• Title/Summary/Keyword: C. neoformans

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The cAMP/Protein Kinase A Pathway and Virulence in Cryptococcus neoformans

  • Kronstad, James W.;Hu, Guang-Gan;Choi, Jae-Hyuk
    • Mycobiology
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    • v.39 no.3
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    • pp.143-150
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    • 2011
  • The basidiomycete fungus Cryptococcus neoformans is an important pathogen of immunocompromised people. The ability of the fungus to sense its environment is critical for proliferation and the generation of infectious propagules, as well as for adaptation to the mammalian host during infection. The conserved cAMP/protein kinase A pathway makes an important contribution to sensing, as demonstrated by the phenotypes of mutants with pathway defects. These phenotypes include loss of the ability to mate and to elaborate the key virulence factors capsule and melanin. This review summarizes recent work that reveals new targets of the pathway, new phenotypic consequences of signaling defects, and a more detailed understanding of connections with other aspects of cryptococcal biology including iron regulation, pH sensing, and stress.

Sensing the Stress: the Role of the Stress-activated p38/Hog1 MAPK Signalling Pathway in Human Pathogenic Fungus Cryptococcus neoformans

  • Bahn, Yong-Sun;Heitman, Joseph
    • Proceedings of the Microbiological Society of Korea Conference
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    • 2007.05a
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    • pp.120-122
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    • 2007
  • All living organisms use numerous signal-transduction pathways to sense and respond to their environments and thereby survive and proliferate in a range of biological niches. Molecular dissection of these signalling networks has increased our understanding of these communication processes and provides a platform for therapeutic intervention when these pathways malfunction in disease states, including infection. Owing to the expanding availability of sequenced genomes, a wealth of genetic and molecular tools and the conservation of signalling networks, members of the fungal kingdom serve as excellent model systems for more complex, multicellular organisms. Here, we employed Cryptococcus neoformans as a model system to understand how fungal-signalling circuits operate at the molecular level to sense and respond to a plethora of environmental stresses, including osmoticshock, UV, high temperature, oxidative stress and toxic drugs/metabolites. The stress-activated p38/Hog1 MAPK pathway is structurally conserved in many organisms as diverse as yeast and mammals, but its regulation is uniquely specialized in a majority of clinical Cryptococcus neoformans serotype A and D strains to control differentiation and virulence factor regulation. C. neoformans Hog1 MAPK is controlled by Pbs2 MAPK kinase (MAPKK). The Pbs2-Hog1 MAPK cascade is controlled by the fungal "two-component" system that is composed of a response regulator, Ssk1, and multiple sensor kinases, including two-component.like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. We also identified and characterized the Ssk2 MAPKKK upstream of the MAPKK Pbs2 and the MAPK Hog1 in C. neoformans. The SSK2 gene was identified as a potential component responsible for differential Hog1 regulation between the serotype D sibling f1 strains B3501 and B3502 through comparative analysis of their meiotic map with the meiotic segregation of Hog1-dependent sensitivity to the fungicide fludioxonil. Ssk2 is the only polymorphic component in the Hog1 MAPK module, including two coding sequence changes between the SSK2 alleles in B3501 and B3502 strains. To further support this finding, the SSK2 allele exchange completely swapped Hog1-related phenotypes between B3501 and B3502 strains. In the serotype A strain H99, disruption of the SSK2 gene dramatically enhanced capsule biosynthesis and mating efficiency, similar to pbs2 and hog1 mutations. Furthermore, ssk2, pbs2, and hog1 mutants are all hypersensitive to a variety of stresses and completely resistant to fludioxonil. Taken together, these findings indicate that Ssk2 is the critical interface protein connecting the two-component system and the Pbs2-Hog1 pathway in C. neoformans.

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Microbe Hunting: A Curious Case of Cryptococcus

