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Pseudohyphal growth of Cryptococcus neoformans is a reversible dimorphic transition in response to ammonium that requires Amt1 and Amt2 ammonium permeases.

ABSTRACT: Cryptococcus neoformans is a human-pathogenic basidiomycete that commonly infects HIV/AIDS patients to cause meningoencephalitis (7, 19). C. neoformans grows as a budding yeast during vegetative growth or as hyphae during sexual reproduction. Pseudohyphal growth of C. neoformans has been observed rarely during murine and human infections but frequently during coculture with amoeba; however, the genetics underlying pseudohyphal growth are largely unknown. Our studies found that C. neoformans displays pseudohyphal growth under nitrogen-limiting conditions, especially when a small amount of ammonium is available as a sole nitrogen source. Pseudohyphal growth was observed with Cryptococcus neoformans serotypes A and D and Cryptococcus gattii. C. neoformans pseudohyphae bud to produce yeast cells and normal smooth hemispherical colonies when transferred to complete media, indicating that pseudohyphal growth is a conditional developmental stage. Subsequent analysis revealed that two ammonium permeases encoded by the AMT1 and AMT2 genes are required for pseudohyphal growth. Both amt1 and amt2 mutants are capable of forming pseudohyphae; however, amt1 amt2 double mutants do not form pseudohyphae. Interestingly, C. gattii pseudohypha formation is irreversible and involves a RAM pathway mutation that drives pseudohyphal development. We also found that pseudohyphal growth is related to the invasive growth into the medium. These results demonstrate that pseudohyphal growth is a common reversible growth pattern in C. neoformans but a mutational genetic event in C. gattii and provide new insights into understanding pseudohyphal growth of Cryptococcus.

SUBMITTER: Lee SC

PROVIDER: S-EPMC3486016 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Pseudohyphal growth of Cryptococcus neoformans is a reversible dimorphic transition in response to ammonium that requires Amt1 and Amt2 ammonium permeases.

Lee Soo Chan SC Phadke Sujal S Sun Sheng S Heitman Joseph J

Eukaryotic cell 20120921 11

Cryptococcus neoformans is a human-pathogenic basidiomycete that commonly infects HIV/AIDS patients to cause meningoencephalitis (7, 19). C. neoformans grows as a budding yeast during vegetative growth or as hyphae during sexual reproduction. Pseudohyphal growth of C. neoformans has been observed rarely during murine and human infections but frequently during coculture with amoeba; however, the genetics underlying pseudohyphal growth are largely unknown. Our studies found that C. neoformans disp ...[more]

PMID: 23002105

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Project description:Fungal opportunistic pathogens colonize various environments, from plants and wood to human and animal tissue. Regarding human pathogens, one great challenge during contrasting niche occupation is the adaptation to different conditions, such as temperature, osmolarity, salinity, pressure, oxidative stress and nutritional availability, which may constitute sources of stress that need to be tolerated and overcome. As an opportunistic pathogen, C. neoformans faces exactly these situations during the transition from the environment to the human host, encountering nutritional constraints. Our previous and current research on amino acid biosynthetic pathways indicates that amino acid permeases are regulated by the presence of the amino acids, nitrogen and temperature. Saccharomyces cerevisiae and Candida albicans have twenty-four and twenty-seven genes encoding amino acid permeases, respectively; conversely, they are scarce in number in Basidiomycetes (C. neoformans, Coprinopsis cinerea and Ustilago maydis), where nine to ten permease genes can be found depending on the species. In this study, we have demonstrated that two amino acid permeases are essential for virulence in C. neoformans. Our data showed that C. neoformans uses two global and redundant amino acid permeases, Aap4 and Aap5 to respond correctly to thermal and oxidative stress. Double deletion of these permeases causes growth arrest in C. neoformans at 37°C and in the presence of hydrogen peroxide. The inability to uptake amino acid at a higher temperature and under oxidative stress also led to virulence attenuation in vivo. Our data showed that thermosensitivity caused by the lack of permeases Aap4 and Aap5 can be remedied by alkaline conditions (higher pH) and salinity. Permeases Aap4 and Aap5 are also required during fluconazole stress and they are the target of the plant secondary metabolite eugenol, a potent antifungal inhibitor that targets amino acid permeases. In summary, our work unravels (i) interesting physiological property of C. neoformans regarding its amino acid uptake system; (ii) an important aspect of virulence, which is the need for amino acid permeases during thermal and oxidative stress resistance and, hence, host invasion and colonization; and (iii) provides a convenient prototype for antifungal development, which are the amino acid permeases Aap4/Aap5 and their inhibitor.

