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Sample records for neoformans var neoformans

  1. Natural habitat of Cryptococcus neoformans var. gattii.

    PubMed Central

    Ellis, D H; Pfeiffer, T J

    1990-01-01

    Environmental isolations have established that Cryptococcus neoformans var. gattii appears to have a specific ecological association with Eucalyptus camaldulensis. So far, we have isolated C. neoformans var. gattii on 35 separate occasions, all from samples associated with E. camaldulensis. The global distribution of E. camaldulensis appears to correspond to the epidemiologic distribution of cryptococcosis caused by C. neoformans var. gattii. No other environmental source for the fungus has yet been detected, and no other eucalypt has the distribution pattern corresponding to reported cases caused by this fungus. These findings may provided an explanation for the high incidence of infections caused by C. neoformans var. gattii in Australian aborigines living in the Northern Territory and for its low worldwide incidence in acquired immunodeficiency syndrome patients. Images PMID:2199524

  2. Characterization of the antigenicity of Cpl1, a surface protein of Cryptococcus neoformans var. neoformans.

    PubMed

    Cai, Jian-Piao; Liu, Ling-Li; To, Kelvin K W; Lau, Candy C Y; Woo, Patrick C Y; Lau, Susanna K P; Guo, Yong-Hui; Ngan, Antonio H Y; Che, Xiao-Yan; Yuen, Kwok-Yung

    2015-01-01

    Cryptococcus neoformans var. neoformans is an important fungal pathogen. The capsule is a well established virulence factor and a target site for diagnostic tests. The CPL1 gene is required for capsular formation and virulence. The protein product Cpl1 has been proposed to be a secreted protein, but the characteristics of this protein have not been reported. Here we sought to characterize Cpl1. Phylogenetic analysis showed that the Cpl1 of C. neoformans var. neoformans and the Cpl1 orthologs identified in C. neoformans var. grubii and C. gattii formed a distinct cluster among related fungi; while the putative ortholog found in Trichosporon asahii was distantly related to the Cryptococcus cluster. We expressed Cpl1 abundantly as a secreted His-tagged protein in Pichia pastoris. The protein was used to immunize guinea pigs and rabbits for high titer mono-specific polyclonal antibody that was shown to be highly specific against the cell wall of C. neoformans var. neoformans and did not cross react with C. gattii, T. asahii, Aspergillus spp., Candida spp. and Penicillium spp. Using the anti-Cpl1 antibody, we detected Cpl1 protein in the fresh culture supernatant of C. neoformans var. neoformans and we showed by immunostaining that the Cpl1 protein was located on the surface. The Cpl1 protein is a specific surface protein of C. neoformans var. neoformans. PMID:25261494

  3. Multilocus sequence typing analysis reveals that Cryptococcus neoformans var. neoformans is a recombinant population

    PubMed Central

    Cogliati, Massimo; Zani, Alberto; Rickerts, Volker; McCormick, Ilka; Desnos-Ollivier, Marie; Velegraki, Aristea; Escandon, Patricia; Ichikawa, Tomoe; Ikeda, Reiko; Bienvenue, Anne-Lise; Tintelnot, Kathrin; Tore, Okan; Akcaglar, Sevim; Lockhart, Shawn; Tortorano, Anna Maria; Varma, Ashok

    2016-01-01

    Cryptococcus neoformans var. neoformans (serotype D) represents about 30% of the clinical isolates in Europe and is present less frequently in the other continents. It is the prevalent etiological agent in primary cutaneous cryptococcosis as well as in cryptococcal skin lesions of disseminated cryptococcosis. Very little is known about the genotypic diversity of this Cryptococcus subtype. The aim of this study was to investigate the genotypic diversity among a set of clinical and environmental C. neoformans var. neoformans isolates and to evaluate the relationship between genotypes, geographical origin and clinical manifestations. A total of 83 globally collected C. neoformans var. neoformans isolates from Italy, Germany, France, Belgium, Denmark, Greece, Turkey, Thailand, Japan, Colombia, and the USA, recovered from different sources (primary and secondary cutaneous cryptococcosis, disseminated cryptococcosis, the environment, and animals), were included in the study. All isolates were confirmed to belong to genotype VNIV by molecular typing and they were further investigated by MLST analysis. Maximum likelihood phylogenetic as well as network analysis strongly suggested the existence of a recombinant rather than a clonal population structure. Geographical origin and source of isolation were not correlated with a specific MLST genotype. The comparison with a set of outgroup C. neoformans var. grubii isolates provided clear evidence that the two varieties have different population structures. PMID:26768709

  4. Isolation of Cryptococcus neoformans var. neoformans from bird droppings, fruits and vegetables in Mexico City.

    PubMed

    López-Martínez, R; Castañón-Olivares, L R

    1995-01-01

    The presence of Cryptococcus neoformans in various natural sources, such as bird droppings, fruits and vegetables, was investigated. A total of 711 samples were analyzed; C. neoformans var. neoformans was isolated from seven out of 74 bird droppings (9.5%), with parrots as one of the most significant sources. Fruits were positive in 9.5% of the 169 samples studied, specially citrus fruits, particularly grapefruit, in which the highest frequency was found. From the 468 vegetable samples, only 20 were positive (4.2%). It is emphasized that five of the positive vegetables species are autochthonous to Mexico: avocado (Nectandra salicifolia), beet (Beta vulgaris var. quinopodiace), chayote (Sechium edule), stringbean (Cassia sp), and nopal (Opuntia ficus-indica). PMID:7617014

  5. Isolation of Cryptococcus neoformans var. gattii from Eucalyptus camaldulensis in India.

    PubMed

    Chakrabarti, A; Jatana, M; Kumar, P; Chatha, L; Kaushal, A; Padhye, A A

    1997-12-01

    Cryptococcus neoformans var. gattii has an ecological association with five Eucalyptus species: E. blakelyi, E. camaldulensis, E. gomphocephala, E. rudis, and E. tereticornis. After human infections due to C. neoformans var. gattii were diagnosed in the states of Punjab, Himachal Pradesh, and Karnataka, India, a study was undertaken to investigate the association of C. neoformans var. gattii with Indian eucalypts, especially in the state of Punjab. A total of 696 specimens collected from E. camaldulensis, E. citriodora and E. tereticornis (hybrid) trees were examined for the presence of C. neoformans var. gattii. Flowers from two trees of E. camaldulensis in the Chak Sarkar forest and one from the village of Periana near the Ferozepur area yielded five isolates of C. neoformans var. gattii. The origin of the trees could be traced to Australia, thus providing evidence that the distribution of E. camaldulensis correlated with the distribution of human cryptococcosis cases caused by C. neoformans var. gattii in northern India. PMID:9399553

  6. In vitro evaluation of combination of fluconazole and flucytosine against Cryptococcus neoformans var. neoformans.

    PubMed Central

    Nguyen, M H; Barchiesi, F; McGough, D A; Yu, V L; Rinaldi, M G

    1995-01-01

    Amphotericin B and fluconazole are current acceptable therapies for cryptococcal meningitis; however, their effect remains suboptimal. The combination of fluconazole and flucytosine has yielded encouraging clinical results in human immunodeficiency virus patients with cryptococcal meningitis. To investigate the biological basis of this finding, we performed in vitro combination testing of fluconazole and flucytosine against 50 clinical strains of Cryptococcus neoformans var. neoformans. Synergy (fractional inhibitory concentration index of < 1.0) was observed in 62% of cases, while antagonism (fractional inhibitory concentration index of > 2.0) was not observed. For cases in which synergy was not achieved (autonomous or additive effects), the beneficial effect of the combination was still seen (i.e., there was still a decrease, although not as dramatic, in the MIC of one or both drugs when used in combination). The in vitro inhibitory action of flucytosine was greatly enhanced by the addition of fluconazole; the flucytosine MICs for Cryptococcus isolates were markedly decreased to concentrations which were severalfold lower than the achievable cerebrospinal fluid flucytosine concentration. On the other hand, the addition of flucytosine did not greatly enhance the in vitro activity of fluconazole if the initial fluconazole MIC for the isolate was > or = 8 micrograms/ml. Controlled clinical studies are warranted to further elucidate the potential utility of fluconazole-flucytosine combination therapy. PMID:7486902

  7. Detection of Cryptococcus neoformans var. grubii in honeybee (Apis mellifera) colonies.

    PubMed

    Ergin, C; Ilkit, M; Kaftanoglu, O

    2004-10-01

    The plant flora has an important role in the ecology of Cryptococcus neoformans. It is estimated that the environmental spreading and contamination of human beings with this yeast occurs via contaminated particles of plants. Cultivation of canopy parts of plants in selective media is the most widely used isolation method of this yeast. Cryptococcus neoformans var. grubii was isolated from honeybee colonies in Eucalyptus forests but was not isolated from the places where this flora did not exist. Our results indicate that the occurrence of C. neoformans in honeybee colonies during the flowering season of Eucalyptus spp. trees can be an important bioindicator for environmental yeast presence. The screening of honeybee colonies is a practical and a rapid method for the monitoring of the C. neoformans presence in flowering plants. PMID:15504129

  8. Genetic and pathological characteristics of Cryptococcus gattii and Cryptococcus neoformans var. neoformans from meningoencephalitis in autochthonous goats and mouflons, Sardinia, Italy.

    PubMed

    Maestrale, Caterina; Masia, Mariangela; Pintus, Davide; Lollai, Stefano; Kozel, Thomas R; Gates-Hollingsworth, Marcellene A; Cancedda, Maria Giovanna; Cabras, Pierangela; Pirino, Salvatore; D'Ascenzo, Vittoria; Ligios, Ciriaco

    2015-06-12

    In this study, we examined in Sardinia the brain of 555 autochthonous sheep, 50 goats, and 4 mouflons which were found affected by neurological signs. We found 6 goats and one mouflon with meningoencephalitis caused by Cryptococcus sp. There was no evidence of cryptococcal infections in any of the examined sheep. MLST genotyping on Cryptococcus sp. isolates identified Cryptococcus gatti genotype AFLP4/VGI and Cryptococcus neoformans var. neoformans genotype AFLP2/VNIV. Phylogenetically, all Cryptococcus gattii isolates fell within the autochthonous animal, human and environmental Mediterranean isolate cluster, forming a distinct branch along with environmental strains from Alicante, in the southern Mediterranean coast of Spain. PMID:25840469

  9. Sexual Cycle of Cryptococcus neoformans var. grubii and Virulence of Congenic a and α Isolates

    PubMed Central

    Nielsen, Kirsten; Cox, Gary M.; Wang, Ping; Toffaletti, Dena L.; Perfect, John R.; Heitman, Joseph

    2003-01-01

    Cryptococcus neoformans is a human-pathogenic fungus that has evolved into three distinct varieties that infect most prominently the central nervous system. A sexual cycle involving haploid cells of a and α mating types has been reported for two varieties (C. neoformans var. neoformans, serotype D, and C. neoformans var. gattii, serotypes B and C), yet the vast majority of infections involve a distinct variety (C. neoformans var. grubii, serotype A) that has been thought to be clonal and restricted to the α mating type. We recently identified the first serotype A isolate of the a mating type which had been thought to be extinct (strain 125.91). Here we report that this unusual strain can mate with a subset of pathogenic serotype A strains to produce a filamentous dikaryon with fused clamp connections, basidia, and viable recombinant basidiospores. One meiotic segregant mated poorly with the serotype A reference strain H99 but robustly with a crg1 mutant that lacks a regulator of G protein signaling and is hyperresponsive to mating pheromone. This meiotic segregant was used to create congenic a and α mating type serotype A strains. Virulence tests with rabbit and murine models of cryptococcal meningitis showed that the serotype A congenic a and α mating type strains had equivalent virulence in animal models, in contrast to previous studies linking the α mating type to increased virulence in congenic serotype D strains. Our studies highlight a role for sexual recombination in the evolution of a human fungal pathogen and provide a robust genetic platform to establish the molecular determinants of virulence. PMID:12933823

  10. Cryptococcosis (C. neoformans)

    MedlinePlus

    ... Foodborne, Waterborne, and Environmental Diseases Mycotic Diseases Branch C. neoformans Infection Recommend on Facebook Tweet Share Compartir ... throughout the world. People can become infected with C. neoformans after breathing in the microscopic fungus, although ...

  11. Experimental infection of almond trees seedlings (Terminalia catappa) with an environmental isolate of Cryptococcus neoformans var. gattii, serotype C.

    PubMed

    Huérfano, S; Castañeda, A; Castañeda, E

    2001-09-01

    Recently, our laboratory reported the isolation of Cryptococcus neoformans var. gattii, serotype C for the first time from almond trees (Terminalia catappa) detritus. The aim of the present study was to establish the survival of C. neoformans in almond trees seedlings. Thirty seedlings were infected in the stems and samples were taken and processed at different times and by different techniques. No morphological alterations (macro or microscopic) were observed in the infected plants. However, C. neoformans was found to be viable for at least 100 days after infection. These data constitute our first approach towards the understanding of the yeast interactions with a host-plant. PMID:15487923

  12. [Experimental inoculation of Terminalia catappa seedlings with an environmental isolate of Cryptococcus neoformans var. gattii serotype C ].

    PubMed

    Escandón, Patricia; Huérfano, Sandra; Castañeda, Elizabeth

    2002-12-01

    In 1997, our laboratory reported for the first time the isolation of Cryptococcus neoformans var. gattii serotype C associated with almond tree (Terminalia catappa) detritus. This finding led to a more detailed follow up of the association between the plant and the yeast. Preliminary data have shown that survival of the yeast in almond trees seedlings goes beyond 100 days. The aim of the present study was to establish if under the conditions previously studied, C. neoformans var. gattii would remain viable for longer periods. A total of 83 almond tree seedings, 20-40 cm high, were inoculated with C. neoformans var. gattii serotype C (INS-755). Assays were carried out inoculating the stem or the soil where the seedlings were planted. Observations were undertaken for a period of up to 12 months. As processing techniques we employed the endophytic fungi procedure (stems), maceration (roots, leaves) and standard suspension method (soils). Additionally, microscopic visualization of the yeast in plant tissues was done with trypan blue plus lactophenol. C. neoformans var. gattii was recovered from the inoculated plants for a period of up to 12 months post-inoculation; additionally, the fungus had the capacity to migrate from the stem to the soil and viceversa, without causing macroscopic or microscopic alterations in the plant tissues. This finding suggests that there appears to be an association between the host plant and C. neoformans var. gattii in the environment. PMID:12596450

  13. First isolation of Cryptococcus neoformans var. gattii, serotype C, from the environment in Colombia.

    PubMed

    Callejas, A; Ordoñez, N; Rodriguez, M C; Castañeda, E

    1998-10-01

    The natural habitat of Cryptococcus neoformans var. gattii, serotype B in the environment was established by Australian investigators who demonstrated its association with species of Eucalyptus. The aim of the present study was to search for the habitat of this variety in a city of Colombia, where clinical cases due to this variety occur with great frequency. For a period of 5 months detritus, vegetable material and air samples in and around 68 almond trees (Terminalia catappa) located in the city were studied. C. neoformans var. gattii serotype C was the only variety isolated from two of the 68 trees sampled. These trees were positive for 4 of the 5 months during which they were studied. From the first positive sample kept under refrigeration, it was possible to isolate the fungus up to 3 months later. This is the first report of the isolation of serotype C from the environment. More studies are required in order to establish the ecological significance of this finding. PMID:10075505

  14. Microsatellite Typing of Clinical and Environmental Cryptococcus neoformans var. grubii Isolates from Cuba Shows Multiple Genetic Lineages

    PubMed Central

    Illnait-Zaragozi, Maria-Teresa; Martínez-Machín, Gerardo F.; Fernández-Andreu, Carlos M.; Boekhout, Teun; Meis, Jacques F.; Klaassen, Corné H. W.

    2010-01-01

    Background Human cryptococcal infections have been associated with bird droppings as a likely source of infection. Studies toward the local and global epidemiology of Cryptococcus spp. have been hampered by the lack of rapid, discriminatory, and exchangeable molecular typing methods. Methodology/Principal Findings We selected nine microsatellite markers for high-resolution fingerprinting from the genome of C. neoformans var. grubii. This panel of markers was applied to a collection of clinical (n = 122) and environmental (n = 68; from pigeon guano) C. neoformans var. grubii isolates from Cuba. All markers proved to be polymorphic. The average number of alleles per marker was 9 (range 5–51). A total of 104 genotypes could be distinguished. The discriminatory power of this panel of markers was 0.993. Multiple clusters of related genotypes could be discriminated that differed in only one or two microsatellite markers. These clusters were assigned as microsatellite complexes. The majority of environmental isolates (>70%) fell into 1 microsatellite complex containing only few clinical isolates (49 environmental versus 2 clinical). Clinical isolates were segregated over multiple microsatellite complexes. Conclusions/Significance A large genotypic variation exists in C. neoformans var. grubii. The genotypic segregation between clinical and environmental isolates from pigeon guano suggests additional source(s) of human cryptococcal infections. The selected panel of microsatellite markers is an excellent tool to study the epidemiology of C. neoformans var. grubii. PMID:20161737

  15. Cryptococcus neoformans var. grubii-Induced Arthritis with Encephalitic Dissemination in a Dog and Review of Published Literature.

    PubMed

    Headley, Selwyn Arlington; Mota, Francisco Claudio D; Lindsay, Scott; de Oliveira, Luiza M; Medeiros, Alessandra Aparecida; Pretto-Giordano, Lucienne Garcia; Saut, João Paulo Elsen; Krockenberger, Mark

    2016-08-01

    This article describes the clinical, pathological, and immunohistochemical findings associated with Cryptococcus neoformans var. grubii in a 4-year-old female Boxer dog from Uberlândia, Minas Gerais, Southeastern Brazil. Clinically, there was a swelling at the right metatarsal region and the hock joint with enlargement of regional lymph nodes. Radiographical evaluation revealed lysis of the tarsal bone; cytology demonstrated cryptococcal intralesional organisms at the swollen joint. Despite empirical antifungals therapeutic, the animal developed neurological cryptococcosis and died spontaneously. Significant pathological alterations included arthritis, lymphadenitis, and encephalitic cryptococcomas associated with numerous intralesional narrow-necked budding encapsulated yeasts. Immunohistochemistry utilising monoclonal antibodies that label C. neoformans sp. complex capsule, characterised the yeasts as C. neoformans var. grubii. Collectively, the pathological and immunohistochemical findings of this dog indicate that the intralesional organisms observed within the articular surface of the hock joint, lymph nodes, and the brain were C. neoformans var. grubii, confirming the participation of this fungal pathogen in the development of cryptococcal arthritis. In this case, the most likely pathogenesis was percutaneous inoculation with resultant abscess-like lesion, which resulted in the draining sinus, swelling of the right hind limb with progression to the articular disease. Thereafter, the fungal pathogen probably compromised the adjacent lymph nodes with subsequent haematogenous distribution to the brain, terminating with cryptococcal arthritis, lymphadenitis, and encephalitis. PMID:27126588

  16. Molecular Genetic Analyses of Mating Pheromones Reveal Intervariety Mating or Hybridization in Cryptococcus neoformans

    PubMed Central

    Chaturvedi, Vishnu; Fan, Jinjiang; Stein, Birgit; Behr, Melissa J.; Samsonoff, William A.; Wickes, Brian L.; Chaturvedi, Sudha

    2002-01-01

    The sexual mating of the pathogenic yeast Cryptococcus neoformans is important for pathogenesis studies because the fungal virulence is linked to the α mating type (MATα). We characterized C. neoformans mating pheromones (MFα 1 and MFa1) from 122 strains to understand intervariety hybridization or mating and intervariety virulence. MFα 1 in three C. neoformans varieties showed (a) specific nucleotide polymorphisms, (b) different copy numbers and chromosomal localizations, and (c) unique deduced amino acids in two geographic populations of C. neoformans var. gattii. MFα 1 of different varieties cross-hybridized in Southern hybridizations. Their phylogenetic analyses showed purifying selection (neutral evolution). These observations suggested that MATα strains from any of the three C. neoformans varieties could mate or hybridize in nature with MATa strains of C. neoformans var. neoformans. A few serotype A/D diploid strains provided evidence for mating or hybridization, while a majority of A/D strains tested positive for haploid MFα 1 identical to that of C. neoformans var. grubii. MFα 1 sequence and copy numbers in diploids were identical to those of C. neoformans var. grubii, while their MFa1 sequences were identical to those of C. neoformans var. neoformans; thus, these strains were hybrids. The mice survival curves and histological lesions revealed A/D diploids to be highly pathogenic, with pathogenicity levels similar to that of the C. neoformans var. grubii type strain and unlike the low pathogenicity levels of C. neoformans var. neoformans strains. In contrast to MFα 1 in three varieties, MFa1 amplicons and hybridization signals could be obtained only from two C. neoformans var. neoformans reference strains and eight A/D diploids. This suggested that a yet undiscovered MFa pheromone(s) in C. neoformans var. gattii and C. neoformans var. grubii is unrelated to, highly divergent from, or rarer than that in C. neoformans var. neoformans. These

  17. Serotyping of Cryptococcus neoformans Isolates from Clinical and Environmental Sources in Spain

    PubMed Central

    Baró, Teresa; Torres-Rodríguez, Josep M.; Morera, Yolanda; Alía, Concepción; López, Olga; Méndez, Raul

    1999-01-01

    We determined biovars and serotypes of 154 isolates of Cryptococcus neoformans from clinical and environmental sources from different areas of Spain. All clinical isolates belonged to C. neoformans var. neoformans. Serotypes showed an irregular distribution. C. neoformans var. gattii serotype B was isolated from necropsy specimens from goats with pulmonary disease. PMID:10074545

  18. The Homeostasis of Iron, Copper, and Zinc in Paracoccidioides Brasiliensis, Cryptococcus Neoformans Var. Grubii, and Cryptococcus Gattii: A Comparative Analysis

    PubMed Central

    Silva, Mirelle Garcia; Schrank, Augusto; Bailão, Elisa Flávia L.C.; Bailão, Alexandre Melo; Borges, Clayton Luiz; Staats, Charley Christian; Parente, Juliana Alves; Pereira, Maristela; Salem-Izacc, Silvia Maria; Mendes-Giannini, Maria José Soares; Oliveira, Rosely Maria Zancopé; Silva, Lívia Kmetzsch Rosa e; Nosanchuk, Joshua D.; Vainstein, Marilene Henning; de Almeida Soares, Célia Maria

    2011-01-01

    Iron, copper, and zinc are essential for all living organisms. Moreover, the homeostasis of these metals is vital to microorganisms during pathogenic interactions with a host. Most pathogens have developed specific mechanisms for the uptake of micronutrients from their hosts in order to counteract the low availability of essential ions in infected tissues. We report here an analysis of genes potentially involved in iron, copper, and zinc uptake and homeostasis in the fungal pathogens Paracoccidioides brasiliensis, Cryptococcus neoformans var. grubii, and Cryptococcus gattii. Although prior studies have identified certain aspects of metal regulation in Cryptococcus species, little is known regarding the regulation of these elements in P. brasiliensis. We also present amino acid sequences analyses of deduced proteins in order to examine possible conserved domains. The genomic data reveals, for the first time, genes associated to iron, copper, and zinc assimilation and homeostasis in P. brasiliensis. Furthermore, analyses of the three fungal species identified homologs to genes associated with high-affinity uptake systems, vacuolar and mitochondrial iron storage, copper uptake and reduction, and zinc assimilation. However, homologs to genes involved in siderophore production were only found in P. brasiliensis. Interestingly, in silico analysis of the genomes of P. brasiliensis Pb01, Pb03, and Pb18 revealed significant differences in the presence and/or number of genes involved in metal homeostasis, such as in genes related to iron reduction and oxidation. The broad analyses of the genomes of P. brasiliensis, C. neoformans var. grubii, and C. gattii for genes involved in metal homeostasis provide important groundwork for numerous interesting future areas of investigation that are required in order to validate and explore the function of the identified genes and gene pathways. PMID:21833306

  19. Biofilm Formation by Cryptococcus neoformans.

    PubMed

    Martinez, Luis R; Casadevall, Arturo

    2015-06-01

    The fungus Cryptococcus neoformans possesses a polysaccharide capsule and can form biofilms on medical devices. The increasing use of ventriculoperitoneal shunts to manage intracranial hypertension associated with cryptococcal meningoencephalitis highlights the importance of investigating the biofilm-forming properties of this organism. Like other microbe-forming biofilms, C. neoformans biofilms are resistant to antimicrobial agents and host defense mechanisms, causing significant morbidity and mortality. This chapter discusses the recent advances in the understanding of cryptococcal biofilms, including the role of its polysaccharide capsule in adherence, gene expression, and quorum sensing in biofilm formation. We describe novel strategies for the prevention or eradication of cryptococcal colonization of medical prosthetic devices. Finally, we provide fresh thoughts on the diverse but interesting directions of research in this field that may result in new insights into C. neoformans biology. PMID:26185073

  20. Cryptococcus neoformans var. grubii Infection in HIV-Seronegative Patients from Northeast India: Report of Two Cases with Review of Literature.

    PubMed

    Nath, Reema; Laskar, Basanta; Ahmed, Jishan; Das, Subhalakshmi; Timung, Longminder; Saikia, Lahari

    2016-04-01

    Cryptococcus neoformans infection can occur in a wide range of hosts ranging from those who are severely immunosuppressed to those who are apparently immunocompetent. Two apparently immunocompetent HIV-seronegative patients with cryptococcal meningitis and multiple skin lesions, both due to C. neoformans var. grubii, are reported. Pregnancy was found as an associated factor in cryptococcal meningitis in a 20-year-old female patient from Arunachal Pradesh. Multiple skin lesions were the presenting feature of an 18-year-old male patient from Dibrugarh, eastern Assam. The organism was identified both phenotypically and by sequencing of ITS1 and ITS2 regions of rRNA gene. The cases are reported because of rarity of this infection in non-HIV-infected patients. PMID:26677012

  1. Cryptococcus neoformans carried by Odontomachus bauri ants.

    PubMed

    Jesus, Mariana Santos de; Rodrigues, William Costa; Barbosa, Glaucia; Trilles, Luciana; Wanke, Bodo; Lazéra, Márcia dos Santos; Silva, Manuela da

    2012-06-01

    Cryptococcus neoformans is the most common causative agent of cryptococcosis worldwide. Although this fungus has been isolated from a variety of organic substrates, several studies suggest that hollow trees constitute an important natural niche for C. neoformans. A previously surveyed hollow of a living pink shower tree (Cassia grandis) positive for C. neoformans in the city of Rio de Janeiro, Brazil, was chosen for further investigation. Odontomachus bauri ants (trap-jaw ants) found inside the hollow were collected for evaluation as possible carriers of Cryptococcus spp. Two out of 10 ants were found to carry phenoloxidase-positive colonies identified as C. neoformans molecular types VNI and VNII. The ants may have acted as a mechanical vector of C. neoformans and possibly contributed to the dispersal of the fungi from one substrate to another. To the best of our knowledge, this is the first report on the association of C. neoformans with ants of the genus Odontomachus. PMID:22666855

  2. Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation

    PubMed Central

    Janbon, Guilhem; Ormerod, Kate L.; Paulet, Damien; Byrnes, Edmond J.; Yadav, Vikas; Chatterjee, Gautam; Mullapudi, Nandita; Hon, Chung-Chau; Billmyre, R. Blake; Brunel, François; Bahn, Yong-Sun; Chen, Weidong; Chen, Yuan; Chow, Eve W. L.; Coppée, Jean-Yves; Floyd-Averette, Anna; Gaillardin, Claude; Gerik, Kimberly J.; Goldberg, Jonathan; Gonzalez-Hilarion, Sara; Gujja, Sharvari; Hamlin, Joyce L.; Hsueh, Yen-Ping; Ianiri, Giuseppe; Jones, Steven; Kodira, Chinnappa D.; Kozubowski, Lukasz; Lam, Woei; Marra, Marco; Mesner, Larry D.; Mieczkowski, Piotr A.; Moyrand, Frédérique; Nielsen, Kirsten; Proux, Caroline; Rossignol, Tristan; Schein, Jacqueline E.; Sun, Sheng; Wollschlaeger, Carolin; Wood, Ian A.; Zeng, Qiandong; Neuvéglise, Cécile; Newlon, Carol S.; Perfect, John R.; Lodge, Jennifer K.; Idnurm, Alexander; Stajich, Jason E.; Kronstad, James W.; Sanyal, Kaustuv; Heitman, Joseph; Fraser, James A.; Cuomo, Christina A.; Dietrich, Fred S.

    2014-01-01

    Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence. PMID:24743168

  3. Application of the DiversiLab system for tracing the source of the mixed infections caused by Cryptococcus neoformans var. grubii from a patient with systemic lupus erythematosus.

    PubMed

    Dou, Hongtao; Xu, Yingchun; Li, Taisheng

    2015-03-01

    Two strains of Cryptococcus neoformans (PU 66 and PU112) were simultaneously isolated from a patient with systemic lupus erythematosus. We aimed to trace the source of the mixed infections. Multi-locus sequence typing (MLST) and the DiversiLab system analyses were performed on the 2 clinical and 23 environmental C. neoformans from pigeon droppings, 11 from the home (H1) the patient visited, 12 from another home (H2) as control. All the strains were uniformly genotyped as C. neoformans var. grubii VNI. Clinical strain PU66 and all the H1 isolates had the same sequence type (ST) - ST5, while for PU112 a new ST was observed - ST265. However, there was only one single base of 7 MLST loci difference between PU66 and PU112. Sequence types of the H2 strains were ST31 and ST297. DiversiLab analysis showed that strain similarity between the two clinical strains was 96.7%. In relation to environmental samples, the highest strain similarity (99.3%) was observed for PU66 and PU70 (H1). However, none of the environmental isolates had similarity over 98.6% comparing to PU112. One source of the mixed infections has been detected, but another needs further investigation. PMID:25591136

  4. Pigment Production on L-Tryptophan Medium by Cryptococcus gattii and Cryptococcus neoformans

    PubMed Central

    Chaskes, Stuart; Cammer, Michael; Nieves, Edward; Casadevall, Arturo

    2014-01-01

    In recent years strains previously grouped within Cryptococcus neoformans have been divided into two species C. neoformans and C. gattii, with Cryptococcus neoformans comprising serotypes A, D, and AD and C. gattii comprising serotypes B and C. Cryptococcus neoformans have also been subdivided into two varieties C. neoformans var. grubii, serotype A, and C. neoformans var. neoformans, serotype D. We analyzed the growth and pigment production characteristics of 139 strains of Cryptococcus spp. in L-tryptophan containing media. Nearly all strains of Cryptococcus, including each variety and serotype tested produced a pink water-soluble pigment (molecular weight of 535.2 Da) from L-tryptophan. Consequently, the partial separation of the species was based on whether the pink pigment was secreted into the medium (extracellular) or retained as an intracellular pigment. On L-tryptophan medium C. neoformans var. grubii and serotype AD produced a pink extracellular pigment. In contrast, for C. gattii, the pink pigment was localized intracellularly and masked by heavy production of brown pigments. Pigment production by C. neoformans var. neoformans was variable with some strains producing the pink extracellular pigment and others retained the pink pigment intracellularly. The pink intracellular pigment produced by strains of C. neoformans var. neoformans was masked by production of brown pigments. Cryptococcus laccase mutants failed to produce pigments from L-tryptophan. This is the first report that the enzyme laccase is involved in tryptophan metabolism. Prior to this report Cryptococcus laccase produced melanin or melanin like-pigments from heterocyclic compounds that contained ortho or para diphenols, diaminobenzenes and aminophenol compounds. The pigments produced from L-tryptophan were not melanin. PMID:24736553

  5. Pigment production on L-tryptophan medium by Cryptococcus gattii and Cryptococcus neoformans.

    PubMed

    Chaskes, Stuart; Cammer, Michael; Nieves, Edward; Casadevall, Arturo

    2014-01-01

    In recent years strains previously grouped within Cryptococcus neoformans have been divided into two species C. neoformans and C. gattii, with Cryptococcus neoformans comprising serotypes A, D, and AD and C. gattii comprising serotypes B and C. Cryptococcus neoformans have also been subdivided into two varieties C. neoformans var. grubii, serotype A, and C. neoformans var. neoformans, serotype D. We analyzed the growth and pigment production characteristics of 139 strains of Cryptococcus spp. in L-tryptophan containing media. Nearly all strains of Cryptococcus, including each variety and serotype tested produced a pink water-soluble pigment (molecular weight of 535.2 Da) from L-tryptophan. Consequently, the partial separation of the species was based on whether the pink pigment was secreted into the medium (extracellular) or retained as an intracellular pigment. On L-tryptophan medium C. neoformans var. grubii and serotype AD produced a pink extracellular pigment. In contrast, for C. gattii, the pink pigment was localized intracellularly and masked by heavy production of brown pigments. Pigment production by C. neoformans var. neoformans was variable with some strains producing the pink extracellular pigment and others retained the pink pigment intracellularly. The pink intracellular pigment produced by strains of C. neoformans var. neoformans was masked by production of brown pigments. Cryptococcus laccase mutants failed to produce pigments from L-tryptophan. This is the first report that the enzyme laccase is involved in tryptophan metabolism. Prior to this report Cryptococcus laccase produced melanin or melanin like-pigments from heterocyclic compounds that contained ortho or para diphenols, diaminobenzenes and aminophenol compounds. The pigments produced from L-tryptophan were not melanin. PMID:24736553

  6. [Phenotype characterization of environmental Cryptococcus neoformans isolates].

    PubMed

    Huérfano, Sandra; Cepero, Maria Caridad; Castañeda, Elizabeth

    2003-09-01

    Cryptococcosis is caused by the three varieties of C. neoformans with physiological and virulence differences, some of which have been studied to determine biological aspects of this microorganism. The phenotypical aspects of environmental isolates from varieties grubii and gattii were evaluated to establish differences associated with their life cycle and virulence. To this end, 28 and 31 strains of C. neoformans serotypes A (var. grubii) and C (var. gattii) were studied. The microscopic and macroscopic morphology on Sabouraud agar and soils, growth rate at 37 degrees C, production of 22 extracellular enzymes, haploid fructification, mating type, killer toxin sensitivity patterns and virulence in BALB/c mice were evaluated. No differences were observed between the two varieties regarding microscopic and macroscopic morphology or growth at 37 degrees C (p > 0.05). However, a decrease in the cellular and capsular sizes of yeast in soil, as compared to Sabouraud, was observed (p < 0.05). Additionally, higher enzimatic activity of proteases, phospholipases, phenoloxidase and beta-glucosidase was observed in var. grubii isolates as compared to var. gattii (p < 0.05). In both varieties, structures related with haploid fruitification were observed and all isolates were mating type alpha. Killer toxin sensitivity patterns of the isolates of var. grubii were I and II; in contrast, in var. gattii, seven different patterns were found: I, V, IX-XIII. In the animal model we found that 12 of 22 (54.5%) isolates of var. grubii caused the death of the mice during the observation period, while none of the 14 var. gattii isolates caused it. The decrease in capsular and cellular sizes of the yeast in soil and the frequency of mating type alpha with structures related to haploid fructification suggest an important mechanism of production of infectious particles in nature. Additionally, greater enzimatic activity of var. grubii can be associated with the virulence in the animal model

  7. A genetic linkage map of Cryptococcus neoformans variety neoformans serotype D (Filobasidiella neoformans).

    PubMed Central

    Marra, Robert E; Huang, Johnny C; Fung, Eula; Nielsen, Kirsten; Heitman, Joseph; Vilgalys, Rytas; Mitchell, Thomas G

    2004-01-01

    To construct a genetic linkage map of the heterothallic yeast, Cryptococcus neoformans (Filobasidiella neoformans), we crossed two mating-compatible strains and analyzed 94 progeny for the segregation of 301 polymorphic markers, consisting of 228 restriction site polymorphisms, 63 microsatellites, two indels, and eight mating-type (MAT)-associated markers. All but six markers showed no significant (P < 0.05) segregation distortion. At a minimum LOD score of 6.0 and a maximum recombination frequency of 0.30, 20 linkage groups were resolved, resulting in a map length of approximately 1500 cM. Average marker density is 5.4 cM (range 1-28.7 cM). Hybridization of selected markers to blots of electrophoretic karyotypes unambiguously assigned all linkage groups to chromosomes and led us to conclude that the C. neoformans genome is approximately 20.2 Mb, comprising 14 chromosomes ranging in size from 0.8 to 2.3 Mb, with a ratio of approximately 13.2 kb/cM averaged across the genome. However, only 2 of 12 ungrouped markers hybridized to chromosome 10. The hybridizations revealed at least one possible reciprocal translocation involving chromosomes 8, 9, and 12. This map has been critical to genome sequence assembly and will be essential for future studies of quantitative trait inheritance. PMID:15238516

  8. Intron retention-dependent gene regulation in Cryptococcus neoformans

    PubMed Central

    Gonzalez-Hilarion, Sara; Paulet, Damien; Lee, Kyung-Tae; Hon, Chung-Chau; Lechat, Pierre; Mogensen, Estelle; Moyrand, Frédérique; Proux, Caroline; Barboux, Rony; Bussotti, Giovanni; Hwang, Jungwook; Coppée, Jean-Yves; Bahn, Yong-Sun; Janbon, Guilhem

    2016-01-01

    The biological impact of alternative splicing is poorly understood in fungi, although recent studies have shown that these microorganisms are usually intron-rich. In this study, we re-annotated the genome of C. neoformans var. neoformans using RNA-Seq data. Comparison with C. neoformans var. grubii revealed that more than 99% of ORF-introns are in the same exact position in the two varieties whereas UTR-introns are much less evolutionary conserved. We also confirmed that alternative splicing is very common in C. neoformans, affecting nearly all expressed genes. We also observed specific regulation of alternative splicing by environmental cues in this yeast. However, alternative splicing does not appear to be an efficient method to diversify the C. neoformans proteome. Instead, our data suggest the existence of an intron retention-dependent mechanism of gene expression regulation that is not dependent on NMD. This regulatory process represents an additional layer of gene expression regulation in fungi and provides a mechanism to tune gene expression levels in response to any environmental modification. PMID:27577684

  9. Intron retention-dependent gene regulation in Cryptococcus neoformans.

    PubMed

    Gonzalez-Hilarion, Sara; Paulet, Damien; Lee, Kyung-Tae; Hon, Chung-Chau; Lechat, Pierre; Mogensen, Estelle; Moyrand, Frédérique; Proux, Caroline; Barboux, Rony; Bussotti, Giovanni; Hwang, Jungwook; Coppée, Jean-Yves; Bahn, Yong-Sun; Janbon, Guilhem

    2016-01-01

    The biological impact of alternative splicing is poorly understood in fungi, although recent studies have shown that these microorganisms are usually intron-rich. In this study, we re-annotated the genome of C. neoformans var. neoformans using RNA-Seq data. Comparison with C. neoformans var. grubii revealed that more than 99% of ORF-introns are in the same exact position in the two varieties whereas UTR-introns are much less evolutionary conserved. We also confirmed that alternative splicing is very common in C. neoformans, affecting nearly all expressed genes. We also observed specific regulation of alternative splicing by environmental cues in this yeast. However, alternative splicing does not appear to be an efficient method to diversify the C. neoformans proteome. Instead, our data suggest the existence of an intron retention-dependent mechanism of gene expression regulation that is not dependent on NMD. This regulatory process represents an additional layer of gene expression regulation in fungi and provides a mechanism to tune gene expression levels in response to any environmental modification. PMID:27577684

  10. Cryptococcus Neoformans Modulates Extracellular Killing by Neutrophils

    PubMed Central

    Qureshi, Asfia; Grey, Angus; Rose, Kristie L.; Schey, Kevin L.; Del Poeta, Maurizio

    2011-01-01

    We recently established a key role for host sphingomyelin synthase (SMS) in regulating the killing activity of neutrophils against Cryptococcus neoformans. In this paper, we studied the effect of C. neoformans on the killing activity of neutrophils and whether SMS would still be a player against C. neoformans in immunocompromised mice lacking T and natural killer (NK) cells (Tgε26 mice). To this end, we analyzed whether C. neoformans would have any effect on neutrophil survival and killing in vitro and in vivo. We show that unlike Candida albicans, neither the presence nor the capsule size of C. neoformans cells have any effect on neutrophil viability. Interestingly, melanized C. neoformans cells totally abrogated the killing activity of neutrophils. We monitored how exposure of neutrophils to C. neoformans cells would interfere with any further killing activity of the conditioned medium and found that pre-incubation with live but not “heat-killed” fungal cells significantly inhibits further killing activity of the medium. We then studied whether activation of SMS at the site of C. neoformans infection is dependent on T and NK cells. Using matrix-assisted laser desorption–ionization tissue imaging in infected lung we found that similar to previous observations in the isogenic wild-type CBA/J mice, SM 16:0 levels are significantly elevated at the site of infection in mice lacking T and NK cells, but only at early time points. This study highlights that C. neoformans may negatively regulate the killing activity of neutrophils and that SMS activation in neutrophils appears to be partially independent of T and/or NK cells. PMID:21960987

  11. Cryptococcus neoformans infection in malignancy.

    PubMed

    Schmalzle, Sarah A; Buchwald, Ulrike K; Gilliam, Bruce L; Riedel, David J

    2016-09-01

    Cryptococcosis is an opportunistic invasive fungal infection that is well described and easily recognised when it occurs as meningitis in HIV-infected persons. Malignancy and its treatment may also confer a higher risk of infection with Cryptococcus neoformans, but this association has not been as well described. A case of cryptococcosis in a cancer patient is presented, and all cases of coincident C. neoformans infection and malignancy in adults published in the literature in English between 1970 and 2014 are reviewed. Data from these cases were aggregated in order to describe the demographics, type of malignancy, site of infection, clinical manifestations, treatment and outcomes of cryptococcosis in patients with cancer. Haematologic malignancies accounted for 82% of cases, with lymphomas over-represented compared to US population data (66% vs. 53% respectively). Cryptococcosis was reported rarely in patients with solid tumours. Haematologic malignancy patients were more likely to have central nervous system (P < 0.001) or disseminated disease (P < 0.001), receive Amphotericin B as part of initial therapy (P = 0.023), and had higher reported mortality rates than those with solid tumours (P = 0.222). Providers should have heightened awareness of the possibility of cryptococcosis in patients with haematologic malignancy presenting with infection. PMID:26932366

  12. Host immunity to Cryptococcus neoformans

    PubMed Central

    Rohatgi, Soma; Pirofski, Liise-anne

    2015-01-01

    Cryptococcosis is caused by the fungal genus Cryptococcus. Cryptococcosis, predominantly meningoencephalitis, emerged with the HIV pandemic, primarily afflicting HIV-infected patients with profound T-cell deficiency. Where in use, combination antiretroviral therapy has markedly reduced the incidence of and risk for disease, but cryptococcosis continues to afflict those without access to therapy, particularly in sub-Saharan Africa and Asia. However, cryptococcosis also occurs in solid organ transplant recipients and patients with other immunodeficiencies as well as those with no known immunodeficiency. This article reviews innate and adaptive immune responses to C. neoformans, with an emphasis on recent studies on the role of B cells, natural IgM and Fc gamma receptor polymorphisms in resistance to cryptococcosis. PMID:25865194

  13. Host immunity to Cryptococcus neoformans.

    PubMed

    Rohatgi, Soma; Pirofski, Liise-Anne

    2015-01-01

    Cryptococcosis is caused by the fungal genus Cryptococcus. Cryptococcosis, predominantly meningoencephalitis, emerged with the HIV pandemic, primarily afflicting HIV-infected patients with profound T-cell deficiency. Where in use, combination antiretroviral therapy has markedly reduced the incidence of and risk for disease, but cryptococcosis continues to afflict those without access to therapy, particularly in sub-Saharan Africa and Asia. However, cryptococcosis also occurs in solid organ transplant recipients and patients with other immunodeficiencies as well as those with no known immunodeficiency. This article reviews innate and adaptive immune responses to C. neoformans, with an emphasis on recent studies on the role of B cells, natural IgM and Fc gamma receptor polymorphisms in resistance to cryptococcosis. PMID:25865194

  14. Identification of QTLs Associated with Virulence Related Traits and Drug Resistance in Cryptococcus neoformans

    PubMed Central

    Vogan, Aaron A.; Khankhet, Jordan; Samarasinghe, Himeshi; Xu, Jianping

    2016-01-01

    Cryptococcus neoformans is a basidiomycete fungus capable of causing deadly meningoenchephilitis, primarily in immunocompromised individuals. Formerly, C. neoformans was composed of two divergent lineages, but these have recently been elevated to species status, now C. neoformans (formerly C. neoformans var. grubii) and C. deneoformans (formerly C. neoformans var. neoformans). While both species can cause deadly infections in humans, C. neoformans is much more prevalent in clinical settings than C. deneoformans. However, the genetic factors contributing to their significant differences in virulence remain largely unknown. Quantitative trait locus (QTL) mapping is a powerful tool that can be used to identify genomic regions associated with phenotypic differences between strains. Here, we analyzed a hybrid cross between these two species and identified a total of 23 QTL, including five for melanin production, six for cell size, one for cell wall thickness, five for the frequency of capsule production, three for minimal inhibitory concentration (MIC) of fluconazole in broth, and three for MIC on solid medium. For the fluconazole resistance-associated QTL, three showed environment and/or concentration-specific effects. Our results provide a large number of candidate gene regions from which to explore the molecular bases for phenotypic differences between C. neoformans and C. deneoformans. PMID:27371951

  15. Decayed wood of Syzygium cumini and Ficus religiosa living trees in Delhi/New Delhi metropolitan area as natural habitat of Cryptococcus neoformans.

