Importance of a developmentally regulated pheromone receptor of Cryptococcus neoformans for virulence.
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ABSTRACT:
SUBMITTER: Chang YC
PROVIDER: S-EPMC187348 | biostudies-literature | 2003 Sep
REPOSITORIES: biostudies-literature
Publications
Importance of a developmentally regulated pheromone receptor of Cryptococcus neoformans for virulence.
Chang Yun C YC Miller Georgina F GF Kwon-Chung K J KJ
Infection and immunity 20030901 9
Cryptococcus neoformans is the etiologic agent of cryptococcosis. Two mating types exist in this fungus, MAT alpha and MATa. The CPRa gene of C. neoformans is a MATa strain-specific gene and encodes a putative seven-transmembrane domain pheromone receptor. Unlike the other reported fungal pheromone receptors, CPRa shows functional diversity. Deletion of CPRa drastically affects mating efficiency but does not abolish mating. CPRa expression is developmentally regulated and is not affected by dele ...[more]
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Project description:Sexual reproduction in fungi requires induction of signaling pheromones within environments that are conducive to mating. The fungus Cryptococcus neoformans is currently the fourth greatest cause of infectious death in regions of Africa and undergoes mating in phytonutrient-rich environments to create spores with infectious potential. Here we show that under conditions where sexual development is inhibited, a ?17-fold excess of MF? pheromone transcript is synthesized and then degraded by a DEAD box protein, Vad1, resulting in low steady state transcript levels. Transfer to mating medium or deletion of the VAD1 gene resulted in high level accumulation of MF? transcripts and enhanced mating, acting in concert with the mating-related HOG1 pathway. We then investigated whether the high metabolic cost of this apparently futile transcriptional cycle could be justified by a more rapid induction of mating. Maintenance of Vad1 activity on inductive mating medium by constitutive expression resulted in repressed levels of MF? that did not prevent but rather prolonged the time to successful mating from 5-6 h to 15 h (p < 0.0001). In sum, these data suggest that VAD1 negatively regulates the sexual cell cycle via degradation of constitutive high levels of MF? transcripts in a synthetic/degradative cycle, providing a mechanism of mRNA induction for time-critical cellular events, such as mating induction.
| S-EPMC2966090 | biostudies-literature
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| S-EPMC5435349 | biostudies-literature
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| S-EPMC118017 | biostudies-literature
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| S-EPMC1462016 | biostudies-other
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.
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Project description:Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.
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Project description:UNLABELLED:While research has identified an important contribution for metals, such as iron, in microbial pathogenesis, the roles of other transition metals, such as copper, remain mostly unknown. Recent evidence points to a requirement for copper homeostasis in the virulence of Cryptococcus neoformans based on a role for a CUF1 copper regulatory factor in mouse models and in a human patient cohort. C. neoformans is an important fungal pathogen that results in an estimated 600,000 AIDS-related deaths yearly. In the present studies, we found that a C. neoformans mutant lacking the CUF1-dependent copper transporter, CTR4, grows normally in rich medium at 37°C but has reduced survival in macrophages and attenuated virulence in a mouse model. This reduced survival and virulence were traced to a growth defect under nutrient-restricted conditions. Expression studies using a full-length CTR4-fluorescent fusion reporter construct demonstrated robust expression in macrophages, brain, and lung, the latter shown by ex vivo fluorescent imaging. Inductively coupled mass spectroscopy (ICP-MS) was used to probe the copper quota of fungal cells grown in defined medium and recovered from brain, which suggested a role for a copper-protective function of CTR4 in combination with cell metallothioneins under copper-replete conditions. In summary, these data suggest a role for CTR4 in copper-related homeostasis and subsequently in fungal virulence. IMPORTANCE:Crytococcus neoformans is a significant global fungal pathogen, and copper homeostasis is a relatively unexplored aspect of microbial pathogenesis that could lead to novel therapeutics. Previous studies correlated expression levels of a Ctr4 copper transporter to development of meningoencephalitis in a patient cohort of solid-organ transplants, but a direct role for Ctr4 in mammalian pathogenesis has not been demonstrated. The present studies utilize a ?ctr4 mutant strain which revealed an important role for CTR4 in C. neoformans infections in mice and relate the gene product to homeostatic control of copper and growth under nutrient-restricted conditions. Robust expression levels of CTR4 during fungal infection were exploited to demonstrate expression and lung cryptococcal disease using ex vivo fluorescence imaging. In summary, these studies are the first to directly demonstrate a role for a copper transporter in fungal disease and provide an ex vivo imaging tool for further study of cryptococcal gene expression and pathogenesis.