Project description:Morphotype switch is a cellular response to external and internal cues. The Cryptococcus neoformans species complex can undergo morphological transitions between the yeast and the hypha form, and such morphological changes profoundly affect cryptococcal interaction with various hosts. Filamentation in Cryptococcus was historically considered a mating response towards pheromone. Recent studies indicate the existence of pheromone-independent signaling pathways but their identity or the effectors remain unknown. Here, we demonstrated that glucosamine stimulated the C. neoformans species complex to undergo self-filamentation. Glucosamine-stimulated filamentation was independent of the key components of the pheromone pathway, which is distinct from pheromone-elicited filamentation. Glucosamine stimulated self-filamentation in H99, a highly virulent serotype A clinical isolate and a widely used reference strain. Through a genetic screen of the deletion sets made in the H99 background, we found that Crz1, a transcription factor downstream of calcineurin, was essential for glucosamine-stimulated filamentation despite its dispensability for pheromone-mediated filamentation. Glucosamine promoted Crz1 translocation from the cytoplasm to the nucleus. Interestingly, multiple components of the high osmolality glycerol response (HOG) pathway, consisting of the phosphorelay system and some of the Hog1 MAPK module, acted as repressors of glucosamine-elicited filamentation through their calcineurin-opposing effect on Crz1's nuclear translocation. Surprisingly, glucosamine-stimulated filamentation did not require Hog1 itself and was distinct from the conventional general stress response. The results demonstrate that Cryptococcus can resort to multiple genetic pathways for morphological transition in response to different stimuli. Given that the filamentous form attenuates cryptococcal virulence and is immune-stimulatory in mammalian models, the findings suggest that morphogenesis is a fertile ground for future investigation into novel means to compromise cryptococcal pathogenesis.

Project description:UnlabelledCryptococcus spp. cause life-threatening fungal infection of the central nervous system (CNS), predominantly in patients with a compromised immune system. Why Cryptococcus neoformans has this remarkable tropism for the CNS is not clear. Recent research on cerebral pathogenesis of C. neoformans revealed a predominantly transcellular migration of cryptococci across the brain endothelium; however, the identities of key fungal virulence factors that function specifically to invade the CNS remain unresolved. Here we found that a novel, secreted metalloprotease (Mpr1) that we identified in the extracellular proteome of C. neoformans (CnMpr1) is required for establishing fungal disease in the CNS. Mpr1 belongs to a poorly characterized M36 class of fungalysins that are expressed in only some fungal species. A strain of C. neoformans lacking the gene encoding Mpr1 (mpr1Δ) failed to breach the endothelium in an in vitro model of the human blood-brain barrier (BBB). A mammalian host infected with the mpr1Δ null strain demonstrated significant improvement in survival due to a reduced brain fungal burden and lacked the brain pathology commonly associated with cryptococcal disease. The in vivo studies further indicate that Mpr1 is not required for fungal dissemination and Mpr1 likely targets the brain endothelium specifically. Remarkably, the sole expression of CnMPR1 in Saccharomyces cerevisiae resulted in a robust migration of yeast cells across the brain endothelium, demonstrating Mpr1's specific activity in breaching the BBB and suggesting that Mpr1 may function independently of the hyaluronic acid-CD44 pathway. This distinct role for Mpr1 may develop into innovative treatment options and facilitate a brain-specific drug delivery platform.ImportanceCryptococcus neoformans is a medically relevant fungal pathogen causing significant morbidity and mortality, particularly in immunocompromised individuals. An intriguing feature is its strong neurotropism, and consequently the hallmark of cryptococcal disease is a brain infection, cryptococcal meningoencephalitis. For C. neoformans to penetrate the central nervous system (CNS), it first breaches the blood-brain barrier via a transcellular pathway; however, the identities of fungal factors required for this transmigration remain largely unknown. In an effort to identify extracellular fungal proteins that could mediate interactions with the brain endothelium, we undertook a proteomic analysis of the extracellular proteome and identified a secreted metalloprotease (Mpr1) belonging to the M36 class of fungalysins. Here we found that Mpr1 promotes migration of C. neoformans across the brain endothelium and into the CNS by facilitating attachment of cryptococci to the endothelium surface, thus underscoring the critical role of M36 proteases in fungal pathogenesis.

Dataset Information

Pseudohyphal growth of Cryptococcus neoformans is a reversible dimorphic transition in response to ammonium that requires Amt1 and Amt2 ammonium permeases.

Publications

Pseudohyphal growth of Cryptococcus neoformans is a reversible dimorphic transition in response to ammonium that requires Amt1 and Amt2 ammonium permeases.

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