    PubMed

    Randhawa, H S; Kowshik, T; Khan, Z U

    2003-06-01

    The isolation is reported of Cryptococcus neoformans var. gattii and C. n. var. neoformans from decayed wood inside trunk hollows of Syzygium cumini and of C. n. var. neoformans from Ficus religiosa trees in the Delhi/New Delhi metropolitan area. Fourteen of sixty-six (21%) S. cumini trees investigated proved to be positive, seven for each variety. The two varieties never co-occurred in the same hollow. C. n. var. neoformans was also isolated from three of seventeen Ficus religiosa-trees. Two of these isolates originated from decayed wood and one from bark. The C. n. var. gattii and C. n. var. neoformans isolates belonged to serotype B and serotype A, respectively. The data strongly supported colonization of S. cumini by both varieties and of F. religiosa trees by C. n. var. neoformans. Evidence of this was found by repeated isolations. For example, in 36/44 (82%) samples for C. n. var. gattii and 22/27 (81%) samples for C. n. var. neoformans, and by a high population density in the tested wood debris (maximally 6 x 10(5) colony-forming units per gram [c.f.u./g] for C. n. var. gattii and 8 x 10(4) c.f.u./g for C. n. var. neoformans). No eucalypt trees were seen near the positive S. cumini and F. religiosa trees. The densities of C. neoformans in these trees exceeded those found previously in Eucalyptus camaldulensis and in other tree species more rarely reported to be sources of C. neoformans in India. S. cumini and F. religiosa appear not to have been reported to date as sources for either C. n. var. gattii or C n. var. neoformans. Our results add to the recently emerging evidence that the natural habitat of C. n. var. gattii and C. n. var. neoformans is not specific to woody or other debris of particular tree species, but instead is more generalized. PMID:12964711

  16. Isolation and Characterization of Cryptococcus neoformans from Environmental Sources in Busan

    PubMed Central

    Oh, Kwang Seok

    2005-01-01

    Twenty nine samples of pigeon droppings (n = 12) and soil contaminated with avian excreta (n = 19), collected from different sites in Busan, were examined for isolation and characterization of Cryptococcus neoformans. Of these samples, 5 strains of C. neoformans were recovered from pigeon droppings (5/12 : 41.7%). All isolates were belonged to C. neoformans var. grubii (serotype A). The extracellular enzyme activities of the strains by using the API-ZYM system showed two different enzymatic patterns. The genetic variability among C. neoformans isolates was analyzed by random amplified polymorphic DNA (RAPD) using three 10-mer primers. Two different RAPD patterns, which clearly distinguished the isolates, were identified. Analysis of RAPD patterns provided a good characterization of environmental strains of C. neoformans serotype A as a heterogeneous group and were in good agreement with enzymatic profiles. PMID:24049499

  17. Ecological niche of Cryptococcus neoformans var. grubii and Cryptococcus gattii in decaying wood of trunk hollows of living trees in Jabalpur City of Central India.

    PubMed

    Grover, N; Nawange, Shesh Rao; Naidu, J; Singh, S M; Sharma, Archana

    2007-10-01

    Cryptococcus neoformans var. grubii and C. gattii were repeatedly isolated from decaying wood of trunk hollows in living trees growing in Jabalpur City in Central India. The isolation of C. gattii has been reported from decayed wood inside trunk hollow of Tamarindus indica (15.6%), Mangifera indica (2.2%), Pithecolobium dulce (12.5%), Syzygium cumini (14%), and one from bark of S. cumini. C. n. var. grubii was isolated from decaying wood debris of T. indica (4.4%), M. indica (13.3%), Terminalia arjuna (25%), S. cumini (2%), Cassia fistula (4.5%), and two from bark of S. cumini. The two species [corrected] never co-occurred in the same hollow. C. gattii [corrected] isolates belonged to serotype B. [corrected] The data strongly supported the colonization of the pathogen in decaying wood hollow of all six-tree species. Evidence of this was found by repeated isolation up to 820 days. P. dulce is being reported for the first time as natural habitat of C. gattii and T. arjuna and C. fistula as natural habitat for C. n. var. grubii. M. indica is being reported for the second time as the natural habitat of both species [corrected] (C. n. var. grubii and C. gattii). The population density of these pathogens from decaying wood debris of various tree species ranged between 0.5 x 10(3) cells/g and 6 x 10(5) cells/g. The seasonal variation has been seen in isolation of these pathogens. [corrected] Our result further reinforce the recently emerging evidence that the natural habitat of C. n. var. grubii and C. gattii is more generalized. PMID:17661160

  18. Molecular Typing of IberoAmerican Cryptococcus neoformans Isolates

    PubMed Central

    Castañeda, Alexandra; Jackson, Stuart; Huynh, Matthew; Castañeda, Elizabeth

    2003-01-01

    A network was established to acquire basic knowledge of Cryptococcus neoformans in IberoAmerican countries. To this effect, 340 clinical, veterinary, and environmental isolates from Argentina, Brazil, Chile, Colombia, Mexico, Peru, Venezuela, Guatemala, and Spain were typed by using M13 polymerase chain reaction-fingerprinting and orotidine monophosphate pyrophosphorylase (URA5) gene restriction fragment length polymorphsm analysis with HhaI and Sau96I in a double digest. Both techniques grouped all isolates into eight previously established molecular types. The majority of the isolates, 68.2% (n=232), were VNI (var. grubii, serotype A), which accords with the fact that this variety causes most human cryptococcal infections worldwide. A smaller proportion, 5.6% (n=19), were VNII (var. grubii, serotype A); 4.1% (n=14), VNIII (AD hybrid), with 9 isolates having a polymorphism in the URA5 gene; 1.8% (n=6), VNIV (var. neoformans, serotype D); 3.5% (n=12), VGI; 6.2% (n=21), VGII; 9.1% (n=31), VGIII, and 1.5% (n=5) VGIV, with all four VG types containing var. gattii serotypes B and C isolates. PMID:12603989

  19. Virulence-Associated Enzymes of Cryptococcus neoformans

    PubMed Central

    Almeida, Fausto; Wolf, Julie M.

    2015-01-01

    Enzymes play key roles in fungal pathogenesis. Manipulation of enzyme expression or activity can significantly alter the infection process, and enzyme expression profiles can be a hallmark of disease. Hence, enzymes are worthy targets for better understanding pathogenesis and identifying new options for combatting fungal infections. Advances in genomics, proteomics, transcriptomics, and mass spectrometry have enabled the identification and characterization of new fungal enzymes. This review focuses on recent developments in the virulence-associated enzymes from Cryptococcus neoformans. The enzymatic suite of C. neoformans has evolved for environmental survival, but several of these enzymes play a dual role in colonizing the mammalian host. We also discuss new therapeutic and diagnostic strategies that could be based on the underlying enzymology. PMID:26453651

  20. Colonization of a voice prosthesis by Cryptococcus neoformans.

    PubMed

    Bauters, T G; Moerman, M; Pini, G; Vermeersch, H; Nelis, H J

    2001-08-01

    Tracheoesophageal voice prostheses in laryngectomized patients commonly deteriorate due to the presence of yeasts, particularly Candida species. We describe the first case of colonization of such a device by Cryptococcus neoformans in a patient with a history of glottic carcinoma. The isolate showed an identical genomic pattern with C. neoformans from pigeon excreta in the patient's environment. PMID:11556769

  1. The expanding host tree species spectrum of Cryptococcus gattii and Cryptococcus neoformans and their isolations from surrounding soil in India.

    PubMed

    Randhawa, H S; Kowshik, T; Chowdhary, Anuradha; Preeti Sinha, K; Khan, Z U; Sun, Sheng; Xu, Jianping

    2008-12-01

    This study reports the widespread prevalence of Cryptococcus neoformans and Cryptococcus gattii in decayed wood inside trunk hollows of 14 species representing 12 families of trees and from soil near the base of various host trees from Delhi and several places in the Indian states of Uttar Pradesh, Haryana, Tamil Nadu and Chandigarh Union Territory. Of the 311 trees from which samples were obtained, 64 (20.5%) were found to contain strains of the C. neoformans species complex. The number of trees positive for C. neoformans var grubii (serotypeA) was 51 (16.3%), for C. gattii (serotype B) 24 (7.7%) and for both C. neoformans and C. gattii 11 (3.5%). The overall prevalence of C. neoformans species complex in decayed wood samples was 19.9% (111/556). There was no obvious correlation between the prevalence of these two yeast species and the species of host trees. The data on prevalence of C. gattii (24%) and C. neoformans (26%) in soil around the base of some host trees indicated that soil is another important ecologic niche for these two Cryptococcus species in India. Among our sampled tree species, eight and six were recorded for the first time as hosts for C. neoformans var grubii and C. gattii, respectively. A longitudinal surveillance of 8 host tree species over 0.7 to 2.5 years indicated long term colonization of Polyalthia longifolia, Mimusops elengi and Manilkara hexandra trees by C. gattii and/or C. neoformans. The mating type was determined for 153 of the isolates, including 98 strains of serotype A and 55 of serotype B and all proved to be mating type alpha (MAT alpha). Our observations document the rapidly expanding spectrum of host tree species for C. gattii and C. neoformans and indicate that decayed woods of many tree species are potentially suitable ecological niches for both pathogens. PMID:18608895

  2. Antibody Response to Cryptococcus neoformans Proteins in Rodents and Humans

    PubMed Central

    Chen, Lin-Chi; Goldman, David L.; Doering, Tamara L.; Pirofski, Liise-anne; Casadevall, Arturo

    1999-01-01

    The prevalence and specificity of serum antibodies to Cryptococcus neoformans proteins was studied in mice and rats with experimental infection, in individuals with or without a history of potential laboratory exposure to C. neoformans, human immunodeficiency virus (HIV)-positive individuals who developed cryptococcosis, in matched samples from HIV-positive individuals who did not develop cryptococcosis, and in HIV-negative individuals. Rodents had little or no serum antibody reactive with C. neoformans proteins prior to infection. The intensity and specificity of the rodent antibody response were dependent on the species, the mouse strain, and the viability of the inoculum. All humans had serum antibodies reactive with C. neoformans proteins regardless of the potential exposure, the HIV infection status, or the subsequent development of cryptococcosis. Our results indicate (i) a high prevalence of antibodies reactive with C. neoformans proteins in the sera of rodents after cryptococcal infection and in humans with or without HIV infection; (ii) qualitative and quantitative differences in the antibody profiles of HIV-positive individuals; and (iii) similarities and differences between humans, mice, and rats with respect to the specificity of the antibodies reactive with C. neoformans proteins. The results are consistent with the view that C. neoformans infections are common in human populations, and the results have implications for the development of vaccination strategies against cryptococcosis. PMID:10225877

  3. Isolation, Identification and Molecular Typing of Cryptococcus neoformans from Pigeon Droppings and Other Environmental Sources in Tripoli, Libya.

    PubMed

    Ellabib, Mohamed S; Aboshkiwa, Mohamed A; Husien, Walid M; D'Amicis, Roberta; Cogliati, Massimo

    2016-08-01

    Cryptococcus neoformans and C. gattii are the major cause of fungal meningitis, a potentially lethal mycosis. Since pigeon excreta and other environmental sources can be considered a significant environmental reservoir of this species in urban areas, 100 samples of pigeon excreta and 420 samples from Eucalyptus camaldulensis and Olea europaea (olive tree) around the city of Tripoli, Libya, were collected. C. neoformans was isolated and identified using standard biochemical assays from 46 samples: 34 from pigeon droppings, 3 from Eucalyptus trees and 9 from olive trees. Molecular typing revealed that all isolates from pigeon droppings belonged to molecular type VNI (C. neoformans var. grubii) and mating type αA, whereas those from trees included also the molecular type VNII and VNIII (AD hybrids). The present study reports, for the first time, information about the distribution of species, mating types and molecular types of C. neoformans/C. gattii species complex in Libya. PMID:26943725

  4. Mathematical modeling of pathogenicity of Cryptococcus neoformans

    PubMed Central

    Garcia, Jacqueline; Shea, John; Alvarez-Vasquez, Fernando; Qureshi, Asfia; Luberto, Chiara; Voit, Eberhard O; Del Poeta, Maurizio

    2008-01-01

    Cryptococcus neoformans (Cn) is the most common cause of fungal meningitis worldwide. In infected patients, growth of the fungus can occur within the phagolysosome of phagocytic cells, especially in non-activated macrophages of immunocompromised subjects. Since this environment is characteristically acidic, Cn must adapt to low pH to survive and efficiently cause disease. In the present work, we designed, tested, and experimentally validated a theoretical model of the sphingolipid biochemical pathway in Cn under acidic conditions. Simulations of metabolic fluxes and enzyme deletions or downregulation led to predictions that show good agreement with experimental results generated post hoc and reconcile intuitively puzzling results. This study demonstrates how biochemical modeling can yield testable predictions and aid our understanding of fungal pathogenesis through the design and computational simulation of hypothetical experiments. PMID:18414484

  5. Mechanisms of inhibition of Cryptococcus neoformans by human lymphocytes.

    PubMed Central

    Levitz, S M; North, E A; Dupont, M P; Harrison, T S

    1995-01-01

    Recently, our laboratory and others have demonstrated that human peripheral blood T and NK lymphocytes directly inhibit the growth of Cryptococcus neoformans. In this study, we further define the conditions under which lymphocyte-mediated fungistasis against C. neoformans occurs and examine whether mechanisms implicated in lymphocyte-mediated activities against other target cells are also involved in anticryptococcal activity. The addition of whole or broken heat-killed C. neoformans modestly inhibited lymphocyte-mediated fungistasis, whereas other particulates had no effect. The hydroxyl radical scavenger catechin, but not diethyl urea or propyl gallate, profoundly inhibited fungistasis. Salicylic acid inhibited fungistasis in a dose-dependent fashion. However, two other cyclooxygenase inhibitors, piroxicam and indomethacin, had no effect, suggesting that the mechanism of inhibition by salicylic acid was cyclooxygenase independent. Reagent prostaglandin E2, at concentrations shown by others to inhibit NK cell-mediated bactericidal and tumorlytic activities, had no effect on lymphocyte-mediated fungistasis. The addition of selected monoclonal antibodies or ligands reactive with receptors on human lymphocytes had no significant effect on lymphocyte-mediated fungistasis. Acapsular, small-capsuled, and large-capsuled C. neoformans organisms were inhibited by lymphocytes to an approximately equal extent. These data demonstrate that lymphocyte-mediated activity against C. neoformans proceeds regardless of the presence of capsule and by mechanisms at least in part dissimilar from those seen with other target cells. PMID:7642290

  6. Cryptococcus neoformans: a sugar-coated killer with designer genes.

    PubMed

    Perfect, John R

    2005-09-01

    Cryptococcus neoformans has become a common central nervous system pathogen as the immunocompromised populations enlarge world-wide. This encapsulated yeast has significant advantages for the study of fungal pathogenesis and these include: (1) a clinically important human pathogen; (2) a tractable genetic system; (3) advanced molecular biology foundation; (4) understanding of several virulence phenotypes; (5) well-studied pathophysiology; and (6) robust animal models. With the use of a sequenced genome and site-directed mutagenesis to produce specific null mutants, the virulence composite of C. neoformans has begun to be identified one gene at a time. Studies into capsule production, melanin synthesis, high temperature growth, metabolic pathways and a variety of signaling pathways have led to understandings of what makes this yeast a pathogen at the molecular level. Multiple principles of molecular pathogenesis have been demonstrated in virulence studies with C. neoformans. These include evolutionary differences between the varieties of C. neoformans in their genes for virulence, quantitative impact of genes on the virulence composite, species and site-specific importance of a virulence gene, gene expression correlation with its functional importance or phenotype and the impact of a pathogenesis gene on the host immune response. C. neoformans has now become a primary model to study molecular fungal pathogenesis with the goal of identifying drug targets or vaccine strategies. PMID:16055314

  7. Isolation and characterization of the Cryptococcus neoformans MATa pheromone gene.

    PubMed Central

    McClelland, Carol M; Fu, Jianmin; Woodlee, Gay L; Seymour, Tara S; Wickes, Brian L

    2002-01-01

    Cryptococcus neoformans is a heterothallic basidiomycete with two mating types, MATa and MATalpha. The mating pathway of this fungus has a number of conserved genes, including a MATalpha-specific pheromone (MFalpha1). A modified differential display strategy was used to identify a gene encoding the MATa pheromone. The gene, designated MFa1, is 42 amino acids in length and contains a conserved farnesylation motif. MFa1 is present in three linked copies that span a 20-kb fragment of MATa-specific DNA and maps to the MAT-containing chromosome. Transformation studies showed that MFa1 induced filament formation only in MATalpha cells, demonstrating that MFa1 is functionally conserved. Sequence analysis of the predicted Mfa1 and Mfalpha1 proteins revealed that, in contrast to other fungi such as Saccharomyces cerevisiae, the C. neoformans pheromone genes are structurally and functionally conserved. However, unlike the MFalpha1 gene, which is found in MATalpha strains of both varieties of C. neoformans, MFa1 is specific for the neoformans variety of C. neoformans. PMID:11901112

  8. Cryptococcus neoformans as a cause of bronchiolitis obliterans organizing pneumonia.

    PubMed

    Kessler, Alexander T; Al Kharrat, Tamim; Kourtis, Athena P

    2010-06-01

    The most frequent manifestations of Cryptococcus neoformans (CN) disease are systemic infections in immunocompromised patients and localized pulmonary disease in immunocompetent individuals. Such pulmonary cryptococcosis can range from asymptomatic infection to frank pneumonia that can be severe. Bronchiolitis obliterans organizing pneumonia (BOOP) is a rare severe form of pneumonitis caused by a variety of infectious and toxic agents or connective tissue diseases. BOOP due to Cryptococcus neoformans has very rarely been reported; there have been only five such case reports, mostly in immunocompromised patients. We report herein on a case of CN-associated BOOP in an immunocompetent individual and discuss the diagnosis and treatment of this entity. PMID:20169387

  9. Isolates of Cryptococcus neoformans from infected animals reveal genetic exchange in unisexual, alpha mating type populations.

    PubMed

    Bui, Tien; Lin, Xiaorong; Malik, Richard; Heitman, Joseph; Carter, Dee

    2008-10-01

    Sexual reproduction and genetic exchange are important for the evolution of fungal pathogens and for producing potentially infective spores. Studies to determine whether sex occurs in the pathogenic yeast Cryptococcus neoformans var. grubii have produced enigmatic results, however: basidiospores are the most likely infective propagules, and clinical isolates are fertile and genetically diverse, consistent with a sexual species, but almost all populations examined consist of a single mating type and have little evidence for genetic recombination. The choice of population is critical when looking for recombination, particularly when significant asexual propagation is likely and when latency may complicate assessing the origin of an isolate. We therefore selected isolates from infected animals living in the region of Sydney, Australia, with the assumption that the relatively short life spans and limited travels of the animal hosts would provide a very defined population. All isolates were mating type alpha and were of molecular genotype VNI or VNII. A lack of linkage disequilibrium among loci suggested that genetic exchange occurred within both genotype groups. Four diploid VNII isolates that produced filaments and basidium-like structures when cultured in proximity to an a mating type strain were found. Recent studies suggest that compatible alpha-alpha unions can occur in C. neoformans var. neoformans populations and in populations of the sibling species Cryptococcus gattii. As a mating type strains of C. neoformans var. grubii have never been found in Australia, or in the VNII molecular type globally, the potential for alpha-alpha unions is evidence that alpha-alpha unisexual mating maintains sexual recombination and diversity in this pathogen and may produce infectious propagules. PMID:18552280

  10. Production of diagnostic pigment by phenoloxidase activity of cryptococcus neoformans.

    PubMed

    Shaw, C E; Kapica, L

    1972-11-01

    Cryptococcus neoformans produces brown pigmented colonies when grown on agar media made from an extract of potatoes and carrots, broad beans (Vicia faba), or Guizotia abyssinica seeds. Since other yeasts do not produce the pigment, these media are useful as differential isolation media for C. neoformans. Similar specific pigment was produced by C. neoformans on chemically defined agar media which contained six different substrates of phenoloxidase (o-diphenol: oxygen oxidoreductase EC 1.10.3.1) an enzyme which catalyses the oxidation of o-diphenols to melanin. Substrates were incorporated singly into the media and included L-3, 4-dihydroxyphenylalanine (L-DOPA), chlorogenic acid, protocatechuic acid, catechol, norepinephrine, and 3-hydroxytyramine hydrochloride (dopamine). No pigment was produced on media without substrate. Phenoloxidase activity in (NH(4))(2)SO(4) precipitates of C. neoformans cell-free extract was assayed by measuring increases in absorbance at 480 nm produced in solutions of L-DOPA. This reaction showed oxygen uptake and was effectively inhibited by copper chelators, but not by catalase. The enzyme also oxidized the five other substrates which induced pigment formation. Electron micrographs of cells incubated in L-DOPA showed deposition of the pigment in the cell wall. PMID:4118328

  11. Cryptococcus neoformans copper detoxification machinery is critical for fungal virulence

    PubMed Central

    Ding, Chen; Festa, Richard A.; Chen, Ying-Lien; Espart, Anna; Palacios, Òscar; Espín, Jordi; Capdevila, Mercè; Atrian, Sílvia; Heitman, Joseph; Thiele, Dennis J.

    2013-01-01

    Summary Copper (Cu) is an essential metal that is toxic at high concentrations. Thus, pathogens often rely on host Cu for growth, but host cells can hyper-accumulate Cu to exert anti-microbial effects. The human fungal pathogen Cryptococcus neoformans encodes various Cu-responsive genes but their role in infection is unclear. We determine that pulmonary C. neoformans infection results in Cu-specific induction of genes encoding the Cu-detoxifying metallothionein (Cmt) proteins. Mutant strains lacking CMTs or expressing Cmt variants defective in Cu-coordination exhibit severely attenuated virulence and reduced pulmonary colonization. Consistent with the up-regulation of Cmt proteins, C. neoformans pulmonary infection results in increased serum Cu concentrations and respectively increases and decreases alveolar macrophage expression of the Cu importer, Ctr1, and ATP7A, a transporter implicated in phagosomal Cu compartmentalization. These studies indicate that the host mobilizes Cu as an innate anti-fungal defense but that C. neoformans senses and neutralizes toxic Cu to promote infection. PMID:23498952

  12. Primary Larynx Cryptococcus neoformans Infection: A Distinctive Clinical Entity.

    PubMed

    Bergeron, Mathieu; Gagné, Andrée-Anne; Côté, Mathieu; Chênevert, Jacinthe; Dubé, Robert; Pelletier, René

    2015-12-01

    Cryptococcus neoformans can directly infect the vocal cords. Endoscopic findings were undistinctive from most infiltrative diseases. Tissue biopsy was essential for the diagnosis. Inhaled corticosteroids can predispose to the infection, and fluconazole 400 mg daily for at least 6 weeks appeared to be minimal to achieve a permanent cure. PMID:26753169

  13. Temporal kinetics and quantitative analysis of Cryptococcus neoformans nonlytic exocytosis.

    PubMed

    Stukes, Sabriya A; Cohen, Hillel W; Casadevall, Arturo

    2014-05-01

    Cryptococcus neoformans is a facultative intracellular pathogen and the causative agent of cryptococcosis, a disease that is often fatal to those with compromised immune systems. C. neoformans has the capacity to escape phagocytic cells through a process known as nonlytic exocytosis whereby the cryptococcal cell is released from the macrophage into the extracellular environment, leaving both the host and pathogen alive. Little is known about the mechanism behind nonlytic exocytosis, but there is evidence that both the fungal and host cells contribute to the process. In this study, we used time-lapse movies of C. neoformans-infected macrophages to delineate the kinetics and quantitative aspects of nonlytic exocytosis. We analyzed approximately 800 macrophages containing intracellular C. neoformans and identified 163 nonlytic exocytosis events that were further characterized into three subcategories: type I (complete emptying of macrophage), type II (partial emptying of macrophage), and type III (cell-to-cell transfer). The majority of type I and II events occurred after several hours of intracellular residence, whereas type III events occurred significantly (P < 0.001) earlier in the course of macrophage infection. Our results show that nonlytic exocytosis is a morphologically and temporally diverse process that occurs relatively rapidly in the course of macrophage infection. PMID:24595144

  14. Radioimmunotherapy of Cryptococcus neoformans spares bystander mammalian cells

    PubMed Central

    Bryan, Ruth A; Jiang, Zewei; Morgenstern, Alfred; Bruchertseifer, Frank; Casadevall, Arturo; Dadachova, Ekaterina

    2013-01-01

    Aim Previously, we showed that radioimmunotherapy (RIT) for cryptococcal infections using radioactively labeled antibodies recognizing the cryptococcal capsule reduced fungal burden and prolonged survival of mice infected with Cryptococcus neoformans. Here, we investigate the effects of RIT on bystander mammalian cells. Materials & methods Heat-killed C. neoformans bound to anticapsular antibodies, unlabeled or labeled with the β-emitter rhenium-188 (16.9-h half-life) or the α-emitter bismuth-213 (46-min half-life), was incubated with macrophage-like J774.16 cells or epithelial-like Chinese hamster ovary cells. Lactate dehydrogenase activity, crystal violet uptake, reduction of tetrazolium dye (2,3)-bis-(2-methoxy-4-nitro-5-sulfenyl)-(2H)-terazolium-5-carboxanilide and nitric oxide production were measured. Results The J774.16 and Chinese hamster ovary cells maintained membrane integrity, viability and metabolic activity following exposure to radiolabeled C. neoformans. Conclusion RIT of C. neoformans is a selective therapy with minimal effects on host cells and these results are consistent with observations that RIT-treated mice with cryptococcal infection lacked RIT-related pathological changes in lungs and brain tissues. PMID:24020737

  15. 21 CFR 866.3165 - Cryptococcus neoformans serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cryptococcus neoformans serological reagents. 866.3165 Section 866.3165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  16. 21 CFR 866.3165 - Cryptococcus neoformans serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Cryptococcus neoformans serological reagents. 866.3165 Section 866.3165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  17. 21 CFR 866.3165 - Cryptococcus neoformans serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Cryptococcus neoformans serological reagents. 866.3165 Section 866.3165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  18. 21 CFR 866.3165 - Cryptococcus neoformans serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Cryptococcus neoformans serological reagents. 866.3165 Section 866.3165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  19. 21 CFR 866.3165 - Cryptococcus neoformans serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Cryptococcus neoformans serological reagents. 866.3165 Section 866.3165 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents §...

  20. Dynamic and Heterogeneous Mutations to Fluconazole Resistance in Cryptococcus neoformans

    PubMed Central

    Xu, Jianping; Onyewu, Chiatogu; Yoell, Heather J.; Ali, Rabia Y.; Vilgalys, Rytas J.; Mitchell, Thomas G.

    2001-01-01

    Infections with the human pathogenic basidiomycetous yeast Cryptococcus neoformans are often treated with fluconazole. Resistance to this antifungal agent has been reported. This study investigated the patterns of mutation to fluconazole resistance in C. neoformans in vitro. The MIC of fluconazole was measured for 21 strains of C. neoformans. The MICs for these 21 strains differed (0.25 to 4.0 μg/ml), but the strains were selected for this study because they exhibited no growth on plates of yeast morphology agar (YMA) containing 8 μg of fluconazole per ml. To determine their mutation rates, six independent cultures from a single original colony were established for each of the 21 strains. Each culture was then spread densely on a YMA plate with 8 μg of fluconazole per ml. A random set of putative mutants was subcultured, and the MIC of fluconazole was determined for each mutant. The 21 strains evinced significant heterogeneity in their mutation rates. The MICs of the putative mutants ranged widely, from their original MIC to 64 μg of fluconazole per ml. However, for this set of 21 strains, there was no significant correlation between the original MIC for a strain and the mutation rate of that strain; the MIC for the mutant could not be predicted from the original MIC. These results suggest that dynamic and heterogeneous mutational processes are involved in generating fluconazole resistance in C. neoformans. PMID:11158735

  1. Temporal Kinetics and Quantitative Analysis of Cryptococcus neoformans Nonlytic Exocytosis

    PubMed Central

    Stukes, Sabriya A.; Cohen, Hillel W.

    2014-01-01

    Cryptococcus neoformans is a facultative intracellular pathogen and the causative agent of cryptococcosis, a disease that is often fatal to those with compromised immune systems. C. neoformans has the capacity to escape phagocytic cells through a process known as nonlytic exocytosis whereby the cryptococcal cell is released from the macrophage into the extracellular environment, leaving both the host and pathogen alive. Little is known about the mechanism behind nonlytic exocytosis, but there is evidence that both the fungal and host cells contribute to the process. In this study, we used time-lapse movies of C. neoformans-infected macrophages to delineate the kinetics and quantitative aspects of nonlytic exocytosis. We analyzed approximately 800 macrophages containing intracellular C. neoformans and identified 163 nonlytic exocytosis events that were further characterized into three subcategories: type I (complete emptying of macrophage), type II (partial emptying of macrophage), and type III (cell-to-cell transfer). The majority of type I and II events occurred after several hours of intracellular residence, whereas type III events occurred significantly (P < 0.001) earlier in the course of macrophage infection. Our results show that nonlytic exocytosis is a morphologically and temporally diverse process that occurs relatively rapidly in the course of macrophage infection. PMID:24595144

  2. Rapid presumptive identification of Cryptococcus neoformans by staphylococcal coagglutination.

    PubMed Central

    Maccani, J E

    1981-01-01

    A coagglutination reagent was prepared by sensitizing the Cowan I strain of Staphylococcus aureus with rabbit immune globulin directed against Cryptococcus neofromans A15 and absorbed with C. laurentii. This reagent was evaluated for its usefulness in differentiating C. neoformans from other yeast colonies rapidly. Antigen-containing extracts were prepared form Sabouraud dextrose agar cultures of 48 C. neoformans, 33 other Cryptococcus species, 21 Candida, 4 Torulopsis, 3 Saccharomyces, and 2 Rhodotorula strains. This was done by suspending a 0.001-ml loopful of colony growth in 0.5 ml of phenolized saline, mixing for 30 s, and then centrifuging. Equal volumes (50 microliters) of coagglutination reagent and yeast extract were mixed within marked circles on a glass slide and then mechanically rotated at 180 rpm for 8 min. Forty-five of the 48 strains of C. neoformans produced strong (3+ to 4+) agglutination, and 3 strains of serotype C produced weak (1+ to 2+) agglutination with the reagent. Other Cryptococcus species which reacted positively were 4 C. albidus subsp. diffluens, 7 C. albidus subsp. albidus, and 2 C. terreus strains; however, false-positive errors in identification were circumvented by performing a supplemental rapid test for nitrate utilization which differentiated these yeasts from C. neoformans. None of the other yeasts tested (including 14 C. laurentii, 2 C. luteolus, and 2 C. uniguttulatus strains) produced any degree of agglutination with the reagent. A commercial cryptococcal latex agglutination reagent (Crypto-Test, Microbiological Associates, Walkersville, Md.) proved less reliable for identifying C. neoformans yeast colonies because of cross-reactions which occurred with all other species of Cryptococcus tested. PMID:7016909

  3. Hybridization probes for conventional DNA fingerprinting used as single primers in the polymerase chain reaction to distinguish strains of Cryptococcus neoformans.

    PubMed Central

    Meyer, W; Mitchell, T G; Freedman, E Z; Vilgalys, R

    1993-01-01

    In conventional DNA fingerprinting, hypervariable and repetitive sequences (minisatellite or microsatellite DNA) are detected with hybridization probes. As demonstrated here, these probes can be used as single primers in the polymerase chain reaction (PCR) to generate individual fingerprints. Several conventional DNA fingerprinting probes were used to prime the PCR, yielding distinctive, hypervariable multifragment profiles for different strains of Cryptococcus neoformans. PCR fingerprinting with the oligonucleotide primers (GTG)5, (GACA)4, and the phage M13 core sequence (GAGGGTGGXGGXTCT), but not with (CA)8 or (CT)8, generated DNA polymorphisms with all 42 strains of C. neoformans investigated. PCR fingerprints produced by priming with (GTG)5, (GACA)4, or the M13 core sequence differentiated the two varieties of C. neoformans, C. neoformans var. neoformans (serotypes A and D) and C. neoformans var. gattii (serotypes B and C). Furthermore, strains of serotypes A, D, and B or C could be distinguished from each other by specific PCR fingerprint patterns. These primers, which also successfully amplified hypervariable DNA segments from other species, provide a convenient method of identification at the species or individual level. Amplification of polymorphic DNA patterns by PCR with these primers offers several advantages over classical DNA fingerprinting techniques, appears to be more reliable than other PCR-based methods for detecting polymorphic DNA, such as analysis of random-amplified polymorphic DNA, and should be applicable to many other organisms. Images PMID:8408543

  4. Cryptococcosis Serotypes Impact Outcome and Provide Evidence of Cryptococcus neoformans Speciation

    PubMed Central

    Desnos-Ollivier, Marie; Patel, Sweta; Raoux-Barbot, Dorothée; Heitman, Joseph

    2015-01-01

    ABSTRACT Cryptococcus neoformans is a human opportunistic fungal pathogen causing severe disseminated meningoencephalitis, mostly in patients with cellular immune defects. This species is divided into three serotypes: A, D, and the AD hybrid. Our objectives were to compare population structures of serotype A and D clinical isolates and to assess whether infections with AD hybrids differ from infections with the other serotypes. For this purpose, we analyzed 483 isolates and the corresponding clinical data from 234 patients enrolled during the CryptoA/D study or the nationwide survey on cryptococcosis in France. Isolates were characterized in terms of ploidy, serotype, mating type, and genotype, utilizing flow cytometry, serotype- and mating type-specific PCR amplifications, and multilocus sequence typing (MLST) methods. Our results suggest that C. neoformans serotypes A and D have different routes of multiplication (primarily clonal expansion versus recombination events for serotype A and serotype D, respectively) and important genomic differences. Cryptococcosis includes a high proportion of proven or probable infections (21.5%) due to a mixture of genotypes, serotypes, and/or ploidies. Multivariate analysis showed that parameters independently associated with failure to achieve cerebrospinal fluid (CSF) sterilization by week 2 were a high serum antigen titer, the lack of flucytosine during induction therapy, and the occurrence of mixed infection, while infections caused by AD hybrids were more likely to be associated with CSF sterilization. Our study provides additional evidence for the possible speciation of C. neoformans var. neoformans and grubii and highlights the importance of careful characterization of causative isolates. PMID:26060271

  5. Distribution of Cryptococcus neoformans in a natural site.

    PubMed Central

    Ruiz, A; Fromtling, R A; Bulmer, G S

    1981-01-01

    Pigeon droppings in a vacant tower were assayed for the number and size of viable cells of Cryptococcus neoformans. The dry, thinly scattered floor debris contained 2.6 x 10(6) viable cells per g--300 times more cells than were cultured from a large, compact pile of pigeon droppings (7.4 x 10(3) cells per g). Aerosols generated from floor debris containing pigeon droppings had an average of 360 viable cells in 31 liters of air; 27 of these cells (7.5%) were 1.1 to 3.3 micrometers in diameter and, therefore, capable of human lung deposition. Environmental factors which may influence the distribution, survival, and proliferation of C. neoformans in nature are discussed. PMID:7012011

  6. Cryptococcus neoformans Host Adaptation: Toward Biological Evidence of Dormancy

    PubMed Central

    Vernel-Pauillac, Frédérique; Sturny-Leclère, Aude; Dromer, Françoise

    2015-01-01

    ABSTRACT Cryptococcosis is an opportunistic infection due to the ubiquitous yeast Cryptococcus neoformans. This yeast interacts closely with innate immune cells, leading to various fates, including fungal persistence within cells, making possible the dissemination of the yeast cells with monocytes via a Trojan horse strategy. In humans, the natural history of the infection begins with primoinfection during childhood, which is followed by dormancy and, in some individuals, reactivation upon immunosuppression. To address the question of dormancy, we studied C. neoformans infection at the macrophage level (in vitro H99-macrophage interaction) and at the organ level in a murine model of cryptococcosis. We analyzed the diversity of yeast adaptation to the host by characterizing several C. neoformans populations with new assays based on flow cytometry (quantitative flow cytometry, multispectral imaging flow cytometry, sorting), microscopy (dynamic imaging), and gene expression analysis. On the basis of parameters of multiplication and stress response, various populations of yeast cells were observed over time in vivo and in vitro. Cell sorting allowed the identification of a subpopulation that was less prone to grow under standard conditions than the other populations, with growth enhanced by the addition of serum. Gene expression analysis revealed that this population had specific metabolic characteristics that could reflect dormancy. Our data suggest that dormant yeast cells could exist in vitro and in vivo. C. neoformans exhibits a huge plasticity and adaptation to hosts that deserves further study. In vitro generation of dormant cells is now the main challenge to overcome the limited number of yeast cells recovered in our models. PMID:25827423

  7. Systematic functional profiling of transcription factor networks in Cryptococcus neoformans

    PubMed Central

    Jung, Kwang-Woo; Yang, Dong-Hoon; Maeng, Shinae; Lee, Kyung-Tae; So, Yee-Seul; Hong, Joohyeon; Choi, Jaeyoung; Byun, Hyo-Jeong; Kim, Hyelim; Bang, Soohyun; Song, Min-Hee; Lee, Jang-Won; Kim, Min Su; Kim, Seo-Young; Ji, Je-Hyun; Park, Goun; Kwon, Hyojeong; Cha, Suyeon; Meyers, Gena Lee; Wang, Li Li; Jang, Jooyoung; Janbon, Guilhem; Adedoyin, Gloria; Kim, Taeyup; Averette, Anna K.; Heitman, Joseph; Cheong, Eunji; Lee, Yong-Hwan; Lee, Yin-Won; Bahn, Yong-Sun

    2015-01-01

    Cryptococcus neoformans causes life-threatening meningoencephalitis in humans, but its overall biological and pathogenic regulatory circuits remain elusive, particularly due to the presence of an evolutionarily divergent set of transcription factors (TFs). Here, we report the construction of a high-quality library of 322 signature-tagged gene-deletion strains for 155 putative TF genes previously predicted using the DNA-binding domain TF database, and examine their in vitro and in vivo phenotypic traits under 32 distinct growth conditions. At least one phenotypic trait is exhibited by 145 out of 155 TF mutants (93%) and ∼85% of them (132/155) are functionally characterized for the first time in this study. The genotypic and phenotypic data for each TF are available in the C. neoformans TF phenome database (http://tf.cryptococcus.org). In conclusion, our phenome-based functional analysis of the C. neoformans TF mutant library provides key insights into transcriptional networks of basidiomycetous fungi and human fungal pathogens. PMID:25849373

  8. The Cryptococcus neoformans Capsule: a Sword and a Shield

    PubMed Central

    O'Meara, Teresa R.

    2012-01-01

    Summary: The human fungal pathogen Cryptococcus neoformans is characterized by its ability to induce a distinct polysaccharide capsule in response to a number of host-specific environmental stimuli. The induction of capsule is a complex biological process encompassing regulation at multiple steps, including the biosynthesis, transport, and maintenance of the polysaccharide at the cell surface. By precisely regulating the composition of its cell surface and secreted polysaccharides, C. neoformans has developed intricate ways to establish chronic infection and dormancy in the human host. The plasticity of the capsule structure in response to various host conditions also underscores the complex relationship between host and parasite. Much of this precise regulation of capsule is achieved through the transcriptional responses of multiple conserved signaling pathways that have been coopted to regulate this C. neoformans-specific virulence-associated phenotype. This review focuses on specific host stimuli that trigger the activation of the signal transduction cascades and on the downstream transcriptional responses that are required for robust encapsulation around the cell. PMID:22763631

  9. Histone deacetylases inhibitors effects on Cryptococcus neoformans major virulence phenotypes

    PubMed Central

    Brandão, Fabiana AS; Derengowski, Lorena S; Albuquerque, Patrícia; Nicola, André M; Silva-Pereira, Ildinete; Poças-Fonseca, Marcio J

    2015-01-01

    Cryptococcus neoformans undergoes phenotypical changes during host infection in order to promote persistence and survival. Studies have demonstrated that such adaptations require alterations in gene transcription networks by distinct mechanisms. Drugs such as the histone deacetylases inhibitors (HDACi) Sodium Butyrate (NaBut) and Trichostatin A (TSA) can alter the chromatin conformation and have been used to modulate epigenetic states in the treatment of diseases such as cancer. In this work, we have studied the effect of NaBut and TSA on the expression of C. neoformans major virulence phenotypes and on the survival rate of an animal model infected with drugs-treated yeasts. Both drugs affected fungal growth at 37°C more intensely than at 30°C; nonetheless, drugs did not affect cell viability at the concentrations we studied. HDACi also provoked the reduction of the fungal capsule expansion. Phospholipases enzyme activity decreased; mating process and melanin synthesis were also affected by both inhibitors. NaBut led to an increase in the population of cells in G2/M. Treated yeast cells, which were washed in order to remove the drugs from the culture medium prior to the inoculation in the Galleria mellonela infection model, did not cause significant difference at the host survival curve when compared to non-treated cells. Overall, NaBut effects on the impairment of C. neoformans main virulence factors were more intense and stable than the TSA effects. PMID:26103530

  10. The capsule of the fungal pathogen Cryptococcus neoformans

    PubMed Central

    Zaragoza, Oscar; Rodrigues, Marcio L.; De Jesus, Magdia; Frases, Susana; Dadachova, Ekaterina; Casadevall, Arturo

    2009-01-01

    The capsule of the fungal pathogen Cryptococcus neoformans has been studied extensively in recent decades, and a large body of information is now available to the scientific community. Well-known aspects of the capsule include its structure, antigenic properties and its function as a virulence factor. The capsule is composed primarily of two polysaccharides, glucuronoxylomannan (GXM) and galactoxylomannan (GalXM), in addition to a smaller proportion of mannoproteins (MP). Most of the studies on the composition of the capsule have focused on GXM, which comprises more than 90% of the capsule's polysaccharide mass. It is GalXM, however, that is of particular scientific interest because of its immunological properties. The molecular structure of these polysaccharides is very complex and has not yet been fully elucidated. Both GXM and GalXM are high molecular mass polymers with the mass of GXM equaling roughly 10 times that of GalXM. Recent findings suggest, however, that the actual Mw might be different to what it has traditionally been thought to be. In addition to their structural roles in the polysaccharide capsule, these molecules have been associated with many deleterious effects on the immune response. Capsular components are therefore considered key virulence determinants in Cryptococcus neoformans, which has motivated their use in vaccines and made them targets for monoclonal antibody treatments. In this review we will provide an update on the current knowledge of the C. neoformans capsule, covering aspects related to its structure, synthesis, and particularly, its role as a virulence factor. PMID:19426855

  11. Antifungal susceptibility of clinical and environmental Cryptococcus neoformans and Cryptococcus gattii isolates in Jabalpur, a city of Madhya Pradesh in Central India

    PubMed Central

    Gutch, Ruchi Sethi; Nawange, Shesh Rao; Singh, Shankar Mohan; Yadu, Ruchika; Tiwari, Aditi; Gumasta, Richa; Kavishwar, Arvind

    2015-01-01

    In this study, we present antifungal susceptibility data of clinical and environmental isolates of Central Indian Cryptococcus neoformans (Serotype A, n = 8 and n = 50 respectively) and Cryptococcus gattii (Serotype B, n = 01 and n = 04 respectively). Susceptibilities to fluconazole, itraconazole and ketoconazole were determined by using NCCLS broth micro-dilution methodology. The total number of resistant strains for fluconazole in case of C. neoformans and C. gattii showed a significant difference by using chi-square test (p < 0.05*), while considering fisher's exact p value was nonsignificant (p > 0.05). However, the total number of resistant strains for itraconazole and ketoconazole was not found statistically significant. A comparison of geometric means of clinical and environmental strains of C. gattii and C. neoformans was not found statistically significant using student ‘t’ test (p value > 0.05 NS). Though less, the antifungal data obtained in this study suggests that primary resistance among environmental and clinical isolates of C. neoformans and C. gattii against tested antifungal was present and C. gattii comparatively was less susceptible than C. neoformans var. grubii isolates to fluconazole than to itraconazole and ketoconazole. A continuous surveillance of antifungal susceptibility of clinical and environmental isolates of C. neoformans and C. gattii is desirable to monitor the emergence of any resistant strains for better management of cryptococcosis patients. PMID:26691471

  12. Decaying wood in tree trunk hollows as a natural substrate for Cryptococcus neoformans and other yeast-like fungi of clinical interest.