  • Bartlett, Karen H.;Kidd, Sarah;Duncan, Colleen;Chow, Yat;Bach, Paxton;Mak, Sunny;MacDougall, Laura;Fyfe, Murray
    • Proceedings of the Korean Environmental Health Society Conference
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    • 2005.06a
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    • pp.45-72
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    • 2005
  • C. neoformans-associated cryptococcosis is primarily a disease of immunocompromised persons, has a world-wide distribution, and is often spread by pigeons in the urban environment. In contrast, C. gattii causes infection in normal hosts, has only been described in tropical and semi-tropical areas of the world, and has a unique niche in river gum Eucalyptus trees. Cryptococcosis is acquired through inhalation of the yeast propagules from the environment. C. gattii has been identified as the cause of an emerging infectious disease centered on Vancouver Island, British Columbia, Canada. No cases of C. gattii-disease were diagnosed prior to 1999; the current incidence rate is 36 cases per million population. A search was initiated in 2001 to find the ecological niche of this basidiomycetous yeast. C. gaftii was found in the environment in treed areas of Vancouver Island. The highest percentage of colonized-tree clusters were found around central Vancouver Island, with decreasing rates of colonization to the north and south. Climate, soil and vegetation cover of this area, called the Coastal Douglas fir biogeoclimatic zone, is unique to British Columbia and Canada. The concentration of airborne C. gattii was highest in the dry summer months, and lowest during late fall, winter, and early spring, months which have heavy rainfall. The study of the emerging colonization of this organism and subsequent cases of environmentally acquired disease will be informative in planning public health management of new routes of exposure to exotic agents in areas impacted by changing climate and land use patterns. Cryptococcosis is an infection associated with an encapsulated, basidiomycetous yeast Cryptococcus neoformans. The route of entry for this organism is through the lungs, with possible systemic spread via the circulatory system to the brain and meninges. There are four cryptococcal serogroups associated with disease in humans and animals, distinguished by capsular polysaccharide antigens. Cryptococcus neoformans: variety grubii (serotype A), variety neoformans (serotype D), and variety gattii (serotypes B and C) (Franzot et at. 1999). C. neoformans variety gattii has recently been elevated to species status, C. gattii. C. neoformans val. grubii and var. neoformans have a world-wide distribution, and are particularly associated with soil and weathered bird droppings. In contrast, C. gattii (CG) is not associated with bird excrement, is primarily found in tropical and subtropical climates, and has a restricted environmental niche associated with specific tree species. (Ellis & Pfiffer 1990) Ellis and Pfeiffer theorize that, as a basidiomycete, CG requires an association with a tree in order to become pathogenic to mammals. In Australia, CG has been found to be associated with five species of Eucalypts, Eucalyptus camaldulensis, E. tereticornis, E. blakelyi, E. gomphocephala, and E. rudis. Eucalypts, although originally native to Australia, now have a world-wide distribution. CG has been found associated with imported eucalypts in India, California, Brazil, and Egypt. In addition, in Brazil and Columbia, where eucalypts have been naturalized, native trees have been shown to harbour CG (Callejas et al. 1998; Montenegro et al. 2000). In British Columbia, Canada, since the beginning of 1999, there have been 120 confirmed cases of cryptococcal mycoses associated with CG in humans, including 4 fatalities (data from British Columbia Centre for Disease Control), and over 200 cases in animal pets in BC (data from Central Laboratory for Veterinarians). What is remarkable about the BC outbreak of C. gattii-cryptococcosis is that all of the cases have been residents of, or visitors to, a narrow area along the eastern coast of Vancouver Island, BC, from the tip of the island in the south (Victoria) to Courtenay on the north-central island as illustrated in Figure 1. Of the first 38 human cases, 58% were male with a mean age of 59.7 years (range 20 - 82): 36 cases (95%) were Caucasian. Ten cases (26%) presented with meningitis, the remainder presented with respiratory symptoms. Cultures recovered from cases of cryptococcosis associated with the outbreak were typed as serogroup B, which is specific to CG (Bartlett et al. 2003). This was the first reported outbreak of CVG in Canada, or indeed, the world. Where infection with CG is endemic, for example, Australia, the incidence of cryptococcosis ranges from 1.8 - 4.7 per million between the southern and northern states (Sorrell 2001). However, the overall incidence of cryptococcosis in immunocompenent individuals has been estimated at 0.2 per million population per year (Kwon-Chung et al. 1984). The population of Vancouver Island is approximately 720,000,consequently, even if the organism were endemic, one would expect a maximum of 0.15 cases of cryptococcal disease annually.

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Effect of Vitamin E Treatments on The Humoral and Cellular Immune Responses in Mice. - Animal experiment for nursing care of vitamin E-deficient patients- (비타민 E 투여가 마우스의 체액성 및 세포성 면역반응에 미치는 영향 -비타민 E 결핍환자의 간호중재 개발을 위한 동물실험 -)

