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Secreted Acb1 Contributes to the Yeast-to-Hypha Transition in Cryptococcus neoformans.

ABSTRACT: Adaptation to stress by eukaryotic pathogens is often accompanied by a transition in cellular morphology. The human fungal pathogen Cryptococcus neoformans is known to switch between the yeast and the filamentous form in response to amoebic predation or during mating. As in the classic dimorphic fungal pathogens, the morphotype is associated with the ability of cryptococci to infect various hosts. Many cryptococcal factors and environmental stimuli, including pheromones (small peptides) and nutrient limitation, are known to induce the yeast-to-hypha transition. We recently discovered that secreted matricellular proteins could also act as intercellular signals to promote the yeast-to-hypha transition. Here we show that the secreted acyl coenzyme A (acyl-CoA)-binding protein Acb1 plays an important role in enhancing this morphotype transition. Acb1 does not possess a signal peptide. Its extracellular secretion and, consequently, its function in filamentation are dependent on an unconventional GRASP (Golgi reassembly stacking protein)-dependent secretion pathway. Surprisingly, intracellular recruitment of Acb1 to the secretory vesicles is independent of Grasp. In addition to Acb1, Grasp possibly controls the secretion of other cargos, because the grasp? mutant, but not the acb1? mutant, is defective in capsule production and macrophage phagocytosis. Nonetheless, Acb1 is likely the major or the sole effector of Grasp in terms of filamentation. Furthermore, we found that the key residue of Acb1 for acyl binding, Y80, is critical for the proper subcellular localization and secretion of Acb1 and for cryptococcal morphogenesis.

SUBMITTER: Xu X

PROVIDER: S-EPMC4751841 | biostudies-other | 2015 Dec

REPOSITORIES: biostudies-other

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Secreted Acb1 Contributes to the Yeast-to-Hypha Transition in Cryptococcus neoformans.

Xu Xinping X Zhao Youbao Y Kirkman Elyssa E Lin Xiaorong X

Applied and environmental microbiology 20151204 4

Adaptation to stress by eukaryotic pathogens is often accompanied by a transition in cellular morphology. The human fungal pathogen Cryptococcus neoformans is known to switch between the yeast and the filamentous form in response to amoebic predation or during mating. As in the classic dimorphic fungal pathogens, the morphotype is associated with the ability of cryptococci to infect various hosts. Many cryptococcal factors and environmental stimuli, including pheromones (small peptides) and nutr ...[more]

PMID: 26637591

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Project description:Cryptococcus neoformans is a major opportunistic fungal pathogen. Like many dimorphic fungal pathogens, C. neoformans can undergo morphological transition from the yeast form to the hypha form, and its morphotype is tightly linked to its virulence. Although some genetic factors controlling morphogenesis have been identified, little is known about the epigenetic regulation in this process. Proteins with the plant homeodomain (PHD) finger, a structurally conserved domain in eukaryotes, were first identified in plants and are known to be involved in reading and effecting chromatin modification. Here, we investigated the role of the PHD finger family genes in Cryptococcus mating and yeast-hypha transition. We found 16 PHD finger domains distributed among 15 genes in the Cryptococcus genome, with two genes, ZNF1? and RUM1?, located in the mating type locus. We deleted these 15 PHD genes and examined the impact of their disruption on cryptococcal morphogenesis. The deletion of five PHD finger genes dramatically affected filamentation. The rum1?? and znf1?? mutants showed enhanced ability to initiate filamentation but impaired ability to maintain filamentous growth. The bye1? and the phd11? mutants exhibited enhanced filamentation, while the set302? mutants displayed reduced filamentation. Ectopic overexpression of these five genes in the corresponding null mutants partially or completely restored the defect in filamentation. Furthermore, we demonstrated that Phd11, a suppressor of filamentation, regulates the yeast-hypha transition through the known master regulator Znf2. The findings indicate the importance of epigenetic regulation in controlling dimorphic transition in C. neoformansIMPORTANCE Morphotype is known to have a profound impact on cryptococcal interaction with various hosts, including mammalian hosts. The yeast form of Cryptococcus neoformans is considered the virulent form, while its hyphal form is attenuated in mammalian models of cryptococcosis. Although some genetic regulators critical for cryptococcal morphogenesis have been identified, little is known about epigenetic regulation in this process. Given that plant homeodomain (PHD) finger proteins are involved in reading and effecting chromatin modification and their functions are unexplored in C. neoformans, we investigated the roles of the 15 PHD finger genes in Cryptococcus mating and yeast-hypha transition. Five of them profoundly affect filamentation as either a suppressor or an activator. Phd11, a suppressor of filamentation, regulates this process via Znf2, a known master regulator of morphogenesis. Thus, epigenetic regulation, coupled with genetic regulation, controls this yeast-hypha transition event.