    PubMed

    Randhawa, H S; Mussa, A Y; Khan, Z U

    2001-01-01

    The occurrence of Cryptococcus neoformans var. neoformans and other yeast-like fungi of clinical interest in decaying wood inside tree trunk hollows, bark and other plant materials is reported. The var. neoformans was isolated from 3 of 45 (6.6%) wood and one of 390 Eucalyptus bark samples. Two of the positive wood samples came from a tree trunk hollow of Butea monosperma (Family: Papilionaceae) growing in Roshan Ara Garden, Old Delhi whereas the third was from a trunk hollow of Tamarindus indica (Family: Papilionaceae) growing outside of Talkatora Garden, New Delhi. The solitary positive Eucalyptus bark sample originated from Amritsar. The isolations of var. neoformans from decaying wood inside trunk hollows of B. monosperma and T indica constitute the first record of the natural occurrence of this pathogen in association with these trees. The observation reinforces the recent evidence for decaying wood inside trunk hollows of some trees to be a new natural habitat of the variety neoformans. Besides, in consonance with their essentially saprobic character, a number of other yeast-like fungi were sporadically isolated. This includes, Cryptoccus laurentii, Cryptococcus albidus, Candida lusitaniae, C. guilliermondii, C. krusei, C. tropicalis, C. zeylanoides, Trichosporon cutaneum, Rhodotorula mucilaginosa, R. glutinis, Geotrichum capitatum, G. klebahnii and Sporobolomyces salmonicolor. Cryptococcus neoformans var. gattii was not found in any of the 702 samples of plant materials, including the bark and detritus of Eucalyptus camaldulensis and E. tereticornis trees. A more extensive environmental survey, covering divergent climatic regions, is warranted to identify the natural reservoirs of var. gattii in India. PMID:11554580

  13. Eucalyptus Tree: A Potential Source of Cryptococcus neoformans in Egyptian Environment

    PubMed Central

    Hamza, Dalia; Elhelw, Rehab; Refai, Mohamed

    2016-01-01

    In Egypt, the River Red Gum (Eucalyptus camaldulensis) is a well-known tree and is highly appreciated by the rural and urban dwellers. The role of Eucalyptus trees in the ecology of Cryptococcus neoformans is documented worldwide. The aim of this survey was to show the prevalence of C. neoformans during the flowering season of E. camaldulensis at the Delta region in Egypt. Three hundred and eleven samples out of two hundred Eucalyptus trees, including leaves, flowers, and woody trunks, were collected from four governorates in the Delta region. Thirteen isolates of C. neoformans were recovered from Eucalyptus tree samples (4.2%). Molecular identification of C. neoformans was done by capsular gene specific primer CAP64 and serotype identification was done depending on LAC1 gene. This study represents an update on the ecology of C. neoformans associated with Eucalyptus tree in Egyptian environment. PMID:26884765

  14. Eucalyptus Tree: A Potential Source of Cryptococcus neoformans in Egyptian Environment.

    PubMed

    Elhariri, Mahmoud; Hamza, Dalia; Elhelw, Rehab; Refai, Mohamed

    2016-01-01

    In Egypt, the River Red Gum (Eucalyptus camaldulensis) is a well-known tree and is highly appreciated by the rural and urban dwellers. The role of Eucalyptus trees in the ecology of Cryptococcus neoformans is documented worldwide. The aim of this survey was to show the prevalence of C. neoformans during the flowering season of E. camaldulensis at the Delta region in Egypt. Three hundred and eleven samples out of two hundred Eucalyptus trees, including leaves, flowers, and woody trunks, were collected from four governorates in the Delta region. Thirteen isolates of C. neoformans were recovered from Eucalyptus tree samples (4.2%). Molecular identification of C. neoformans was done by capsular gene specific primer CAP64 and serotype identification was done depending on LAC1 gene. This study represents an update on the ecology of C. neoformans associated with Eucalyptus tree in Egyptian environment. PMID:26884765

  15. Morphotype-specific effector functions of Cryptococcus neoformans PUM1

    PubMed Central

    Kaur, Jan Naseer; Panepinto, John C.

    2016-01-01

    The basidiomycete fungal pathogen Cryptococcus neoformans requires the PUF protein, Pum1, for hyphal morphogenesis during sexual development. In this study we found that Pum1 was auto-repressive under growth as yeast, but that auto-repression was relieved during filamentous growth through utilization of an alternative transcription start site driven by the master filamentation regulator Znf2. In addition, Pum1 was required to stabilize the ZNF2 mRNA through an indirect mechanism suggesting that Znf2 and Pum1 each positively regulate the expression of the other to achieve the filamentous morphotype required for sexual development in Cryptococcus. PMID:27008977

  16. Antimicrobial activity of Hymenaea martiana towards dermatophytes and Cryptococcus neoformans.

    PubMed

    de Souza, Ana Cristina Machado; Kato, Lucilia; da Silva, Cleuza Conceição; Cidade, Amanda Feitosa; de Oliveira, Cecilia Maria Alves; Silva, Maria do Rosário Rodrigues

    2010-11-01

    The biological activity of crude extract and fractions of Hymenaea martiana was evaluated against a panel of human pathogenic fungi. The crude extracts and hydroalcoholic fractions (E) showed a high activity against Cryptococcus neoformans species complex isolates with MICs between 2 and 64 μg ml(-1). The methanolic (C) and butanolic (D) fractions were the most active against Trichopyton rubrum, Trichopyton mentagrophytes and Microsporum canis with MICs between 8 and 256 μg ml(-1). None of the extracts was active against the yeast Malassezia furfur, Malassezia obtusa and Malassezia sympodialis. PMID:19563478

  17. Multicenter Comparison of Three Different Analytical Systems for Evaluation of DNA Banding Patterns from Cryptococcus neoformans

    PubMed Central

    Cardinali, Gianluigi; Martini, Alessandro; Preziosi, Roberta; Bistoni, Francesco; Baldelli, Franco

    2002-01-01

    The enormous improvement of molecular typing techniques for epidemiological and clinical studies has not always been matched by an equivalent effort in applying optimal criteria for the analysis of both phenotypic and molecular data. In spite of the availability of a large collection of statistical and phylogenetic methods, the vast majority of commercial packages are limited by using only the unweighted pair group method with arithmetic mean algorithm to construct trees and by considering electrophoretic pattern only as migration distances. The latter method has serious drawbacks when different runs (separate gels) of the same molecular analysis are to be compared. This work presents a multicenter comparison of three different systems of banding pattern analysis on random amplified polymorphic DNA, (GACA)4, and contour-clamped homogeneous electric field patterns from strains of Cryptococcus neoformans var. neoformans isolated in different clinical and geographical situations and a standard Saccharomyces cerevisiae strain employed as an outgroup. The systems considered were evaluated for their actual ability to(i) recognize identities, (ii) define complete differences (i.e., the ability to place S. cerevisiae out of the C. neoformans cluster), and (iii) estimate the extent of similarity among different strains. The ability to cluster strains according to the patient from which they were isolated was also evaluated. The results indicate that different algorithms do indeed produce divergent trees, both in overall topology and in clustering of individual strains, thus suggesting that care must be taken by individual investigators to use the most appropriate procedure and by the scientific community in defining a consensus system. PMID:12037071

  18. Cryptococcus neoformans: paradigm for the role of antibody immunity against fungi?

    PubMed

    Pirofski, L A; Casadevall, A

    1996-08-01

    Cryptococcus neoformans is an encapsulated fungus that is a frequent cause of life-threatening infections in patients with AIDS. C. neoformans has many similarities with encapsulated bacteria such as S. pneumoniae and H. influenzae for which antibody immunity is important in protection. However the role of antibody immunity in protection against C. neoformans has been controversial. Experiments with polyclonal sera have produced conflicting evidence for and against the importance of antibody immunity in host defense. Experiments with monoclonal antibodies (mAb) to the C. neoformans capsular polysaccharide (CPS) have revealed the existence of protective, non-protective and disease-enhancing mAbs, suggesting that the divergent results obtained with polyclonal preparations may be a result of relative proportion of protective and non-protective antibodies in immune sera. Administration of protective mAbs can prolong survival, decrease organ fungal burden, and reduce serum polysaccharide antigen. In vitro experiments suggests that protective mAbs modify the course of infection by enhancing effector cell function against C. neoformans. Addition of mAb to antifungal drugs enhances their efficacy against C. neoformans in vivo and in vitro. Human-mouse chimeric antibodies with activity against C. neoformans have been constructed. A highly immunogenic capsular polysaccharide-protein vaccine has been synthesized that elicits protective antibodies in mice. Antibody immunity elicited by conjugate vaccines or provided by passive administration may be useful in the prevention treatment of human cryptococcal infections. PMID:8899968

  19. STAT1 signaling within macrophages is required for antifungal activity against Cryptococcus neoformans.

    PubMed

    Leopold Wager, Chrissy M; Hole, Camaron R; Wozniak, Karen L; Olszewski, Michal A; Mueller, Mathias; Wormley, Floyd L

    2015-12-01

    Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an opportunistic fungal pathogen that primarily affects AIDS patients and patients undergoing immunosuppressive therapy. In immunocompromised individuals, C. neoformans can lead to life-threatening meningoencephalitis. Studies using a virulent strain of C. neoformans engineered to produce gamma interferon (IFN-γ), denoted H99γ, demonstrated that protection against pulmonary C. neoformans infection is associated with the generation of a T helper 1 (Th1)-type immune response and signal transducer and activator of transcription 1 (STAT1)-mediated classical (M1) macrophage activation. However, the critical mechanism by which M1 macrophages mediate their anti-C. neoformans activity remains unknown. The current studies demonstrate that infection with C. neoformans strain H99γ in mice with macrophage-specific STAT1 ablation resulted in severely increased inflammation of the pulmonary tissue, a dysregulated Th1/Th2-type immune response, increased fungal burden, deficient M1 macrophage activation, and loss of protection. STAT1-deficient macrophages produced significantly less nitric oxide (NO) than STAT1-sufficient macrophages, correlating with an inability to control intracellular cryptococcal proliferation, even in the presence of reactive oxygen species (ROS). Furthermore, macrophages from inducible nitric oxide synthase knockout mice, which had intact ROS production, were deficient in anticryptococcal activity. These data indicate that STAT1 activation within macrophages is required for M1 macrophage activation and anti-C. neoformans activity via the production of NO. PMID:26351277

  20. Phospholipids Trigger Cryptococcus neoformans Capsular Enlargement during Interactions with Amoebae and Macrophages

    PubMed Central

    Chrisman, Cara J.; Albuquerque, Patricia; Guimaraes, Allan J.; Nieves, Edward; Casadevall, Arturo

    2011-01-01

    A remarkable aspect of the interaction of Cryptococcus neoformans with mammalian hosts is a consistent increase in capsule volume. Given that many aspects of the interaction of C. neoformans with macrophages are also observed with amoebae, we hypothesized that the capsule enlargement phenomenon also had a protozoan parallel. Incubation of C. neoformans with Acanthamoeba castellanii resulted in C. neoformans capsular enlargement. The phenomenon required contact between fungal and protozoan cells but did not require amoeba viability. Analysis of amoebae extracts showed that the likely stimuli for capsule enlargement were protozoan polar lipids. Extracts from macrophages and mammalian serum also triggered cryptococcal capsular enlargement. C. neoformans capsule enlargement required expression of fungal phospholipase B, but not phospholipase C. Purified phospholipids, in particular, phosphatidylcholine, and derived molecules triggered capsular enlargement with the subsequent formation of giant cells. These results implicate phospholipids as a trigger for both C. neoformans capsule enlargement in vivo and exopolysaccharide production. The observation that the incubation of C. neoformans with phospholipids led to the formation of giant cells provides the means to generate these enigmatic cells in vitro. Protozoan- or mammalian-derived polar lipids could represent a danger signal for C. neoformans that triggers capsular enlargement as a non-specific defense mechanism against potential predatory cells. Hence, phospholipids are the first host-derived molecules identified to trigger capsular enlargement. The parallels apparent in the capsular response of C. neoformans to both amoebae and macrophages provide additional support for the notion that certain aspects of cryptococcal virulence emerged as a consequence of environmental interactions with other microorganisms such as protists. PMID:21637814

  1. Lipophilic dye staining of Cryptococcus neoformans extracellular vesicles and capsule.

    PubMed

    Nicola, André Moraes; Frases, Susana; Casadevall, Arturo

    2009-09-01

    Cryptococcus neoformans is an encapsulated yeast that causes systemic mycosis in immunosuppressed individuals. Recent studies have determined that this fungus produces vesicles that are released to the extracellular environment both in vivo and in vitro. These vesicles contain assorted cargo that includes several molecules associated with virulence and implicated in host-pathogen interactions, such as capsular polysaccharides, laccase, urease, and other proteins. To date, visualization of extracellular vesicles has relied on transmission electron microscopy, a time-consuming technique. In this work we report the use of fluorescent membrane tracers to stain lipophilic structures in cryptococcal culture supernatants and capsules. Two dialkylcarbocyanine probes with different spectral characteristics were used to visualize purified vesicles by fluorescence microscopy and flow cytometry. Dual staining of vesicles with dialkylcarbocyanine and RNA-selective nucleic acid dyes suggested that a fraction of the vesicle population carried RNA. Use of these dyes to stain whole cells, however, was hampered by their possible direct binding to capsular polysaccharide. A fluorescent phospholipid was used as additional membrane tracer to stain whole cells, revealing punctate structures on the edge of the capsule which are consistent with vesicular trafficking. Lipophilic dyes provide new tools for the study of fungal extracellular vesicles and their content. The finding of hydrophobic regions in the capsule of C. neoformans adds to the growing evidence for a structurally complex structure composed of polysaccharide and nonpolysaccharide components. PMID:19465562

  2. Extensive Genetic Diversity within the Dutch Clinical Cryptococcus neoformans Population

    PubMed Central

    Hagen, Ferry; Illnait-Zaragozí, María-Teresa; Meis, Jacques F.; Chew, William H. M.; Curfs-Breuker, Ilse; Mouton, Johan W.; Hoepelman, Andy I. M.; Spanjaard, Lodewijk; Verweij, Paul E.; Kampinga, Greetje A.; Kuijper, Ed J.; Klaassen, Corné H. W.

    2012-01-01

    A set of 300 Dutch Cryptococcus neoformans isolates, obtained from 237 patients during 1977 to 2007, was investigated by determining the mating type, serotype, and AFLP and microsatellite genotype and susceptibility to seven antifungal compounds. Almost half of the studied cases were from HIV-infected patients, followed by a patient group of individuals with other underlying diseases and immunocompetent individuals. The majority of the isolates were mating type α and serotype A, followed by αD isolates and other minor categories. The most frequently observed genotype was AFLP1, distantly followed by AFLP2 and AFLP3. Microsatellite typing revealed a high genetic diversity among serotype A isolates but a lower diversity within the serotype D set of isolates. One patient was infected by multiple AFLP genotypes. Fluconazole and flucytosine had the highest geometric mean MICs of 2.9 and 3.5 μg/ml, respectively, while amphotericin B (0.24 μg/ml), itraconazole (0.08 μg/ml), voriconazole (0.07 μg/ml), posaconazole (0.06 μg/ml), and isavuconazole (0.03 μg/ml) had much lower geometric mean MICs. One isolate had a high flucytosine MIC (>64 μg/ml), while decreased susceptibility (≥16 μg/ml) for flucytosine and fluconazole was found in 9 and 10 C. neoformans isolates, respectively. PMID:22442325

  3. Transcriptional control of sexual development in Cryptococcus neoformans.

    PubMed

    Mead, Matthew E; Hull, Christina M

    2016-05-01

    Developmental processes are essential for the normal life cycles of many pathogenic fungi, and they can facilitate survival in challenging environments, including the human host. Sexual development of the human fungal pathogen Cryptococcus neoformans not only produces infectious particles (spores) but has also enabled the evolution of new disease-related traits such as drug resistance. Transcription factor networks are essential to the development and pathogenesis of C. neoformans, and a variety of sequence-specific DNA-binding proteins control both key developmental transitions and virulence by regulating the expression of their target genes. In this review we discuss the roles of known transcription factors that harbor important connections to both development and virulence. Recent studies of these transcription factors have identified a common theme in which metabolic, stress, and other responses that are required for sexual development appear to have been co-opted for survival in the human host, thus facilitating pathogenesis. Future work elucidating the connection between development and pathogenesis will provide vital insights into the evolution of complex traits in eukaryotes as well as mechanisms that may be used to combat fungal pathogens. PMID:27095452

  4. Mitochondrial Protein Nfu1 Influences Homeostasis of Essential Metals in the Human Fungal Pathogen Cryptococcus neoformans

    PubMed Central

    Kim, Jeongmi; Park, Minji; Do, Eunsoo

    2014-01-01

    Mitochondrial protein Nfu1 plays an important role in the assembly of mitochondrial Fe-S clusters and intracellular iron homeostasis in the model yeast Saccharomyces cerevisiae. In this study, we identified the Nfu1 ortholog in the human fungal pathogen Cryptococcus neoformans. Our data showed that C. neoformans Nfu1 localized in the mitochondria and influenced homeostasis of essential metals such as iron, copper and manganese. Marked growth defects were observed in the mutant lacking NFU1, which suggests a critical role of Nfu1 in Fe-S cluster biosynthesis and intracellular metal homeostasis in C. neoformans. PMID:25606020

  5. The Cryptococcus neoformans capsule: lessons from the use of optical tweezers and other biophysical tools

    PubMed Central

    Pontes, Bruno; Frases, Susana

    2015-01-01

    The fungal pathogen Cryptococcus neoformans causes life-threatening infections in immunocompromised individuals, representing one of the leading causes of morbidity and mortality in AIDS patients. The main virulence factor of C. neoformans is the polysaccharide capsule; however, many fundamental aspects of capsule structure and function remain poorly understood. Recently, important capsule properties were uncovered using optical tweezers and other biophysical techniques, including dynamic and static light scattering, zeta potential and viscosity analysis. This review provides an overview of the latest findings in this emerging field, explaining the impact of these findings on our understanding of C. neoformans biology and resistance to host immune defenses. PMID:26157436

  6. Role of Mannoprotein in Induction and Regulation of Immunity to Cryptococcus neoformans

    PubMed Central

    Pietrella, Donatella; Cherniak, Robert; Strappini, Carla; Perito, Stefano; Mosci, Paolo; Bistoni, Francesco; Vecchiarelli, Anna

    2001-01-01

    Our previous observations showed that mannoprotein (MP) induces early and massive production of interleukin-12 (IL-12) in vitro. This study was designed to investigate whether this phenomenon could be applied in vivo and to determine the biological significance of MP in Cryptococcus neoformans infection. The results reported here show that MP treatment induces IL-12 secretion by splenic macrophages and IL-12 p40 mRNA in the brain. During C. neoformans infection, MP reinforced IL-12 and IFN-γ secretion that coincided with enhanced antifungal activity of natural effector cells, early resolution of the inflammatory process, and clearance of fungal load from the brain. These studies show that MP is a key inflammatory mediator that induces a protective immune response against C. neoformans infection. This information can be used to facilitate the design of a rational approach to manipulate the immune response to C. neoformans. PMID:11292692

  7. Genotypes of Clinical and Environmental Isolates of Cryptococcus neoformans and Cryptococcus gattii in Korea

    PubMed Central

    Park, So Hae; Choi, Seok Cheol; Lee, Kyung Won; Kim, Mi-Na

    2015-01-01

    Multilocus sequence typing analysis was applied to determine the genotypes of 147 (137 clinical and 10 environmental) Cryptococcus neoformans and three clinical Cryptococcus gattii isolates from 1993 to 2014 in Korea. Among the 137 clinical isolates of C. neoformans, the most prevalent genotype was ST5 (n = 131), followed by ST31 (n = 5) and ST127 (n = 1). Three C. gattii strains were identified as ST57, ST7, and ST113. All environmental isolates were identified as C. neoformans with two genotypes, ST5 (n = 7) and ST31 (n = 3). Our results show that C. neoformans isolates in Korea are genetically homogeneous, and represent a close genetic relationship between clinical and environmental isolates. PMID:26539057

  8. Aging as an emergent factor that contributes to phenotypic variation in Cryptococcus neoformans.

    PubMed

    Bouklas, Tejas; Fries, Bettina C

    2015-05-01

    Cryptococcus neoformans, similar to other eukaryotes, undergoes replicative aging. Replicative life spans have been determined for clinical C. neoformans strains, and although they are a reproducible trait, life spans vary considerably among strains. C. neoformans has been proposed as an ideal model organism to investigate the contribution of replicative aging in a fungal pathogen population to emerging phenotypic variation during chronic cryptococcal infections. C. neoformans cells of advanced generational age manifest a distinct phenotype; specifically, a larger cell size, a thicker cell wall, drug resistance, as well as resistance to hydrogen peroxide-mediated killing. Consequently, old cells are selected in the host environment during chronic infection and aging could be an unanticipated mechanism of pathogen adaptation that contributes to persistent disease. Aging as a natural process of phenotypic variation should be further studied as it likely is also relevant for other eukaryotic pathogen populations that undergo asymmetric replicative aging. PMID:25307541

  9. Magnesium Ion Acts as a Signal for Capsule Induction in Cryptococcus neoformans

    PubMed Central

    Rathore, Sudarshan S.; Raman, Thiagarajan; Ramakrishnan, Jayapradha

    2016-01-01

    Cryptococcal meningitis caused by Cryptococcus neoformans, is a common opportunistic neural infection in immunocompromised individuals. Cryptococcus meningitis is associated with fungal burden with larger capsule size in cerebrospinal fluid (CSF). To understand the role of CSF constituents in capsule enlargement, we have evaluated the effect of artificial CSF on capsule induction in comparison with various other capsule inducing media. Two different strains of C. neoformans, an environmental and a clinical isolates were used in the present study. While comparing the various capsule inducing media for the two different strains of C. neoformans, it was observed that the capsule growth was significantly increased when grown in artificial CSF at pH 5.5, temperature 34°C for ATCC C. neoformans and 37°C for Clinical C. neoformans and with an incubation period of 72 h. In addition, artificial CSF supports biofilm formation in C. neoformans. While investigating the individual components of artificial CSF, we found that Mg2+ ions influence the capsule growth in both environmental and clinical strains of C. neoformans. To confirm our results we studied the expression of four major CAP genes namely, CAP10, CAP59, CAP60, and CAP64 in various capsule inducing media and in different concentrations of Mg2+ and Ca2+. Our results on gene expression suggest that, Mg2+ does have an effect on CAP gene expression, which are important for capsule biosynthesis and virulence. Our findings on the role of Mg2+ ion as a signal for capsule induction will promote a way to elucidate the control mechanisms for capsule biosynthesis in C. neoformans. PMID:27014245

  10. Genetic study of oxygen resistance and melanization in Cryptococcus neoformans.

    PubMed Central

    Emery, H S; Shelburne, C P; Bowman, J P; Fallon, P G; Schulz, C A; Jacobson, E S

    1994-01-01

    Genetic analysis of oxygen-sensitive mutants of Cryptococcus neoformans revealed two loci (oxy1 and oxy2) linking hyperoxia sensitivity to production of melanin, a known virulence factor. Hyperoxia-sensitive strain 562 (oxy1 oxy2) is albino and avirulent. oxy2-defective strains lacking the oxy1 defect are melanin deficient but show normal hyperoxia resistance. Mutants defective at three additional mapped melanin loci fail to show hyperoxia sensitivity in the oxy1 background. Revertants of strain 562, which regain the ability to synthesize melanin by mutation at suppressor sites unlinked to oxy2, retain the oxygen sensitivity conferred by their oxy1 and oxy2 defects. These data identify the melanin gene oxy2 as unique in its association of hyperoxia resistance and melanization. Images PMID:7960156

  11. “Virulence Mechanisms and Cryptococcus neoformans pathogenesis”

    PubMed Central

    Alspaugh, J. Andrew

    2014-01-01

    The human fungal pathogen Cryptococcus neoformans is able to rapidly and effectively adapt to varying conditions, favoring its survival in the environment and in the infected host. Many microbial phenotypes have been specifically correlated with virulence in this opportunistic pathogen, such as capsule production, melanin formation, and the secretion of various proteins. Additionally, cellular features such as the cell wall and morphogenesis play important roles in the interaction of this fungus with host immune recognition and response pathways. Survival in the face of host stress also requires maintaining RNA/DNA integrity. Additionally, aging and senescence of the fungal cells determines resistance to host-derived stresses. New mechanisms regulating the expression of these virulence-associated phenotypes have been recently explored. Importantly, human clinical studies are now confirming the roles of specific microbial factors in human infections. PMID:25256589

  12. Immunity to Cryptococcus neoformans and C. gattii during cryptococcosis

    PubMed Central

    Gibson, Josie F.; Johnston, Simon A.

    2015-01-01

    The vast majority of infection with cryptococcal species occurs with Cryptococcus neoformans in the severely immunocompromised. A significant exception to this is the infections of those with apparently normal immune systems by Cryptococcus gattii. Susceptibility to cryptococcosis can be broadly categorised as a defect in adaptive immune responses, especially in T cell immunity. However, innate immune cells such as macrophages play a key role and are likely the primary effector cell in the killing and ultimate clearance of cryptococcal infection. In this review we discuss the current state of our understanding of how the immune system responds to cryptococcal infection in health and disease, with reference to the work communicated at the 9th International Conference on Cryptococcus and Cryptococcosis (ICCC9). We have focussed on cell mediated responses, particularly early in infection, but with the aim of presenting a broad overview of our understanding of immunity to cryptococcal infection, highlighting some recent advances and offering some perspectives on future directions. PMID:25498576

  13. Occurrence and susceptibilities to disinfectants of Cryptococcus neoformans in fecal droppings from pigeons in Bangkok, Thailand.

    PubMed

    Krangvichain, Prathomporn; Niyomtham, Waree; Prapasarakul, Nuvee

    2016-03-01

    Cryptococcus neoformans is an opportunistic pathogenic yeast that causes meningoencephalitis and deep skin dermatitis in humans and animals. A hygienic strategy using disinfectants on environmental samples can reduce the risk to the public. The objectives were to survey the distribution of C. neoformans in pigeon fecal droppings collected in 11 districts in Bangkok during 2011-2012 and to evaluate the efficacy of three commercial disinfectant products (based on potassium monopersulfate, sodium hypochlorite and quaternary ammonium compounds, respectively). These were evaluated against pure C. neoformans and yeasts resuspended in sterile pigeon feces using the dilution-neutralization method [Europäische NORM (EN) 1656]. In total, 18 of 164 (11%) samples were positive for C. neoformans. These came from only three of the 11 districts, with a prevalence of between 13-56%. Using multiplex PCR, serotype A was the sole group found. For all disinfectants, C. neoformans mixed in feces was tolerated at a higher dose and time exposure than pure isolates. The most effective disinfectant in this study was a 0.12% quaternary ammonium compound that could rapidly eradicate the yeasts mixed in feces. This finding highlights the occurrence and distribution of C. neoformans in the capital city of Thailand and the need to prolong the duration of exposure to disinfectants with pigeon feces. PMID:26596636

  14. Cryptococcus neoformans Mates on Pigeon Guano: Implications for the Realized Ecological Niche and Globalization▿

    PubMed Central

    Nielsen, Kirsten; De Obaldia, Anna L.; Heitman, Joseph

    2007-01-01

    The ecological niche that a species can occupy is determined by its resource requirements and the physical conditions necessary for survival. The niche to which an organism is most highly adapted is the realized niche, whereas the complete range of habitats that an organism can occupy represents the fundamental niche. The growth and development of Cryptococcus neoformans and Cryptococcus gattii on pigeon guano were examined to determine whether these two species occupy the same or different ecological niches. C. neoformans is a cosmopolitan pathogenic yeast that infects predominantly immunocompromised individuals, exists in two varieties (grubii [serotype A] and neoformans [serotype D]), and is commonly isolated from pigeon guano worldwide. By contrast, C. gattii often infects immunocompetent individuals and is associated with geographically restricted environments, most notably, eucalyptus trees. Pigeon guano supported the growth of both species, and a brown pigment related to melanin, a key virulence factor, was produced. C. neoformans exhibited prolific mating on pigeon guano, whereas C. gattii did not. The observations that C. neoformans completes the life cycle on pigeon guano but that C. gattii does not indicates that pigeon guano could represent the realized ecological niche for C. neoformans. Because C. gattii grows on pigeon guano but cannot sexually reproduce, pigeon guano represents a fundamental but not a realized niche for C. gattii. Based on these studies, we hypothesize that an ancestral Cryptococcus strain gained the ability to sexually reproduce in pigeon guano and then swept the globe. PMID:17449657

  15. Occurrence and susceptibilities to disinfectants of Cryptococcus neoformans in fecal droppings from pigeons in Bangkok, Thailand

    PubMed Central

    KRANGVICHAIN, Prathomporn; NIYOMTHAM, Waree; PRAPASARAKUL, Nuvee

    2015-01-01

    Cryptococcus neoformans is an opportunistic pathogenic yeast that causes meningoencephalitis and deep skin dermatitis in humans and animals. A hygienic strategy using disinfectants on environmental samples can reduce the risk to the public. The objectives were to survey the distribution of C. neoformans in pigeon fecal droppings collected in 11 districts in Bangkok during 2011–2012 and to evaluate the efficacy of three commercial disinfectant products (based on potassium monopersulfate, sodium hypochlorite and quaternary ammonium compounds, respectively). These were evaluated against pure C. neoformans and yeasts resuspended in sterile pigeon feces using the dilution-neutralization method [Europäische NORM (EN) 1656]. In total, 18 of 164 (11%) samples were positive for C. neoformans. These came from only three of the 11 districts, with a prevalence of between 13–56%. Using multiplex PCR, serotype A was the sole group found. For all disinfectants, C. neoformans mixed in feces was tolerated at a higher dose and time exposure than pure isolates. The most effective disinfectant in this study was a 0.12% quaternary ammonium compound that could rapidly eradicate the yeasts mixed in feces. This finding highlights the occurrence and distribution of C. neoformans in the capital city of Thailand and the need to prolong the duration of exposure to disinfectants with pigeon feces. PMID:26596636

  16. Production of tumor necrosis factor alpha in human leukocytes stimulated by Cryptococcus neoformans.

    PubMed Central

    Levitz, S M; Tabuni, A; Kornfeld, H; Reardon, C C; Golenbock, D T

    1994-01-01

    Tumor necrosis factor alpha (TNF-alpha) is a key mediator of inflammation and may promote human immunodeficiency virus replication in latently infected cells. Since cryptococcosis often is associated with aberrations in the host inflammatory response and occurs preferentially in persons with AIDS, we defined the conditions under which human leukocytes produce TNF-alpha when stimulated by Cryptococcus neoformans. Peripheral blood mononuclear cells (PBMC) produced comparable amounts of TNF-alpha following stimulation with C. neoformans and lipopolysaccharide. Detectable TNF-alpha release in response to C. neoformans occurred only when fungi with small-sized capsules were used and complement-sufficient serum was added. Fractionation of PBMC established that monocytes were the predominant source of TNF-alpha. TNF-alpha gene expression and release occurred significantly later in PBMC stimulated with C. neoformans than in PBMC stimulated with LPS. C. neoformans was also a potent inducer of TNF-alpha from freshly isolated bronchoalveolar macrophages (BAM). Upon in vitro culture, BAM and monocytes bound greater numbers of fungal cells, yet their capacity to produce TNF-alpha following cryptococcal stimulation declined by 74 to 100%. However, this decline was reversed if the BAM and monocytes were cultured with gamma interferon. These data establish that C. neoformans can potently stimulate TNF-alpha release from human leukocytes. However, several variables profoundly affected the amount of TNF-alpha released, including the type of leukocyte and its state of activation, the size of the cryptococcal capsule, and the availability of opsonins. PMID:8168965

  17. Peroxisomal and Mitochondrial β-Oxidation Pathways Influence the Virulence of the Pathogenic Fungus Cryptococcus neoformans

    PubMed Central

    Kretschmer, Matthias; Wang, Joyce

    2012-01-01

    An understanding of the connections between metabolism and elaboration of virulence factors during host colonization by the human-pathogenic fungus Cryptococcus neoformans is important for developing antifungal therapies. Lipids are abundant in host tissues, and fungal pathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. In addition, lipids are important signaling molecules in both fungi and mammals. In this report, we demonstrate that defects in the peroxisomal and mitochondrial β-oxidation pathways influence the growth of C. neoformans on fatty acids as well as the virulence of the fungus in a mouse inhalation model of cryptococcosis. Disease attenuation may be due to the cumulative influence of altered carbon source acquisition or processing, interference with secretion, changes in cell wall integrity, and an observed defect in capsule production for the peroxisomal mutant. Altered capsule elaboration in the context of a β-oxidation defect was unexpected but is particularly important because this trait is a major virulence factor for C. neoformans. Additionally, analysis of mutants in the peroxisomal pathway revealed a growth-promoting activity for C. neoformans, and subsequent work identified oleic acid and biotin as candidates for such factors. Overall, this study reveals that β-oxidation influences virulence in C. neoformans by multiple mechanisms that likely include contributions to carbon source acquisition and virulence factor elaboration. PMID:22707485

  18. Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean basin.

    PubMed

    Cogliati, Massimo; D'Amicis, Roberta; Zani, Alberto; Montagna, Maria Teresa; Caggiano, Giuseppina; De Giglio, Osvalda; Balbino, Stella; De Donno, Antonella; Serio, Francesca; Susever, Serdar; Ergin, Cagri; Velegraki, Aristea; Ellabib, Mohamed S; Nardoni, Simona; Macci, Cristina; Oliveri, Salvatore; Trovato, Laura; Dipineto, Ludovico; Rickerts, Volker; McCormick-Smith, Ilka; Akcaglar, Sevim; Tore, Okan; Mlinaric-Missoni, Emilija; Bertout, Sebastien; Mallié, Michele; Martins, Maria da Luz; Vencà, Ana C F; Vieira, Maria L; Sampaio, Ana C; Pereira, Cheila; Griseo, Giuseppe; Romeo, Orazio; Ranque, Stéphane; Al-Yasiri, Mohammed H Y; Kaya, Meltem; Cerikcioglu, Nilgun; Marchese, Anna; Vezzulli, Luigi; Ilkit, Macit; Desnos-Ollivier, Marie; Pasquale, Vincenzo; Korem, Maya; Polacheck, Itzhack; Scopa, Antonio; Meyer, Wieland; Ferreira-Paim, Kennio; Hagen, Ferry; Theelen, Bart; Boekhout, Teun; Lockhart, Shawn R; Tintelnot, Kathrin; Tortorano, Anna Maria; Dromer, Françoise; Varma, Ashok; Kwon-Chung, Kyung J; Inácio, Joäo; Alonso, Beatriz; Colom, Maria F

    2016-06-01

    In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-2015. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus and two Olea trees. Cryptococcus neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates, VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent an important niche for these yeasts. PMID:27188887

  19. Cryptococcus neoformans capsule protects cell from oxygen reactive species generated by antimicrobial photodynamic inactivation

    NASA Astrophysics Data System (ADS)

    Prates, Renato Araujo; Hamblin, Michael R.; Kato, Ilka T.; Fuchs, Beth; Mylonakis, Eleytherios; Simões Ribeiro, Martha; Tegos, George

    2011-03-01

    Antimicrobial photodynamic inactivation (APDI) is based on the utilization of substances that can photosensitize biological tissues and are capable of being activated in the presence of light. Cryptococcus neoformans is an yeast surrounded by a capsule composed primarily of glucoronoxylomannan that plays an important role in its virulence. This yeast causes infection on skin, lungs and brain that can be associated with neurological sequelae and neurosurgical interventions, and its conventional treatment requires prolonged antifungal therapy, which presents important adverse effects. The aim of this study was to evaluate the protective effect of Cryptococcus neoformans capsule against reactive oxygen species generated by APDI. Cryptococcus neoformans KN99α, which is a strain able to produce capsule, and CAP59 that does not present capsule production were submitted to APDI using methylene blue (MB), rose bengal (RB), and pL-ce6 as photosensitizers (PS). Then microbial inactivation was evaluated by counting colony form units following APDI and confocal laser scanning microscopy (CLSM) illustrated localization as well as the preferential accumulation of PS into the fungal cells. C. neoformans KN99α was more resistant to APDI than CAP59 for all PSs tested. CLSM showed incorporation of MB and RB into the cytoplasm and a preferential uptake in mitochondria. A nuclear accumulation of MB was also observed. Contrarily, pL-ce6 appears accumulated in cell wall and cell membrane and minimal florescence was observed inside the fungal cells. In conclusion, the ability of C. neoformans to form capsule enhances survival following APDI.

  20. Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens

    SciTech Connect

    Hast, Michael A.; Nichols, Connie B.; Armstrong, Stephanie M.; Kelly, Shannon M.; Hellinga, Homme W.; Alspaugh, J. Andrew; Beese, Lorena S.

    2012-09-17

    Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections.

  1. Neutrophil swarming toward Cryptococcus neoformans is mediated by complement and leukotriene B4.

    PubMed

    Sun, Donglei; Shi, Meiqing

    2016-09-01

    Swarming behavior of neutrophils has been noticed in both sterile injury and infection models and the mechanisms are being unveiled. So far, no in vitro model has been established to study neutrophil swarming to microbes. In the current study, using live-cell imaging, we observed in vitro neutrophil swarming toward Cryptococcus neoformans, a fungal pathogen causing human meningoencephalitis. Complement C3 and CD11b expression are essential for neutrophils to form cell swarms surrounding C. neoformans. Leukotriene B4 (LTB4) was quickly released by neutrophils during their interactions with C. neoformans. Blockade of LTB4 synthesis inhibited the swarming response to C. neoformans. Importantly, blockade of LTB4 synthesis also significantly reduced neutrophil recruitment in the lung vasculature of mice infected intravenously with C. neoformans, demonstrating a critical role of LTB4 in intravascular neutrophil swarming during infection. Together, this is the first report of neutrophil dynamics of swarming toward a microorganism in vitro, mediated by complement and LTB4. PMID:27402276

  2. Susceptibility profile and epidemiological cut-off values of Cryptococcus neoformans species complex from Argentina.

    PubMed

    Córdoba, Susana; Isla, Maria G; Szusz, Wanda; Vivot, Walter; Altamirano, Rodrigo; Davel, Graciela

    2016-06-01

    Epidemiological cut-off values (ECVs) based on minimal inhibitory concentration (MIC) distribution have been recently proposed for some antifungal drug/Cryptococcus neoformans combinations. However, these ECVs vary according to the species studied, being serotypes and the geographical origin of strains, variables to be considered. The aims were to define the wild-type (WT) population of the C. neoformans species complex (C. neoformans) isolated from patients living in Argentina, and to propose ECVs for six antifungal drugs. A total of 707 unique C. neoformans isolates obtained from HIV patients suffering cryptococcal meningitis were studied. The MIC of amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and posaconazole was determined according to the EDef 7.2 (EUCAST) reference document. The MIC distribution, MIC50 , MIC90 and ECV for each of these drugs were calculated. The highest ECV, which included ≥95% of the WT population modelled, was observed for flucytosine and fluconazole (32 μg ml(-1) each). For amphotericin B, itraconazole, voriconazole and posaconazole, the ECVs were: 0.5, 0.5, 0.5 and 0.06 μg ml(-1) respectively. The ECVs determined in this study may aid in identifying the C. neoformans strains circulating in Argentina with decreased susceptibility to the antifungal drugs tested. PMID:26865081

  3. Fungicidal mechanism of action of D0870 against Cryptococcus neoformans under acidic conditions.

    PubMed Central

    Yamada, H; Watanabe, T; Kato, K; Mochizuki, H

    1997-01-01

    The fungicidal mechanism of the triazole D0870 against Cryptococcus neoformans under acidic conditions was investigated. D0870 reduced the intracellular K+ content of C. neoformans at pH 4 to about half the value at pH 7 after 12 h of incubation. The 50% inhibitory concentrations of D0870 for ergosterol biosynthesis were almost the same at both pH 4 (0.017 microg/ml) and 7 (0.014 microg/ml); however, D0870 caused a marked accumulation of an unknown lipid and methylated sterols in C. neoformans cultured at pH 4. Extracted fractions containing the unknown lipid or methylated sterols showed strong fungicidal activities against C. neoformans both at pH 4 and 7 in phosphate-citrate buffer not containing D0870. Gas chromatographic-mass spectrometric analysis showed that the unknown lipid was obtusifolione. These results suggest that D0870 kills C. neoformans by disturbing the permeability of the cell membrane through the accumulation of obtusifolione and methylated sterols in the cell membrane under acidic conditions. PMID:9420043

  4. Microevolution of a Standard Strain of Cryptococcus neoformans Resulting in Differences in Virulence and Other Phenotypes

    PubMed Central

    Franzot, Sarah P.; Mukherjee, Jean; Cherniak, Robert; Chen, Lin-Chi; Hamdan, Junia S.; Casadevall, Arturo

    1998-01-01

    Cryptococcus neoformans is a major fungal pathogen for patients with debilitated immune systems. However, no information is available on the stability of virulence or of phenotypes associated with virulence for C. neoformans laboratory strains. A serendipitous observation in our laboratory that one isolate of C. neoformans ATCC 24067 (strain 52D) became attenuated after continuous in vitro culture prompted us to perform a comparative study of nine strain 24067 isolates obtained from six different research laboratories. Each isolate was characterized by DNA typing, virulence for mice, proteinase production, extracellular protein synthesis, melanin synthesis, carbon assimilation pattern, antifungal drug susceptibility, colony morphology, growth rate, agglutination titers, phagocytosis by murine macrophages, capsule size, and capsular polysaccharide structure. All isolates had similar DNA typing patterns consistent with their assignment to the same strain, although minor chromosome size polymorphisms were observed in the electrophoretic karyotypes of two isolates. Several isolates had major differences in phenotypes that may be associated with virulence, including growth rate, capsule size, proteinase production, and melanization. These findings imply that C. neoformans is able to undergo rapid changes in vitro, probably as a result of adaptation to laboratory conditions, and suggest the need for careful attention to storage and maintenance conditions. In summary, our results indicate that C. neoformans (i) can become attenuated by in vitro culture and (ii) is capable of microevolution in vitro with the emergence of variants exhibiting new genotypic and phenotypic characteristics. PMID:9423844

  5. Structures of Cryptococcus neoformans Protein Farnesyltransferase Reveal Strategies for Developing Inhibitors That Target Fungal Pathogens*

    PubMed Central

    Hast, Michael A.; Nichols, Connie B.; Armstrong, Stephanie M.; Kelly, Shannon M.; Hellinga, Homme W.; Alspaugh, J. Andrew; Beese, Lorena S.