  • 김금재
    • Journal of Korean Academy of Nursing
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    • v.23 no.4
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    • pp.528-543
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    • 1993
  • Vitamin E, which has its advocates in the treatment of diabetes mellitus. autoimmune disease, cancer and peripheral vascular and thromboembolic disease, has now been alleged to have a powerful antioxident effect and to affect various biological activities such as fertility factor, inhibition of human platelet aggregation and stabilization of biological membranes. The present study was designed to test whether vitamin I(alpha-tocopherol) can : (1) enhance the hemagglutinin response to sheep red blood cells (SRBC), (2) modulate Arthus and delayed type hypersensitivity(DTH) to SRBC and contact hypersensitivity to dinitrofluorobenzene (DNFB). (3) enhance the mitogenic response of murine splenocyte, (4) decrease the recovery of Cryptococcus neoformans from brain, lung, liver, spleen and kidney of infected mice and (5) have an inhibitory or enhancing effect on the induction of active systemic anaphylaxis(ASA) induced by chicken-gamma globulin (CGG) in mice. Mice were given either intramuscular injections of 0.3ml (300mg) of vitamin I before immunization or were infection for 10 consecutive days or were given by vitamin I esophageal intubation, 0.1ml(100mg), for 20 days before sacrifice for the mitogenic response experiments. It was found that vitamin E treated mice showed a significant enhancement in hemagglutinin response, Arthus reaction and DTH to SRBC and contact hypersensitivity to DNFB. There was no significant difference in the mitogenic response to phytohemagglutinin(PHA), but the response to concanavalin A(ConA) or pokeweed mitogem(PWM) was increased in vitamin E-treated mice. Interestingly, the vitamin E administration before C. neoformans infection decreased significantly the recovery of C. neoformans from brain lung, liver, spleen and kidney of the infected mice as compared with that of the control mice, strongly suggesting that vitamin E pretreatment may increase the resistance of mice to the fungal infection. Unexpectedly, vitamin E administration enhanced the production of CGG -induced ASA. Taken together, it can be concluded that vitamin I administration may in-crease the humoral and cellular immune response and resistance. to C. neoformans infection, but enhance the induction of ASA to CGG. Further studies are necessary to clarify the underlying mechanism accounting for these effects.

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Molecular Characterization of Adenylyl Cyclase Complex Proteins Using Versatile Protein-Tagging Plasmid Systems in Cryptococcus neoformans

  • So, Yee-Seul;Yang, Dong-Hoon;Jung, Kwang-Woo;Huh, Won-Ki;Bahn, Yong-Sun
    • Journal of Microbiology and Biotechnology
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    • v.27 no.2
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    • pp.357-364
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    • 2017
  • In this study, we aimed to generate a series of versatile tagging plasmids that can be used in diverse molecular biological studies of the fungal pathogen Cryptococcus neoformans. We constructed 12 plasmids that can be used to tag a protein of interest with a GFP, mCherry, $4{\times}FLAG$, or $6{\times}HA$, along with nourseothricin-, neomycin-, or hygromycin-resistant selection markers. Using this tagging plasmid set, we explored the adenylyl cyclase complex (ACC), consisting of adenylyl cyclase (Cac1) and its associated protein Aca1, in the cAMP-signaling pathway, which is critical for the pathogenicity of C. neoformans. We found that Cac1-mCherry and Aca1-GFP were mainly colocalized as punctate forms in the cell membrane and non-nuclear cellular organelles. We also demonstrated that Cac1 and Aca1 interacted in vivo by co-immunoprecipitation, using $Cac1-6{\times}HA$ and $Aca1-4{\times}FLAG$ tagging strains. Bimolecular fluorescence complementation further confirmed the in vivo interaction of Cac1 and Aca1 in live cells. Finally, protein pull-down experiments using $aca1{\Delta}$::ACA1-GFP and $aca1{\Delta}$::ACA1-GFP $cac1{\Delta}$ strains and comparative mass spectrometry analysis identified Cac1 and a number of other novel ACC-interacting proteins. Thus, this versatile tagging plasmid system will facilitate diverse mechanistic studies in C. neoformans and further our understanding of its biology.

Antifungal Mechanism of Action of Lauryl Betaine Against Skin-Associated Fungus Malassezia restricta

  • Do, Eunsoo;Lee, Hyun Gee;Park, Minji;Cho, Yong-Joon;Kim, Dong Hyeun;Park, Se-Ho;Eun, Daekyung;Park, Taehun;An, Susun;Jung, Won Hee
    • Mycobiology
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    • v.47 no.2
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    • pp.242-249
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    • 2019
  • Betaine derivatives are considered major ingredients of shampoos and are commonly used as antistatic and viscosity-increasing agents. Several studies have also suggested that betaine derivatives can be used as antimicrobial agents. However, the antifungal activity and mechanism of action of betaine derivatives have not yet been fully understood. In this study, we investigated the antifungal activity of six betaine derivatives against Malassezia restricta, which is the most frequently isolated fungus from the human skin and is implicated in the development of dandruff. We found that, among the six betaine derivatives, lauryl betaine showed the most potent antifungal activity. The mechanism of action of lauryl betaine was studied mainly using another phylogenetically close model fungal organism, Cryptococcus neoformans, because of a lack of available genetic manipulation and functional genomics tools for M. restricta. Our genome-wide reverse genetic screening method using the C. neoformans gene deletion mutant library showed that the mutants with mutations in genes for cell membrane synthesis and integrity, particularly ergosterol synthesis, are highly sensitive to lauryl betaine. Furthermore, transcriptome changes in both C. neoformans and M. restricta cells grown in the presence of lauryl betaine were analyzed and the results indicated that the compound mainly affected cell membrane synthesis, particularly ergosterol synthesis. Overall, our data demonstrated that lauryl betaine influences ergosterol synthesis in C. neoformans and that the compound exerts a similar mechanism of action on M. restricta.

