhibit signal transduction. In T cells and Saccharomyces cerevisiae, CsA and FK506 bind to the immunophilins cyclophilin A and FKBP12 and the resulting complexes inhibit the Ca2+-regulated protein phosphatase calcineurin. We find that growth of the opportunistic fungal pathogen Cryptococcus neoformans is sensitive to CsA and FK506 at 37 degrees C but not at 24 degrees C, suggesting that CsA and FK506 inhibit a protein required for C. neoformans growth at elevated temperature. Genetic evidence supports a model in which immunophilin-drug complexes inhibit calcineurin to prevent growth at 37 degrees C. The gene encoding the C. neoformans calcineurin A catalytic subunit was cloned and disrupted by homologous recombination. Calcineurin mutant strains are viable but do not survive in vitro conditions that mimic the host environment (elevated temperature, 5% CO2 or alkaline pH) and are no longer pathogenic in an animal model of cryptococcal meningitis. Introduction of the wild-type calcineurin A gene complemented these growth defects and restored virulence. Our findings demonstrate that calcineurin is required for C. neoformans virulence and may define signal transduction elements required for fungal pathogenesis that could be targets for therapeutic intervention.