    2011-01-01

    Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections in immunocompromised individuals, including AIDS patients and transplant recipients. Few antifungals can treat C. neoformans infections, and drug resistance is increasing. Protein farnesyltransferase (FTase) catalyzes post-translational lipidation of key signal transduction proteins and is essential in C. neoformans. We present a multidisciplinary study validating C. neoformans FTase (CnFTase) as a drug target, showing that several anticancer FTase inhibitors with disparate scaffolds can inhibit C. neoformans and suggesting structure-based strategies for further optimization of these leads. Structural studies are an essential element for species-specific inhibitor development strategies by revealing similarities and differences between pathogen and host orthologs that can be exploited. We, therefore, present eight crystal structures of CnFTase that define the enzymatic reaction cycle, basis of ligand selection, and structurally divergent regions of the active site. Crystal structures of clinically important anticancer FTase inhibitors in complex with CnFTase reveal opportunities for optimization of selectivity for the fungal enzyme by modifying functional groups that interact with structurally diverse regions. A substrate-induced conformational change in CnFTase is observed as part of the reaction cycle, a feature that is mechanistically distinct from human FTase. Our combined structural and functional studies provide a framework for developing FTase inhibitors to treat invasive fungal infections. PMID:21816822

  6. Cryptococcal transmigration across a model brain blood-barrier: evidence of the Trojan horse mechanism and differences between Cryptococcus neoformans var. grubii strain H99 and Cryptococcus gattii strain R265.

    PubMed

    Sorrell, Tania C; Juillard, Pierre-Georges; Djordjevic, Julianne T; Kaufman-Francis, Keren; Dietmann, Anelia; Milonig, Alban; Combes, Valery; Grau, Georges E R

    2016-01-01

    Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg) cause neurological disease and cross the BBB as free cells or in mononuclear phagocytes via the Trojan horse mechanism, although evidence for the latter is indirect. There is emerging evidence that Cn and the North American outbreak Cg strain (R265) more commonly cause neurological and lung disease, respectively. We have employed a widely validated in vitro model of the BBB, which utilizes the hCMEC/D3 cell line derived from human brain endothelial cells (HBEC) and the human macrophage-like cell line, THP-1, to investigate whether transport of dual fluorescence-labelled Cn and Cg across the BBB occurs within macrophages. We showed that phagocytosis of Cn by non-interferon (IFN)-γ stimulated THP-1 cells was higher than that of Cg. Although Cn and Cg-loaded THP-1 bound similarly to TNF-activated HBECs under shear stress, more Cn-loaded macrophages were transported across an intact HBEC monolayer, consistent with the predilection of Cn for CNS infection. Furthermore, Cn exhibited a higher rate of expulsion from transmigrated THP-1 compared with Cg. Our results therefore provide further evidence for transmigration of both Cn and Cg via the Trojan horse mechanism and a potential explanation for the predilection of Cn to cause CNS infection. PMID:26369713

  7. Macrophage mitochondrial and stress response to ingestion of Cryptococcus neoformans

    PubMed Central

    Coelho, Carolina; Souza, Ana Camila Oliveira; Derengowski, Lorena da Silveira; de Leon-Rodriguez, Carlos; Wang, Bo; Leon-Rivera, Rosiris; Bocca, Anamelia Lorenzetti; Gonçalves, Teresa; Casadevall, Arturo

    2015-01-01

    Human infection with Cryptococcus neoformans (Cn), a common fungal pathogen follows deposition of yeast spores in the lung alveoli. The subsequent host-pathogen interaction can result in either eradication, latency or extra-pulmonary dissemination. Successful control of Cn infection is dependent on host macrophages but macrophages display little ability to kill Cn in vitro. Recently, we reported that ingestion of Cn by mouse macrophages induces early cell cycle progression followed by mitotic arrest, an event that almost certainly reflects host cell damage. The goal of the present work was to understand macrophage pathways affected by Cn toxicity. Infection of macrophages by Cn was associated with alterations in protein translation rate and activation of several stress pathways such as Hypoxia Inducing Factor-1α (HIF-1α), Receptor-interacting Protein 1 (RIP1) and Apoptosis Inducing Factor (AIF). Concomitantly we observed mitochondrial depolarization in infected macrophages, an observation that was replicated in vivo. We also observed differences in the stress pathways activated depending on macrophage cell type, consistent with the non-specific nature of Cn virulence known to infect phylogenetically distant hosts. Our results indicate that Cn infection impairs multiple host cellular functions and undermines the health of these critical phagocytic cells, which can potentially interfere with their ability to clear this fungal pathogen. PMID:25646306

  8. Interaction of Cryptococcus neoformans Extracellular Vesicles with the Cell Wall

    PubMed Central

    Wolf, Julie M.; Espadas-Moreno, Javier; Luque-Garcia, Jose L.

    2014-01-01

    Cryptococcus neoformans produces extracellular vesicles containing a variety of cargo, including virulence factors. To become extracellular, these vesicles not only must be released from the plasma membrane but also must pass through the dense matrix of the cell wall. The greatest unknown in the area of fungal vesicles is the mechanism by which these vesicles are released to the extracellular space given the presence of the fungal cell wall. Here we used electron microscopy techniques to image the interactions of vesicles with the cell wall. Our goal was to define the ultrastructural morphology of the process to gain insights into the mechanisms involved. We describe single and multiple vesicle-leaving events, which we hypothesized were due to plasma membrane and multivesicular body vesicle origins, respectively. We further utilized melanized cells to “trap” vesicles and visualize those passing through the cell wall. Vesicle size differed depending on whether vesicles left the cytoplasm in single versus multiple release events. Furthermore, we analyzed different vesicle populations for vesicle dimensions and protein composition. Proteomic analysis tripled the number of proteins known to be associated with vesicles. Despite separation of vesicles into batches differing in size, we did not identify major differences in protein composition. In summary, our results indicate that vesicles are generated by more than one mechanism, that vesicles exit the cell by traversing the cell wall, and that vesicle populations exist as a continuum with regard to size and protein composition. PMID:24906412

  9. Hydroxyurea treatment inhibits proliferation of Cryptococcus neoformans in mice.

    PubMed

    Tripathi, Kaushlendra; Mor, Visesato; Bairwa, Narendra K; Del Poeta, Maurizio; Mohanty, Bidyut K

    2012-01-01

    The fungal pathogen Cryptococcus neoformans (Cn) is a serious threat to immunocompromised individuals, especially for HIV patients who develop meningoencephalitis. For effective cryptococcal treatment, novel antifungal drugs or innovative combination therapies are needed. Recently, sphingolipids have emerged as important bioactive molecules in the regulation of microbial pathogenesis. Previously we reported that the sphingolipid pathway gene, ISC1, which is responsible for ceramide production, is a major virulence factor in Cn infection. Here we report our studies of the role of ISC1 during genotoxic stress induced by the antineoplastic hydroxyurea (HU) and methyl methanesulfonate (MMS), which affect DNA replication and genome integrity. We observed that Cn cells lacking ISC1 are highly sensitive to HU and MMS in a rich culture medium. HU affected cell division of Cn cells lacking the ISC1 gene, resulting in cell clusters. Cn ISC1, when expressed in a Saccharomyces cerevisiae (Sc) strain lacking its own ISC1 gene, restored HU resistance. In macrophage-like cells, although HU affected the proliferation of wild type (WT) Cn cells by 50% at the concentration tested, HU completely inhibited Cn isc1Δ cell proliferation. Interestingly, our preliminary data show that mice infected with WT or Cn isc1Δ cells and subsequently treated with HU had longer lifespans than untreated, infected control mice. Our work suggests that the sphingolipid pathway gene, ISC1, is a likely target for combination therapy with traditional drugs such as HU. PMID:22783238

  10. Surface-Associated Plasminogen Binding of Cryptococcus neoformans Promotes Extracellular Matrix Invasion

    PubMed Central

    Fox, Deborah

    2009-01-01

    Background The fungal pathogen Cryptococcus neoformans is a leading cause of illness and death in persons with predisposing factors, including: malignancies, solid organ transplants, and corticosteroid use. C. neoformans is ubiquitous in the environment and enters into the lungs via inhalation, where it can disseminate through the bloodstream and penetrate the central nervous system (CNS), resulting in a difficult to treat and often-fatal infection of the brain, called meningoencephalitis. Plasminogen is a highly abundant protein found in the plasma component of blood and is necessary for the degradation of fibrin, collagen, and other structural components of tissues. This fibrinolytic system is utilized by cancer cells during metastasis and several pathogenic species of bacteria have been found to manipulate the host plasminogen system to facilitate invasion of tissues during infection by modifying the activation of this process through the binding of plasminogen at their surface. Methodology The invasion of the brain and the central nervous system by penetration of the protective blood-brain barrier is a prerequisite to the establishment of meningoencephalitis by the opportunistic fungal pathogen C. neoformans. In this study, we examined the ability of C. neoformans to subvert the host plasminogen system to facilitate tissue barrier invasion. Through a combination of biochemical, cell biology, and proteomic approaches, we have shown that C. neoformans utilizes the host plasminogen system to cross tissue barriers, providing support for the hypothesis that plasminogen-binding may contribute to the invasion of the blood-brain barrier by penetration of the brain endothelial cells and underlying matrix. In addition, we have identified the cell wall-associated proteins that serve as plasminogen receptors and characterized both the plasminogen-binding and plasmin-activation potential for this significant human pathogen. Conclusions The results of this study provide

  11. Lipid Flippase Subunit Cdc50 Mediates Drug Resistance and Virulence in Cryptococcus neoformans

    PubMed Central

    Huang, Wei; Liao, Guojian; Baker, Gregory M.; Wang, Yina; Lau, Richard; Paderu, Padmaja; Perlin, David S.

    2016-01-01

    ABSTRACT Cryptococcus neoformans is a human fungal pathogen and a major cause of fungal meningitis in immunocompromised individuals. Treatment options for cryptococcosis are limited. Of the two major antifungal drug classes, azoles are active against C. neoformans but exert a fungistatic effect, necessitating long treatment regimens and leaving open an avenue for emergence of azole resistance. Drugs of the echinocandin class, which target the glucan synthase and are fungicidal against a number of other fungal pathogens, such as Candida species, are ineffective against C. neoformans. Despite the sensitivity of the target enzyme to the drug, the reasons for the innate resistance of C. neoformans to echinocandins remain unknown. To understand the mechanism of echinocandin resistance in C. neoformans, we screened gene disruption and gene deletion libraries for mutants sensitive to the echinocandin-class drug caspofungin and identified a mutation of CDC50, which encodes the β-subunit of membrane lipid flippase. We found that the Cdc50 protein localized to membranes and that its absence led to plasma membrane defects and enhanced caspofungin penetration into the cell, potentially explaining the increased caspofungin sensitivity. Loss of CDC50 also led to hypersensitivity to the azole-class drug fluconazole. Interestingly, in addition to functioning in drug resistance, CDC50 was also essential for fungal resistance to macrophage killing and for virulence in a murine model of cryptococcosis. Furthermore, the surface of cdc50Δ cells contained increased levels of phosphatidylserine, which has been proposed to act as a macrophage recognition signal. Together, these results reveal a previously unappreciated role of membrane lipid flippase in C. neoformans drug resistance and virulence. PMID:27165800

  12. Cryptococcus neoformans Directly Stimulates Perforin Production and Rearms NK Cells for Enhanced Anticryptococcal Microbicidal Activity▿

    PubMed Central

    Marr, Kaleb J.; Jones, Gareth J.; Zheng, Chunfu; Huston, Shaunna M.; Timm-McCann, Martina; Islam, Anowara; Berenger, Byron M.; Ma, Ling Ling; Wiseman, Jeremy C. D.; Mody, Christopher H.

    2009-01-01

    NK cells, in addition to possessing antitumor and antiviral activity, exhibit perforin-dependent microbicidal activity against the opportunistic pathogen Cryptococcus neoformans. However, the factors controlling this response, particularly whether the pathogen itself provides an activation or rearming signal, are largely unknown. The current studies were performed to determine whether exposure to this fungus alters subsequent NK cell anticryptococcal activity. NK cells lost perforin and mobilized lysosome-associated membrane protein 1 to the cell surface following incubation with the fungus, indicating that degranulation had occurred. Despite a reduced perforin content during killing, NK cells acquired an enhanced ability to kill C. neoformans, as demonstrated using auxotrophs that allowed independent assessment of the killing of two strains. De novo protein synthesis was required for optimal killing; however, there was no evidence that a soluble factor contributed to the enhanced anticryptococcal activity. Exposure of NK cells to C. neoformans caused the cells to rearm, as demonstrated by increased perforin mRNA levels and enhanced loss of perforin when transcription was blocked. Degranulation alone was insufficient to provide the activation signal as NK cells lost anticryptococcal activity following treatment with strontium chloride. However, NK cells regained the activity upon prolonged exposure to C. neoformans, which is consistent with activation by the microbe. The enhanced cytotoxicity did not extend to tumor killing since NK cells exposed to C. neoformans failed to kill NK-sensitive tumor targets (K562 cells). These studies demonstrate that there is contact-mediated microbe-specific rearming and activation of microbicidal activity that are necessary for optimal killing of C. neoformans. PMID:19307209

  13. Transcriptional Regulation by Protein Kinase A in Cryptococcus neoformans

    PubMed Central

    Hu, Guanggan; Steen, Barbara R; Lian, Tianshun; Sham, Anita P; Tam, Nicola; Tangen, Kristin L; Kronstad, James W

    2007-01-01

    A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5′-monophosphate (cAMP)–dependent protein kinase A (PKA) is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential targets for

  14. Cryptococcus neoformans: Tripping on Acid in the Phagolysosome

    PubMed Central

    DeLeon-Rodriguez, Carlos M.; Casadevall, Arturo

    2016-01-01

    Cryptococcus neoformans (Cn) is a basidiomycetous pathogenic yeast that is a frequent cause of meningoencephalitis in immunocompromised individuals. Cn is a facultative intracellular pathogen in mammals, insects and amoeba. Cn infection occurs after inhalation of spores or desiccated cells from the environment. After inhalation Cn localizes to the lungs where it can be phagocytosed by alveolar macrophages. Cn is surrounded by a polysaccharide capsule that helps the fungus survive in vivo by interfering with phagocytosis, quenching free radical bursts and shedding polysaccharides that negatively modulates the immune system. After phagocytosis, Cn resides within the phagosome that matures to become a phagolysosome, a process that results in the acidification of the phagolysosomal lumen. Cn replicates at a higher rate inside macrophages than in the extracellular environment, possibly as a result that the phagosomal pH is near that optimal for growth. Cn increases the phagolysosomal pH and modulates the dynamics of Rab GTPases interaction with the phagolysosome. Chemical manipulation of the phagolysosomal pH with drugs can result in direct and indirect killing of Cn and reduced non-lytic exocytosis. Phagolysosomal membrane damage after Cn infection occurs both in vivo and in vitro, and is required for Cn growth and survival. Macrophage treatment with IFN-γ reduces the phagolysosomal damage and increases intracellular killing of Cn. Studies on mice and humans show that treatment with IFN-γ can improve host control of the disease. However, the mechanism by which Cn mediates phagolysosomal membrane damage remains unknown but likely candidates are phospholipases and mechanical damage from an enlarging capsule. Here we review Cn intracellular interaction with a particular emphasis on phagosomal interactions and develop the notion that the extent of damage of the phagosomal membrane is a key determinant of the outcome of the Cn-macrophage interaction. PMID:26925039

  15. Cryptococcus neoformans induces antimicrobial responses and behaves as a facultative intracellular pathogen in the non mammalian model Galleria mellonella

    PubMed Central

    Trevijano-Contador, Nuria; Herrero-Fernández, Inés; García-Barbazán, Irene; Scorzoni, Liliana; Rueda, Cristina; Rossi, Suélen Andreia; García-Rodas, Rocío; Zaragoza, Oscar

    2015-01-01

    Cryptococcus neoformans is an encapsulated opportunistic fungal pathogen that is found in multiple niches in the environment and that can cause fatal meningoencephalitis in susceptible patients, mainly HIV+ individuals. Cryptococcus also infects environmental hosts such as nematodes, insects and plants. In particular, C. neoformans can kill the lepidopteran Galleria mellonella, which offers a useful tool to study microbial virulence and drug efficacy. Galleria mellonella immunity relies on innate responses based on melanization, accumulation of antimicrobial peptides, and cellular responses as phagocytosis or multicellular encapsulation. In this work we have investigated the immune response of G. mellonella during cryptococcal infection. We found that G. mellonella infected with C. neoformans had a high lytic activity in their hemolymph. This response was temperature- and capsule-dependent. During interaction with phagocytic cells, C. neoformans behaved as an intracellular pathogen since it could replicate within hemocytes. Non-lytic events were also observed. In contrast to Candida species, C. neoformans did not induce melanization of G. mellonella after infection. Finally, passage of C. neoformans through G. mellonella resulted in changes in capsule structure as it has been also reported during infection in mammals. Our results highlight that G. mellonella is an optimal model to investigate innate immune responses against C. neoformans. PMID:25531532

  16. Pleiotropic Roles of the Msi1-Like Protein Msl1 in Cryptococcus neoformans

    PubMed Central

    Yang, Dong-Hoon; Maeng, Shinae; Strain, Anna K.; Floyd, Anna; Nielsen, Kirsten; Heitman, Joseph

    2012-01-01

    Msi1-like (MSIL) proteins contain WD40 motifs and have a pleiotropic cellular function as negative regulators of the Ras/cyclic AMP (cAMP) pathway and components of chromatin assembly factor 1 (CAF-1), yet they have not been studied in fungal pathogens. Here we identified and characterized an MSIL protein, Msl1, in Cryptococcus neoformans, which causes life-threatening meningoencephalitis in humans. Notably, Msl1 plays pleiotropic roles in C. neoformans in both cAMP-dependent and -independent manners largely independent of Ras. Msl1 negatively controls antioxidant melanin production and sexual differentiation, and this was repressed by the inhibition of the cAMP-signaling pathway. In contrast, Msl1 controls thermotolerance, diverse stress responses, and antifungal drug resistance in a Ras/cAMP-independent manner. Cac2, which is the second CAF-1 component, appears to play both redundant and distinct functions compared to the functions of Msl1. Msl1 is required for the full virulence of C. neoformans. Transcriptome analysis identified a group of Msl1-regulated genes, which include stress-related genes such as HSP12 and HSP78. In conclusion, this study demonstrates pleiotropic roles of Msl1 in the human fungal pathogen C. neoformans, providing insight into a potential novel antifungal therapeutic target. PMID:23042129

  17. Cryptococcus neoformans Yap1 is required for normal fluconazole and oxidative stress resistance

    PubMed Central

    Paul, Sanjoy; Doering, Tamara L.; Moye-Rowley, W. Scott

    2014-01-01

    Cryptococcus neoformans is a pathogen that is the most common cause of fungal meningitis. As with most fungal pathogens, the most prevalent clinical antifungal used to treat Cryptococcosis is orally administered fluconazole. Resistance to this antifungal is an increasing concern in treatment of fungal disease in general. Our knowledge of the specific determinants involved in fluconazole resistance in Cryptococcus is limited. Here we report the identification of an important genetic determinant of fluconazole resistance in Cryptococcus neoformans that encodes a basic region-leucine zipper transcription factor homologous to Saccharomyces cerevisiae Yap1. Expression of a codon-optimized form of the Cn YAP1 cDNA in S. cerevisiae complemented defects caused by loss of the endogenous S. cerevisiae YAP1 gene and activated transcription from a reporter gene construct. Mutant strains of C. neoformans lacking YAP1 were hypersensitive to a range of oxidative stress agents but importantly also to fluconazole. Loss of Yap1 homologues from other fungal pathogens like Candida albicans or Aspergillus fumigatus was previously found to cause oxidant hypersensitivity but had no detectable effect on fluconazole resistance. Our data provide evidence for a unique biological role of Yap1 in wild-type fluconazole resistance in C. neoformans. PMID:25445311

  18. ISOLATION OF Cryptococcus neoformans FROM ENVIRONMENTAL SAMPLES COLLECTED IN SOUTHEASTERN NIGERIA.

    PubMed

    Nweze, Emeka I; Kechia, Fred A; Dibua, Uju E; Eze, Charles; Onoja, Uwakwe S

    2015-01-01

    Cryptococcosis caused by Cryptococcus neoformans is the second most common fungal opportunistic pathogen and a life-threatening infection with serious clinical manifestations especially in HIV/AIDS and other immunocompromised patients. In Nigeria, HIV/AIDS infection has reached an alarming level. Despite this, information on the presence of this fungus in clinical and environmental samples is very scanty in Nigeria and many other parts of Africa. We set out to evaluate the presence of Cryptococcus neoformans or C. gattiiin pigeon droppings obtained from Southeastern Nigeria. One hundred and seventy-seven samples of pigeon droppings from six sample types were collected. The area covered comprised of ten cities and other locations spanning across five States in Nigeria. Using established techniques, Cryptococcus neoformans was isolated from 39 of the 177 (22.0%) samples overall. No C. gattiiwas isolated. Most of the isolates (32.4%) were recovered from dovecotes (11 of 34) followed closely by samples taken from markets (31.8%; seven of 22) and least from the church (4.0%; one of 25). The highest isolation rate (38.9%) was found in samples from Enugu-Ezike(seven of 23) while the least came from Afikpo and the other locations each with 9.1% isolation rate. This is the first large-scale screening of Cryptococcus neoformans from pigeon droppings in Nigeria. The ecological and epidemiological significance of these findings are discussed. PMID:26422152

  19. Cryptococcus neoformans meningitis with negative cryptococcal antigen: Evaluation of a new immunochromatographic detection assay

    PubMed Central

    Opota, O.; Desgraz, B.; Kenfak, A.; Jaton, K.; Cavassini, M.; Greub, G.; Prod'hom, G.; Giulieri, S.

    2014-01-01

    Detection of cryptococcal antigen in serum or cerebrospinal fluid allows cryptococcal meningitis diagnosis within few hours with >90% sensitivity. In an HIV-positive patient with Cryptococcus neoformans meningitis, initial antigen detection by immunoagglutination was negative. We thus evaluated a new immunochromatographic detection assay that exhibited a higher sensitivity. PMID:25755893

  20. A Multi-Host Approach for the Systematic Analysis of Virulence Factors in Cryptococcus neoformans

    PubMed Central

    Desalermos, Athanasios; Tan, Xiaojiang; Rajamuthiah, Rajmohan; Arvanitis, Marios; Wang, Yan; Li, Dedong; Kourkoumpetis, Themistoklis K.; Fuchs, Beth Burgwyn; Mylonakis, Eleftherios

    2015-01-01

    A multi-host approach was followed to screen a library of 1201 signature-tagged deletion strains of Cryptococcus neoformans mutants to identify previously unknown virulence factors. The primary screen was performed using a Caenorhabditis elegans–C. neoformans infection assay. The hits among these strains were reconfirmed as less virulent than the wild type in the insect Galleria mellonella–C. neoformans infection assay. After this 2-stage screen, and to prioritize hits, we performed serial evaluations of the selected strains, using the C. elegans model. All hit strains identified through these studies were validated in a murine model of systemic cryptococcosis. Twelve strains were identified through a stepwise screening assay. Among them, 4 (CSN1201, SRE1, RDI1, and YLR243W) were previously discovered, providing proof of principle for this approach, while the role of the remaining 8 genes (CKS101, CNC5600, YOL003C, CND1850, MLH3, HAP502, MSL5, and CNA2580) were not previously described in cryptococcal virulence. The multi-host approach is an efficient method of studying the pathogenesis of C. neoformans. We used diverse model hosts, C. elegans, G. mellonella, and mice, with physiological differences and identified 12 genes associated with mammalian infection. Our approach may be suitable for large pathogenesis screens. PMID:25114160

  1. Multilocus sequence typing of Cryptococcus neoformans in non-HIV associated cryptococcosis in Nagasaki, Japan.

    PubMed

    Mihara, Tomo; Izumikawa, Koichi; Kakeya, Hiroshi; Ngamskulrungroj, Popchai; Umeyama, Takashi; Takazono, Takahiro; Tashiro, Masato; Nakamura, Shigeki; Imamura, Yoshifumi; Miyazaki, Taiga; Ohno, Hideaki; Yamamoto, Yoshihiro; Yanagihara, Katsunori; Miyzaki, Yoshitsugu; Kohno, Shigeru

    2013-04-01

    Cryptococcosis is primarily caused by two Cryptococcus species, i.e., Cryptococcus neoformans and C. gattii. Both include several genetically diverse subgroups that can be differentiated using various molecular strain typing methods. Since little is known about the molecular epidemiology of the C. neoformans/C. gattii species complex in Japan, we conducted a molecular epidemiological analysis of 35 C. neoformans isolates from non-HIV patients in Nagasaki, Japan and 10 environmental isolates from Thailand. All were analyzed using URA5-restriction fragment length polymorphism (RFLP) and multilocus sequence typing (MLST). Combined sequence data for all isolates were evaluated with the neighbor-joining method. All were found to be serotype A and mating type MATα. Thirty-two of the 35 clinical isolates molecular type VNI, while the three remaining isolates were VNII as determined through the URA5-RFLP method. Thirty-one of the VNI isolates were identified as MLST sequence type (ST) 5, the remaining one was ST 32 and the three VNII isolates were found to be ST 43. All the environmental isolates were identified as molecular type VNI (four MLST ST 5 and six ST 4). Our study shows that C. neoformans isolates in Nagasaki are genetically homogeneous, with most of the isolates being ST 5. PMID:22901045

  2. The Role of Amino Acid Permeases and Tryptophan Biosynthesis in Cryptococcus neoformans Survival

    PubMed Central

    Fernandes, João Daniel Santos; Martho, Kevin; Tofik, Veridiana; Vallim, Marcelo A.; Pascon, Renata C.

    2015-01-01

    Metabolic diversity is an important factor during microbial adaptation to different environments. Among metabolic processes, amino acid biosynthesis has been demonstrated to be relevant for survival for many microbial pathogens, whereas the association between pathogenesis and amino acid uptake and recycling are less well-established. Cryptococcus neoformans is an opportunistic fungal pathogen with many habitats. As a result, it faces frequent metabolic shifts and challenges during its life cycle. Here we studied the C. neoformans tryptophan biosynthetic pathway and found that the pathway is essential. RNAi indicated that interruptions in the biosynthetic pathway render strains inviable. However, auxotroph complementation can be partially achieved by tryptophan uptake when a non preferred nitrogen source and lower growth temperature are applied, suggesting that amino acid permeases may be the target of nitrogen catabolism repression (NCR). We used bioinformatics to search for amino acid permeases in the C. neoformans and found eight potential global permeases (AAP1 to AAP8). The transcriptional profile of them revealed that they are subjected to regulatory mechanisms which are known to respond to nutritional status in other fungi, such as (i) quality of nitrogen (Nitrogen Catabolism Repression, NCR) and carbon sources (Carbon Catabolism Repression, CCR), (ii) amino acid availability in the extracellular environment (SPS-sensing) and (iii) nutritional deprivation (Global Amino Acid Control, GAAC). This study shows that C. neoformans has fewer amino acid permeases than other model yeasts, and that these proteins may be subjected to complex regulatory mechanisms. Our data suggest that the C. neoformans tryptophan biosynthetic pathway is an excellent pharmacological target. Furthermore, inhibitors of this pathway cause Cryptococcus growth arrest in vitro. PMID:26162077

  3. Photodynamic therapy can kill Cryptococcus neoformans in in vitro and in vivo models

    NASA Astrophysics Data System (ADS)

    Prates, Renato A.; da Silva, Eriques G.; Chaves, Priscila F.; Santos, Antônio José S.; Paula, Claudete R.; Ribeiro, Martha S.

    2009-02-01

    Cryptococcosis is an infection caused by the encapsulated yeast Cryptococcus neoformans and the most afflicted sites are lung, skin and central nervous system. A range of studies had reported that photodynamic therapy (PDT) can inactivate yeast cells; however, the in vivo experimental models of cryptococcosis photoinactivation are not commonly reported. The aim of this study was to investigate the ability of methylene blue (MB) combined with a low-power red laser to inactivate Cryptococcus neoformans in in vitro and in vivo experimental models. To perform the in vitro study, suspension of Cryptococcus neoformans ATCC-90112 (106cfu/mL) was used. The light source was a laser (Photon Lase III, DMC, SÃ#o Carlos, Brazil) emitting at λ660nm with output power of 90mW for 6 and 9min of irradiation, resulting fluences at 108 and 162J/cm². As photosensitizer, 100μM MB was used. For the in vivo study, 10 BALB/c mice had the left paw inoculated with C. neoformans ATCC-90112 (107cfu). Twenty-four hours after inoculation, PDT was performed using 150μM MB and 100mW red laser with fluence at 180J/cm2. PDT was efficient in vitro against C. neoformans in both parameters used: 3 log reduction with 108J/cm² and 6 log reduction with 162J/cm². In the in vivo experiment, PDT was also effective; however, its effect was less expressive than in the in vitro study (about 1 log reduction). In conclusion, PDT seems to be a helpful alternative to treat dermal cryptococcosis; however, more effective parameters must be found in in vivo studies.

  4. Molecular typing of environmental Cryptococcus neoformans/C. gattii species complex isolates from Manaus, Amazonas, Brazil.

    PubMed

    Alves, Gleica Soyan Barbosa; Freire, Ana Karla Lima; Bentes, Amaury Dos Santos; Pinheiro, José Felipe de Souza; de Souza, João Vicente Braga; Wanke, Bodo; Matsuura, Takeshi; Jackisch-Matsuura, Ani Beatriz

    2016-08-01

    Cryptococcus neoformans and Cryptococcus gattii are the main causative agents of cryptococcosis, a systemic fungal disease that affects internal organs and skin, and which is acquired by inhalation of spores or encapsulated yeasts. It is currently known that the C. neoformans/C. gattii species complex has a worldwide distribution, however, some molecular types seem to prevail in certain regions. Few environmental studies of Cryptococcus have been conducted in the Brazilian Amazon. This is the first ecological study of the pathogenic fungi C. neoformans/C. gattii species complex in the urban area of Manaus, Amazonas, Brazil. A total of 506 samples from pigeon droppings (n = 191), captive bird droppings (n = 60) and tree hollows (n = 255) were collected from June 2012 to January 2014 at schools and public buildings, squares, pet shops, households, the zoo and the bus station. Samples were plated on niger seed agar (NSA) medium supplemented with chloramphenicol and incubated at 25°C for 5 days. Dark-brown colonies were isolated and tested for thermotolerance at 37°C, cycloheximide resistance and growth on canavanine-glycine-bromothymol blue agar. Molecular typing was done by PCR-RFLP. Susceptibility to the antifungal drugs amphotericin B, fluconazole, itraconazole and ketoconazole was tested using Etest(®) strips. In total, 13 positive samples were obtained: one tree hollow (C. gattiiVGII), nine pigeon droppings (C. neoformansVNI) and three captive bird droppings (C. neoformansVNI). The environmental cryptococcal isolates found in this study were of the same molecular types as those responsible for infections in Manaus. PMID:27005969

  5. Comparative analyses of clinical and environmental populations of Cryptococcus neoformans in Botswana.

    PubMed

    Chen, Yuan; Litvintseva, Anastasia P; Frazzitta, Aubrey E; Haverkamp, Miriam R; Wang, Liuyang; Fang, Charles; Muthoga, Charles; Mitchell, Thomas G; Perfect, John R

    2015-07-01

    Cryptococcus neoformans var. grubii (Cng) is the most common cause of fungal meningitis, and its prevalence is highest in sub-Saharan Africa. Patients become infected by inhaling airborne spores or desiccated yeast cells from the environment, where the fungus thrives in avian droppings, trees and soil. To investigate the prevalence and population structure of Cng in southern Africa, we analysed isolates from 77 environmental samples and 64 patients. We detected significant genetic diversity among isolates and strong evidence of geographic structure at the local level. High proportions of isolates with the rare MATa allele were observed in both clinical and environmental isolates; however, the mating-type alleles were unevenly distributed among different subpopulations. Nearly equal proportions of the MATa and MATα mating types were observed among all clinical isolates and in one environmental subpopulation from the eastern part of Botswana. As previously reported, there was evidence of both clonality and recombination in different geographic areas. These results provide a foundation for subsequent genomewide association studies to identify genes and genotypes linked to pathogenicity in humans. PMID:26053414

  6. Triclosan Demonstrates Synergic Effect with Amphotericin B and Fluconazole and Induces Apoptosis-Like Cell Death in Cryptococcus neoformans

    PubMed Central

    Movahed, Elaheh; Tan, Grace Min Yi; Munusamy, Komathy; Yeow, Tee Cian; Tay, Sun Tee; Wong, Won Fen; Looi, Chung Yeng

    2016-01-01

    Objectives: Cryptococcus neoformans is an opportunistic fungus that causes fatal meningoencephalitis especially in AIDS patients. There is an increasing need for discovery of new anti-cryptococcal drugs due to emergence of resistance cases in recent years. In this study, we aim to elucidate the antifungal effect of triclosan against C. neoformans. Methods: Minimal inhibitory concentration (MIC) of triclosan in different C. neoformans strains was first examined. The in vitro interactions between triclosan and two standard anti-fungal drugs (amphotericin B and fluconazole) were further evaluated by microdilution checkerboard assay. Mechanism of triclosan fungicidal activity was then investigated by viewing the cell morphology under transmission electron microscope. Results: We reported that triclosan potently inhibited the growth of C. neoformans. A combination of triclosan with amphotericin B or with fluconazole enhanced their fungicidal effects. Triclosan-treated C. neoformans displayed characteristics such as nuclear chromatin condensation, extensive intracellular vacuolation and mitochondrial swelling, indicating that triclosan triggered apoptosis-like cell death. Conclusion: In summary, our report suggests triclosan as an independent drug or synergent for C. neoformans treatment. PMID:27047474

  7. Induction of Brain Microvascular Endothelial Cell Urokinase Expression by Cryptococcus neoformans Facilitates Blood-Brain Barrier Invasion

    PubMed Central

    Stie, Jamal; Fox, Deborah

    2012-01-01

    The invasive ability of the blood-borne fungal pathogen Cryptococcus neoformans can be enhanced through interactions with host plasma components, such as plasminogen. Previously we showed by in vitro studies that plasminogen coats the surface of C. neoformans and is converted to the active serine protease, plasmin, by host plasminogen activators. Viable, but not formaldehyde- or sodium azide-killed, cryptococcal strains undergo brain microvascular endothelial cell-dependent plasminogen-to-plasmin activation, which results in enhanced, plasmin-dependent cryptococcal invasion of primary bovine brain microvascular endothelial cells and fungal ability to degrade plasmin substrates. In the present work, brain microvascular endothelial cells cultured with viable, but not killed, cryptococcal strains led to significant increases in both urokinase mRNA transcription and cell-associated urokinase protein expression. Soluble urokinase was also detected in conditioned medium from brain microvascular endothelial cells cultured with viable, but not killed, C. neoformans. Exposure of plasminogen pre-coated viable C. neoformans to conditioned medium from strain-matched brain microvascular endothelial cell-fungal co-cultures resulted in plasminogen-to-plasmin activation and plasmin-dependent cryptococcal invasion. siRNA-mediated silencing of urokinase gene expression or the use of specific inhibitors of urokinase activity abrogated both plasminogen-to-plasmin activation on C. neoformans and cryptococcal-brain microvascular endothelial cell invasion. Our results suggest that pathogen exploitation of the host urokinase-plasmin(ogen) system may contribute to C. neoformans virulence during invasive cryptococcosis. PMID:23145170

  8. Usefulness of silkworm as a host animal for understanding pathogenicity of Cryptococcus neoformans.

    PubMed

    Ishii, Masaki; Matsumoto, Yasuhiko; Sekimizu, Kazuhisa

    2016-02-01

    We propose Cryptococcus neoformans infection model using silkworm for understanding cryptococcosis and screening of therapeutically effective antibiotics. Silkworm is an insect whose rearing methods were established through a long history of the sericulture industry. Silkworm facilitates experiments using a large number of individuals because of low cost for rearing and few ethical problems caused by killing animals. Silkworm can be reared at 37˚C to perform infection experiments at same temperature to human body. Injection of accurate amounts of samples into hemolymph of silkworm by usual syringes is easy to be done since silkworm has an appropriate size to handle. Moreover two injection methods, injection into hemolymph and intestine, are distinguishable for silkworms. The former is correspondent to intravenous injection, and the latter is to oral administration in humans. Taking these advantages of silkworms as host animals, it is possible to evaluate the virulence factors in C. neoformans and the therapeutic efficacy of antifungal agents. PMID:26902902

  9. 3-Bromopyruvate: a novel antifungal agent against the human pathogen Cryptococcus neoformans.

    PubMed

    Dyląg, Mariusz; Lis, Paweł; Niedźwiecka, Katarzyna; Ko, Young H; Pedersen, Peter L; Goffeau, Andre; Ułaszewski, Stanisław

    2013-05-01

    We have investigated the antifungal activity of the pyruvic acid analogue: 3-bromopyruvate (3-BP). Growth inhibition by 3-BP of 110 strains of yeast-like and filamentous fungi was tested by standard spot tests or microdilution method. The human pathogen Cryptococcus neoformans exhibited a low Minimal Inhibitory Concentration (MIC) of 0.12-0.15 mM 3-BP. The high toxicity of 3-BP toward C. neoformans correlated with high intracellular accumulation of 3-BP and also with low levels of intracellular ATP and glutathione. Weak cytotoxicity towards mammalian cells and lack of resistance conferred by the PDR (Pleiotropic Drug Resistance) network in the yeast Saccharomyces cerevisiae, are other properties of 3-BP that makes it a novel promising anticryptococcal drug. PMID:23541578

  10. A Small Protein Associated with Fungal Energy Metabolism Affects the Virulence of Cryptococcus neoformans in Mammals.

    PubMed

    McClelland, Erin E; Ramagopal, Udupi A; Rivera, Johanna; Cox, James; Nakouzi, Antonio; Prabu, Moses M; Almo, Steven C; Casadevall, Arturo

    2016-09-01

    The pathogenic yeast Cryptococcus neoformans causes cryptococcosis, a life-threatening fungal disease. C. neoformans has multiple virulence mechanisms that are non-host specific, induce damage and interfere with immune clearance. Microarray analysis of C. neoformans strains serially passaged in mice associated a small gene (CNAG_02591) with virulence. This gene, hereafter identified as HVA1 (hypervirulence-associated protein 1), encodes a protein that has homologs of unknown function in plant and animal fungi, consistent with a conserved mechanism. Expression of HVA1 was negatively correlated with virulence and was reduced in vitro and in vivo in both mouse- and Galleria-passaged strains of C. neoformans. Phenotypic analysis in hva1Δ and hva1Δ+HVA1 strains revealed no significant differences in established virulence factors. Mice infected intravenously with the hva1Δ strain had higher fungal burden in the spleen and brain, but lower fungal burden in the lungs, and died faster than mice infected with H99W or the hva1Δ+HVA1 strain. Metabolomics analysis demonstrated a general increase in all amino acids measured in the disrupted strain and a block in the TCA cycle at isocitrate dehydrogenase, possibly due to alterations in the nicotinamide cofactor pool. Macrophage fungal burden experiments recapitulated the mouse hypervirulent phenotype of the hva1Δ strain only in the presence of exogenous NADPH. The crystal structure of the Hva1 protein was solved, and a comparison of structurally similar proteins correlated with the metabolomics data and potential interactions with NADPH. We report a new gene that modulates virulence through a mechanism associated with changes in fungal metabolism. PMID:27583447

  11. Restricted Substrate Specificity for the Geranylgeranyltransferase-I Enzyme in Cryptococcus neoformans: Implications for Virulence

    PubMed Central

    Selvig, Kyla; Ballou, Elizabeth R.; Nichols, Connie B.