Mon1 Is Essential for Fungal Virulence and Stress Survival in Cryptococcus neoformans

  • Son, Ye-Eun;Jung, Won-Hee;Oh, Sang-Hun;Kwak, Jin-Hwan;Cardenas, Maria E.;Park, Hee-Soo
    • Mycobiology
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    • v.46 no.2
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    • pp.114-121
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    • 2018
  • Mon1 is a guanine nucleotide exchange factor subunit that activates the Ypt7 Rab GTPase and is essential for vacuole trafficking and autophagy in eukaryotic organisms. Here, we identified and characterized the function of Mon1, an ortholog of Saccharomyces cerevisiae Mon1, in a human fungal pathogen, Cryptococcus neoformans. Mutation in mon1 resulted in hypersensitivity to thermal stress. The mon1 deletion mutant exhibited increased sensitivity to cell wall and endoplasmic reticulum stress. However, the mon1 deletion mutant showed more resistance to the antifungal agent fluconazole. In vivo studies demonstrated that compared to the wild-type strain, the mon1 deletion mutant attenuated virulence in the Galleria mellonella insect model. Moreover, the mon1 deletion mutant was avirulent in the murine inhalation model. These results demonstrate that Mon1 plays a crucial role in stress survival and pathogenicity in C. neoformans.

Ultrastructure of Cryptococcus neoformans in the Skin Tissue (조직내 Cryptococcus neoformans의 전자현미경적 관찰)

  • Seo, Young-Hoon;Kwon, Tae-Jung;Kim, Chung-Sook
    • Applied Microscopy
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    • v.12 no.1
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    • pp.49-56
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    • 1982
  • A case of systemic cryptococcosis developed in 4 year old boy was described and illustrated by light and electron microscope. Light microscopically, the upper dermis of the skin showed chronic nonspecific inflammation with numerous spherical spores surrounded by a clear halo created by the wide gelatinous capsule. Ultrastructurally, the C. neoformans showed the wide capsule containing microfibrils that appeared to radiate from the cell wall and to coil and interwine in various directions. The cell was uninucleate with a single nucleolus. Along the inner nuclear envelope, numerous small vesicles were present. In addition, C. neoformans presented membranous organelles derived from the plasma membrane and comparable to bacterial mesosomes.

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Inhibition of the Calcineurin Pathway by Two Flavonoids Isolated from Miliusa sinensis Finet & Gagnep.

  • Lee, Won Jeong;Moon, Jae Sun;Kim, Young Tae;Bach, Tran The;Hai, Do Van;Kim, Sung Uk
    • Journal of Microbiology and Biotechnology
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    • v.26 no.10
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    • pp.1696-1700
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    • 2016
  • In order to discover plant-derived signaling pathway inhibitors with antifungal properties, a two-component screening system utilizing the calcineurin and Hog1 mitogen-activated protein kinase pathways responsible for the virulence networks of Cryptococcus neoformans was employed, owing to the counter-regulatory actions of these pathways. Of the 1,000 plant extracts tested, two bioactive compounds from Miliusa sinensis were found to act specifically on the calcineurin pathway of C. neoformans. These compounds, identified as pashanone and 5-hydroxy-6,7-dimethoxyflavanone, exhibited potent antifungal activities against various human pathogenic fungi with minimum inhibitory concentration values ranging from 4.0 to >128 μg/ml.

A Phenylpropanoid Glycoside as a Calcineurin Inhibitor Isolated from Magnolia obovata Thunb.

  • Lee, Won Jeong;Moon, Jae Sun;Kim, Sung In;Bahn, Yong-Sun;Lee, Hanna;Kang, Tae Hoon;Shin, Heung Mook;Kim, Sung Uk
    • Journal of Microbiology and Biotechnology
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    • v.25 no.9
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    • pp.1429-1432
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    • 2015
  • To identify plant-derived cell signaling inhibitors with antifungal properties, a twocomponent screening system using both wild-type Cryptococcus neoformans and a calcineurin mutant was employed owing to their counter-regulatory actions on the Hog1 mitogenactivated protein kinase and calcineurin pathways. Of the 2,000 plant extracts evaluated, a single bioactive compound from M. obovata Thunb. was found to act specifically on the calcineurin pathway of C. neoformans. This compound was identified as magnoloside A, and had potent antifungal activities against various Cryptococcus strains with minimum inhibitory concentration values ranging from 1.0 to 4.0 μg/ml.