Project description:Kinetochores facilitate interaction between chromosomes and the spindle apparatus. The formation of a metazoan trilayered kinetochore is an ordered event in which inner, middle, and outer layers assemble during disassembly of the nuclear envelope during mitosis. The existence of a similar strong correlation between kinetochore assembly and nuclear envelope breakdown in unicellular eukaryotes is unclear. Studies in the hemiascomycetous budding yeasts Saccharomyces cerevisiae and Candida albicans suggest that an ordered kinetochore assembly may not be evolutionarily conserved. Here, we utilized high-resolution time-lapse microscopy to analyze the localization patterns of a series of putative kinetochore proteins in the basidiomycetous budding yeast Cryptococcus neoformans, a human pathogen. Strikingly, similar to most metazoa but atypical of yeasts, the centromeres are not clustered but positioned adjacent to the nuclear envelope in premitotic C. neoformans cells. The centromeres gradually coalesce to a single cluster as cells progress toward mitosis. The mitotic clustering of centromeres seems to be dependent on the integrity of the mitotic spindle. To study the dynamics of the nuclear envelope, we followed the localization of two marker proteins, Ndc1 and Nup107. Fluorescence microscopy of the nuclear envelope and components of the kinetochore, along with ultrastructure analysis by transmission electron microscopy, reveal that in C. neoformans, the kinetochore assembles in an ordered manner prior to mitosis in concert with a partial opening of the nuclear envelope. Taken together, the results of this study demonstrate that kinetochore dynamics in C. neoformans is reminiscent of that of metazoans and shed new light on the evolution of mitosis in eukaryotes.Successful propagation of genetic material in progeny is essential for the survival of any organism. A proper kinetochore-microtubule interaction is crucial for high-fidelity chromosome segregation. An error in this process can lead to loss or gain of chromosomes, a common feature of most solid cancers. Several proteins assemble on centromere DNA to form a kinetochore. However, significant differences in the process of kinetochore assembly exist between unicellular yeasts and multicellular metaozoa. Here, we examined the key events that lead to formation of a proper kinetochore in a basidiomycetous budding yeast, Cryptococcus neoformans. We found that, during the progression of the cell cycle, nonclustered centromeres gradually clustered and kinetochores assembled in an ordered manner concomitant with partial opening of the nuclear envelope in this organism. These events have higher similarity to mitotic events of metazoans than to those previously described in other yeasts.

Project description:UNLABELLED:Cryptococcus 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 (mpr1?) failed to breach the endothelium in an in vitro model of the human blood-brain barrier (BBB). A mammalian host infected with the mpr1? 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. IMPORTANCE:Cryptococcus 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.

Project description:UnlabelledCryptococcus neoformans strains isolated from patients with AIDS secrete acid phosphatase, but the identity and role of the enzyme(s) responsible have not been elucidated. By combining a one-dimensional electrophoresis step with mass spectrometry, a canonically secreted acid phosphatase, CNAG_02944 (Aph1), was identified in the secretome of the highly virulent serotype A strain H99. We created an APH1 deletion mutant (?aph1) and showed that ?aph1-infected Galleria mellonella and mice survived longer than those infected with the wild type (WT), demonstrating that Aph1 contributes to cryptococcal virulence. Phosphate starvation induced APH1 expression and secretion of catalytically active acid phosphatase in the WT, but not in the ?aph1 mutant, indicating that Aph1 is the major extracellular acid phosphatase in C. neoformans and that it is phosphate repressible. DsRed-tagged Aph1 was transported to the fungal cell periphery and vacuoles via endosome-like structures and was enriched in bud necks. A similar pattern of Aph1 localization was observed in cryptococci cocultured with THP-1 monocytes, suggesting that Aph1 is produced during host infection. In contrast to Aph1, but consistent with our previous biochemical data, green fluorescent protein (GFP)-tagged phospholipase B1 (Plb1) was predominantly localized at the cell periphery, with no evidence of endosome-mediated export. Despite use of different intracellular transport routes by Plb1 and Aph1, secretion of both proteins was compromised in a ?sec14-1 mutant. Secretions from the WT, but not from ?aph1, hydrolyzed a range of physiological substrates, including phosphotyrosine, glucose-1-phosphate, ?-glycerol phosphate, AMP, and mannose-6-phosphate, suggesting that the role of Aph1 is to recycle phosphate from macromolecules in cryptococcal vacuoles and to scavenge phosphate from the extracellular environment.ImportanceInfections with the AIDS-related fungal pathogen Cryptococcus neoformans cause more than 600,000 deaths per year worldwide. Strains of Cryptococcus neoformans isolated from patients with AIDS secrete acid phosphatase; however, the identity and role of the enzyme(s) are unknown. We have analyzed the secretome of the highly virulent serotype A strain H99 and identified Aph1, a canonically secreted acid phosphatase. By creating an APH1 deletion mutant and an Aph1-DsRed-expressing strain, we demonstrate that Aph1 is the major extracellular and vacuolar acid phosphatase in C. neoformans and that it is phosphate repressible. Furthermore, we show that Aph1 is produced in cryptococci during coculture with THP-1 monocytes and contributes to fungal virulence in Galleria mellonella and mouse models of cryptococcosis. Our findings suggest that Aph1 is secreted to the environment to scavenge phosphate from a wide range of physiological substrates and is targeted to vacuoles to recycle phosphate from the expendable macromolecules.

Dataset Information

Secreted Acb1 Contributes to the Yeast-to-Hypha Transition in Cryptococcus neoformans.

Publications

Secreted Acb1 Contributes to the Yeast-to-Hypha Transition in Cryptococcus neoformans.

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