    2013-01-01

    Proper cellular localization is required for the function of many proteins. The CaaX prenyltransferases (where CaaX indicates a cysteine followed by two aliphatic amino acids and a variable amino acid) direct the subcellular localization of a large group of proteins by catalyzing the attachment of hydrophobic isoprenoid moieties onto C-terminal CaaX motifs, thus facilitating membrane association. This group of enzymes includes farnesyltransferase (Ftase) and geranylgeranyltransferase-I (Ggtase-1). Classically, the variable (X) amino acid determines whether a protein will be an Ftase or Ggtase-I substrate, with Ggtase-I substrates often containing CaaL motifs. In this study, we identify the gene encoding the β subunit of Ggtase-I (CDC43) and demonstrate that Ggtase-mediated activity is not essential. However, Cryptococcus neoformans CDC43 is important for thermotolerance, morphogenesis, and virulence. We find that Ggtase-I function is required for full membrane localization of Rho10 and the two Cdc42 paralogs (Cdc42 and Cdc420). Interestingly, the related Rac and Ras proteins are not mislocalized in the cdc43Δ mutant even though they contain similar CaaL motifs. Additionally, the membrane localization of each of these GTPases is dependent on the prenylation of the CaaX cysteine. These results indicate that C. neoformans CaaX prenyltransferases may recognize their substrates in a unique manner from existing models of prenyltransferase specificity. It also suggests that the C. neoformans Ftase, which has been shown to be more important for C. neoformans proliferation and viability, may be the primary prenyltransferase for proteins that are typically geranylgeranylated in other species. PMID:24014765

  12. Mouse-human immunoglobulin G1 chimeric antibodies with activities against Cryptococcus neoformans.

    PubMed Central

    Zebedee, S L; Koduri, R K; Mukherjee, J; Mukherjee, S; Lee, S; Sauer, D F; Scharff, M D; Casadevall, A

    1994-01-01

    Passive antibody administration is a potentially useful approach for the therapy of human Cryptococcus neoformans infections. To evaluate the efficacy of the human immunoglobulin G1 (IgG1) constant region against C. neoformans and to construct murine antibody derivatives with reduced immunogenicities and longer half-lives in humans, two mouse-human IgG1 chimeric antibodies were generated from the protective murine monoclonal antibodies 2D10 (IgM) and 18B7 (IgG1). The 2D10 mouse-human IgG1 chimeric antibody (ch2D10) had significantly lower binding affinity than its parent murine antibody (m2D10), presumably because of a loss of avidity contribution on switching from IgM to IgG. The 18B7 mouse-human IgG1 chimeric antibody (ch18B7) had higher affinity for cryptococcal polysaccharide antigen than its parent murine antibody (m18B7). ch18B7 and ch2D10 promoted phagocytosis of C. neoformans by primary human microglial cells and the murine J774.16 macrophage-like cell line. ch18B7 and m18B7 enhanced fungistatic or fungicidal activity of J774.16 cells and prolonged the survival of lethally infected mice. We conclude that the human IgG1 constant chain can be effective in mediating antifungal activity against C. neoformans. ch18B7 or similar antibodies are potential candidates for passive antibody therapy of human cryptococcosis. PMID:7979280

  13. In Vitro Analysis of Metabolites Secreted during Infection of Lung Epithelial Cells by Cryptococcus neoformans.

    PubMed

    Liew, Kah Leong; Jee, Jap Meng; Yap, Ivan; Yong, Phelim Voon Chen

    2016-01-01

    Cryptococcus neoformans is an encapsulated basidiomycetous yeast commonly associated with pigeon droppings and soil. The opportunistic pathogen infects humans through the respiratory system and the metabolic implications of C. neoformans infection have yet to be explored. Studying the metabolic profile associated with the infection could lead to the identification of important metabolites associated with pulmonary infection. Therefore, the aim of the study was to simulate cryptococcal infection at the primary site of infection, the lungs, and to identify the metabolic profile and important metabolites associated with the infection at low and high multiplicity of infections (MOI). The culture supernatant of lung epithelial cells infected with C. neoformans at MOI of 10 and 100 over a period of 18 hours were analysed using gas chromatography mass spectrometry. The metabolic profiles obtained were further analysed using multivariate analysis and the pathway analysis tool, MetaboAnalyst 2.0. Based on the results from the multivariate analyses, ten metabolites were selected as the discriminatory metabolites that were important in both the infection conditions. The pathways affected during early C. neoformans infection of lung epithelial cells were mainly the central carbon metabolism and biosynthesis of amino acids. Infection at a higher MOI led to a perturbance in the β-alanine metabolism and an increase in the secretion of pantothenic acid into the growth media. Pantothenic acid production during yeast infection has not been documented and the β-alanine metabolism as well as the pantothenate and CoA biosynthesis pathways may represent underlying metabolic pathways associated with disease progression. Our study suggested that β-alanine metabolism and the pantothenate and CoA biosynthesis pathways might be the important pathways associated with cryptococcal infection. PMID:27054608

  14. In Vitro Analysis of Metabolites Secreted during Infection of Lung Epithelial Cells by Cryptococcus neoformans

    PubMed Central

    2016-01-01

    Cryptococcus neoformans is an encapsulated basidiomycetous yeast commonly associated with pigeon droppings and soil. The opportunistic pathogen infects humans through the respiratory system and the metabolic implications of C. neoformans infection have yet to be explored. Studying the metabolic profile associated with the infection could lead to the identification of important metabolites associated with pulmonary infection. Therefore, the aim of the study was to simulate cryptococcal infection at the primary site of infection, the lungs, and to identify the metabolic profile and important metabolites associated with the infection at low and high multiplicity of infections (MOI). The culture supernatant of lung epithelial cells infected with C. neoformans at MOI of 10 and 100 over a period of 18 hours were analysed using gas chromatography mass spectrometry. The metabolic profiles obtained were further analysed using multivariate analysis and the pathway analysis tool, MetaboAnalyst 2.0. Based on the results from the multivariate analyses, ten metabolites were selected as the discriminatory metabolites that were important in both the infection conditions. The pathways affected during early C. neoformans infection of lung epithelial cells were mainly the central carbon metabolism and biosynthesis of amino acids. Infection at a higher MOI led to a perturbance in the β-alanine metabolism and an increase in the secretion of pantothenic acid into the growth media. Pantothenic acid production during yeast infection has not been documented and the β-alanine metabolism as well as the pantothenate and CoA biosynthesis pathways may represent underlying metabolic pathways associated with disease progression. Our study suggested that β-alanine metabolism and the pantothenate and CoA biosynthesis pathways might be the important pathways associated with cryptococcal infection. PMID:27054608

  15. Activity of tannins from Stryphnodendron adstringens on Cryptococcus neoformans: effects on growth, capsule size and pigmentation

    PubMed Central

    Ishida, Kelly; Rozental, Sonia; de Mello, João Carlos Palazzo; Nakamura, Celso Vataru

    2009-01-01

    Background Stryphnodendron adstringens (Mart.) Coville, Leguminosae, also known in Brazil as barbatimão, is rich in tannins and many flavan-3-ols and proanthocyanidins such as prodelphinidins and prorobinetinidins. Previous studies have demonstrated several pharmacological properties of tannins from barbatimão, including anti-candidal activity. Methods The antifungal activity of proanthocyanidin polymeric tannins from Stryphnodendron adstringens (subfraction F2.4) was evaluated against three strains of Cryptococcus neoformans with different capsule expressions, using the broth microdilution technique, light microscopy and transmission electron microscopy. The effect of subfraction F2.4 on C. neoformans and melanoma mammalian cells pigmentation was also evaluated. Results Although susceptibility assays revealed MIC values quite similar (between 2.5 and 5.0 μg/ml), analyses of MFC values revealing that the acapsular mutant Cap 67 was more susceptible to be killed by the subfraction F2.4 (MFC = 20 μg/ml) than the two tested capsular strains (T1-444 and ATCC 28957) (MFC > 160 μg/ml). Optical and electron microscopy experiments revealed relevant alterations in cell shape and size in all strains treated with 1 and 2.5 μg/ml of subfraction F2.4. Capsule size of the capsular strains decreased drastically after subfraction F2.4 treatment. In addition, ultrastructural alterations such as cell wall disruption, cytoplasm extraction, mitochondria swelling, increase in the number of cytoplasmic vacuoles and formation of membranous structures in the cytoplasm were also observed in treated yeasts. Incubation with subfraction F2.4 also decreased C. neoformans pigmentation, however, did not interfere in melanization of B16F10 mammalian cells. Conclusion Our data indicate that tannins extracted from S. adstringens interfered with growth, capsule size and pigmentation, all important virulence factors of C. neoformans, and may be considered as a putative candidate for the

  16. Scavenger Receptor A Modulates the Immune Response to Pulmonary Cryptococcus neoformans Infection

    PubMed Central

    Qiu, Yafeng; Dayrit, Jeremy K.; Davis, Michael J.; Carolan, Jacob F.; Osterholzer, John J.; Curtis, Jeffrey L.; Olszewski, Michal A.

    2014-01-01

    Scavenger receptors represent an important class of pattern recognition receptors shown to mediate both beneficial and detrimental roles in host defense against microbial pathogens. The role of the major macrophage scavenger receptor, scavenger receptor A (SRA), in the immune response against the pathogenic fungus, Cryptococcus neoformans, is unknown. To evaluate the role of SRA in anticryptococcal host defenses, SRA+/+ mice and SRA−/− mice were infected intratracheally with C. neoformans. Results show that infection of SRA−/− mice resulted in a reduction in the pulmonary fungal burden at the efferent phase (3 wk) compared with SRA+/+ mice. Improved fungal clearance in SRA−/− mice was associated with decreased accumulation of eosinophils and greater accumulation of CD4+ T cells and CD11b+ dendritic cells. Additional parameters were consistent with enhanced anti-cryptococcal immunity in the infected SRA−/− mice: 1) increased expression of the costimulatory molecules CD80 and CD86 by lung APCs, 2) decreased expression of Th2 cytokines (IL-4 and IL-13) and IL-10 in lung leukocytes and in cryptococcal Ag-pulsed splenocytes, 3) diminished IgE production in sera, and 4) increased hallmarks of classical pulmonary macrophage activation. These effects were preceded by increased expression of early pro-Th1 genes in pulmonary lymph nodes at the afferent phase (1 wk). Collectively, our data show that SRA can be exploited by C. neoformans to interfere with the early events of the afferent responses that support Th1 immune polarization. This results in amplification of Th2 arm of the immune response and subsequently impaired adaptive control of C. neoformans in the infected lungs. PMID:23733871

  17. Invasion of the Central Nervous System by Cryptococcus neoformans Requires a Secreted Fungal Metalloprotease

    PubMed Central

    Vu, Kiem; Tham, Rick; Uhrig, John P.; Thompson, George R.; Na Pombejra, Sarisa; Jamklang, Mantana; Bautos, Jennifer M.

    2014-01-01

    ABSTRACT 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 (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. PMID:24895304

  18. Distinct and redundant roles of exonucleases in Cryptococcus neoformans: Implications for virulence and mating

    PubMed Central

    Wollschlaeger, Carolin; Trevijano-Contador, Nuria; Wang, Xuying; Legrand, Mélanie; Zaragoza, Oscar; Heitman, Joseph; Janbon, Guilhem

    2015-01-01

    Opportunistic pathogens like Cryptococcus neoformans are constantly exposed to changing environments, in their natural habitat as well as when encountering a human host. This requires a coordinated program to regulate gene expression that can act at the levels of mRNA synthesis and also mRNA degradation. Here, we find that deletion of the gene encoding the major cytoplasmic 5’→3’ exonuclease Xrn1p in C. neoformans has important consequences for virulence associated phenotypes such as growth at 37°C, capsule and melanin. In an invertebrate model of cryptococcosis the alteration of these virulence properties corresponds to avirulence of the xrn1Δ mutant strains. Additionally, deletion of XRN1 impairs uni- and bisexual mating. On a molecular level, the absence of XRN1 is associated with the upregulation of other major exonuclease encoding genes (i.e. XRN2 and RRP44). Using inducible alleles of RRP44 and XRN2, we show that artificial overexpression of these genes alters LAC1 gene expression and mating. Our data thus suggest the existence of a complex interdependent regulation of exonuclease encoding genes that impact upon virulence and mating in C. neoformans. PMID:25267175

  19. Binding of Cryptococcus neoformans by human cultured macrophages. Requirements for multiple complement receptors and actin.

    PubMed Central

    Levitz, S M; Tabuni, A

    1991-01-01

    We studied the receptors on human cultured macrophages (MO-M phi) responsible for binding encapsulated and isogenic mutant acapsular strains of Cryptococcus neoformans, and whether such binding leads to a phagocytic event. Both strains required opsonization with complement components in normal human serum in order for binding to occur. Binding of the acapsular, but not the encapsulated, strain led to phagocytosis. MAb directed against any of the three defined complement receptors (CR) on MO-M phi (CR1, CR3, and CR4) profoundly inhibited binding of serum-opsonized encapsulated (and to a lesser extent acapsular) organisms to MO-M phi. Immunofluorescence studies demonstrated migration of CR to the area of the cryptococcal binding site. Trypsin and elastase inhibited binding of encapsulated and, to a lesser extent, acapsular yeasts to MO-M phi. Binding of encapsulated C. neoformans was profoundly inhibited by incubation in the cold or by inhibitors of receptor capping and actin microfilaments. Thus, multiple CR appear to contribute to binding of serum-opsonized encapsulated C. neoformans by MO-M phi. Binding is an energy-dependent process that requires conformational changes in actin yet does not lead to phagocytosis of the organism. In contrast, energy is not required for binding of acapsular yeasts by MO-M phi and binding triggers phagocytosis. Images PMID:1991837

  20. Sec6-dependent sorting of fungal extracellular exosomes and laccase of Cryptococcus neoformans.

    PubMed

    Panepinto, John; Komperda, Kazimierz; Frases, Susana; Park, Yoon-Dong; Djordjevic, Julianne T; Casadevall, Arturo; Williamson, Peter R

    2009-03-01

    The cell wall of pathogenic fungi such as Cryptococcus neoformans, provides a formidable barrier to secrete virulence factors that produce host cell damage. To study secretion of virulence factors to the cell periphery, sec6 RNAi mutant strains of C. neoformans were tested for virulence factor expression. The studies reported here show that SEC6 RNAi mutant strains were defective in a number of virulence factors including laccase, urease as well as soluble polysaccharide and demonstrated attenuated virulence in mice. Further analysis by transmission electron microscopy detected the production of abundant extracellular exosomes in wild-type strains containing empty plasmid, but a complete absence in the iSEC6 strain. In addition, a green fluorescent protein-laccase fusion protein demonstrated aberrant localization within cytoplasmic vesicles in iSEC6 strains. In contrast, iSEC6 strains retained normal growth at 37 degrees C, as well as substantially normal capsule formation, phospholipase activity and total secreted protein. These results provide the first molecular evidence for the existence of fungal exosomes and associate these vesicles with the virulence of C. neoformans. PMID:19210702

  1. Unravelling secretion in Cryptococcus neoformans: more than one way to skin a cat.

    PubMed

    Rodrigues, Marcio L; Djordjevic, Julianne T

    2012-06-01

    Secretion pathways in fungi are essential for the maintenance of cell wall architecture and for the export of a number of virulence factors. In the fungal pathogen, Cryptococcus neoformans, much evidence supports the existence of more than one route taken by secreted molecules to reach the cell periphery and extracellular space, and a significant degree of crosstalk between conventional and non-conventional secretion routes. The need for such complexity may be due to differences in the nature of the exported cargo, the spatial and temporal requirements for constitutive and non-constitutive protein secretion, and/or as a means of compensating for the extra burden on the secretion machinery imposed by the elaboration of the polysaccharide capsule. This review focuses on the role of specific components of the C. neoformans secretion machinery in protein and/or polysaccharide export, including Sec4, Sec6, Sec14, Golgi reassembly and stacking protein and extracellular exosome-like vesicles. We also address what is known about traffic of the lipid, glucosylceramide, a target of therapeutic antibodies and an important regulator of C. neoformans pathogenicity, and the role of signalling pathways in the regulation of secretion. PMID:21898146

  2. Multicenter evaluation of broth microdilution method for susceptibility testing of Cryptococcus neoformans against fluconazole.

    PubMed Central

    Sanati, H; Messer, S A; Pfaller, M; Witt, M; Larsen, R; Espinel-Ingroff, A; Ghannoum, M

    1996-01-01

    We have developed a microdilution method for measuring the susceptibility of Cryptococcus neoformans to fluconazole. The present study evaluated the interlaboratory agreement of the results for the microdilution method obtained at three different sites and compared this method with the National Committee for Clinical Laboratory Standards M27-P reference method. Excellent interlaboratory agreement among the results obtained at the three sites was achieved with this method (83 and 96% agreement within 1 and 2 log2 dilutions, respectively). An overall agreement of 90% between the microdilution method and the M27-P method was observed, demonstrating the comparability of the two methods. However, there are inherent problems with the M27-P method in relation to measuring C. neoformans susceptibility, including suboptimal growth of the organism in RPMI 1640, a longer incubation period, and a narrow range of MICs. On the basis of these data, the microdilution method tested in this study is recommended for inclusion in the National Committee for Laboratory Standards method for testing the antifungal susceptibility of C. neoformans. PMID:8727919

  3. Cryptococcus neoformans Requires a Functional Glycolytic Pathway for Disease but Not Persistence in the Host

    PubMed Central

    Price, Michael S.; Betancourt-Quiroz, Marisol; Price, Jennifer L.; Toffaletti, Dena L.; Vora, Haily; Hu, Guanggan; Kronstad, James W.; Perfect, John R.

    2011-01-01

    ABSTRACT Cryptococcus neoformans is an important fungal pathogen of immunocompromised individuals, with a close relative, Cryptococcus gattii, emerging as a serious threat for the immunocompetent. During initial infection, C. neoformans colonizes the airspaces of the lungs, resulting in pneumonia, and subsequently migrates to the central nervous system (CNS). We sought to understand fungal carbon utilization during colonization of these fundamentally different niches within the host, in particular the roles of gluconeogenesis and glycolysis. We created mutants at key points in the gluconeogenesis/glycolysis metabolic pathways that are restricted for growth on lactate and glucose, respectively. A phosphoenolpyruvate carboxykinase mutant (the pck1∆ mutant), blocked for entry of 2- and 3-carbon substrates into gluconeogenesis and attenuated for virulence in a murine inhalation model, showed wild-type (WT) persistence in a rabbit cerebrospinal fluid (CSF) model of cryptococcosis. Conversely, both the pyruvate kinase (pyk1∆) and the hexose kinase I and II (hxk1∆/hxk2∆) mutants, which show impaired glucose utilization, exhibited severely attenuated virulence in the murine inhalation model of cryptococcosis and decreased persistence in the CNS in both the rabbit CSF and the murine inhalation models while displaying adequate persistence in the lungs of mice. These data suggest that glucose utilization is critical for virulence of C. neoformans and persistence of the yeast in the CNS. PMID:21652778

  4. Genome-Wide Transcription Study of Cryptococcus neoformans H99 Clinical Strain versus Environmental Strains

    PubMed Central

    Movahed, Elaheh; Munusamy, Komathy; Tan, Grace Min Yi; Looi, Chung Yeng; Tay, Sun Tee; Wong, Won Fen

    2015-01-01

    The infection of Cryptococcus neoformans is acquired through the inhalation of desiccated yeast cells and basidiospores originated from the environment, particularly from bird’s droppings and decaying wood. Three environmental strains of C. neoformans originated from bird droppings (H4, S48B and S68B) and C. neoformans reference clinical strain (H99) were used for intranasal infection in C57BL/6 mice. We showed that the H99 strain demonstrated higher virulence compared to H4, S48B and S68B strains. To examine if gene expression contributed to the different degree of virulence among these strains, a genome-wide microarray study was performed to inspect the transcriptomic profiles of all four strains. Our results revealed that out of 7,419 genes (22,257 probes) examined, 65 genes were significantly up-or down-regulated in H99 versus H4, S48B and S68B strains. The up-regulated genes in H99 strain include Hydroxymethylglutaryl-CoA synthase (MVA1), Mitochondrial matrix factor 1 (MMF1), Bud-site-selection protein 8 (BUD8), High affinity glucose transporter 3 (SNF3) and Rho GTPase-activating protein 2 (RGA2). Pathway annotation using DAVID bioinformatics resource showed that metal ion binding and sugar transmembrane transporter activity pathways were highly expressed in the H99 strain. We suggest that the genes and pathways identified may possibly play crucial roles in the fungal pathogenesis. PMID:26360021

  5. Relationship of the Glyoxylate Pathway to the Pathogenesis of Cryptococcus neoformans

    PubMed Central

    Rude, Thomas H.; Toffaletti, Dena L.; Cox, Gary M.; Perfect, John R.

    2002-01-01

    Functional genomics has become a major focus in the study of microbial pathogenesis. This study used a functional genomic tool, differential display reverse transcription-PCR, to identify a transcriptional profile of Cryptococcus neoformans cells as they produced meningitis in an immunosuppressed host. This serial global gene expression during infection allowed for the identification of up- and down-regulated genes during infection. During this profiling, a single gene for the enzyme isocitrate lyase (ICL1) was found to be up regulated at 1 week of infection in a rabbit meningitis model and during a time of maximum host cellular response. The finding suggested that this enzyme and the glyoxylate shunt pathway are important to this yeast's energy production during infection. However, site-directed icl1 mutants had no apparent virulence defect in two animal models and no growth defect within macrophages. These observations suggest that although the yeast responded to a certain environmental cue(s) by an increase in ICL1 expression during infection, this gene was not necessary for progression of a C. neoformans infection. Compounds that specifically target only ICL1 are unlikely to cripple C. neoformans growth in vivo. PMID:12228298

  6. Cryptococcus neoformans Intracellular Proliferation and Capsule Size Determines Early Macrophage Control of Infection.

    PubMed

    Bojarczuk, Aleksandra; Miller, Katie A; Hotham, Richard; Lewis, Amy; Ogryzko, Nikolay V; Kamuyango, Alfred A; Frost, Helen; Gibson, Rory H; Stillman, Eleanor; May, Robin C; Renshaw, Stephen A; Johnston, Simon A

    2016-01-01

    Cryptococcus neoformans is a significant fungal pathogen of immunocompromised patients. Many questions remain regarding the function of macrophages in normal clearance of cryptococcal infection and the defects present in uncontrolled cryptococcosis. Two current limitations are: 1) The difficulties in interpreting studies using isolated macrophages in the context of the progression of infection, and 2) The use of high resolution imaging in understanding immune cell behavior during animal infection. Here we describe a high-content imaging method in a zebrafish model of cryptococcosis that permits the detailed analysis of macrophage interactions with C. neoformans during infection. Using this approach we demonstrate that, while macrophages are critical for control of C. neoformans, a failure of macrophage response is not the limiting defect in fatal infections. We find phagocytosis is restrained very early in infection and that increases in cryptococcal number are driven by intracellular proliferation. We show that macrophages preferentially phagocytose cryptococci with smaller polysaccharide capsules and that capsule size is greatly increased over twenty-four hours of infection, a change that is sufficient to severely limit further phagocytosis. Thus, high-content imaging of cryptococcal infection in vivo demonstrates how very early interactions between macrophages and cryptococci are critical in the outcome of cryptococcosis. PMID:26887656

  7. Radiological Studies Reveal Radial Differences in the Architecture of the Polysaccharide Capsule of Cryptococcus neoformans

    PubMed Central

    Bryan, R. A.; Zaragoza, O.; Zhang, T.; Ortiz, G.; Casadevall, A.; Dadachova, E.

    2005-01-01

    The polysaccharide capsule of the pathogenic fungus Cryptococcus neoformans is an important virulence factor, but relatively little is known about its architecture. We applied a combination of radiological, chemical, and serological methods to investigate the structure of this polysaccharide capsule. Exposure of C. neoformans cells to gamma radiation, dimethyl sulfoxide, or radiolabeled monoclonal antibody removed a significant part of the capsule. Short intervals of gamma irradiation removed the outer portion of the cryptococcal capsule without killing cells, which could subsequently repair their capsules. Survival analysis of irradiated wild-type, acapsular mutant, and complemented mutant strains demonstrated that the capsule contributed to radioprotection and had a linear attenuation coefficient higher than that of lead. The capsule portions remaining after dimethyl sulfoxide or gamma radiation treatment were comparable in size, 65 to 66 μm3, and retained immunoreactivity for a monoclonal antibody to glucuronoxylomannan. Simultaneous or sequential treatment of the cells with dimethyl sulfoxide and radiation removed the remaining capsule so that it was not visible by light microscopy. The capsule could be protected against radiation by either of the free radical scavengers ascorbic acid and sorbitol. Sugar composition analysis of polysaccharide removed from the outer and inner parts of the capsule revealed significant differences in glucuronic acid and xylose molar ratios, implying differences in the chemical structure of the constituent polysaccharides. Our results provide compelling evidence for the existence of two zones in the C. neoformans capsule that differ in susceptibility to dimethyl sulfoxide and radiation and, possibly, in packing and composition. PMID:15701808

  8. Capsular polysaccharides from Cryptococcus neoformans modulate production of neutrophil extracellular traps (NETs) by human neutrophils.

    PubMed

    Rocha, Juliana D B; Nascimento, Michelle T C; Decote-Ricardo, Debora; Côrte-Real, Suzana; Morrot, Alexandre; Heise, Norton; Nunes, Marise P; Previato, José Osvaldo; Mendonça-Previato, Lucia; DosReis, George A; Saraiva, Elvira M; Freire-de-Lima, Célio G

    2015-01-01

    In the present study, we characterized the in vitro modulation of NETs (neutrophil extracellular traps) induced in human neutrophils by the opportunistic fungus Cryptococcus neoformans, evaluating the participation of capsular polysaccharides glucuronoxylomanan (GXM) and glucuronoxylomannogalactan (GXMGal) in this phenomenon. The mutant acapsular strain CAP67 and the capsular polysaccharide GXMGal induced NET production. In contrast, the wild-type strain and the major polysaccharide GXM did not induce NET release. In addition, C. neoformans and the capsular polysaccharide GXM inhibited PMA-induced NET release. Additionally, we observed that the NET-enriched supernatants induced through CAP67 yeasts showed fungicidal activity on the capsular strain, and neutrophil elastase, myeloperoxidase, collagenase and histones were the key components for the induction of NET fungicidal activity. The signaling pathways associated with NET induction through the CAP67 strain were dependent on reactive oxygen species (ROS) and peptidylarginine deiminase-4 (PAD-4). Neither polysaccharide induced ROS production however both molecules blocked the production of ROS through PMA-activated neutrophils. Taken together, the results demonstrate that C. neoformans and the capsular component GXM inhibit the production of NETs in human neutrophils. This mechanism indicates a potentially new and important modulation factor for this fungal pathogen. PMID:25620354

  9. Cryptococcus neoformans Intracellular Proliferation and Capsule Size Determines Early Macrophage Control of Infection

    PubMed Central

    Bojarczuk, Aleksandra; Miller, Katie A.; Hotham, Richard; Lewis, Amy; Ogryzko, Nikolay V.; Kamuyango, Alfred A.; Frost, Helen; Gibson, Rory H.; Stillman, Eleanor; May, Robin C.; Renshaw, Stephen A.; Johnston, Simon A.

    2016-01-01

    Cryptococcus neoformans is a significant fungal pathogen of immunocompromised patients. Many questions remain regarding the function of macrophages in normal clearance of cryptococcal infection and the defects present in uncontrolled cryptococcosis. Two current limitations are: 1) The difficulties in interpreting studies using isolated macrophages in the context of the progression of infection, and 2) The use of high resolution imaging in understanding immune cell behavior during animal infection. Here we describe a high-content imaging method in a zebrafish model of cryptococcosis that permits the detailed analysis of macrophage interactions with C. neoformans during infection. Using this approach we demonstrate that, while macrophages are critical for control of C. neoformans, a failure of macrophage response is not the limiting defect in fatal infections. We find phagocytosis is restrained very early in infection and that increases in cryptococcal number are driven by intracellular proliferation. We show that macrophages preferentially phagocytose cryptococci with smaller polysaccharide capsules and that capsule size is greatly increased over twenty-four hours of infection, a change that is sufficient to severely limit further phagocytosis. Thus, high-content imaging of cryptococcal infection in vivo demonstrates how very early interactions between macrophages and cryptococci are critical in the outcome of cryptococcosis. PMID:26887656

  10. Leu1 plays a role in iron metabolism and is required for virulence in Cryptococcus neoformans

    PubMed Central

    Do, Eunsoo; Hu, Guanggan; Caza, Mélissa; Oliveira, Debora; Kronstad, James W.; Jung, Won Hee

    2015-01-01

    Amino acid biosynthetic pathways that are absent in mammals are considered an attractive target for antifungal therapy. Leucine biosynthesis is one such target pathway, consisting of a five-step conversion process starting from the valine precursor 2-keto-isovalerate. Isopropylmalate dehydrogenase (Leu1) is an Fe-S cluster protein that is required for leucine biosynthesis in the model fungus Saccharomyces cerevisiae. The human pathogenic fungus Cryptococcus neoformans possesses an ortholog of S. cerevisiae Leu1, and our previous transcriptome data showed that the expression of LEU1 is regulated by iron availability. In this study, we characterized the role of Leu1 in iron homeostasis and the virulence of C. neoformans. We found that deletion of LEU1 caused leucine auxotrophy and that Leu1 may play a role in the mitochondrial-cytoplasmic Fe-S cluster balance. Whereas cytoplasmic Fe-S protein levels were not affected, mitochondrial Fe-S proteins were up- regulated in the leu1 mutant, suggesting that Leu1 mainly influences mitochondrial iron metabolism. The leu1 mutant also displayed increased sensitivity to oxidative stress and cell wall/membrane disrupting agents, which may have been caused by mitochondrial dysfunction. Furthermore, the leu1 mutant was deficient in capsule formation and showed attenuated virulence in a mouse inhalation model of cryptococcosis. Overall, our results indicate that Leu1 plays a role in iron metabolism and is required for virulence in C. neoformans. PMID:25554701

  11. Essential Roles of the Kar2/BiP Molecular Chaperone Downstream of the UPR Pathway in Cryptococcus neoformans

    PubMed Central

    Jung, Kwang-Woo; Kang, Hyun Ah; Bahn, Yong-Sun

    2013-01-01

    The endoplasmic reticulum (ER) is a central hub where secreted or membrane-bound proteins are maturated and folded properly in eukaryotes. Maintenance of ER homeostasis is particularly important for human fungal pathogens, such as Cryptococcus neoformans, which encounter a plethora of host-mediated stresses during infection. Our previous study demonstrated that the unfolded protein response (UPR) pathway, composed of the evolutionarily conserved Ire1 kinase and the unique Hxl1 transcription factor, has pleiotropic roles in ER stress response, thermotolerance, antifungal drug resistance, and virulence in C. neoformans. Here, we functionally characterized an ER-resident molecular chaperone, Kar2/BiP, in C. neoformans. Conditional expression of KAR2 by the copper-regulated promoter revealed that Kar2 is essential for the viability of C. neoformans. Constitutive expression of KAR2 by the strong histone H3 promoter partially restores resistance to ER stress, cell wall stress, thermotolerance, and genotoxic stress in ire1Δ and hxl1Δ mutants, suggesting that Kar2 mainly functions downstream of the UPR pathway. Furthermore, Kar2 appears to control azole resistance in C. neoformans downstream of the UPR pathway without regulation of ERG11 or ERG3. Interestingly, we discovered that azole treatment is sensed as ER-stress and subsequently activates the Ire1-dependent Hxl1 splicing event and induction of KAR2 by the UPR pathway. In contrast, the constitutive expression of Kar2 is not sufficient to restore the Ire1-mediated regulation of capsule production in C. neoformans UPR mutants. In conclusion, this study demonstrates that Kar2 is not only essential for vegetative growth but also required for response and adaptation to the environmental stresses and antifungal drugs downstream of the UPR pathway in C. neoformans. PMID:23484059

  12. Relative Contributions of Prenylation and Postprenylation Processing in Cryptococcus neoformans Pathogenesis.

    PubMed

    Esher, Shannon K; Ost, Kyla S; Kozubowski, Lukasz; Yang, Dong-Hoon; Kim, Min Su; Bahn, Yong-Sun; Alspaugh, J Andrew; Nichols, Connie B

    2016-01-01

    Prenyltransferase enzymes promote the membrane localization of their target proteins by directing the attachment of a hydrophobic lipid group at a conserved C-terminal CAAX motif. Subsequently, the prenylated protein is further modified by postprenylation processing enzymes that cleave the terminal 3 amino acids and carboxymethylate the prenylated cysteine residue. Many prenylated proteins, including Ras1 and Ras-like proteins, require this multistep membrane localization process in order to function properly. In the human fungal pathogen Cryptococcus neoformans, previous studies have demonstrated that two distinct forms of protein prenylation, farnesylation and geranylgeranylation, are both required for cellular adaptation to stress, as well as full virulence in animal infection models. Here, we establish that the C. neoformans RAM1 gene encoding the farnesyltransferase β-subunit, though not strictly essential for growth under permissive in vitro conditions, is absolutely required for cryptococcal pathogenesis. We also identify and characterize postprenylation protease and carboxyl methyltransferase enzymes in C. neoformans. In contrast to the prenyltransferases, deletion of the genes encoding the Rce1 protease and Ste14 carboxyl methyltransferase results in subtle defects in stress response and only partial reductions in virulence. These postprenylation modifications, as well as the prenylation events themselves, do play important roles in mating and hyphal transitions, likely due to their regulation of peptide pheromones and other proteins involved in development. IMPORTANCE Cryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular membranes

  13. Relative Contributions of Prenylation and Postprenylation Processing in Cryptococcus neoformans Pathogenesis

    PubMed Central

    Esher, Shannon K.; Ost, Kyla S.; Kozubowski, Lukasz; Yang, Dong-Hoon; Kim, Min Su; Bahn, Yong-Sun; Nichols, Connie B.

    2016-01-01

    ABSTRACT Prenyltransferase enzymes promote the membrane localization of their target proteins by directing the attachment of a hydrophobic lipid group at a conserved C-terminal CAAX motif. Subsequently, the prenylated protein is further modified by postprenylation processing enzymes that cleave the terminal 3 amino acids and carboxymethylate the prenylated cysteine residue. Many prenylated proteins, including Ras1 and Ras-like proteins, require this multistep membrane localization process in order to function properly. In the human fungal pathogen Cryptococcus neoformans, previous studies have demonstrated that two distinct forms of protein prenylation, farnesylation and geranylgeranylation, are both required for cellular adaptation to stress, as well as full virulence in animal infection models. Here, we establish that the C. neoformans RAM1 gene encoding the farnesyltransferase β-subunit, though not strictly essential for growth under permissive in vitro conditions, is absolutely required for cryptococcal pathogenesis. We also identify and characterize postprenylation protease and carboxyl methyltransferase enzymes in C. neoformans. In contrast to the prenyltransferases, deletion of the genes encoding the Rce1 protease and Ste14 carboxyl methyltransferase results in subtle defects in stress response and only partial reductions in virulence. These postprenylation modifications, as well as the prenylation events themselves, do play important roles in mating and hyphal transitions, likely due to their regulation of peptide pheromones and other proteins involved in development. IMPORTANCE Cryptococcus neoformans is an important human fungal pathogen that causes disease and death in immunocompromised individuals. The growth and morphogenesis of this fungus are controlled by conserved Ras-like GTPases, which are also important for its pathogenicity. Many of these proteins require proper subcellular localization for full function, and they are directed to cellular

  14. Essential Gene Discovery in the Basidiomycete Cryptococcus neoformans for Antifungal Drug Target Prioritization

    PubMed Central

    Ianiri, Giuseppe

    2015-01-01

    ABSTRACT Fungal diseases represent a major burden to health care globally. As with other pathogenic microbes, there is a limited number of agents suitable for use in treating fungal diseases, and resistance to these agents can develop rapidly. Cryptococcus neoformans is a basidiomycete fungus that causes cryptococcosis worldwide in both immunocompromised and healthy individuals. As a basidiomycete, it diverged from other common pathogenic or model ascomycete fungi more than 500 million years ago. Here, we report C. neoformans genes that are essential for viability as identified through forward and reverse genetic approaches, using an engineered diploid strain and genetic segregation after meiosis. The forward genetic approach generated random insertional mutants in the diploid strain, the induction of meiosis and sporulation, and selection for haploid cells with counterselection of the insertion event. More than 2,500 mutants were analyzed, and transfer DNA (T-DNA) insertions in several genes required for viability were identified. The genes include those encoding the thioredoxin reductase (Trr1), a ribosome assembly factor (Rsa4), an mRNA-capping component (Cet1), and others. For targeted gene replacement, the C. neoformans homologs of 35 genes required for viability in ascomycete fungi were disrupted, meiosis and sporulation were induced, and haploid progeny were evaluated for their ability to grow on selective media. Twenty-one (60%) were found to be required for viability in C. neoformans. These genes are involved in mitochondrial translation, ergosterol biosynthesis, and RNA-related functions. The heterozygous diploid mutants were evaluated for haploinsufficiency on a number of perturbing agents and drugs, revealing phenotypes due to the loss of one copy of an essential gene in C. neoformans. This study expands the knowledge of the essential genes in fungi using a basidiomycete as a model organism. Genes that have no mammalian homologs and are essential

  15. Identification and Characterization of CPS1 as a Hyaluronic Acid Synthase Contributing to the Pathogenesis of Cryptococcus neoformans Infection▿

    PubMed Central

    Jong, Ambrose; Wu, Chun-Hua; Chen, Han-Min; Luo, Feng; Kwon-Chung, Kyung J.; Chang, Yun C.; LaMunyon, Craig W.; Plaas, Anna; Huang, Sheng-He

    2007-01-01

    Cryptococcus neoformans is a pathogenic yeast that often causes devastating meningoencephalitis in immunocompromised individuals. We have previously identified the C. neoformans CPS1 gene, which is required for a capsular layer on the outer cell wall. In this report, we investigate the function of the CPS1 gene and its pathogenesis. We demonstrated that treatment of yeast with either 4-methylumbelliferone or hyaluronidase resulted in a reduction of the level of C. neoformans binding to human brain microvascular endothelial cells (HBMEC). Yeast extracellular structures were also altered accordingly in hyaluronidase-treated cells. Furthermore, observation of yeast strains with different hyaluronic acid contents showed that the ability to bind to HBMEC is proportional to the hyaluronic acid content. A killing assay with Caenorhabditis elegans demonstrated that the CPS1 wild-type strain is more virulent than the cps1Δ strain. When CPS1 is expressed in Saccharomyces cerevisiae and Escherichia coli, hyaluronic acid can be detected in the cells. Additionally, we determined by fluorophore-assisted carbohydrate electrophoretic analysis that hyaluronic acid is a component of the C. neoformans capsule. The size of hyaluronic acid molecules is evaluated by gel filtration and transmission electron microscopy studies. Together, our results support that C. neoformans CPS1 encodes hyaluronic acid synthase and that its product, hyaluronic acid, plays a role as an adhesion molecule during the association of endothelial cells with yeast. PMID:17545316

  16. Vesicular Polysaccharide Export in Cryptococcus neoformans Is a Eukaryotic Solution to the Problem of Fungal Trans-Cell Wall Transport▿

    PubMed Central

    Rodrigues, Marcio L.; Nimrichter, Leonardo; Oliveira, Débora L.; Frases, Susana; Miranda, Kildare; Zaragoza, Oscar; Alvarez, Mauricio; Nakouzi, Antonio; Feldmesser, Marta; Casadevall, Arturo

    2007-01-01

    The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule, and key lipids, such as glucosylceramide and sterols. A correlation between GXM-containing vesicles and capsule expression was observed. The results imply a novel mechanism for the release of the major virulence factor of C. neoformans whereby polysaccharide packaged in lipid vesicles crosses the cell wall and the capsule network to reach the extracellular environment. PMID:17114598

  17. Cryptococcus neoformans Thermotolerance to Avian Body Temperature Is Sufficient For Extracellular Growth But Not Intracellular Survival In Macrophages

    PubMed Central

    Johnston, Simon A.; Voelz, Kerstin; May, Robin C.

    2016-01-01

    Cryptococcus neoformans is a fatal fungal pathogen of humans that efficiently parasitises macrophages. Birds can be colonised by cryptococci and can transmit cryptococcosis to humans via inhalation of inoculated bird excreta. However, colonisation of birds appears to occur in the absence of symptomatic infection. Here, using a pure population of primary bird macrophages, we demonstrate a mechanism for this relationship. We find that bird macrophages are able to suppress the growth of cryptococci seen in mammalian cells despite C. neoformans being able to grow at bird body temperature, and are able to escape from bird macrophages by vomocytosis. A small subset of cryptococci are able to adapt to the inhibitory intracellular environment of bird macrophages, exhibiting a large cell phenotype that rescues growth suppression. Thus, restriction of intracellular growth combined with survival at bird body temperature explains the ability of birds to efficiently spread C. neoformans in the environment whilst avoiding systemic disease. PMID:26883088

  18. Identification and properties of plasma membrane azole efflux pumps from the pathogenic fungi Cryptococcus gattii and Cryptococcus neoformans

    PubMed Central

    Basso, Luiz R.; Gast, Charles E.; Bruzual, Igor; Wong, Brian

    2015-01-01

    Objectives Cryptococcus gattii from the North American Northwest (NW) have higher azole MICs than do non-NW C. gattii or Cryptococcus neoformans. Since mechanisms of azole resistance in C. gattii are not known, we identified C. gattii and C. neoformans plasma membrane azole efflux pumps and characterized their properties. Methods The C. gattii R265 genome was searched for orthologues of known fungal azole efflux genes, expression of candidate genes was assessed by RT–PCR and the expressed genes' cDNAs were cloned and expressed in Saccharomyces cerevisiae. Azole MICs and intracellular [3H]fluconazole were measured in C. gattii and C. neoformans and in S. cerevisiae expressing each cDNA of interest, as was [3H]fluconazole uptake by post-Golgi vesicles (PGVs) isolated from S. cerevisiae sec6-4 mutants expressing each cDNA of interest. Results Intracellular [3H]fluconazole concentrations were inversely correlated with fluconazole MICs only in 25 NW C. gattii strains. S. cerevisiae expressing three C. gattii cDNAs (encoded by orthologues of C. neoformans AFR1 and MDR1 and the previously unstudied gene AFR2) and their C. neoformans counterparts had higher azole MICs and lower intracellular [3H]fluconazole concentrations than did empty-vector controls. PGVs from S. cerevisiae expressing all six Cryptococcus cDNAs also accumulated more [3H]fluconazole than did controls, and [3H]fluconazole transport by all six transporters of interest was ATP dependent and was inhibited by excess unlabelled fluconazole, voriconazole, itraconazole and posaconazole. Conclusions We conclude that C. gattii and C. neoformans AFR1, MDR1 and AFR2 encode ABC transporters that pump multiple azoles out of S. cerevisiae cells, thereby causing azole resistance. PMID:25630649

  19. Molecular characterization and evaluation of virulence factors of Cryptococcus laurentii and Cryptococcus neoformans strains isolated from external hospital areas.

    PubMed

    Andrade-Silva, Leonardo; Ferreira-Paim, Kennio; Silva-Vergara, Mario León; Pedrosa, André Luiz

    2010-01-01

    Cryptococcosis is a common opportunistic fungal infection that is mainly caused by the species Cryptococcus neoformans and Cryptococcus gattii, but there have recently been several reports of infection by non-neoformans Cryptococcus species. The aims of this study were to genetically characterize Cryptococcus spp. isolated from external hospital areas in Minas Gerais State, Brazil, and to evaluate their pathogenic potential, analyzing their phospholipase and melanin production and the capacity for capsule enlargement. Seventy-three different samples were collected: 62 from bird droppings and 11 from tree detritus. C. neoformans alone was isolated from 43.8% of the samples, Cryptococcus laurentii alone from 23.3% and both fungi were found together in 10.9%. C. laurentii was exclusively isolated from 45% (5/11) of the tree samples (Anacardium occidentale, Guazuma ulmifolia, Mangifera indica and Ficus benjamina). Among the 51 C. neoformans isolates, 47 were classified as type VNI and four as type VNII. All of the C. neoformans isolates were of MATα type. Among the 21 isolates of C. laurentii genotyped using the URA5-RFLP technique, 16 amplified a 1.6kb amplicon which produced a specific restriction profile in 15 isolates. In C. neoformans, 76.4% of the isolates were capable of capsule enlargement in the induction medium and 92.1% were phospholipase producers. In C. laurentii, 7.4% of the isolates were capable of capsule enlargement and 85.1% were phospholipase producers. Characterization of the genotypes and the pathogenic potential of the Cryptococcus spp. isolates studied may contribute towards better understanding of the epidemiology of cryptococcosis and the ecology of agents causing this disease in our region. PMID:20943154

  20. Mitochondria are inherited from the MATa parent in crosses of the basidiomycete fungus Cryptococcus neoformans.

    PubMed Central

    Yan, Zhun; Xu, Jianping

    2003-01-01

    Previous studies demonstrated that mitochondrial DNA (mtDNA) was uniparentally transmitted in laboratory crosses of the pathogenic yeast Cryptococcus neoformans. To begin understanding the mechanisms, this study examined the potential role of the mating-type locus on mtDNA inheritance in C. neoformans. Using existing isogenic strains (JEC20 and JEC21) that differed only at the mating-type locus and a clinical strain (CDC46) that possessed a mitochondrial genotype different from JEC20 and JEC21, we constructed strains that differed only in mating type and mitochondrial genotype. These strains were then crossed to produce hyphae and sexual spores. Among the 206 single spores analyzed from six crosses, all but one inherited mtDNA from the MATa parents. Analyses of mating-type alleles and mtDNA genotypes of natural hybrids from clinical and natural samples were consistent with the hypothesis that mtDNA is inherited from the MATa parent in C. neoformans. To distinguish two potential mechanisms, we obtained a pair of isogenic strains with different mating-type alleles, mtDNA types, and auxotrophic markers. Diploid cells from mating between these two strains were selected and 29 independent colonies were genotyped. These cells did not go through the hyphal stage or the meiotic process. All 29 colonies contained mtDNA from the MATa parent. Because no filamentation, meiosis, or spore formation was involved in generating these diploid cells, our results suggest a selective elimination of mtDNA from the MATalpha parent soon after mating. To our knowledge, this is the first demonstration that mating type controls mtDNA inheritance in fungi. PMID:12702677

  1. The ZIP family zinc transporters support the virulence of Cryptococcus neoformans.

    PubMed

    Do, Eunsoo; Hu, Guanggan; Caza, Mélissa; Kronstad, James W; Jung, Won Hee

    2016-08-01

    Zinc is an essential element in living organisms and a cofactor for various metalloproteins. To disseminate and survive, a pathogenic microbe must obtain zinc from the host, which is an environment with extremely limited zinc availability. In this study, we investigated the roles of the ZIP family zinc transporters Zip1 and Zip2 in the human pathogenic fungus Cryptococcus neoformans Zip1 and Zip2 are homologous to Zrt1 and Zrt2 of the model fungus, Saccharomyces cerevisiae, respectively. We found that the expression of ZIP1 was regulated by the zinc concentration in the environment. Furthermore, the mutant lacking ZIP1 displayed a severe growth defect under zinc-limited conditions, while the mutant lacking ZIP2 displayed normal growth. Inductively coupled plasma-atomic emission spectroscopy analysis showed that the absence of Zip1 expression significantly reduced total cellular zinc levels relative to that in the wild type, while overexpression of Zip1 was associated with increased cellular zinc levels. These findings suggested that Zip1 plays roles in zinc uptake in C. neoformans We also constructed a Zip1-FLAG fusion protein and found, by immunofluorescence, not only that the protein was localized to the periphery implying it is a membrane transporter, but also that the protein was N-glycosylated. Furthermore, the mutant lacking ZIP1 showed attenuated virulence in a murine inhalation model of cryptococcosis and reduced survival within murine macrophages. Overall, our data suggest that Zip1 plays essential roles in zinc transport and the virulence of C. neoformans. PMID:27118799

  2. Cryptococcus neoformans varieties from material under the canopies of eucalyptus trees and pigeon dropping samples from four major cities in Jordan.