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Project description:Infection with Cryptococcus neoformans begins when desiccated yeast cells or spores are inhaled and lodge in the alveoli of the lungs. A subset of cryptococcal cells in the lungs differentiate into enlarged cells, referred to as titan cells. Titan cells can be as large as 50 to 100 ?m in diameter and exhibit a number of features that may affect interactions with host immune defenses. To characterize the effect of titan cell formation on the host-pathogen interaction, we utilized a previously described C. neoformans mutant, the gpr4? gpr5? mutant, which has minimal titan cell production in vivo. The gpr4? gpr5? mutant strain had attenuated virulence, a lower CFU, and reduced dissemination compared to the wild-type strain. Titan cell production by the wild-type strain also resulted in increased eosinophil accumulation and decreased phagocytosis in the lungs compared to those with the gpr4? gpr5? mutant strain. Phagocytosed cryptococcal cells exhibited less viability than nonphagocytosed cells, which potentially explains the reduced cell survival and overall attenuation of virulence in the absence of titan cells. These data show that titan cell formation is a novel virulence factor in C. neoformans that promotes establishment of the initial pulmonary infection and plays a key role in disease progression.
| S-EPMC3486048 | biostudies-literature
Project description:The basidiomycete fungus Cryptococcus neoformans is an opportunistic pathogen of worldwide importance that causes meningitis, leading to death in immunocompromised individuals. Unlike many basidiomycete fungi, C. neoformans is thermotolerant, and its ability to grow at 37 degrees C is considered to be a virulence factor. We used serial analysis of gene expression (SAGE) to characterize the transcriptomes of C. neoformans strains that represent two varieties with different polysaccharide capsule serotypes. These include a serotype D strain of the C. neoformans variety neoformans and a serotype A strain of variety grubii. In this report, we describe the construction and characterization of SAGE libraries from each strain grown at 25 degrees C and 37 degrees C. The SAGE data reveal transcriptome differences between the two strains, even at this early stage of analysis, and identify sets of genes with higher transcript levels at 25 degrees C or 37 degrees C. Notably, growth at the lower temperature increased transcript levels for histone genes, indicating a general influence of temperature on chromatin structure. At 37 degrees C, we noted elevated transcript levels for several genes encoding heat shock proteins and translation machinery. Some of these genes may play a role in temperature-regulated phenotypes in C. neoformans, such as the adaptation of the fungus to growth in the host and the dimorphic transition between budding and filamentous growth. Overall, this work provides the most comprehensive gene expression data available for C. neoformans; this information will be a critical resource both for gene discovery and genome annotation in this pathogen.
| S-EPMC186651 | biostudies-literature
Project description:Crg1 and Crg2 are regulators of G-protein signaling homologs found in the human fungal pathogen Cryptococcus neoformans. Crg1 negatively regulates pheromone responses and mating through direct inhibition of Galpha subunits Gpa2 and Gpa3. It has also been proposed that Crg2 has a role in mating, as genetic crosses involving Deltacrg2 mutants resulted in formation of hyperfilaments. We found that mutation of Gpa2 and Gpa3 partially suppressed the hyperfilamentation, mutation of Gpa3 alleviated Deltacrg2-specfic cell swelling, and mutation of the mitogen-activated protein kinase Cpk1 blocked both processes. These findings indicate that Gpa2 and Gpa3 function downstream of Crg2 and that Gpa3 is also epistatic to Crg2 in a Cpk1-dependent morphogenesis process linked to mating. Significantly, we found that Deltacrg2 mutants formed enlarged capsules that mimic cells expressing a constitutively active GPA1(Q284L) allele and that the levels of intracellular cyclic AMP (cAMP) were also elevated, suggesting that Crg2 also negatively regulates the Gpa1-cAMP signaling pathway. We further showed that Crg2 interacted with Gpa3 and Gpa1, but not Gpa2, in a pulldown assay and that Crg2 maintained a higher in vitro GTPase-activating protein activity toward Gpa3 and Gpa1 than to Gpa2. Finally, we found that dysregulation of cAMP due to the Crg2 mutation attenuated virulence in a murine model of cryptococcosis. Taken together, our study reveals Crg2 as an RGS (regulator of G-protein signaling) protein of multiregulatory function, including one that controls mating distinctly from Crg1 and one that serves as a novel inhibitor of Gpa1-cAMP signaling.
| S-EPMC2547063 | biostudies-literature