    PubMed

    Hamasha, Akram Mohammad Saad; Yildiran, Sinasi Taner; Gonlum, Ahmet; Saracli, Mehmet Ali; Doganci, Levent

    2004-08-01

    To our best knowledge, any study related to the ecological distribution of Cryptococcus neoformans in Jordan does not exist in the medical literature. In order to determine the environmental occurrence of both varieties of Cryptococcus neoformans in Jordan, pigeon droppings and material under the canopies of eucalyptus trees were collected from four major cities of this country. For the isolation of Cryptococcus neoformans variety gattii from environmental sources, 500 samples of the mixed soil debris, including tree materials, under the eucalyptus trees from cities of Amman, Irbid, Jerash, and Ajlun were collected. Also, 509 samples of pigeon droppings were collected from the same cities for the isolation of Cryptococcus neoformans variety neoformans. After inoculating the samples onto modified Staib agar medium in Petri dishes, a total of 336 melanoid yeast colonies were picked up during screening process. At the end of serial mycological studies, none of these isolates was identified as Cryptococcus neoformans, but all were Cryptococcus species other than C. neoformans. For determining the exact status, more extensive environmental studies need to be done in the future. PMID:15518348

  3. Real-Time Imaging of Interactions of Neutrophils with Cryptococcus neoformans Demonstrates a Crucial Role of Complement C5a-C5aR Signaling

    PubMed Central

    Sun, Donglei; Zhang, Mingshun; Liu, Gongguan; Wu, Hui; Zhu, Xiaoping; Zhou, Hong

    2015-01-01

    Neutrophils have been shown to efficiently kill Cryptococcus neoformans, a causative agent of meningoencephalitis. Here, using live-cell imaging, we characterize the dynamic interactions of neutrophils with C. neoformans and the underlying mechanisms in real time. Neutrophils were directly seen to chase C. neoformans cells and then rapidly internalize them. Complement C5a-C5aR signaling guided neutrophils to migrate to the yeast cells, resulting in optimal phagocytosis and subsequent killing of the organisms. The addition of recombinant complement C5a enhanced neutrophil movement but did not induce chemotaxis, suggesting that the C5a gradient is crucial. Incubation with C. neoformans resulted in enhanced activation of Erk and p38 mitogen-activated protein (MAP) kinases (MAPKs) in neutrophils. Inhibition of the p38 MAPK pathway, but not the Erk pathway, significantly impaired neutrophil migration and its subsequent killing of C. neoformans. Deficiency of CD11b or blocking of CD11b did not affect the migration of neutrophils toward C. neoformans but almost completely abolished phagocytosis and killing of the organisms by neutrophils. C5a-C5aR signaling induced enhanced surface expression of CD11b. Interestingly, the original surface expression of CD11b was essential and sufficient for neutrophils to attach to C. neoformans but was unable to mediate phagocytosis. In contrast, the enhanced surface expression of CD11b induced by C5a-C5aR signaling was essential for neutrophil phagocytosis and subsequent killing of yeast cells. Collectively, this is the first report of the dynamic interactions of neutrophils with C. neoformans, demonstrating a crucial role of C5a-C5aR signaling in neutrophil killing of C. neoformans in real time. PMID:26502909

  4. Effect of Virulence Factors on the Photodynamic Inactivation of Cryptococcus neoformans

    PubMed Central

    Prates, Renato A.; Fuchs, Beth Burgwyn; Mizuno, Kazue; Naqvi, Qurat; Kato, Ilka T.; Ribeiro, Martha S.; Mylonakis, Eleftherios; Tegos, George P.; Hamblin, Michael R.

    2013-01-01

    Opportunistic fungal pathogens may cause an array of superficial infections or serious invasive infections, especially in immunocompromised patients. Cryptococcus neoformans is a pathogen causing cryptococcosis in HIV/AIDS patients, but treatment is limited due to the relative lack of potent antifungal agents. Photodynamic inactivation (PDI) uses the combination of non-toxic dyes called photosensitizers and harmless visible light, which produces singlet oxygen and other reactive oxygen species that produce cell inactivation and death. We report the use of five structurally unrelated photosensitizers (methylene blue, Rose Bengal, selenium derivative of a Nile blue dye, a cationic fullerene and a conjugate between poly-L-lysine and chlorin(e6)) combined with appropriate wavelengths of light to inactivate C. neoformans. Mutants lacking capsule and laccase, and culture conditions that favoured melanin production were used to probe the mechanisms of PDI and the effect of virulence factors. The presence of cell wall, laccase and melanin tended to protect against PDI, but the choice of the appropriate photosensitizers and dosimetry was able to overcome this resistance. PMID:23349872

  5. Carbonic anhydrase and CO2 sensing during Cryptococcus neoformans growth, differentiation, and virulence.

    PubMed

    Bahn, Yong-Sun; Cox, Gary M; Perfect, John R; Heitman, Joseph

    2005-11-22

    The gas carbon dioxide (CO2) plays a critical role in microbial and mammalian respiration, photosynthesis in algae and plants, chemoreception in insects, and even global warming . However, how CO2 is transported, sensed, and metabolized by microorganisms is largely not understood. For instance, CO2 is known to induce production of polysaccharide capsule virulence determinants in pathogenic bacteria and fungi via unknown mechanisms . Therefore, we studied CO2 actions in growth, differentiation, and virulence of the basidiomycetous human fungal pathogen Cryptococcus neoformans. The CAN2 gene encoding beta-carbonic anhydrase in C. neoformans was found to be essential for growth in environmental ambient conditions but dispensable for in vivo proliferation and virulence at the high CO2 levels in the host. The can2Delta mutant in vitro growth defect is largely attributable to defective fatty acid synthesis. CO2 was found to inhibit cell-cell fusion but not filamentation during sexual reproduction. The can2 mutation restored early mating events in high CO2 but not later steps (fruiting body formation, sporulation), indicating a major role for carbonic anhydrase and CO2/HCO3- in this developmental cascade leading to the production of infectious spores. Our studies illustrate diverse roles of an ancient enzyme class in enabling environmental survival of a ubiquitous human pathogen. PMID:16303560

  6. Identification of the galactosyltransferase of Cryptococcus neoformans involved in the biosynthesis of basidiomycete-type glycosylinositolphosphoceramide

    PubMed Central

    Wohlschlager, Therese; Buser, Reto; Skowyra, Michael L; Haynes, Brian C; Henrissat, Bernard; Doering, Tamara L; Künzler, Markus; Aebi, Markus

    2013-01-01

    The pathogenic fungus Cryptococcus neoformans synthesizes a complex family of glycosylinositolphosphoceramide (GIPC) structures. These glycosphingolipids (GSLs) consist of mannosylinositolphosphoceramide (MIPC) extended by β1-6-linked galactose, a unique structure that has to date only been identified in basidiomycetes. Further extension by up to five mannose residues and a branching xylose has been described. In this study, we identified and determined the gene structure of the enzyme Ggt1, which catalyzes the transfer of a galactose residue to MIPC. Deletion of the gene in C. neoformans resulted in complete loss of GIPCs containing galactose, a phenotype that could be restored by the episomal expression of Ggt1 in the deletion mutant. The entire annotated open reading frame, encoding a C-terminal GT31 galactosyltransferase domain and a large N-terminal domain of unknown function, was required for complementation. Notably, this gene does not encode a predicted signal sequence or transmembrane domain. The demonstration that Ggt1 is responsible for the transfer of a galactose residue to a GSL thus raises questions regarding the topology of this biosynthetic pathway and the function of the N-terminal domain. Phylogenetic analysis of the GGT1 gene shows conservation in hetero- and homobasidiomycetes but no homologs in ascomycetes or outside of the fungal kingdom. PMID:23926231

  7. Global Molecular Epidemiology of Cryptococcus neoformans and Cryptococcus gattii: An Atlas of the Molecular Types

    PubMed Central

    Cogliati, Massimo

    2013-01-01

    Cryptococcosis is a fungal disease affecting more than one million people per year worldwide. The main etiological agents of cryptococcosis are the two sibling species Cryptococcus neoformans and Cryptococcus gattii that present numerous differences in geographical distribution, ecological niches, epidemiology, pathobiology, clinical presentation and molecular characters. Genotyping of the two Cryptococcus species at subspecies level supplies relevant information to understand how this fungus has spread worldwide, the nature of its population structure, and how it evolved to be a deadly pathogen. At present, nine major molecular types have been recognized: VNI, VNII, VNB, VNIII, and VNIV among C. neoformans isolates, and VGI, VGII, VGIII, and VGIV among C. gattii isolates. In this paper all the information available in the literature concerning the isolation of the two Cryptococcus species has been collected and analyzed on the basis of their geographical origin, source of isolation, level of identification, species, and molecular type. A detailed analysis of the geographical distribution of the major molecular types in each continent has been described and represented on thematic maps. This study represents a useful tool to start new epidemiological surveys on the basis of the present knowledge. PMID:24278784

  8. Cluster of Cryptococcus neoformans Infections in Intensive Care Unit, Arkansas, USA, 2013

    PubMed Central

    Haselow, Dirk; Lloyd, Spencer; Lockhart, Shawn; Moulton-Meissner, Heather; Lester, Laura; Wheeler, Gary; Gladden, Linda; Garner, Kelley; Derado, Gordana; Park, Benjamin; Harris, Julie R.

    2015-01-01

    We investigated an unusual cluster of 6 patients with Cryptococcus neoformans infection at a community hospital in Arkansas during April–December 2013, to determine source of infection. Four patients had bloodstream infection and 2 had respiratory infection; 3 infections occurred within a 10-day period. Five patients had been admitted to the intensive care unit (ICU) with diagnoses other than cryptococcosis; none had HIV infection, and 1 patient had a history of organ transplantation. We then conducted a retrospective cohort study of all patients admitted to the ICU during April–December 2013 to determine risk factors for cryptococcosis. Four patients with C. neoformans infection had received a short course of steroids; this short-term use was associated with increased risk for cryptococcosis (rate ratio 19.1; 95% CI 2.1–170.0; p<0.01). Although long-term use of steroids is a known risk factor for cryptococcosis, the relationship between short-term steroid use and disease warrants further study PMID:26403080

  9. Chemotaxigenesis and activation of the alternative complement pathway by encapsulated and non-encapsulated Cryptococcus neoformans.

    PubMed Central

    Laxalt, K A; Kozel, T R

    1979-01-01

    In the presence of serum, whole cells of encapsulated and non-encapsulated Cryptococcus neoformans generated a chemotactic response by neutrophils. Heat inactivation of serum ablated all chemotactic activity. Cryptococcal polysaccharide was not chemotaxigenic. Assays for alternative complement pathway activation such as depletion of alternative complement pathway factor B or electrophoretic conversion of factor B closely paralleled chemotaxis assays. Cells of encapsulated and non-encapsulated C. neoformans activated the alternative complement pathway, whereas cryptococcal polysaccharide was inactive. Failure of the capsular material to activate the alternative pathway was not due to serotype specificity because polysaccharide of several serotypes failed to achieve activation. The results suggest that chemotaxigenesis and alternative complement pathway activation are functions of the yeast cell wall. The results support our proposal that the cryptococcal capsul does not prevent potential opsonins from reaching binding and activation sites at the yeast cell wall or the release of biologically active soluble cleavage products into the surrounding medium; however, cell wall-bound cleavage products remain bound to the cell wall beneath the capsule. Therefore, they are unable to participate as opsonins in phagocytosis. PMID:397927

  10. Diagnostic Challenges of Cryptococcus neoformans in an Immunocompetent Individual Masquerading as Chronic Hydrocephalus.

    PubMed

    Mahajan, Kedar R; Roberts, Amity L; Curtis, Mark T; Fortuna, Danielle; Dharia, Robin; Sheehan, Lori

    2016-01-01

    Cryptococcus neoformans can cause disseminated meningoencephalitis and evade immunosurveillance with expression of a major virulence factor, the polysaccharide capsule. Direct diagnostic assays often rely on the presence of the cryptococcal glucuronoxylomannan capsular antigen (CrAg) or visualization of the capsule. Strain specific phenotypic traits and environmental conditions influence differences in expression that can thereby compromise detection and timely diagnosis. Immunocompetent hosts may manifest clinical signs and symptoms indolently, often expanding the differential and delaying appropriate treatment and diagnosis. We describe a 63-year-old man who presented with a progressive four-year history of ambulatory dysfunction, headache, and communicating hydrocephalus. Serial lumbar punctures (LPs) revealed elevated protein (153-300 mg/dL), hypoglycorrhachia (19-47 mg/dL), lymphocytic pleocytosis (89-95% lymphocyte, WBC 67-303 mg/dL, and RBC 34-108 mg/dL), and normal opening pressure (13-16 cm H2O). Two different cerebrospinal fluid (CSF) CrAg assays were negative. A large volume CSF fungal culture grew unencapsulated C. neoformans. He was initiated on induction therapy with amphotericin B plus flucytosine and consolidation/maintenance therapy with flucytosine, but he died following discharge due to complications. Elevated levels of CSF Th1 cytokines and decreased IL6 may have affected the virulence and detection of the pathogen. PMID:27525140

  11. Thiazole compounds with activity against Cryptococcus gattii and Cryptococcus neoformans in vitro.

    PubMed

    Pereira de Sá, Nívea; Lino, Cleudiomar Inácio; Fonseca, Nayara Cristina; Borelli, Beatriz Martins; Ramos, Jonas Pereira; Souza-Fagundes, Elaine Maria; Rosa, Carlos Augusto; Santos, Daniel Assis; Barbosa de Oliveira, Renata; Johann, Susana

    2015-09-18

    Human cryptococcosis can occur as a primary or opportunistic infection and develop as an acute, subacute, or chronic, systemic infection involving different host organs. We evaluated the antifungal activity of thirteen compounds against Cryptococcus gattii and Cryptococcus neoformans in vitro, by assessing the toxicity of the compounds showing the greatest antifungal activity in VERO cells and murine macrophages. From these results, four compounds were considered promising for further studies because they displayed low cytotoxicity and significant antifungal activity. The heterocyclic compounds 1b, 1c, 1d, and 1m have antifungal activity levels between that of amphotericin B and fluconazole in vitro. The death curve of Cryptococcus spp. treated with these four compounds was similar to the curve obtained for amphotericin B, in that we observed a significant reduction in cell viability within the first 24 h of treatment. Additionally, we found that there was no effect when these compounds were combined with amphotericin and fluconazole, except for 1c, which antagonized the effect of amphotericin B against C. gattii, also reflected in the reduction of the post-antifungal effect (PAFE); however, this interaction did not alter the ergosterol content. The results shown in this paper reveal the discovery of novel thiazole compounds, which are easy to synthesize, and with potentially exhibit antifungal activity, and display low cytotoxicity in normal mammalian cells. These compounds can be used as prototypes for the design of new antifungal drugs against C. gattii and C. neoformans. PMID:26276437

  12. CHARACTERIZATION OF THE PYROGENICITY OF CANDIDA ALBICANS, SACCHAROMYCES CEREVISIAE, AND CRYPTOCOCCUS NEOFORMANS.

    PubMed

    KOBAYASHI, G S; FRIEDMAN, L

    1964-09-01

    Kobayashi, George S. (Tulane University, New Orleans, La.), and Lorraine Friedman. Characterization of the pyrogenicity of Candida albicans, Saccharomyces cerevisiae, and Cryptococcus neoformans. J. Bacteriol. 88:660-666. 1964.-The intravenous injection into rabbits of 10(9) yeast cells of Candida albicans, Saccharomyces cerevisiae, or Cryptococcus neoformans (both slightly and heavily encapsulated forms) induced a febrile response indistinguishable from that elicited by gram-negative bacterial endotoxin. There was a brisk rise in body temperature which began as early as 30 min after injection, peaked once or twice, and then returned to normal after about 10 hr. With viable C. albicans, the febrile response did not return to normal but remained elevated for several days and terminated at death of the animal. Of three extraction procedures employed in attempts to isolate the endotoxin-like pyrogenically active substances from C. albicans, only one, the phenol extraction method, was successful. Pyrogenic substances were more easily extractable from S. cerevisiae, but extracted cells of both species were still highly pyrogenic. It was concluded that the particulate nature of the yeast cell did not contribute to the induction of fever, for latex particles of a similar size were nonpyrogenic. Viable or heat-killed C. albicans, phenol extract of C. albicans, zymosan, and polystyrene latex particles all failed to induce in rabbits increased dermal reactivity to epinephrine. PMID:14208504

  13. Diagnostic Challenges of Cryptococcus neoformans in an Immunocompetent Individual Masquerading as Chronic Hydrocephalus

    PubMed Central

    Fortuna, Danielle; Dharia, Robin

    2016-01-01

    Cryptococcus neoformans can cause disseminated meningoencephalitis and evade immunosurveillance with expression of a major virulence factor, the polysaccharide capsule. Direct diagnostic assays often rely on the presence of the cryptococcal glucuronoxylomannan capsular antigen (CrAg) or visualization of the capsule. Strain specific phenotypic traits and environmental conditions influence differences in expression that can thereby compromise detection and timely diagnosis. Immunocompetent hosts may manifest clinical signs and symptoms indolently, often expanding the differential and delaying appropriate treatment and diagnosis. We describe a 63-year-old man who presented with a progressive four-year history of ambulatory dysfunction, headache, and communicating hydrocephalus. Serial lumbar punctures (LPs) revealed elevated protein (153–300 mg/dL), hypoglycorrhachia (19–47 mg/dL), lymphocytic pleocytosis (89–95% lymphocyte, WBC 67–303 mg/dL, and RBC 34–108 mg/dL), and normal opening pressure (13–16 cm H2O). Two different cerebrospinal fluid (CSF) CrAg assays were negative. A large volume CSF fungal culture grew unencapsulated C. neoformans. He was initiated on induction therapy with amphotericin B plus flucytosine and consolidation/maintenance therapy with flucytosine, but he died following discharge due to complications. Elevated levels of CSF Th1 cytokines and decreased IL6 may have affected the virulence and detection of the pathogen. PMID:27525140

  14. Effects of CTR4 deletion on virulence and stress response in Cryptococcus neoformans.

    PubMed

    Zhang, Ping; Zhang, Defa; Zhao, Xueru; Wei, Dongsheng; Wang, Yu; Zhu, Xudong

    2016-08-01

    Roles of the high-affinity copper transporter Ctr4 in the virulence of Cryptococcus neoformans remain to be fully determined. Here we demonstrate that Ctr4 plays a necessary role in virulence and tolerance to a number of stress conditions. We first observed, with the method of flame atomic absorption spectrometry, that deletion of CTR4 resulted in a significant decrease in intracellular copper level, confirming the role of Ctr4 as a copper transporter in C. neoformans. Furthermore, CTR4 was critical for the yeast to survive at both elevated and low temperatures, as the growth rate of the ctr4Δ mutant at 4 and 37 °C was significantly decreased. The mutant ctr4Δ also exhibited hypersensitivity to osmotic stress imposed by 2 M NaCl or KCl, indicating the possible crosstalk of Ctr4 with the HOG signalling pathway. Moreover, cell wall and plasma membrane integrity appeared to be impaired in the ctr4Δ strain. The virulence of ctr4Δ in two mouse cryptococcosis models was remarkably reduced either via an intranasal or intravenous inoculation. Our work confirms the roles of Ctr4 in virulence and copper homeostasis as well as other additional novel functions. PMID:27317510

  15. Cryptococcosis in the era of AIDS--100 years after the discovery of Cryptococcus neoformans.

    PubMed Central

    Mitchell, T G; Perfect, J R

    1995-01-01

    Although Cryptococcus neoformans and cryptococcosis have existed for several millennia, a century has passed since the discovery of this encapsulated yeast and its devastating disease. With the advent of the AIDS pandemic, cryptococcal meningitis has emerged as a leading cause of infectious morbidity and mortality and a frequently life-threatening opportunistic mycosis among patients with AIDS. Both basic and clinical research have accelerated in the 1990s, and this review attempts to highlight some of these advances. The discussion covers recent findings, current concepts, controversies, and unresolved issues related to the ecology and genetics of C. neoformans; the surface structure of the yeast; and the mechanisms of host defense. Regarding cell-mediated immunity, CD4+ T cells are crucial for successful resistance, but CD8+ T cells may also participate significantly in the cytokine-mediated activation of anticryptococcal effector cells. In addition to cell-mediated immunity, monoclonal antibodies to the major capsular polysaccharide, the glucuronoxylomannan, offer some protection in murine models of cryptococcosis. Clinical concepts are presented that relate to the distinctive features of cryptococcosis in patients with AIDS and the diagnosis, treatment, and prevention of cryptococcosis in AIDS patients. PMID:8665468

  16. Capsule Growth in Cryptococcus neoformans Is Coordinated with Cell Cycle Progression

    PubMed Central

    García-Rodas, Rocío; Cordero, Radames J. B.; Trevijano-Contador, Nuria; Janbon, Guilhem; Moyrand, Frédérique; Casadevall, Arturo

    2014-01-01

    ABSTRACT The fungal pathogen Cryptococcus neoformans has several virulence factors, among which the most important is a polysaccharide capsule. The size of the capsule is variable and can increase significantly during infection. In this work, we investigated the relationship between capsular enlargement and the cell cycle. Capsule growth occurred primarily during the G1 phase. Real-time visualization of capsule growth demonstrated that this process occurred before the appearance of the bud and that capsule growth arrested during budding. Benomyl, which arrests the cells in G2/M, inhibited capsule growth, while sirolimus (rapamycin) addition, which induces G1 arrest, resulted in cells with larger capsule. Furthermore, we have characterized a mutant strain that lacks a putative G1/S cyclin. This mutant showed an increased capacity to enlarge the capsule, both in vivo (using Galleria mellonella as the host model) and in vitro. In the absence of Cln1, there was a significant increase in the production of extracellular vesicles. Proteomic assays suggest that in the cln1 mutant strain, there is an upregulation of the glyoxylate acid cycle. Besides, this cyclin mutant is avirulent at 37°C, which correlates with growth defects at this temperature in rich medium. In addition, the cln1 mutant showed lower intracellular replication rates in murine macrophages. We conclude that cell cycle regulatory elements are involved in the modulation of the expression of the main virulence factor in C. neoformans. PMID:24939886

  17. Activity of sertraline against Cryptococcus neoformans: in vitro and in vivo assays.

    PubMed

    Treviño-Rangel, Rogelio de J; Villanueva-Lozano, Hiram; Hernández-Rodríguez, Pedro; Martínez-Reséndez, Michel F; García-Juárez, Jaime; Rodríguez-Rocha, Humberto; González, Gloria M

    2016-03-01

    Cryptococcus neoformans infection is an important cause of meningitis in HIV/AIDS endemic regions. Antifungals for its management include amphotericin B, flucytosine, and fluconazole. Recently, treatment of this mycosis with sertraline has been studied with variable clinical outcomes. The aim of the study was to assess the in vitro antifungal effect of sertraline against clinical isolates of Cryptococcus spp. as well as its in vivo activity in a murine model of cryptococcal meningoencephalitis. The in vitro susceptibility to fluconazole, amphotericin B, voriconazole and sertraline of 153 Cryptococcus spp. strains were evaluated according to CLSI procedures. Fungal tissue burden, serum antigenaemia and histopathology, together with the therapeutic efficacy of amphotericin B (3 mg/kg), fluconazole (15 mg/kg), and sertraline (3, 10, and 15 mg/kg) were evaluated in mice intracranially inoculated with one isolate of Cryptococcus neoformans. All strains were susceptible to the antifungals studied and exhibited growth inhibition with sertraline at clinically relevant concentrations. Sertraline at a dose of 15 mg/kg reduced the fungal burden in the brain and spleen with an efficacy comparable to that of fluconazole. In conclusion, sertraline exhibited an excellent in vitro-in vivo anti-cryptococcal activity, representing a possible new alternative for the clinical management of meningeal cryptococcosis. PMID:26705833

  18. Impact of Resistance to Fluconazole on Virulence and Morphological Aspects of Cryptococcus neoformans and Cryptococcus gattii Isolates

    PubMed Central

    Rossi, Suélen A.; Trevijano-Contador, Nuria; Scorzoni, Liliana; Mesa-Arango, Ana Cecilia; de Oliveira, Haroldo C.; Werther, Karin; de Freitas Raso, Tânia; Mendes-Giannini, Maria J. S.; Zaragoza, Oscar; Fusco-Almeida, Ana M.

    2016-01-01

    Cryptococcus sp. are responsible for around 1 million cases of meningitis every year. Fluconazole (FLU) is commonly used in the treatment of cryptococcosis, mainly in immunocompromised patients and the resistance is usually reported after long periods of treatment. In this study, the morphological characterization and virulence profile of FLU-susceptible and FLU-resistant clinical and environmental isolates of C. neoformans and C. gattii were performed both in vitro and in vivo using the Galleria mellonella model. FLU-susceptible isolates from C. neoformans were significantly more virulent than the FLU-resistant isolates. FLU-susceptible C. gattii isolates showed a different virulence profile from C. neoformans isolates where only the environmental isolate, CL, was more virulent compared with the resistant isolates. Cell morphology and capsule size were analyzed and the FLU-resistant isolates did not change significantly compared with the most sensitive isolates. Growth at 37°C was also evaluated and in both species, the resistant isolates showed a reduced growth at this temperature, indicating that FLU resistance can affect their growth. Based on the results obtained is possible suggest that FLU resistance can influence the morphology of the isolates and consequently changed the virulence profiles. The most evident results were observed for C. neoformans showing that the adaptation of isolates to antifungal selective pressure influenced the loss of virulence. PMID:26909069

  19. The 14-3-3 Gene Function of Cryptococcus neoformans Is Required for its Growth and Virulence.

    PubMed

    Li, Jingbo; Chang, Yun C; Wu, Chun-Hua; Liu, Jennifer; Kwon-Chung, Kyung J; Huang, Sheng-He; Shimada, Hiro; Fante, Rob; Fu, Xiaowei; Jong, Ambrose

    2016-05-28

    Cryptococcus neoformans is a life-threatening pathogenic yeast that causes devastating meningoencephalitis. The mechanism of cryptococcal brain invasion is largely unknown, and recent studies suggest that its extracellular microvesicles may be involved in the invasion process. The 14-3-3 protein is abundant in the extracellular microvesicles of C. neoformans, and the 14-3-3-GFP fusion has been used as the microvesicle's marker. However, the physiological role of 14-3-3 has not been explored. In this report, we have found that C. neoformans contains a single 14-3-3 gene that apparently is an essential gene. To explore the functions of 14-3-3, we substituted the promoter region of the 14-3-3 with the copper-controllable promoter CTR4. The CTR4 regulatory strain showed an enlarged cell size, drastic changes in morphology, and a decrease in the thickness of the capsule under copper-enriched conditions. Furthermore, the mutant cells produced a lower amount of total proteins in their extracellular microvesicles and reduced adhesion to human brain microvascular endothelial cells in vitro. Proteomic analyses of the protein components under 14-3-3-overexpressed and -suppressed conditions revealed that the 14-3-3 function(s) might be associated with the microvesicle biogenesis. Our results support that 14-3-3 has diverse pertinent roles in both physiology and pathogenesis in C. neoformans. Its gene functions are closely relevant to the pathogenesis of this fungus. PMID:26437944

  20. Binding of the wheat germ lectin to Cryptococcus neoformans chitooligomers affects multiple mechanisms required for fungal pathogenesis

    PubMed Central

    Fonseca, Fernanda L.; Guimarães, Allan J.; Kmetzsch, Lívia; Dutra, Fabianno F.; Silva, Fernanda D.; Taborda, Carlos P.; Araujo, Glauber de S.; Frases, Susana; Staats, Charley C.; Bozza, Marcelo T.; Schrank, Augusto; Vainstein, Marilene H.; Nimrichter, Leonardo; Casadevall, Arturo; Rodrigues, Marcio L.

    2015-01-01

    The principal capsular component of Cryptococcus neoformans, glucuronoxylomannan (GXM), interacts with surface glycans, including chitin-like oligomers. Although the role of GXM in cryptococcal infection has been well explored, there is no information on how chitooligomers affect fungal pathogenesis. In this study, surface chitooligomers of C. neoformans were blocked through the use of the wheat germ lectin (WGA) and the effects on animal pathogenesis, interaction with host cells, fungal growth and capsule formation were analyzed. Treatment of C. neoformans cells with WGA followed by infection of mice delayed mortality relative to animals infected with untreated fungal cells. This observation was associated with reduced brain colonization by lectin-treated cryptococci. Blocking chitooligomers also rendered yeast cells less efficient in their ability to associate with phagocytes. WGA did not affect fungal viability, but inhibited GXM release to the extracellular space and capsule formation. In WGA-treated yeast cells, genes that are involved in capsule formation and GXM traffic had their transcription levels decreased in comparison with untreated cells. Our results suggest that cellular pathways required for capsule formation and pathogenic mechanisms are affected by blocking chitin-derived structures at the cell surface of C. neoformans. Targeting chitooligomers with specific ligands may reveal new therapeutic alternatives to control cryptococcosis. PMID:23608320

  1. Role of Sterylglucosidase 1 (Sgl1) on the pathogenicity of Cryptococcus neoformans: potential applications for vaccine development

    PubMed Central

    Rella, Antonella; Mor, Visesato; Farnoud, Amir M.; Singh, Ashutosh; Shamseddine, Achraf A.; Ivanova, Elitza; Carpino, Nicholas; Montagna, Maria T.; Luberto, Chiara; Del Poeta, Maurizio

    2015-01-01

    Cryptococcosis caused by Cryptococcus neoformans and Cryptococcus gattii affects a large population and is a cause of significant morbidity and mortality. Despite its public health burden, there are currently no vaccines against cryptococcosis and new strategies against such infections are needed. In this study, we demonstrate that C. neoformans has the biochemical ability to metabolize sterylglucosides (SGs), a class of immunomodulatory glycolipids. Genetic manipulations that eliminate cryptococccal sterylglucosidase lead to the accumulation of SGs and generate a mutant strain (Δsgl1) that is non-pathogenic in the mouse models of cryptococcosis. Interestingly, this mutant strain acts as a vaccine strain and protects mice against cryptococcosis following infection with C. neoformans or C. gattii. The immunity induced by the Δsgl1 strain is not CD4+ T-cells dependent. Immunocompromised mice, which lack CD4+ T-cells, are able to control the infection by Δsgl1 and acquire immunity against the challenge by wild-type C. neoformans following vaccination with the Δsgl1 strain. These findings are particularly important in the context of HIV/AIDS immune deficiency and suggest that the Δsgl1 strain might provide a potential vaccination strategy against cryptococcosis. PMID:26322039

  2. [Evaluation of a new medium, eggplant (Solanum melongena) agar as a screening medium for Cryptococcus neoformans in environmental samples].

    PubMed

    Sengul, Mustafa; Ergin, Cağrı; Kartal, Tuğba

    2014-04-01

    Cryptococcus neofomans is an encapsulated yeast-like fungus that causes life-threatening infections, especially in immunosuppresive patients. C.neoformans infection is believed to be acquired via inhalation of aerosolized particles from the environment. Avian guano, decaying tree hollows and soil are the related known environmental niches. Brown pigmented yeast growth from the precursors in growth media is an important step for the identification and isolation of C.neoformans. Seeds of plants in nature are preferred owing to easy accessibility and low costs for the preparation of such media. Guizotia abysinicca (Niger seed) as Staib agar, Helianthus annus (Sunflower) as Pal's medium, Brassica nigra (Mustard) agar, tobacco agar, Mucuna pruriens (Velvet bean) seed agar, Perilla frutescens (Beefsteak plant) seed agar, Rubus fruticosus (Blackberry) agar and ground red hot pepper agar are pigment-based selective media for the differentiation of C.neoformans. The aim of this study was to observe the pigment production of C.neoformans in a new medium based on eggplant (Solanum melongena) and also to compare its performance with the simplified Staib, Pal's and tobacco agar for isolation from the environment. Three different eggplant-based medium (S.melongena Melanzaza viserba, S.melongena Pinstripe F1 and S.ovigerum Ivory F1) were included in the study. Pigment-forming eggplant medium, simplified Staib agar, Pal's agar and tobacco agar were used for the cultivation of the environmental swabbed samples from 19 Eucalyptus camaldulensis trunk hollows in continuous colonization region. While pigment formation were observed with S.melongena Melanzaza viserba and S.melongena Pinstripe F1 containing media, S.ovigerum Ivory F1 medium was found to be non-reactive. In colonization area (Gökova-Akyaka, Turkey), 11 (57.9%) out of 19 E.camaldulensis samples were positive with simplified Staib agar, Pal's agar and eggplant agar while 10 (52.6%) of them are positive with tobacco agar. C.neoformans

  3. Deletion of Cryptococcus neoformans AIF Ortholog Promotes Chromosome Aneuploidy and Fluconazole-Resistance in a Metacaspase-Independent Manner

    PubMed Central

    Semighini, Camile P.; Averette, Anna F.; Perfect, John R.; Heitman, Joseph

    2011-01-01

    Apoptosis is a form of programmed cell death critical for development and homeostasis in multicellular organisms. Apoptosis-like cell death (ALCD) has been described in several fungi, including the opportunistic human pathogen Cryptococcus neoformans. In addition, capsular polysaccharides of C. neoformans are known to induce apoptosis in host immune cells, thereby contributing to its virulence. Our goals were to characterize the apoptotic signaling cascade in C. neoformans as well as its unique features compared to the host machinery to exploit the endogenous fungal apoptotic pathways as a novel antifungal strategy in the future. The dissection of apoptotic pathways revealed that apoptosis-inducing factor (Aif1) and metacaspases (Mca1 and Mca2) are independently required for ALCD in C. neoformans. We show that the apoptotic pathways are required for cell fusion and sporulation during mating, indicating that apoptosis may occur during sexual development. Previous studies showed that antifungal drugs induce ALCD in fungi and that C. neoformans adapts to high concentrations of the antifungal fluconazole (FLC) by acquisition of aneuploidy, especially duplication of chromosome 1 (Chr1). Disruption of aif1, but not the metacaspases, stimulates the emergence of aneuploid subpopulations with Chr1 disomy that are resistant to fluconazole (FLCR) in vitro and in vivo. FLCR isolates in the aif1 background are stable in the absence of the drug, while those in the wild-type background readily revert to FLC sensitivity. We propose that apoptosis orchestrated by Aif1 might eliminate aneuploid cells from the population and defects in this pathway contribute to the selection of aneuploid FLCR subpopulations during treatment. Aneuploid clinical isolates with disomies for chromosomes other than Chr1 exhibit reduced AIF1 expression, suggesting that inactivation of Aif1 might be a novel aneuploidy-tolerating mechanism in fungi that facilitates the selection of antifungal drug resistance

  4. Growth and pigment production on D-tryptophan medium by Cryptococcus gattii, Cryptococcus neoformans, and Candida albicans.

    PubMed

    Chaskes, Stuart; Frases, Susana; Cammer, Michael; Gerfen, Gary; Casadevall, Arturo

    2008-01-01

    Given the increasing prevalence of cryptococcosis caused by Cryptococcus gattii (serotypes B and C) strains, there is a need for rapid and reliable tests that discriminate C. gattii from Cryptococcus neoformans (serotypes A, D, and AD). Seventy-two C. neoformans strains, sixty-seven C. gattii strains, and five Candida albicans strains were analyzed for their ability to grow and produce pigment on minimal D-tryptophan D-proline (m-DTDP) medium, on yeast carbon base D-tryptophan D-proline (YCB-DTDP) medium, and on fructose D-tryptophan glycine (m-FDTG) medium. Of the C. gattii and C. neoformans isolates, 94% and 0% grew on m-DTDP agar, respectively, and 98% and 0% grew in YCB-DTDP medium, respectively. C. gattii produced large amounts of brown intracellular pigment(s) on m-DTDP agar and smaller amounts of yellow-brown (amber) extracellular pigment(s). C. albicans grew on both media and produced a pink photoactivated pigment on m-DTDP agar. C. gattii produced large amounts of brown intracellular pigments on the differential medium m-FDTG, whereas C. neoformans produced smaller amounts of the brown pigments and C. albicans produced a pink pigment. The pigments produced by C. gattii from D-tryptophan were distinct and were not related to melanin formation from 3,4-dihydroxyphenylalanine. Thin-layer chromatography of the methanol-extracted C. gattii cells detected four different pigments, including brown (two types), yellow, and pink-purple compounds. We conclude that tryptophan-derived pigments are not melanins and that growth on m-DTDP or YCB-DTDP agar can be used to rapidly differentiate C. gattii from C. neoformans. PMID:17989195

  5. Applying Genetics and Molecular Biology to the Study of the Human Pathogen Cryptococcus neoformans

    PubMed Central

    Chun, Cheryl D.; Madhani, Hiten D.

    2013-01-01

    The basidiomycete yeast Crytococcus neoformans is a prominent human pathogen. It primarily infects immunocompromised individuals producing a meningoencephalitis that is lethal if untreated. Recent advances in its genetics and molecular biology have made it a model system for understanding both the Basidiomycota phylum and mechanisms of fungal pathogenesis. The relative ease of experimental manipulation coupled with the development of murine models for human disease allow for powerful studies in the mechanisms of virulence and host responses. This chapter introduces the organism and its life cycle and then provides detailed step-by-step protocols for culture, manipulation of the genome, analysis of nucleic acids and proteins, and assessment of virulence and expression of virulence factors. PMID:20946836

  6. Networks of fibers and factors: regulation of capsule formation in Cryptococcus neoformans.

    PubMed

    Ding, Hao; Mayer, François L; Sánchez-León, Eddy; de S Araújo, Glauber R; Frases, Susana; Kronstad, James W

    2016-01-01

    The ability of the pathogenic fungus Cryptococcus neoformans to cause life-threatening meningoencephalitis in immunocompromised individuals is due in large part to elaboration of a capsule consisting of polysaccharide fibers. The size of the cell-associated capsule is remarkably responsive to a variety of environmental and host conditions, but the mechanistic details of the regulation, synthesis, trafficking, and attachment of the polysaccharides are poorly understood. Recent studies reveal a complex network of transcription factors that influence capsule elaboration in response to several different signals of relevance to disease (e.g., iron deprivation). The emerging complexity of the network is consistent with the diversity of conditions that influence the capsule and illustrates the responsiveness of the fungus to both the environment and mammalian hosts. PMID:27516877

  7. Networks of fibers and factors: regulation of capsule formation in Cryptococcus neoformans

    PubMed Central

    de S. Araújo, Glauber R.; Frases, Susana; Kronstad, James W.

    2016-01-01

    The ability of the pathogenic fungus Cryptococcus neoformans to cause life-threatening meningoencephalitis in immunocompromised individuals is due in large part to elaboration of a capsule consisting of polysaccharide fibers. The size of the cell-associated capsule is remarkably responsive to a variety of environmental and host conditions, but the mechanistic details of the regulation, synthesis, trafficking, and attachment of the polysaccharides are poorly understood. Recent studies reveal a complex network of transcription factors that influence capsule elaboration in response to several different signals of relevance to disease (e.g., iron deprivation). The emerging complexity of the network is consistent with the diversity of conditions that influence the capsule and illustrates the responsiveness of the fungus to both the environment and mammalian hosts. PMID:27516877

  8. All about that fat: Lipid modification of proteins in Cryptococcus neoformans

    PubMed Central

    Santiago-Tirado, Felipe H.; Doering, Tamara L.

    2016-01-01

    Lipid modification of proteins is a widespread, essential process whereby fatty acids, cholesterol, isoprenoids, phospholipids, or glycosylphospholipids are attached to polypeptides. These hydrophobic groups may affect protein structure, function, localization, and/or stability; as a consequence such modifications play critical regulatory roles in cellular systems. Recent advances in chemical biology and proteomics have allowed the profiling of modified proteins, enabling dissection of the functional consequences of lipid addition. The enzymes that mediate lipid modification are specific for both the lipid and protein substrates, and are conserved from fungi to humans. In this article we review these enzymes, their substrates, and the processes involved in eukaryotic lipid modification of proteins. We further focus on its occurrence in the fungal pathogen Cryptococcus neoformans, highlighting unique features that are both relevant for the biology of the organism and potentially important in the search for new therapies. PMID:26920881

  9. How Sweet it is! Cell Wall Biogenesis and Polysaccharide Capsule Formation in Cryptococcus neoformans

    PubMed Central

    Doering, Tamara Lea

    2010-01-01

    Cryptococcus neoformans is a pathogenic fungus responsible for severe opportunistic infections. The most prominent feature of this yeast is its elaborate polysaccharide capsule, a complex structure that is required for virulence. The capsule is intimately associated with the cell wall, which underlies the capsule and offers the organism strength and flexibility in potentially hostile environments. Both structures are primarily composed of polysaccharides, offering a glimpse of the tremendous variation inherent in natural carbohydrate structures and their multiple biological functions. The steps in cell wall and capsule biosynthesis and assembly pose fascinating questions of metabolism, enzymology, cell biology, and regulation; the answers have potential application to treatment of a deadly infection. This article reviews current knowledge of cryptococcal cell wall and capsule biosynthesis and outstanding questions for the future. PMID:19575556

  10. Massive cerebral edema resulting in brain death as a complication of Cryptococcus neoformans meningitis

    PubMed Central

    Orsini, Jose; Blaak, Christa; Mahmoud, Dalia; Young-Gwang, Jeong

    2015-01-01

    Despite the widespread use of highly active antiretroviral therapy, cryptococcal meningoencephalitis has emerged as the second leading cause of infectious morbidity and mortality in HIV-infected patients worldwide. It presents usually as subacute or chronic disease but occasionally may be fulminant. Common clinical presentations included headache, fever, and depressed level of consciousness. The infection affects both the subarachnoid space and brain parenchyma, and is characterized by a paucity of inflammation and a large fungal burden in the cerebrospinal fluid at the time of diagnosis. Infection is usually lethal without treatment, thus the prompt diagnosis and therapy might improve the outcome. We report a case of brain death caused by Cryptococcus neoformans meningitis that was diagnosed based on clinical neurological examinations and supported by the absence of cerebral blood flow on brain angiography. PMID:25656669

  11. Polyethylene sulfonate: a tight-binding inhibitor of 6-phosphogluconate dehydrogenase of Cryptococcus neoformans.

    PubMed

    Niehaus, W G; White, R H; Richardson, S B; Bourne, A; Ray, W K

    1995-12-20

    Polyethylene sulfonate (PES) or polyvinyl sulfonate was found to be a potent inhibitor of a number of fungal enzymes, including 6-phosphogluconate dehydrogenase from Cryptococcus neoformans. The inhibition was apparently competitive versus either NADP or 6-phosphogluconate, with 50% inhibition at PES concentrations below 10 nM. Replots of slopes of double-reciprocal plots versus inhibitor concentration were sharply concave upward, whereas replots of slope versus [PES]3 were linear. The inhibition was freely reversible upon dilution of the enzyme-PES complex. A model is presented that involves initial binding of the long (M(r) 50,000) polyanionic PES at a remote site on the enzyme, followed by interaction of the end of the tethered polymer with the binding site for NADP or for 6-phosphogluconate. PMID:8554324

  12. Analysis of Cell Cycle and Replication of Mouse Macrophages after In Vivo and In Vitro Cryptococcus neoformans Infection Using Laser Scanning Cytometry

    PubMed Central

    Tesfa, Lydia; Zhang, Jinghang; Rivera, Johanna; Gonçalves, Teresa; Casadevall, Arturo

    2012-01-01

    We investigated the outcome of the interaction of Cryptococcus neoformans with murine macrophages using laser scanning cytometry (LSC). Previous results in our lab had shown that phagocytosis of C. neoformans promoted cell cycle progression. LSC allowed us to simultaneously measure the phagocytic index, macrophage DNA content, and 5-ethynyl-2′-deoxyuridine (EdU) incorporation such that it was possible to study host cell division as a function of phagocytosis. LSC proved to be a robust, reliable, and high-throughput method for quantifying phagocytosis. Phagocytosis of C. neoformans promoted cell cycle progression, but infected macrophages were significantly less likely to complete mitosis. Hence, we report a new cytotoxic effect associated with intracellular C. neoformans residence that manifested itself in impaired cell cycle completion as a consequence of a block in the G2/M stage of the mitotic cell cycle. Cell cycle arrest was not due to increased cell membrane permeability or DNA damage. We investigated alveolar macrophage replication in vivo and demonstrated that these cells are capable of low levels of cell division in the presence or absence of C. neoformans infection. In summary, we simultaneously studied phagocytosis, the cell cycle state of the host cell and pathogen-mediated cytotoxicity, and our results demonstrate a new cytotoxic effect of C. neoformans infection on murine macrophages: fungus-induced cell cycle arrest. Finally, we provide evidence for alveolar macrophage proliferation in vivo. PMID:22252872

  13. The Cryptococcus neoformans Alkaline Response Pathway: Identification of a Novel Rim Pathway Activator

    PubMed Central

    Ost, Kyla S.; O’Meara, Teresa R.; Huda, Naureen; Esher, Shannon K.; Alspaugh, J. Andrew

    2015-01-01

    The Rim101/PacC transcription factor acts in a fungal-specific signaling pathway responsible for sensing extracellular pH signals. First characterized in ascomycete fungi such as Aspergillus nidulans and Saccharomyces cerevisiae, the Rim/Pal pathway maintains conserved features among very distantly related fungi, where it coordinates cellular adaptation to alkaline pH signals and micronutrient deprivation. However, it also directs species-specific functions in fungal pathogens such as Cryptococcus neoformans, where it controls surface capsule expression. Moreover, disruption of the Rim pathway central transcription factor, Rim101, results in a strain that causes a hyper-inflammatory response in animal infection models. Using targeted gene deletions, we demonstrate that several genes encoding components of the classical Rim/Pal pathway are present in the C. neoformans genome. Many of these genes are in fact required for Rim101 activation, including members of the ESCRT complex (Vps23 and Snf7), ESCRT-interacting proteins (Rim20 and Rim23), and the predicted Rim13 protease. We demonstrate that in neutral/alkaline pH, Rim23 is recruited to punctate regions on the plasma membrane. This change in Rim23 localization requires upstream ESCRT complex components but does not require other Rim101 proteolysis components, such as Rim20 or Rim13. Using a forward genetics screen, we identified the RRA1 gene encoding a novel membrane protein that is also required for Rim101 protein activation and, like the ESCRT complex, is functionally upstream of Rim23-membrane localization. Homologs of RRA1 are present in other Cryptococcus species as well as other basidiomycetes, but closely related genes are not present in ascomycetes. These findings suggest that major branches of the fungal Kingdom developed different mechanisms to sense and respond to very elemental extracellular signals such as changing pH levels. PMID:25859664

  14. Unisexual reproduction drives meiotic recombination and phenotypic and karyotypic plasticity in Cryptococcus neoformans.

    PubMed

    Sun, Sheng; Billmyre, R Blake; Mieczkowski, Piotr A; Heitman, Joseph

    2014-12-01

    In fungi, unisexual reproduction, where sexual development is initiated without the presence of two compatible mating type alleles, has been observed in several species that can also undergo traditional bisexual reproduction, including the important human fungal pathogens Cryptococcus neoformans and Candida albicans. While unisexual reproduction has been well characterized qualitatively, detailed quantifications are still lacking for aspects of this process, such as the frequency of recombination during unisexual reproduction, and how this compares with bisexual reproduction. Here, we analyzed meiotic recombination during α-α unisexual and a-α bisexual reproduction of C. neoformans. We found that meiotic recombination operates in a similar fashion during both modes of sexual reproduction. Specifically, we observed that in α-α unisexual reproduction, the numbers of crossovers along the chromosomes during meiosis, recombination frequencies at specific chromosomal regions, as well as meiotic recombination hot and cold spots, are all similar to those observed during a-α bisexual reproduction. The similarity in meiosis is also reflected by the fact that phenotypic segregation among progeny collected from the two modes of sexual reproduction is also similar, with transgressive segregation being observed in both. Additionally, we found diploid meiotic progeny were also produced at similar frequencies in the two modes of sexual reproduction, and transient chromosomal loss and duplication likely occurs frequently and results in aneuploidy and loss of heterozygosity that can span entire chromosomes. Furthermore, in both α-α unisexual and a-α bisexual reproduction, we observed biased allele inheritance in regions on chromosome 4, suggesting the presence of fragile chromosomal regions that might be vulnerable to mitotic recombination. Interestingly, we also observed a crossover event that occurred within the MAT locus during α-α unisexual reproduction. Our results

  15. Cryptococcus neoformans serotype A glucuronoxylomannan-protein conjugate vaccines: synthesis, characterization, and immunogenicity.

    PubMed

    Devi, S J; Schneerson, R; Egan, W; Ulrich, T J; Bryla, D; Robbins, J B; Bennett, J E

    1991-10-01

    We synthesized Cryptococcus neoformans serotype A glucuronoxylomannan (GXM) conjugate vaccines under conditions suitable for human use to prevent disseminated cryptococcosis. The purified, sonicated GXM was derivatized with adipic acid dihydrazide through either hydroxyl or carboxyl groups and then covalently bound to tetanus toxoid (TT) or Pseudomonas aeruginosa exoprotein A (rEPA). The immunogenicity of these conjugates was evaluated in BALB/c and general purpose mice by subcutaneous injection in saline. The conjugates elicited higher GXM antibody responses than GXM alone. Booster immunoglobulin G (IgG) and IgM responses were elicited by all conjugates in BALB/c mice. The conjugates prepared through hydroxyl activation (GXM-TT2 and GXM-rEPA) were more immunogenic than the one prepared through carboxyl activation (GXM-TT1). GXM antibody response was enhanced by the administration of monophosphoryl lipid A 2 days following the injection of GXM-TT2 (P less than 0.03). The conjugates also elicited IgG antibodies to the carrier proteins. Gel diffusion tests using conjugate-induced hyperimmune sera and chemically modified GXMs suggested that the specificity of GXM-TT1-induced antibodies was conferred by the O-acetyl groups. Hyperimmune sera generated by GXM-TT2 precipitated with the chemically unmodified and the de-O-acetylated GXMs but not with the carboxyl-reduced and de-O-acetylated GXM. GXM-TT2-induced hyperimmune serum also precipitated with the capsular polysaccharides of C. neoformans serotypes D, B, and C. The conjugate vaccines prepared through hydroxyl activation of the GXM are sufficiently immunogenic and appear to be suitable for clinical evaluation. PMID:1716613

  16. Antibodies to the Cryptococcus neoformans capsular glucuronoxylomannan are ubiquitous in serum from HIV+ and HIV- individuals.

    PubMed Central

    Deshaw, M; Pirofski, L A

    1995-01-01

    Murine MoAbs to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) polysaccharide are protective in mice in vivo and in vitro. The prevalence of protective anti-GXM antibodies in human serum is unknown. To provide further insight into the human antibody response to C. neoformans we determined the prevalence, isotype, and IgG subclass utilization of human anti-GXM antibodies in HIV+ and HIV- sera by a sensitive antigen capture FLISA assay. One hundred and twenty-three sera from the Bronx Municipal Hospital Centre serum bank were studied retrospectively. Seventy were from HIV+ individuals, 10 with a history of cryptococcal meningitis (CM), and 53 were from HIV- individuals. Serum GXM determinations were also performed on 61 HIV+ sera. Our results demonstrated that anti-GXM IgG, IgA, and IgM are ubiquitous in both HIV+ (including those with CM), and HIV- sera. Anti-GXM IgA titres and total serum IgA concentration were elevated in HIV+ sera. Anti-GXM IgG antibodies were almost exclusively isotype-restricted to the IgG2 subclass. Our data also demonstrated elevations of anti-bovine serum albumin (BSA) titres in HIV+ sera. Taken together, our findings confirm hypergammaglobulinaemia and expansion of anti-protein (BSA) antibodies in HIV+ individuals and isotype restriction of human anti-carbohydrate (GXM) antibodies to the IgG2 subclass. Our report of ubiquitous anti-GXM antibodies of the IgG and IgA isotypes suggests that anti-GXM antibodies exist before HIV infection. PMID:7882565

  17. The Gamma Interferon Receptor Is Required for the Protective Pulmonary Inflammatory Response to Cryptococcus neoformans

    PubMed Central

    Chen, Gwo-Hsiao; McDonald, Roderick A.; Wells, Jason C.; Huffnagle, Gary B.; Lukacs, Nicholas W.; Toews, Galen B.

    2005-01-01

    Mice with a null deletion mutation in the gamma interferon (IFN-γ) receptor gene were used to study the role of IFN-γ responsiveness during experimental pulmonary cryptococcosis. Cryptococcus neoformans was inoculated intratracheally into mice lacking the IFN-γ receptor gene (IFN-γR−/−) and into control mice (IFN-γR+/+). The numbers of CFU in lung, spleen, and brain were determined to assess clearance; cytokines produced by lung leukocytes were measured, and survival curves were generated. In the present study, we demonstrate the following points. (i) IFN-γR−/− mice are markedly more susceptible to C. neoformans infection than IFN-γR+/+ mice. (ii) In the absence of IFN-γ signaling, pulmonary CFU continue to increase over the course of infection, and the infection disseminates to the brain. (iii) In the absence of IFN-γ receptor, recruitment of inflammatory cells in response to pulmonary cryptococcal infection is not impaired. (iv) At week 5 postinfection, IFN-γR−/− mice have recruited greater numbers of leukocytes into their lungs, with neutrophils, eosinophils, and lymphocytes accounting for this cellular increase. (v) IFN-γ signaling is required for the development of a T1 over a T2 immune response in the lung following cryptococcal infection. These results indicate that in the absence of IFN- γ responsiveness, even though the recruitment of pulmonary inflammatory cells is not impaired and the secretion of IFN-γ is not affected, IFN-γR−/− mice do not have the ability to resolve the cryptococcal infection. In conclusion, our data suggest that proper functional IFN-γ signaling, possibly through a mechanism which inhibits the potentially disease-promoting T2 response, is required for mice to confine the cryptococcal infection. PMID:15731080

  18. Crystal structure of Gib2, a signal-transducing protein scaffold associated with ribosomes in Cryptococcus neoformans

    NASA Astrophysics Data System (ADS)

    Ero, Rya; Dimitrova, Valya Tenusheva; Chen, Yun; Bu, Wenting; Feng, Shu; Liu, Tongbao; Wang, Ping; Xue, Chaoyang; Tan, Suet Mien; Gao, Yong-Gui

    2015-03-01

    The atypical Gβ-like/RACK1 Gib2 protein promotes cAMP signalling that plays a central role in regulating the virulence of Cryptococcus neoformans. Gib2 contains a seven-bladed β transducin structure and is emerging as a scaffold protein interconnecting signalling pathways through interactions with various protein partners. Here, we present the crystal structure of Gib2 at a 2.2-Å resolution. The structure allows us to analyse the association between Gib2 and the ribosome, as well as to identify the Gib2 amino acid residues involved in ribosome binding. Our studies not only suggest that Gib2 has a role in protein translation but also present Gib2 as a physical link at the crossroads of various regulatory pathways important for the growth and virulence of C. neoformans.

  19. High-Throughput Screen in Cryptococcus neoformans Identifies a Novel Molecular Scaffold That Inhibits Cell Wall Integrity Pathway Signaling

    PubMed Central

    2015-01-01

    Cryptococcus neoformans is one of the most important human fungal pathogens; however, no new therapies have been developed in over 50 years. Fungicidal activity is crucially important for an effective anticryptococal agent and, therefore, we screened 361,675 molecules against C. neoformans using an adenylate kinase release assay that specifically detects fungicidal activity. A set of secondary assays narrowed the set of hits to molecules that interfere with fungal cell wall integrity and identified three benzothioureas with low in vitro mammalian toxicity and good in vitro anticryptococcal (minimum inhibitory concentration = 4 μg/mL). This scaffold inhibits signaling through the cell wall integrity MAP kinase cascade. Structure–activity studies indicate that the thiocarbonyl moiety is crucial for activity. Genetic and biochemical data suggest that benzothioureas inhibit signaling upstream of the kinase cascade. Thus, the benzothioureas appear to be a promising new scaffold for further exploration in the search for new anticryptococcal agents. PMID:26807437

  20. A Role for LHC1 in Higher Order Structure and Complement Binding of the Cryptococcus neoformans Capsule

    PubMed Central

    Park, Yoon-Dong; Shin, Soowan; Panepinto, John; Ramos, Jeanie; Qiu, Jin; Frases, Susana; Albuquerque, Patricia; Cordero, Radames J. B.; Zhang, Nannan; Himmelreich, Uwe; Beenhouwer, David; Bennett, John E.; Casadevall, Arturo; Williamson, Peter R.

    2014-01-01

    Polysaccharide capsules are important virulence factors for many microbial pathogens including the opportunistic fungus Cryptococcus neoformans. In the present study, we demonstrate an unusual role for a secreted lactonohydrolase of C. neoformans, LHC1 in capsular higher order structure. Analysis of extracted capsular polysaccharide from wild-type and lhc1Δ strains by dynamic and static light scattering suggested a role for the LHC1 locus in altering the capsular polysaccharide, both reducing dimensions and altering its branching, density and solvation. These changes in the capsular structure resulted in LHC1-dependent alterations of antibody binding patterns, reductions in human and mouse complement binding and phagocytosis by the macrophage-like cell line J774, as well as increased virulence in mice. These findings identify a unique molecular mechanism for tertiary structural changes in a microbial capsule, facilitating immune evasion and virulence of a fungal pathogen. PMID:24789368

  1. Solid-state NMR Reveals the Carbon-based Molecular Architecture of Cryptococcus neoformans Fungal Eumelanins in the Cell Wall*

    PubMed Central

    Chatterjee, Subhasish; Prados-Rosales, Rafael; Itin, Boris; Casadevall, Arturo; Stark, Ruth E.

    2015-01-01

    Melanin pigments protect against both ionizing radiation and free radicals and have potential soil remediation capabilities. Eumelanins produced by pathogenic Cryptococcus neoformans fungi are virulence factors that render the fungal cells resistant to host defenses and certain antifungal drugs. Because of their insoluble and amorphous characteristics, neither the pigment bonding framework nor the cellular interactions underlying melanization of C. neoformans have yielded to comprehensive molecular-scale investigation. This study used the C. neoformans requirement of exogenous obligatory catecholamine precursors for melanization to produce isotopically enriched pigment “ghosts” and applied 2D 13C-13C correlation solid-state NMR to reveal the carbon-based architecture of intact natural eumelanin assemblies in fungal cells. We demonstrated that the aliphatic moieties of solid C. neoformans melanin ghosts include cell-wall components derived from polysaccharides and/or chitin that are associated proximally with lipid membrane constituents. Prior to development of the mature aromatic fungal pigment, these aliphatic moieties form a chemically resistant framework that could serve as the scaffold for melanin synthesis. The indole-based core aromatic moieties show interconnections that are consistent with proposed melanin structures consisting of stacked planar assemblies, which are associated spatially with the aliphatic scaffold. The pyrrole aromatic carbons of the pigments bind covalently to the aliphatic framework via glycoside or glyceride functional groups. These findings establish that the structure of the pigment assembly changes with time and provide the first biophysical information on the mechanism by which melanin is assembled in the fungal cell wall, offering vital insights that can advance the design of bioinspired conductive nanomaterials and novel therapeutics. PMID:25825492

  2. In Vitro Comparative Efficacy of Voriconazole and Itraconazole against Fluconazole-Susceptible and -Resistant Cryptococcus neoformans Isolates

    PubMed Central

    Nguyen, M. Hong; Yu, Christine Y.

    1998-01-01

    In vitro susceptibility testing for 50 clinical isolates of fluconazole-susceptible or -resistant Cryptococcus neoformans was performed with itraconazole and voriconazole. Voriconazole was more potent than itraconazole for fluconazole-susceptible isolates and as potent as itraconazole for fluconazole-susceptible dose-dependent isolates and for fluconazole-resistant isolates. For fluconazole-resistant isolates, the voriconazole and itraconazole MICs ranged from 1 to 2 μg/ml. PMID:9527812

  3. The Investigational Fungal Cyp51 Inhibitor VT-1129 Demonstrates Potent In Vitro Activity against Cryptococcus neoformans and Cryptococcus gattii.

    PubMed

    Lockhart, Shawn R; Fothergill, Annette W; Iqbal, Naureen; Bolden, Carol B; Grossman, Nina T; Garvey, Edward P; Brand, Stephen R; Hoekstra, William J; Schotzinger, Robert J; Ottinger, Elizabeth; Patterson, Thomas F; Wiederhold, Nathan P

    2016-04-01

    Thein vitroactivities of the novel fungal Cyp51 inhibitor VT-1129 were evaluated against a large panel ofCryptococcus neoformansandCryptococcus gattiiisolates. VT-1129 demonstrated potent activities against bothCryptococcusspecies as demonstrated by low MIC50and MIC90values. ForC. gattii, thein vitropotency was maintained against all genotypes. In addition, significantly lower geometric mean MICs were observed for VT-1129 than for fluconazole againstC. neoformans, including isolates with reduced fluconazole susceptibility. PMID:26787697

  4. Requirement for CD4+ T Lymphocytes in Host Resistance against Cryptococcus neoformans in the Central Nervous System of Immunized Mice

    PubMed Central

    Buchanan, Kent L.; Doyle, Hester A.

    2000-01-01

    The importance of cell-mediated immunity (CMI) and CD4+ T lymphocytes in host resistance against Cryptococcus neoformans is well documented and is exemplified by the high susceptibility to progressive infection with this pathogen of AIDS patients with reduced CD4+ T-cell numbers. Although much has been learned about the role of CMI in the clearance of C. neoformans from the lungs and other internal organs, less is known about the protective mechanisms in the brain, the organ most frequently involved with a fatal outcome of cryptococcosis. We hypothesized that host resistance mechanisms against C. neoformans in the central nervous system (CNS) were similar to those outside the CNS (i.e., gamma interferon [IFN-γ], CD4+ T cells, and others). To test this hypothesis, we used a murine model of cryptococcal meningitis whereby cryptococci are introduced directly into the CNS. In experiments where mice were immunized to mount an anticryptococcal CMI response, our results indicate that immunization induced protective mechanisms that could be detected in the CNS by inhibition of the growth of viable yeast cells. Flow cytometric analyses of leukocytes in brain and spinal cord homogenates revealed that T lymphocytes, macrophages, and neutrophils accumulated in C. neoformans-infected brains of immune mice. In vivo depletion of CD4+ T cells, but not CD8+ T cells, resulted in significantly reduced leukocyte accumulation in the brains of immune mice. Furthermore, depletion of CD4+ T cells or neutralization of IFN-γ exacerbated CNS infection in immune mice, suggesting a critical role for CMI mechanisms in acquired protection in the CNS. PMID:10639404

  5. Cryptococcus neoformans Requires the ESCRT Protein Vps23 for Iron Acquisition from Heme, for Capsule Formation, and for Virulence

    PubMed Central

    Hu, Guanggan; Caza, Mélissa; Cadieux, Brigitte; Chan, Vivienne; Liu, Victor

    2013-01-01

    Iron availability is a key regulator of virulence factor elaboration in Cryptococcus neoformans, the causative agent of fungal meningoencephalitis in HIV/AIDS patients. In addition, iron is an essential nutrient for pathogen proliferation in mammalian hosts but little is known about the mechanisms of iron sensing and uptake in fungal pathogens that attack humans. In this study, we mutagenized C. neoformans by Agrobacterium-mediated T-DNA insertion and screened for mutants with reduced growth on heme as the sole iron source. Among 34 mutants, we identified a subset with insertions in the gene for the ESCRT-I (endosomal sorting complex required for transport) protein Vps23 that resulted in a growth defect on heme, presumably due to a defect in uptake via endocytosis or misregulation of iron acquisition from heme. Remarkably, vps23 mutants were also defective in the elaboration of the cell-associated capsular polysaccharide that is a major virulence factor, while overexpression of Vps23 resulted in cells with a slightly enlarged capsule. These phenotypes were mirrored by a virulence defect in the vps23 mutant in a mouse model of cryptococcosis and by hypervirulence of the overexpression strain. Overall, these results reveal an important role for trafficking via ESCRT functions in both heme uptake and capsule formation, and they further reinforce the connection between iron and virulence factor deployment in C. neoformans. PMID:23132495

  6. Characterization of Lipids and Proteins Associated to the Cell Wall of the Acapsular Mutant Cryptococcus neoformans Cap 67.

    PubMed

    Longo, Larissa V G; Nakayasu, Ernesto S; Pires, Jhon H S; Gazos-Lopes, Felipe; Vallejo, Milene C; Sobreira, Tiago J P; Almeida, Igor C; Puccia, Rosana

    2015-01-01

    Cryptococcus neoformans is an opportunistic human pathogen that causes life-threatening meningitis. In this fungus, the cell wall is exceptionally not the outermost structure due to the presence of a surrounding polysaccharide capsule, which has been highly studied. Considering that there is little information about C. neoformans cell wall composition, we aimed at describing proteins and lipids extractable from this organelle, using as model the acapsular mutant C. neoformans cap 67. Purified cell wall preparations were extracted with either chloroform/methanol or hot sodium dodecyl sulfate. Total lipids fractionated in silica gel 60 were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS), while trypsin digested proteins were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We detected 25 phospholipid species among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Two glycolipid species were identified as monohexosyl ceramides. We identified 192 noncovalently linked proteins belonging to different metabolic processes. Most proteins were classified as secretory, mainly via nonclassical mechanisms, suggesting a role for extracellular vesicles (EV) in transwall transportation. In concert with that, orthologs from 86% of these proteins have previously been reported both in fungal cell wall and/or in EV. The possible role of the presently described structures in fungal-host relationship is discussed. PMID:25733123

  7. Cryptococcus neoformans Is Internalized by Receptor-Mediated or ‘Triggered’ Phagocytosis, Dependent on Actin Recruitment

    PubMed Central

    Guerra, Caroline Rezende; Seabra, Sergio Henrique; de Souza, Wanderley; Rozental, Sonia

    2014-01-01

    Cryptococcosis by the encapsulated yeast Cryptococcus neoformans affects mostly immunocompromised individuals and is a frequent neurological complication in AIDS patients. Recent studies support the idea that intracellular survival of Cryptococcus yeast cells is important for the pathogenesis of cryptococcosis. However, the initial steps of Cryptococcus internalization by host cells remain poorly understood. Here, we investigate the mechanism of Cryptococcus neoformans phagocytosis by peritoneal macrophages using confocal and electron microscopy techniques, as well as flow cytometry quantification, evaluating the importance of fungal capsule production and of host cell cytoskeletal elements for fungal phagocytosis. Electron microscopy analyses revealed that capsular and acapsular strains of C. neoformans are internalized by macrophages via both ‘zipper’ (receptor-mediated) and ‘trigger’ (membrane ruffle-dependent) phagocytosis mechanisms. Actin filaments surrounded phagosomes of capsular and acapsular yeasts, and the actin depolymerizing drugs cytochalasin D and latrunculin B inhibited yeast internalization and actin recruitment to the phagosome area. In contrast, nocodazole and paclitaxel, inhibitors of microtubule dynamics decreased internalization but did not prevent actin recruitment to the site of phagocytosis. Our results show that different uptake mechanisms, dependent on both actin and tubulin dynamics occur during yeast internalization by macrophages, and that capsule production does not affect the mode of Cryptococcus uptake by host cells. PMID:24586631

  8. αADα Hybrids of Cryptococcus neoformans: Evidence of Same-Sex Mating in Nature and Hybrid Fitness

    PubMed Central

    Lin, Xiaorong; Litvintseva, Anastasia P; Nielsen, Kirsten; Patel, Sweta; Floyd, Anna; Mitchell, Thomas G; Heitman, Joseph

    2007-01-01

    Cryptococcus neoformans is a ubiquitous human fungal pathogen that causes meningoencephalitis in predominantly immunocompromised hosts. The fungus is typically haploid, and sexual reproduction involves two individuals with opposite mating types/sexes, α and a. However, the overwhelming predominance of mating type (MAT) α over a in C. neoformans populations limits α–a mating in nature. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions, especially between α isolates. Whether same-sex mating occurs in nature and contributes to the current population structure was unknown. In this study, natural αADα hybrids that arose by fusion between two α cells of different serotypes (A and D) were identified and characterized, providing definitive evidence that same-sex mating occurs naturally. A novel truncated allele of the mating-type-specific cell identity determinant SXI1α was also identified as a genetic factor likely involved in this process. In addition, laboratory-constructed αADα strains exhibited hybrid vigor both in vitro and in vivo, providing a plausible explanation for their relative abundance in nature despite the fact that AD hybrids are inefficient in meiosis/sporulation and are trapped in the diploid state. These findings provide insights on the origins, genetic mechanisms, and fitness impact of unisexual hybridization in the Cryptococcus population. PMID:17953489

  9. A novel experimental model of Cryptococcus neoformans-related immune reconstitution inflammatory syndrome (IRIS) provides insights into pathogenesis.

    PubMed

    Eschke, Maria; Piehler, Daniel; Schulze, Bianca; Richter, Tina; Grahnert, Andreas; Protschka, Martina; Müller, Uwe; Köhler, Gabriele; Höfling, Corinna; Rossner, Steffen; Alber, Gottfried

    2015-12-01

    Antiretroviral therapy (ART) has yielded major advances in fighting the HIV pandemic by restoring protective immunity. However, a significant proportion of HIV patients co-infected with the opportunistic fungal pathogen Cryptococcus neoformans paradoxically develops a life-threatening immune reconstitution inflammatory syndrome (IRIS) during antiretroviral therapy. Despite several clinical studies, the underlying pathomecha-nisms are poorly understood. Here, we present the first mouse model of cryptococcal IRIS that allows for a detailed analysis of disease development. Lymphocyte-deficient RAG-1(-/-) mice are infected with C. neoformans and 4 weeks later adoptively transferred with purified CD4(+) T cells. Reconstitution of CD4(+) T cells is sufficient to induce a severe inflammatory disease similar to clinical IRIS in C. neoformans-infected RAG-1(-/-) mice of different genetic backgrounds and immunological phenotypes (i.e. C57BL/6 and BALB/c). Multiorgan inflammation is accompanied by a systemic release of distinct proinflammatory cytokines, i.e. IFN-γ, IL-6, and TNF-α. IRIS development is characterized by infection-dependent activation of donor CD4(+) T cells, which are the source of IFN-γ. Interestingly, IFN-γ-mediated effects are not required for disease induction. Taken together, this novel mouse model of cryptococcal IRIS provides a useful tool to verify potential mechanisms of pathogenesis, revealing targets for diagnosis and therapeutic interventions. PMID:26381487

  10. Characterization of the Chromosome 4 Genes That Affect Fluconazole-Induced Disomy Formation in Cryptococcus neoformans

    PubMed Central

    Ngamskulrungroj, Popchai; Chang, Yun; Hansen, Bryan; Bugge, Cliff; Fischer, Elizabeth; Kwon-Chung, Kyung J.

    2012-01-01

    Heteroresistance in Cryptococcus neoformans is an intrinsic adaptive resistance to azoles and the heteroresistant phenotype is associated with disomic chromosomes. Two chromosome 1 (Chr1) genes, ERG11, the fluconazole target, and AFR1, a drug transporter, were reported as major factors in the emergence of Chr1 disomy. In the present study, we show Chr4 to be the second most frequently formed disomy at high concentrations of fluconazole (FLC) and characterize the importance of resident genes contributing to disomy formation. We deleted nine Chr4 genes presumed to have functions in ergosterol biosynthesis, membrane composition/integrity or drug transportation that could influence Chr4 disomy under FLC stress. Of these nine, disruption of three genes homologous to Sey1 (a GTPase), Glo3 and Gcs2 (the ADP-ribosylation factor GTPase activating proteins) significantly reduced the frequency of Chr4 disomy in heteroresistant clones. Furthermore, FLC resistant clones derived from sey1Δglo3Δ did not show disomy of either Chr4 or Chr1 but instead had increased the copy number of the genes proximal to ERG11 locus on Chr1. Since the three genes are critical for the integrity of endoplasmic reticulum (ER) in Saccharomyces cerevisiae, we used Sec61ß-GFP fusion as a marker to study the ER in the mutants. The cytoplasmic ER was found to be elongated in sey1Δ but without any discernable alteration in gcs2Δ and glo3Δ under fluorescence microscopy. The aberrant ER morphology of all three mutant strains, however, was discernable by transmission electron microscopy. A 3D reconstruction using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) revealed considerably reduced reticulation in the ER of glo3Δ and gcs2Δ strains. In sey1Δ, ER reticulation was barely detectable and cisternae were expanded extensively compared to the wild type strains. These data suggest that the genes required for maintenance of ER integrity are important for the formation of disomic chromosomes in C

  11. First report on Cryptococcus neoformans in pigeon excreta from public and residential locations in the metropolitan area of Cuiabá, State of Mato Grosso, Brazil.

    PubMed

    Takahara, Doracilde Terumi; Lazéra, Márcia dos Santos; Wanke, Bodo; Trilles, Luciana; Dutra, Valéria; Paula, Daphine Ariadne Jesus de; Nakazato, Luciano; Anzai, Mariana Caselli; Leite Júnior, Diniz Pereira; Paula, Claudete Rodrigues; Hahn, Rosane Christine

    2013-01-01

    Cryptococcosis is a severe systemic mycosis caused by two species of Cryptococcus that affect humans and animals: C. neoformans and C. gattii. Cosmopolitan and emergent, the mycosis results from the interaction between a susceptible host and the environment. The occurrence of C. neoformans was evaluated in 122 samples of dried pigeon excreta collected in 49 locations in the City of Cuiabá, State of Mato Grosso, Brazil, including public squares (n = 5), churches (n = 4), educational institutions (n = 3), health units (n = 8), open areas covered with asbestos (n = 4), residences (n = 23), factory (n = 1) and a prison (n = 1). Samples collected from July to December of 2010 were seeded on Niger seed agar (NSA). Dark brown colonies were identified by urease test, carbon source assimilation tests and canavanine-glycine-bromothymol blue medium. Polymerase chain reaction primer pairs specific for C. neoformans were also used for identification. Cryptococcus neoformans associated to pigeon excreta was isolated from eight (6.6%) samples corresponding to six (12.2%) locations. Cryptococcus neoformans was isolated from urban areas, predominantly in residences, constituting a risk of acquiring the disease by immunocompromised and immunocompetent individuals. PMID:24213188

  12. Dual Infections with Pigmented and Albino Strains of Cryptococcus neoformans in Patients with or without Human Immunodeficiency Virus Infection in India

    PubMed Central

    Mandal, Piyali; Banerjee, Uma; Casadevall, Arturo; Nosanchuk, Joshua D.

    2005-01-01

    Cryptococcus neoformans is an encapsulated yeast-like fungus of worldwide distribution. Melanin production is an important virulence factor of C. neoformans. We report the identification of distinct cryptococcal isolates with either pigmented or white colony phenotypes on l-dihydroxyphenylalanine agar plates in three patients who presented with meningitis to the All India Institute of Medical Sciences in India. Two of the patients were also infected with human immunodeficiency virus. Biochemical studies, India ink analysis, immunofluorescence with antibodies specific to capsular antigen, and serotyping confirmed that the melanotic and albino strains were C. neoformans serotypes A and D, respectively. Genotyping with M13 and [GACA]4 primers revealed that all the C. neoformans isolates were genetically different. The CNLAC1 gene associated with melanin production was identified in all the strains by PCR. Standard MIC testing revealed that the strains had similar susceptibilities to amphotericin B, but time-kill assays with the antifungal showed reduced susceptibility in melanin-producing strains. Infection studies with A/Jcr mice showed that the melanin-lacking yeast were less virulent than melanin-producing isolates. These findings indicate that these patients had dual infections with pigmented and albino strains of C. neoformans that were phenotypically and biologically different. Continued surveillance of primary isolates from patients with cryptococcosis by analyzing phenotypic differences and by molecular methods may reveal that mixed infections occur more commonly than is currently realized. PMID:16145139

  13. A Flucytosine-Responsive Mbp1/Swi4-Like Protein, Mbs1, Plays Pleiotropic Roles in Antifungal Drug Resistance, Stress Response, and Virulence of Cryptococcus neoformans

    PubMed Central

    Song, Min-Hee; Lee, Jang-Won; Kim, Min Su; Yoon, Ja-Kyung; White, Theodore C.; Floyd, Anna; Heitman, Joseph; Strain, Anna K.; Nielsen, Judith N.; Nielsen, Kirsten

    2012-01-01

    Cryptococcosis, caused by the basidiomycetous fungus Cryptococcus neoformans, is responsible for more than 600,000 deaths annually in AIDS patients. Flucytosine is one of the most commonly used antifungal drugs for its treatment, but its resistance and regulatory mechanisms have never been investigated at the genome scale in C. neoformans. In the present study, we performed comparative transcriptome analysis by employing two-component system mutants (tco1Δ and tco2Δ) exhibiting opposing flucytosine susceptibility. As a result, a total of 177 flucytosine-responsive genes were identified, and many of them were found to be regulated by Tco1 or Tco2. Among these, we discovered an APSES-like transcription factor, Mbs1 (Mbp1- and Swi4-like protein 1). Expression analysis revealed that MBS1 was regulated in response to flucytosine in a Tco2/Hog1-dependent manner. Supporting this, C. neoformans with the deletion of MBS1 exhibited increased susceptibility to flucytosine. Intriguingly, Mbs1 played pleiotropic roles in diverse cellular processes of C. neoformans. Mbs1 positively regulated ergosterol biosynthesis and thereby affected polyene and azole drug susceptibility. Mbs1 was also involved in genotoxic and oxidative stress responses. Furthermore, Mbs1 promoted production of melanin and capsule and thereby was required for full virulence of C. neoformans. In conclusion, Mbs1 is considered to be a novel antifungal therapeutic target for treatment of cryptococcosis. PMID:22080454

  14. Susceptibility of Intact Germinating Arabidopsis thaliana to Human Fungal Pathogens Cryptococcus neoformans and C. gattii

    PubMed Central

    Park, Yoon-Dong

    2013-01-01

    The fungus Cryptococcus contributes a large global burden of infectious death in both HIV-infected and healthy individuals. As Cryptococcus is an opportunistic pathogen, much of the evolutionary pressure shaping virulence occurs in environments in contact with plants and soil. The present studies investigated inoculation of intact seeds of the common weed Arabidopsis thaliana with fungal cells over a 21-day period. C. gattii was the more virulent plant pathogen, resulting in disrupted germination as well as increased stem lodging, fungal burden, and plant tissue colocalization. C. neoformans was a less virulent plant pathogen but exhibited prolonged tissue residence within the cuticle and vascular spaces. Arabidopsis mutants of the PRN1 gene, which is involved in abiotic and biotic signaling affecting phenylalanine-derived flavonoids, showed altered susceptibility to cryptoccocal infections, suggesting roles for this pathway in cryptococcal defense. The fungal virulence factor laccase was also implicated in plant pathogenesis, as a cryptococcal lac1Δ strain was less virulent than wild-type fungi and was unable to colonize seedlings. In conclusion, these studies expand knowledge concerning the ecological niche of Cryptococcus by demonstrating the pathogenic capacity of the anamorphic form of cryptococcal cells against healthy seedlings under physiologically relevant conditions. In addition, an important role of laccase in plant as well as human virulence may suggest mechanisms for laccase retention and optimization during evolution of this fungal pathogen. PMID:23435895

  15. Antibody-Mediated Immobilization of Cryptococcus neoformans Promotes Biofilm Formation▿ †

    PubMed Central

    Robertson, Emma J.; Casadevall, Arturo

    2009-01-01

    Most microbes, including the fungal pathogen Cryptococcus neoformans, can grow as biofilms. Biofilms confer upon microbes a range of characteristics, including an ability to colonize materials such as shunts and catheters and increased resistance to antibiotics. Here, we provide evidence that coating surfaces with a monoclonal antibody to glucuronoxylomannan, the major component of the fungal capsular polysaccharide, immobilizes cryptococcal cells to a surface support and, subsequently, promotes biofilm formation. We used time-lapse microscopy to visualize the growth of cryptococcal biofilms, generating the first movies of fungal biofilm growth. We show that when fungal cells are immobilized using surface-attached specific antibody to the capsule, the initial stages of biofilm formation are significantly faster than those on surfaces with no antibody coating or surfaces coated with unspecific monoclonal antibody. Time-lapse microscopy revealed that biofilm growth was a dynamic process in which cells shuffled position during budding and was accompanied by emergence of planktonic variant cells that left the attached biofilm community. The planktonic variant cells exhibited mobility, presumably by Brownian motion. Our results indicate that microbial immobilization by antibody capture hastens biofilm formation and suggest that antibody coating of medical devices with immunoglobulins must exclude binding to common pathogenic microbes and the possibility that this effect could be exploited in industrial microbiology. PMID:19251903

  16. Visualizing non-lytic exocytosis of Cryptococcus neoformans from macrophages using digital light microscopy.

    PubMed

    Stukes, Sabriya; Casadevall, Arturo

    2014-01-01

    Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon. PMID:25350860

  17. Intra-strain variability of Cryptococcus neoformans can be detected on phloxin B medium.

    PubMed

    Kucsera, Judit; Yarita, Kyoko; Takeo, Kanji; Yoshida, Soichi; Gácser, Attila; Hamari, Zsuzsanna; Avasi, Zoltán; Kevei, Ferenc

    2002-01-01

    A method was devised for easy detection of intra-strain variability of the human pathogenic yeast Cryptococcus neoformans. Cultivation of strains on a medium containing Phloxin B resulted in different coloured colonies. Generally, colonies were either pink or red; however there were also several colony-colour segregant in which both colours could be observed. A number of these segregants were isolated and analysed. Virulence factors such as the cell and capsule sizes were measured; further temperature sensitivity, growth rates, mating-types and melanin production were also studied. Segregants were examined by random amplified polymorphic DNA (RAPD) fingerprinting and electrophoretic karyotyping by pulsed-field gel electrophoresis (CHEF). They showed both phenotypic and genotypic differences. The main differences appeared in phenotypic characters and RAPD patterns; while the chromosomal patterns remained unchanged. Reversion frequency analysis revealed that the reason for this segregation could be due to phenotypic switching. The physiological reason for the colour changes was also investigated and was attributed to the differential ability of the cells to accumulate Phloxin B either into their capsules or into their cells. The method described here is potentially applicable for the detection of strain heterogeneity in both basic and clinical microbiology laboratories. PMID:11981875

  18. The ESCRT machinery influences haem uptake and capsule elaboration in Cryptococcus neoformans

    PubMed Central

    Hu, Guanggan; Caza, Mélissa; Cadieux, Brigitte; Bakkeren, Erik; Do, Eunsoo; Jung, Won Hee; Kronstad, James W.

    2015-01-01

    Summary Iron availability is a key determinant of virulence in the pathogenic fungus Cryptococcus neoformans. Previous work revealed that the ESCRT (endosomal sorting complex required for transport) protein Vps23 functions in iron acquisition, capsule formation and virulence. Here, we further characterized the ESCRT machinery to demonstrate that defects in the ESCRT-II and III complexes caused reduced capsule attachment, impaired growth on haem and resistance to non-iron metalloprotoporphyrins. The ESCRT mutants shared several phenotypes with a mutant lacking the pH-response regulator Rim101 and, in other fungi, the ESCRT machinery is known to activate Rim101 via proteolytic cleavage. We therefore expressed a truncated and activated version of Rim101 in the ESCRT mutants and found that this allele restored capsule formation but not growth on haem, thus suggesting a Rim101-independent contribution to haem uptake. We also demonstrated that the ESCRT machinery acts downstream of the cAMP/protein kinase A pathway to influence capsule elaboration. Defects in the ESCRT components also attenuated virulence in macrophage survival assays and a mouse model of cryptococcosis to a greater extent than reported for loss of Rim101. Overall, these results indicate that the ESCRT complexes function in capsule elaboration, haem uptake and virulence via Rim101-dependent and independent mechanisms. PMID:25732100

  19. Cryptococcus neoformans Variants Generated by Phenotypic Switching Differ in Virulence through Effects on Macrophage Activation▿ †

    PubMed Central

    Guerrero, A.; Jain, N.; Wang, X.; Fries, B. C.

    2010-01-01

    Macrophages have a central role in the pathogenesis of cryptococcosis since they are an important line of defense, serve as a site for fungal replication, and also can contribute to tissue damage. The objective of this study was to investigate the interaction of macrophages with cells from smooth-colony variants (SM) and mucoid-colony variants (MC) arising from phenotypic switching of Cryptococcus neoformans. Alveolar macrophages (AMs) isolated from SM- and MC-infected mice exhibited differences in gene and surface expression of PD-L1, PD-L2, and major histocompatibility class II (MHC-II). PD-L1 and PD-L2 are the ligands for PD1 and are differentially regulated in Th1- and Th2-type cells. In addition, macrophage activation in SM- and MC-infected mice was characterized as alternatively activated. Flow cytometric and cytokine analysis demonstrated that MC infection was associated with the emergence of Th17 cells and higher levels of interleukin-17 (IL-17) in lung tissue, which were reduced by AM depletion. In conclusion, our results indicate that macrophages play a significant role in maintaining damage-promoting inflammation in the lung during MC infection, which ultimately results in death. PMID:20048044

  20. Molecular epidemiology of isolates of the Cryptococcus neoformans species complex from Spain.

    PubMed

    Frasés, Susana; Ferrer, Consuelo; Sánchez, Manuel; Colom-Valiente, María Francisca

    2009-06-30

    To study genetic diversity of Cryptococcus neoformans species complex in Spain, 97 isolates of the yeast recovered from human, animal and environmental samples have been analysed using three molecular epidemiological techniques. One of these, URA5 gene fragment length polymorphism (RFLP) analysis, has been previously described as a molecular epidemiology tool. Thus, standard profiles and reference strains have been defined for it. In addition, 5S rDNA/IGS RFLP and [GACA](4) microsatellite PCR fingerprinting were also used. Our results show five of the previously defined URA5 genotypes with a high frequency (33%) of the VNI type, which is in concordance with other studies. The high presence of VNIII pattern (28.9%) among our strains is remarkable and could be a specific feature of the isolates from our country. 5S rDNA/IGS RFLP showed a low intra-species discriminative power. Three different molecular profiles (S1-3), which showed a good correlation with the different species, varieties and genotypes, were obtained. [GACA](4) microsatellite PCR-fingerprinting analysis showed a high variability of patterns among the studied strains. Molecular profiles represented in a dendrogram clustered strains in four main groups related with the source of the yeast and also in concordance with some of the described genotypes (VNI-IV and VGI). PMID:19631160

  1. Cryptococcus neoformans-induced macrophage lysosome damage crucially contributes to fungal virulence1

    PubMed Central

    Davis, Michael J.; Eastman, Alison J.; Qiu, Yafeng; Gregorka, Brian; Kozel, Thomas R.; Osterholzer, John J.; Curtis, Jeffrey L.; Swanson, Joel A.; Olszewski, Michal A.

    2015-01-01

    Upon ingestion by macrophages, Cryptococcus neoformans (Cn) can survive and replicate intracellularly unless the macrophages become classically activated. The mechanism enabling intracellular replication is not fully understood; neither are the mechanisms which allow classical activation to counteract replication. Cn-induced lysosome damage was observed in infected murine bone marrow-derived macrophages, increased with time and required yeast viability. To demonstrate lysosome damage in the infected host, we developed a novel flow-cytometric method for measuring lysosome damage. Increased lysosome damage was found in Cn-containing lung cells compared to Cn–free cells. Among Cn-containing myeloid cells, recently recruited cells displayed lower damage than resident cells, consistent with the protective role of recruited macrophages. The magnitude of lysosome damage correlated with increased Cn replication. Experimental induction of lysosome damage increased Cn replication. Activation of macrophages with IFN-γ abolished macrophage lysosome damage and enabled increased killing of Cn. We conclude that induction of lysosome damage is an important Cn survival strategy and that classical activation of host macrophages counters replication by preventing damage. Thus, therapeutic strategies which decrease lysosomal damage, or increase resistance to such damage, could be valuable in treating cryptococcal infections. PMID:25637026

  2. Involvement of PDK1, PKC and TOR signaling pathways in basal fluconazole tolerance in Cryptococcus neoformans

    PubMed Central

    Lee, Hyeseung; Lamichhane, Ami Khanal; Garraffo, H. Martin; Kwon-Chung, Kyung J.; Chang, Yun C.

    2012-01-01

    Summary This study shows the importance of PDK1, TOR and PKC signaling pathways to the basal tolerance of Cryptococcus neoformans toward fluconazole, the widely used drug for treatment of cryptococcosis. Mutations in genes integral to these pathway resulted in hypersensitivity to the drug. Upon fluconazole treatment, Mpk1, the downstream target of PKC was phosphorylated and its phosphorylation required Pdk1. We show genetically that the PDK1 and TOR phosphorylation sites in Ypk1 as well as the kinase activity of Ypk1 are required for the fluconazole basal tolerance. The involvement of these pathways in fluconazole basal tolerance was associated with sphingolipid homeostasis. Deletion of PDK1, SIN1, or YPK1 but not MPK1 affected cell viability in the presence of sphingolipid biosynthesis inhibitors. Concurrently, pdk1Δ, sinΔ1, ypk1Δ, and mpk1Δ exhibited altered sphingolipid content and elevated fluconazole accumulation compared with the wild-type. The fluconazole hypersensitivity phenotype of these mutants, therefore, appears to be the result of malfunction of the influx/efflux systems due to modifications of membrane sphingolipid content. Interestingly, the reduced virulence of these strains in mice suggests that the cryptococcal PDK1, PKC, and likely the TOR pathways play an important role in managing stress exerted either by fluconazole or by the host environment. PMID:22339665

  3. The lysine biosynthetic enzyme Lys4 influences iron metabolism, mitochondrial function and virulence in Cryptococcus neoformans.

    PubMed

    Do, Eunsoo; Park, Minji; Hu, Guanggan; Caza, Mélissa; Kronstad, James W; Jung, Won Hee

    2016-09-01

    The lysine biosynthesis pathway via α-aminoadipate in fungi is considered an attractive target for antifungal drugs due to its absence in mammalian hosts. The iron-sulfur cluster-containing enzyme homoaconitase converts homocitrate to homoisocitrate in the lysine biosynthetic pathway, and is encoded by LYS4 in the model yeast Saccharomyces cerevisiae. In this study, we identified the ortholog of LYS4 in the human fungal pathogen, Cryptococcus neoformans, and found that LYS4 expression is regulated by iron levels and by the iron-related transcription factors Hap3 and HapX. Deletion of the LYS4 gene resulted in lysine auxotrophy suggesting that Lys4 is essential for lysine biosynthesis. Our study also revealed that lysine uptake was mediated by two amino acid permeases, Aap2 and Aap3, and influenced by nitrogen catabolite repression (NCR). Furthermore, the lys4 mutant showed increased sensitivity to oxidative stress, agents that challenge cell wall/membrane integrity, and azole antifungal drugs. We showed that these phenotypes were due in part to impaired mitochondrial function as a result of LYS4 deletion, which we propose disrupts iron homeostasis in the organelle. The combination of defects are consistent with our observation that the lys4 mutant was attenuated virulence in a mouse inhalation model of cryptococcosis. PMID:27353379

  4. De novo GTP Biosynthesis Is Critical for Virulence of the Fungal Pathogen Cryptococcus neoformans

    PubMed Central

    Morrow, Carl A.; Valkov, Eugene; Stamp, Anna; Chow, Eve W. L.; Lee, I. Russel; Wronski, Ania; Williams, Simon J.; Hill, Justine M.; Djordjevic, Julianne T.; Kappler, Ulrike; Kobe, Bostjan; Fraser, James A.

    2012-01-01

    We have investigated the potential of the GTP synthesis pathways as chemotherapeutic targets in the human pathogen Cryptococcus neoformans, a common cause of fatal fungal meningoencephalitis. We find that de novo GTP biosynthesis, but not the alternate salvage pathway, is critical to cryptococcal dissemination and survival in vivo. Loss of inosine monophosphate dehydrogenase (IMPDH) in the de novo pathway results in slow growth and virulence factor defects, while loss of the cognate phosphoribosyltransferase in the salvage pathway yielded no phenotypes. Further, the Cryptococcus species complex displays variable sensitivity to the IMPDH inhibitor mycophenolic acid, and we uncover a rare drug-resistant subtype of C. gattii that suggests an adaptive response to microbial IMPDH inhibitors in its environmental niche. We report the structural and functional characterization of IMPDH from Cryptococcus, revealing insights into the basis for drug resistance and suggesting strategies for the development of fungal-specific inhibitors. The crystal structure reveals the position of the IMPDH moveable flap and catalytic arginine in the open conformation for the first time, plus unique, exploitable differences in the highly conserved active site. Treatment with mycophenolic acid led to significantly increased survival times in a nematode model, validating de novo GTP biosynthesis as an antifungal target in Cryptococcus. PMID:23071437

  5. Cryptococcus neoformans Dual GDP-Mannose Transporters and Their Role in Biology and Virulence

    PubMed Central

    Wang, Zhuo A.; Griffith, Cara L.; Skowyra, Michael L.; Salinas, Nichole; Williams, Matthew; Maier, Ezekiel J.; Gish, Stacey R.; Liu, Hong; Brent, Michael R.

    2014-01-01

    Cryptococcus neoformans is an opportunistic yeast responsible for lethal meningoencephalitis in humans. This pathogen elaborates a polysaccharide capsule, which is its major virulence factor. Mannose constitutes over one-half of the capsule mass and is also extensively utilized in cell wall synthesis and in glycosylation of proteins and lipids. The activated mannose donor for most biosynthetic reactions, GDP-mannose, is made in the cytosol, although it is primarily consumed in secretory organelles. This compartmentalization necessitates specific transmembrane transporters to make the donor available for glycan synthesis. We previously identified two cryptococcal GDP-mannose transporters, Gmt1 and Gmt2. Biochemical studies of each protein expressed in Saccharomyces cerevisiae showed that both are functional, with similar kinetics and substrate specificities in vitro. We have now examined these proteins in vivo and demonstrate that cells lacking Gmt1 show significant phenotypic differences from those lacking Gmt2 in terms of growth, colony morphology, protein glycosylation, and capsule phenotypes. Some of these observations may be explained by differential expression of the two genes, but others suggest that the two proteins play overlapping but nonidentical roles in cryptococcal biology. Furthermore, gmt1 gmt2 double mutant cells, which are unexpectedly viable, exhibit severe defects in capsule synthesis and protein glycosylation and are avirulent in mouse models of cryptococcosis. PMID:24747214

  6. Novel imidazo[2,1-b]-1,3,4-thiadiazoles as promising antifungal agents against clinical isolate of Cryptococcus neoformans.

    PubMed

    Alwan, Wesam S; Karpoormath, Rajshekhar; Palkar, Mahesh B; Patel, Harun M; Rane, Rajesh A; Shaikh, Mahamadhanif S; Kajee, Afsana; Mlisana, Koleka P

    2015-05-01

    We herein report the synthesis and in vitro antimicrobial evaluation of twenty five novel hybrid derivatives of imidazo [2,1-b]-1,3,4-thiadiazole containing chalcones (5a-o) and Schiff bases (6a-j) against three fungal strains (Candida albicans, Cryptococcus neoformans and Aspergillus niger). Most of the tested compounds displayed substantial anti-fungal activity with MICs ranging between 1.56 and 100 μg/mL. Compounds 5a, 5b and 5n exhibited promising activity against C. neoformans at a MIC 1.56 μg/mL. In addition, compound 5n also demonstrated significant antifungal activity against the clinical isolates of C. neoformans at MIC 3.125 μg/mL. However, moderate activity was observed for these compounds against four bacterial strains (Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa) and Mycobacterium tuberculosis (H37Rv). PMID:25847769

  7. Importance of the association of molecular and immunological diagnosis in immunocompetent patient with Histoplasma capsulatum and Cryptoccocus neoformans infection: a case report

    PubMed Central

    2014-01-01

    This case reports an immunocompetent 29-year-old woman with suspected pneumonia, suggestive of fungal infection. Immunoblotting analysis reactivity against Histoplasma capsulatum and Paracoccidioides brasiliensis were observed. Nested-PCR in blood employing species-specific primers was positive for H. capsulatum and Cryptococcus neoformans. The evaluation of paucisymptomatic patients with positive results for H. capsulatum and C. neoformans could be relevant for the prevention as well as the possible evaluation of the reactivated quiescent foci. In conclusion, the associated methodology may have contributed to the monitoring endogenous reactivation of these diseases. PMID:25180029

  8. Inheritance of Immune Polarization Patterns Is Linked to Resistance versus Susceptibility to Cryptococcus neoformans in a Mouse Model▿

    PubMed Central

    Chen, Gwo-hsiao; McNamara, David A.; Hernandez, Yadira; Huffnagle, Gary B.; Toews, Galen B.; Olszewski, Michal A.

    2008-01-01

    Genetic background variation between inbred strains accounts for different levels of susceptibility to Cryptococcus neoformans in the mouse infection model. To elucidate the inheritance of immunophenotypic traits and their associations with clearance outcomes during cryptococcal infection, we compared C57BL/6, BALB/c, and their first-generation hybrid, CB6F1 (F1), mice. Mice from each group were infected with C. neoformans (104 CFU) and analyzed at weekly intervals over a 6-week period. BALB/c mice progressively cleared the cryptococcal infection in the lungs and showed a Th1-skewed immune response: a Th1-shifted cytokine profile, modest lung pathology, and no significant elevation in the systemic immunoglobulin E (IgE) level. In contrast, C57BL/6 mice developed a chronic infection with a Th2-skewed immune response: a Th2-shifted cytokine profile, pulmonary eosinophilia, severe lung pathology, elevated serum IgE, fungemia, and cryptococcal dissemination in the central nervous system. F1 mice demonstrated intermediate resistance to C. neoformans, with a stronger resemblance to the immunophenotype of the resistant (BALB/c) mice. F1 mice also demonstrated enhanced pulmonary recruitment of lymphocytes, especially CD8+ T cells, in comparison to both parental strains, suggesting positive heterosis. We conclude that the inheritance of traits responsible for early cytokine induction in the infected lungs and dendritic-cell maturation/activation status in draining nodes is responsible for the intermediate immune response polarization and clearance outcome observed initially in the lungs of F1 mice. The enhanced pulmonary lymphocyte recruitment could be responsible for a gradual shutdown of the undesirable Th2 arm of the immune response and subsequently improved anticryptococcal resistance in F1 mice. PMID:18391002

  9. Lipoxin Signaling in Murine Lung Host Responses to Cryptococcus neoformans Infection.

    PubMed

    Colby, Jennifer K; Gott, Katherine M; Wilder, Julie A; Levy, Bruce D

    2016-01-01

    Lipoxins (LX) are proresolving mediators that augment host defense against bacterial infection. Here, we investigated roles for LX in lung clearance of the fungal pathogen Cryptococcus neoformans (Cne). After intranasal inoculation of 5,000 CFU Cne, C57BL/6 and C.B-17 mice exhibited strain-dependent differences in Cne clearance, immunologic responses, and lipoxin A4 (LXA4) formation and receptor (ALX/FPR2) expression. Compared with C.B-17 mice, C57BL/6 lungs had increased and persistent Cne infection 14 days after inoculation, increased eosinophils, and distinct profiles of inflammatory cytokines. Relative to C.B-17 mice, bronchoalveolar lavage fluid levels of LXA4 were increased before and after infection in C57BL/6. The kinetics for 15-epi-LXA4 production were similar in both strains. Lung basal expression of the LX biosynthetic enzyme Alox12/15 (12/15-lipoxygenase) was increased in C57BL/6 mice and further increased after Cne infection. In contrast, lung basal expression of the LXA4 receptor Alx/Fpr2 was higher in C.B-17 relative to C57BL/6 mice, and after Cne infection, Alx/Fpr2 expression was significantly increased in only C.B-17 mice. Heat-killed Cne initiated lung cell generation of IFN-γ and IL-17 and was further increased in C.B-17 mice by 15-epi-LXA4. A trend toward reduced Cne clearance and IFN-γ production was observed upon in vivo administration of an ALX/FPR2 antagonist. Together, these findings provide the first evidence that alterations in cellular immunity against Cne are associated with differences in LXA4 production and receptor expression, suggesting an important role for ALX/FPR2 signaling in the regulation of pathogen-mediated inflammation and antifungal lung host defense. PMID:26039320

  10. Characterization of a flocculation-like phenotype in Cryptococcus neoformans and its effects on pathogenesis.

    PubMed

    Li, Li; Zaragoza, Oscar; Casadevall, Arturo; Fries, Bettina C

    2006-11-01

    We investigated the phenomenon of cell-cell aggregation (flocculation) in a serotype D strain of Cryptococcus neoformans (ATCC 24067, isolate RC-2). Cell aggregation into clumps of 5-40 cells (clump+ cells) occurred during the early log phase and disappeared in the beginning of the stationary phase (clump- cells). The cell aggregation phenomenon was medium dependent. Clump+ cells could be dispersed by either vortexing or proteinase K digestion. Most importantly, the transient change in cellular phenotype changed several important host-pathogen interactions. Adherence of clump+ cells to murine macrophage-like cells J774.16 was significantly (P < 0.001) enhanced compared with adherence of clump- cells. Furthermore, complement-mediated phagocytosis efficacy of dispersed clump+ cells was significantly higher (P < 0.001) compared with clump- cells. Similar findings were documented with an in vivo phagocytosis assay. Infection of mice with a low inoculum (10(4)) of clump+ cells resulted in lower fungal burden when compared with mice infected with clump- cells. Accordingly, mice infected with clump+ cells survived significantly longer than mice infected with clump- cells. These results indicate that the cellular phenotype undergoes significant changes that result in a transient flocculation-like phenotype. We hypothesize that this cell-cell aggregation is the result of changes in protein content in the polysaccharide capsule. We conclude from our data that the change in cellular phenotype has a dramatic effect on cell adherence, and on complement-mediated phagocytosis, both of which can affect the pathogenesis of the disease in the host. Our results underscore the complexity of studies that investigate host pathogen interactions and may explain differences and inconsistencies observed in in vitro and in vivo assays. PMID:16759224

  11. The Role of Host Gender in the Pathogenesis of Cryptococcus neoformans Infections

    PubMed Central

    McClelland, Erin E.; Hobbs, Letizia M.; Rivera, Johanna; Casadevall, Arturo; Potts, Wayne K.; Smith, Jennifer M.; Ory, Jeramia J.

    2013-01-01

    Cryptococcus neoformans (Cn) is a pathogenic yeast and the cause of cryptococcal meningitis. Prevalence of disease between males and females is skewed, with males having an increased incidence of disease. Based on the reported gender susceptibility differences to Cn in the literature, we used clinical isolates from Botswanan HIV-infected patients to test the hypothesis that different gender environments exerted different selective pressures on Cn. When we examined this data set, we found that men had significantly higher risk of death despite having significantly higher CD4+ T lymphocyte counts upon admittance to the hospital. These observations suggested that Cn strains are uniquely adapted to different host gender environments and that the male immune response may be less efficient in controlling Cn infection. To discriminate between these possibilities, we tested whether there were phenotypic differences between strains isolated from males and females and whether there was an interaction between Cn and the host immune response. Virulence phenotypes showed that Cn isolates from females had longer doubling times and released more capsular glucoronoxylomannan (GXM). The presence of testosterone but not 17-β estradiol was associated with higher levels of GXM release for a laboratory strain and 28 clinical isolates. We also measured phagocytic efficiency, survival of Cn, and amount of killing of human macrophages by Cn after incubation with four isolates. While macrophages from females phagocytosed more Cn than macrophages from males, male macrophages had a higher fungal burden and showed increased killing by Cn. These data are consistent with the hypothesis that differential interaction between Cn and macrophages within different gender environments contribute to the increased prevalence of cryptococcosis in males. This could be related to differential expression of cryptococcal virulence genes and capsule metabolism, changes in Cn phagocytosis and increased death

  12. A 'suicide' CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans.

    PubMed

    Wang, Yu; Wei, Dongsheng; Zhu, Xiangyang; Pan, Jiao; Zhang, Ping; Huo, Liang; Zhu, Xudong

    2016-01-01

    Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This 'suicide' CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans. PMID:27503169

  13. Antifungal activity against Cryptococcus neoformans strains and genotoxicity assessment in human leukocyte cells of Euphorbia tirucalli L

    PubMed Central

    de Oliveira, Luís Flávio Souza; Fuentefria, Alexandre Meneghello; Klein, Fernanda da Silva; Machado, Michel Mansur

    2014-01-01

    In the last times, focus on plant research has increased all over the world. Euphorbia tirucalli L., a plant known popularly as Aveloz, and originally used in Africa, has been drawing attention for its use in the United States and Latin America, both for use as an ornamental plant and as a medicinal plant. E. tirucalli L. is a member of the family Euphorbiaceae and contains many diterpenoids and triterpenoids, in particular phorbol esters, apparently the main constituent of this plant, which are assumed to be responsible for their activities in vivo and in vitro. The in vitro antifungal activities of Euphorbia tirucalli (L.) against opportunistic yeasts were studied using microbroth dilution assay. The results showed that aqueous extract and latex preparation were effective against ten clinical strains of Cryptococcus neoformans in vitro (Latex and extract MIC range of 3.2 – > 411 μg/mL). Aiming the safe use in humans, the genotoxic effects of E. tirucalli were evaluated in human leukocytes cells. Our data show that both aqueous extract and latex preparation have no genotoxic effect in human leukocytes cells in vitro. Although the results cannot be extrapolated by itself for use in vivo, they suggest a good perspective for a therapeutic application in future. In conclusion, our results show that the aqueous extract and latex preparation from E. tirucalli L. are antifungal agents effectives against several strains of C. neoformans and do not provoke DNA damage in human leukocyte cells, considering the concentrations tested. PMID:25763040

  14. Serotyping of 467 Cryptococcus neoformans Isolates from Clinical and Environmental Sources in Brazil: Analysis of Host and Regional Patterns

    PubMed Central

    Nishikawa, Marília M.; Lazera, Márcia S.; Barbosa, Glaucia G.; Trilles, Luciana; Balassiano, Beatriz R.; Macedo, Regina C. L.; Bezerra, Cláudia C. F.; Pérez, Maurício A.; Cardarelli, Paola; Wanke, Bodo

    2003-01-01

    Cryptococcus neoformans is an important zoopathogen, and it is one of the most prevalent lethal mycotic agents. Its polysaccharide capsule, synthesized in vivo and in vitro, is a virulence factor, contains predominantly glucuronoxylomannan, and is responsible for the antigenic differentiation of serotypes A, B, C, D, and AD. A total of 467 isolates of C. neoformans obtained from clinical and environmental sources from Brazilian regions were studied serologically by using the Crypto Check Iatron RM 304-K kit. Serotyping of the clinical isolates showed the following prevalences of the serotypes: A (77.95%), followed by B (18.2%), AD (1.3%), D (0.4%), C (0.2%), and untypeable (1.93%). The epidemiology of serotype A in the Brazilian southern and southeastern regions reproduces the picture observed worldwide. In contrast, serotype B was the most frequent agent of cryptococcosis in the northeastern region, occurring nearly equally in male and female healthy hosts. Among the isolates from environmental sources, serotypes A and B were found to occur in the hollows of tropical trees of the genera Cassia, Ficus, and Moquillea. The few isolates from Eucalyptus camaldulensis debris were serotypes A and B and untypeable. Overall, no association with a specific host tree was identified for these serotypes, denoting a distinct ecoepidemiological regional pattern. The one serotype C isolate was recovered from a human immunodeficiency virus-negative host. Serotype AD predominated over serotype D among both clinical and environmental isolates. PMID:12517828

  15. Serotyping of 467 Cryptococcus neoformans isolates from clinical and environmental sources in Brazil: analysis of host and regional patterns.

    PubMed

    Nishikawa, Marília M; Lazera, Márcia S; Barbosa, Glaucia G; Trilles, Luciana; Balassiano, Beatriz R; Macedo, Regina C L; Bezerra, Cláudia C F; Pérez, Maurício A; Cardarelli, Paola; Wanke, Bodo

    2003-01-01

    Cryptococcus neoformans is an important zoopathogen, and it is one of the most prevalent lethal mycotic agents. Its polysaccharide capsule, synthesized in vivo and in vitro, is a virulence factor, contains predominantly glucuronoxylomannan, and is responsible for the antigenic differentiation of serotypes A, B, C, D, and AD. A total of 467 isolates of C. neoformans obtained from clinical and environmental sources from Brazilian regions were studied serologically by using the Crypto Check Iatron RM 304-K kit. Serotyping of the clinical isolates showed the following prevalences of the serotypes: A (77.95%), followed by B (18.2%), AD (1.3%), D (0.4%), C (0.2%), and untypeable (1.93%). The epidemiology of serotype A in the Brazilian southern and southeastern regions reproduces the picture observed worldwide. In contrast, serotype B was the most frequent agent of cryptococcosis in the northeastern region, occurring nearly equally in male and female healthy hosts. Among the isolates from environmental sources, serotypes A and B were found to occur in the hollows of tropical trees of the genera Cassia, Ficus, and MOQUILLEA: The few isolates from Eucalyptus camaldulensis debris were serotypes A and B and untypeable. Overall, no association with a specific host tree was identified for these serotypes, denoting a distinct ecoepidemiological regional pattern. The one serotype C isolate was recovered from a human immunodeficiency virus-negative host. Serotype AD predominated over serotype D among both clinical and environmental isolates. PMID:12517828

  16. A ‘suicide’ CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans

    PubMed Central

    Wang, Yu; Wei, Dongsheng; Zhu, Xiangyang; Pan, Jiao; Zhang, Ping; Huo, Liang; Zhu, Xudong

    2016-01-01

    Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This ‘suicide’ CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans. PMID:27503169

  17. Using Solid-state NMR to Monitor the Molecular Consequences of Cryptococcus neoformans Melanization with Different Catecholamine Precursors

    PubMed Central

    Chatterjee, Subhasish; Prados-Rosales, Rafael; Frases, Susana; Itin, Boris; Casadevall, Arturo; Stark, Ruth E.

    2012-01-01

    Melanins are a class of natural pigments associated with a wide range of biological functions, including microbial virulence, energy transduction, and protection against solar radiation. Because of their insolubility and structural heterogeneity, solid-state nuclear magnetic resonance (NMR) spectroscopy provides an unprecedented means to define the molecular architecture of these enigmatic pigments. The requirement of obligatory catecholamines for melanization of the pathogenic fungus Cryptococcus neoformans also offers unique opportunities for investigating melanin development. In the current study, pigments produced with L-dopa, methyl-L-dopa, epinephrine, and norepinephrine precursors are compared structurally using 13C and 1H magic-angle spinning (MAS) NMR. Striking structural differences were observed for both aromatic and aliphatic molecular constituents of the mature fungal pigment assemblies, thus making it possible to redefine the molecular prerequisites for formation of the aromatic domains of insoluble indole-based biopolymers, to rationalize their distinctive physical characteristics, and to delineate the role of cellular constituents in assembly of the melanized macromolecules with polysaccharides and fatty acyl chain-containing moieties. By achieving an augmented understanding of the mechanisms of C. neoformans melanin biosynthesis and cellular assembly, such studies can guide future drug discovery efforts related to melanin-associated virulence, resistance to tumor therapy, and production of melanin mimetics under cell-free conditions. PMID:22765382

  18. Variable Region Identical IgA and IgE to Cryptococcus neoformans Capsular Polysaccharide Manifest Specificity Differences*

    PubMed Central

    Janda, Alena; Eryilmaz, Ertan; Nakouzi, Antonio; Pohl, Mary Ann; Bowen, Anthony; Casadevall, Arturo

    2015-01-01

    In recent years several groups have shown that isotype switching from IgM to IgG to IgA can affect the affinity and specificity of antibodies sharing identical variable (V) regions. However, whether the same applies to IgE is unknown. In this study we compared the fine specificity of V region-identical IgE and IgA to Cryptococcus neoformans capsular polysaccharide and found that these differed in specificity from each other. The IgE and IgA paratopes were probed by nuclear magnetic resonance spectroscopy with 15N-labeled peptide mimetics of cryptococcal polysaccharide antigen (Ag). IgE was found to cleave the peptide at a much faster rate than V region-identical IgG subclasses and IgA, consistent with an altered paratope. Both IgE and IgA were opsonic for C. neoformans and protected against infection in mice. In summary, V-region expression in the context of the ϵ constant (C) region results in specificity changes that are greater than observed for comparable IgG subclasses. These results raise the possibility that expression of certain V regions in the context of α and ϵ C regions affects their function and contributes to the special properties of those isotypes. PMID:25778397

  19. A synthetic strategy to xylose-containing thioglycoside tri- and tetrasaccharide building blocks corresponding to Cryptococcus neoformans capsular polysaccharide structures.

    PubMed

    Guazzelli, Lorenzo; Ulc, Rebecca; Rydner, Lina; Oscarson, Stefan

    2015-06-21

    As part of an ongoing project aimed at developing vaccine candidates against Cryptococcus neoformans the preparation of tri- and tetrasaccharide thioglycoside building blocks, to be used in construction of structurally defined part structures of C. neoformans GXM capsular polysaccharide, was investigated. Using a naphthalenylmethyl (NAP) ether as a temporary protecting group and trichloroacetimidate donors in optimized glycosylations the target building blocks, ethyl 6-O-acetyl-2,4-di-O-benzyl-3-O-(2-naphthalenylmethyl)-α-D-mannopyranosyl-(1→3)-[2,3,4-tri-O-benzyl-β-D-xylopyranosyl-(1→2)]-4,6-di-O-benzyl-1-thio-α-D-mannopyranoside (16) and ethyl 2,3,4-tri-O-benzyl-β-D-xylopyranosyl-(1→2)-4,6-di-O-benzyl-3-O-(2-naphthalenylmethyl)-α-D-mannopyranosyl-(1→3)-[2,3,4-tri-O-benzyl-β-D-xylopyra-nosyl-(1→2)]-6-O-acetyl-4-O-benzyl-1-thio-α-D-mannopyranoside (21), were efficiently prepared. These synthesized thiosaccharide building blocks were then used as donors in high-yielding (~90%) DMTST promoted glycosylations to a spacer-containing acceptor to, after deprotection, afford GXM polysaccharide part structures ready for protein conjugation to give vaccine candidates. Also, the NAP groups in the building blocks were removed to obtain tri- and tetrasaccharide acceptors suitable for further elongation towards larger thiosaccharide building blocks. PMID:25986781

  20. Antifungal Activity of Plasmacytoid Dendritic Cells against Cryptococcus neoformans In Vitro Requires Expression of Dectin-3 (CLEC4D) and Reactive Oxygen Species.

    PubMed

    Hole, Camaron R; Leopold Wager, Chrissy M; Mendiola, Andrew S; Wozniak, Karen L; Campuzano, Althea; Lin, Xin; Wormley, Floyd L

    2016-09-01

    Conventional dendritic cells (cDCs) are critical for protection against pulmonary infection with the opportunistic fungal pathogen Cryptococcus neoformans; however, the role of plasmacytoid dendritic cells (pDCs) is unknown. We show for the first time that murine pDCs have direct activity against C. neoformans via reactive oxygen species (ROS), a mechanism different from that employed to control Aspergillus fumigatus infections. The anticryptococcal activity of murine pDCs is independent of opsonization but appears to require the C-type lectin receptor Dectin-3, a receptor not previously evaluated during cryptococcal infections. Human pDCs can also inhibit cryptococcal growth by a mechanism similar to that of murine pDCs. Experimental pulmonary infection of mice with a C. neoformans strain that induces protective immunity demonstrated that recruitment of pDCs to the lungs is CXCR3 dependent. Taken together, our results show that pDCs inhibit C. neoformans growth in vitro via the production of ROS and that Dectin-3 is required for optimal growth-inhibitory activity. PMID:27324480

  1. Inhibition of Cryptococcus neoformans replication by nitrogen oxides supports the role of these molecules as effectors of macrophage-mediated cytostasis

    SciTech Connect

    Alspaugh, J.A.; Granger, D.L. )

    1991-07-01

    Activated macrophages are able to inhibit the replication of intracellular microbes and tumor cells. In the murine system, this cytostatic effect is associated with the oxidation of L-arginine to L-citrulline, nitrite, and nitrate and is thought to be mediated by an intermediate of this reaction, possibly nitric oxide (NO.). By exposing replicating Cryptococcus neoformans cells to conditions under which NO. is chemically generated, we have observed a cytostatic effect similar to that caused by activated murine macrophages. Nitric oxide is formed as a decomposition product of nitrite salts in acidic, aqueous solutions. Although C. neoformans replicates well in the presence of high nitrite concentrations at physiologic pH, its growth in acidic media can be inhibited by the addition of low concentrations of sodium nitrite. The degree of cytostasis is dependent on both the pH and the nitrite concentration of the NO. generating solution. The cytostatic effector molecule appears to be a gas since, in addition to inhibiting C. neoformans replication in solution, it is able to exert its inhibitory effect across a gas-permeable but ion-impermeable membrane. At high nitrite concentrations, a fungicidal effect occurs. We propose that the growth inhibition of C. neoformans upon exposure to chemically generated NO. or some related oxide of nitrogen represents a cell-free system simulating the cytostatic effect of activated murine macrophages.

  2. Role of Granulocyte Macrophage Colony-Stimulating Factor in Host Defense Against Pulmonary Cryptococcus neoformans Infection during Murine Allergic Bronchopulmonary Mycosis

    PubMed Central

    Chen, Gwo-Hsiao; Olszewski, Michal A.; McDonald, Roderick A.; Wells, Jason C.; Paine, Robert; Huffnagle, Gary B.; Toews, Galen B.

    2007-01-01

    We investigated the role of granulocyte macrophage colony-stimulating factor (GM-CSF) in host defense in a murine model of pulmonary cryptococcosis induced by intratracheal inoculation of Cryptococcus neoformans. Pulmonary C. neoformans infection of C57BL/6 mice is an established model of an allergic bronchopulmonary mycosis. Our objective was to determine whether GM-CSF regulates the pulmonary Th2 immune response in C. neoformans-infected C57BL/6 mice. Long-term pulmonary fungistasis was lost in GM-CSF knockout (GM−/−) mice, resulting in increased pulmonary burden of fungi between weeks 3 and 5. GM-CSF was required for the early influx of macrophages and CD4 and CD8 T cells into the lungs but was not required later in the infection. Lack of GM-CSF also resulted in reduced eosinophil recruitment and delayed recruitment of mononuclear cells into the airspace. Macrophages from GM+/+ mice showed numerous hallmarks of alternatively activated macrophages: higher numbers of intracellular cryptococci, YM1 crystals, and induction of CCL17. These hallmarks are absent in macrophages from GM−/− mice. Mucus-producing goblet cells were abundantly present within the bronchial epithelial layer in GM+/+ mice but not in GM−/− mice at week 5 after infection. Production of both Th1 and Th2 cytokines was impaired in the absence of GM-CSF, consistent with both reduced C. neoformans clearance and absence of allergic lung pathology. PMID:17322386

  3. Induction of Protective Immunity to Cryptococcal Infection in Mice by a Heat-Killed, Chitosan-Deficient Strain of Cryptococcus neoformans

    PubMed Central

    Upadhya, Rajendra; Lam, Woei C.; Maybruck, Brian; Specht, Charles A.; Levitz, Stuart M.

    2016-01-01

    ABSTRACT Cryptococcus neoformans is a major opportunistic fungal pathogen that causes fatal meningoencephalitis in immunocompromised individuals and is responsible for a large proportion of AIDS-related deaths. The fungal cell wall is an essential organelle which undergoes constant modification during various stages of growth and is critical for fungal pathogenesis. One critical component of the fungal cell wall is chitin, which in C. neoformans is predominantly deacetylated to chitosan. We previously reported that three chitin deacetylase (CDA) genes have to be deleted to generate a chitosan-deficient C. neoformans strain. This cda1Δ2Δ3Δ strain was avirulent in mice, as it was rapidly cleared from the lungs of infected mice. Here, we report that clearance of the cda1Δ2Δ3Δ strain was associated with sharply spiked concentrations of proinflammatory molecules that are known to be critical mediators of the orchestration of a protective Th1-type adaptive immune response. This was followed by the selective enrichment of the Th1-type T cell population in the cda1Δ2Δ3Δ strain-infected mouse lung. Importantly, this response resulted in the development of robust protective immunity to a subsequent lethal challenge with a virulent wild-type C. neoformans strain. Moreover, protective immunity was also induced in mice vaccinated with heat-killed cda1Δ2Δ3Δ cells and was effective in multiple mouse strains. The results presented here provide a strong framework to develop the cda1Δ2Δ3Δ strain as a potential vaccine candidate for C. neoformans infection. PMID:27165801

  4. Expression of inducible nitric oxide synthase in rat pulmonary Cryptococcus neoformans granulomas.

    PubMed Central

    Goldman, D.; Cho, Y.; Zhao, M.; Casadevall, A.; Lee, S. C.

    1996-01-01

    Rats, like humans, have extremely effective immune mechanisms for controlling pulmonary Cryptococcus neoformans infection. The mechanism(s) responsible for efficient immunity in rat experimental infection is unknown. Recently, induction of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) have been implicated as an important microbicidal mechanism by which activated macrophages effect cytotoxicity against microbes. In this report, we investigated the expression of iNOS in rat pulmonary cryptococcosis. Localization and regulation of NO production was studied by immunohistochemistry for iNOS in conjunction with immunohistochemistry for cell markers, cytokines, and cryptococcal capsular polysaccharide. iNOS immunoreactivity was detected in macrophages, neutrophils, vascular endothelium, and respiratory epithelium. Double-immunolabeling studies revealed that the most prominent iNOS immunoreactivity was localized to epithelioid macrophages (CD11b/c+) within granulomas; CD4+ and CD8+ T cells were numerous around granulomas but did not express iNOS. iNOS immunoreactivity was detected in a selective population of epithelioid macrophages within some granulomas but not others. iNOS- granulomas were identical to iNOS+ granulomas with respect to morphology and immunohistochemical profiles. Macrophage iNOS immunoreactivity was detected 1 week after infection in one out of four rats and was strongly expressed in all rats at 2 weeks (in up to 50 percent of the granulomas) but declined considerably by 25 days. iNOS expression coincided with granuloma formation and preceded a decrease in lung fungal burden, suggesting an anticryptococcal role for NO. By double labeling, cytokines that have been shown to promote (interferon-gamma, granulocyte/macrophage colony-stimulating factor) and inhibit (transforming growth factor-beta) macrophage iNOS expression were detected around iNOS+ granuloma. iNOS immunoreactivity was expressed in selected neutrophils (1 and 2 weeks) and

  5. The Zinc Finger Protein Mig1 Regulates Mitochondrial Function and Azole Drug Susceptibility in the Pathogenic Fungus Cryptococcus neoformans.

    PubMed

    Caza, Mélissa; Hu, Guanggan; Price, Michael; Perfect, John R; Kronstad, James W

    2016-01-01

    The opportunistic pathogen Cryptococcus neoformans causes fungal meningoencephalitis in immunocompromised individuals. In previous studies, we found that the Hap complex in this pathogen represses genes encoding mitochondrial respiratory functions and tricarboxylic acid (TCA) cycle components under low-iron conditions. The orthologous Hap2/3/4/5 complex in Saccharomyces cerevisiae exerts a regulatory influence on mitochondrial functions, and Hap4 is subject to glucose repression via the carbon catabolite repressor Mig1. In this study, we explored the regulatory link between a candidate ortholog of the Mig1 protein and the HapX component of the Hap complex in C. neoformans. This analysis revealed repression of MIG1 by HapX and activation of HAPX by Mig1 under low-iron conditions and Mig1 regulation of mitochondrial functions, including respiration, tolerance for reactive oxygen species, and expression of genes for iron consumption and iron acquisition functions. Consistently with these regulatory functions, a mig1Δ mutant had impaired growth on inhibitors of mitochondrial respiration and inducers of ROS. Furthermore, deletion of MIG1 provoked a dysregulation in nutrient sensing via the TOR pathway and impacted the pathway for cell wall remodeling. Importantly, loss of Mig1 increased susceptibility to fluconazole, thus further establishing a link between azole antifungal drugs and mitochondrial function. Mig1 and HapX were also required together for survival in macrophages, but Mig1 alone had a minimal impact on virulence in mice. Overall, these studies provide novel insights into a HapX/Mig1 regulatory network and reinforce an association between mitochondrial dysfunction and drug susceptibility that may provide new targets for the development of antifungal drugs. IMPORTANCE Fungal pathogens of humans are difficult to treat, and there is a pressing need to identify new targets for antifungal drugs and to obtain a detailed understanding of fungal proliferation in

  6. The Zinc Finger Protein Mig1 Regulates Mitochondrial Function and Azole Drug Susceptibility in the Pathogenic Fungus Cryptococcus neoformans

    PubMed Central

    Caza, Mélissa; Hu, Guanggan; Price, Michael; Perfect, John R.

    2016-01-01

    ABSTRACT The opportunistic pathogen Cryptococcus neoformans causes fungal meningoencephalitis in immunocompromised individuals. In previous studies, we found that the Hap complex in this pathogen represses genes encoding mitochondrial respiratory functions and tricarboxylic acid (TCA) cycle components under low-iron conditions. The orthologous Hap2/3/4/5 complex in Saccharomyces cerevisiae exerts a regulatory influence on mitochondrial functions, and Hap4 is subject to glucose repression via the carbon catabolite repressor Mig1. In this study, we explored the regulatory link between a candidate ortholog of the Mig1 protein and the HapX component of the Hap complex in C. neoformans. This analysis revealed repression of MIG1 by HapX and activation of HAPX by Mig1 under low-iron conditions and Mig1 regulation of mitochondrial functions, including respiration, tolerance for reactive oxygen species, and expression of genes for iron consumption and iron acquisition functions. Consistently with these regulatory functions, a mig1Δ mutant had impaired growth on inhibitors of mitochondrial respiration and inducers of ROS. Furthermore, deletion of MIG1 provoked a dysregulation in nutrient sensing via the TOR pathway and impacted the pathway for cell wall remodeling. Importantly, loss of Mig1 increased susceptibility to fluconazole, thus further establishing a link between azole antifungal drugs and mitochondrial function. Mig1 and HapX were also required together for survival in macrophages, but Mig1 alone had a minimal impact on virulence in mice. Overall, these studies provide novel insights into a HapX/Mig1 regulatory network and reinforce an association between mitochondrial dysfunction and drug susceptibility that may provide new targets for the development of antifungal drugs. IMPORTANCE Fungal pathogens of humans are difficult to treat, and there is a pressing need to identify new targets for antifungal drugs and to obtain a detailed understanding of fungal

  7. Cryptococcal meningitis due to Cryptococcus neoformans genotype AFLP1/VNI in Iran: a review of the literature.

    PubMed

    Badali, Hamid; Alian, Shahriar; Fakhim, Hamed; Falahatinejad, Mahsa; Moradi, Ali; Mohammad Davoudi, Mehrnaz; Hagen, Ferry; Meis, Jacques F

    2015-12-01

    Cryptococcal meningitis is the most important opportunistic fungal infection with a high mortality in HIV-patients in less developed regions. Here, we report a case of cryptococcal meningitis in a 49-year-old HIV-positive female due to Cryptococcus neoformans (serotype A, mating-type alpha, genotype AFLP1/VNI) in Sari, Iran. In vitro antifungal susceptibility tests showed MICs of isavuconazole (0.016 μg ml(-1) ), voriconazole (0.031 μg ml(-1) ), posaconazole (0.031 μg ml(-1) ), itraconazole (0.063 μg ml(-1) ), amphotericin B (0.125 μg ml(-1) ) and fluconazole (8 μg ml(-1) ). Despite immediate antifungal therapy, the patient died 4 days later due to respiratory failure. Cryptococcal infections have been infrequently reported from Iran and therefore we analysed all published cases of cryptococcosis in Iran since the first reported case from 1969. PMID:26444438

  8. Dithiocarbamates are strong inhibitors of the beta-class fungal carbonic anhydrases from Cryptococcus neoformans, Candida albicans and Candida glabrata.

    PubMed

    Monti, Simona Maria; Maresca, Alfonso; Viparelli, Francesca; Carta, Fabrizio; De Simone, Giuseppina; Mühlschlegel, Fritz A; Scozzafava, Andrea; Supuran, Claudiu T

    2012-01-15

    A series of N-mono- and N,N-disubstituted dithiocarbamates have been investigated as inhibitors of three β-carbonic anhydrases (CAs, EC 4.2.1.1) from the fungal pathogens Cryptococcus neoformans, Candida albicans and Candida glabrata, that is, Can2, CaNce103 and CgNce103, respectively. These enzymes were inhibited with efficacies between the subnanomolar to the micromolar range, depending on the substitution pattern at the nitrogen atom from the dithiocarbamate zinc-binding group. This new class of β-CA inhibitors may have the potential for developing antifungal agents with a diverse mechanism of action compared to the clinically used drugs for which drug resistance was reported, and may also explain the efficacy of dithiocarbamates as agricultural antifungal agents. PMID:22209456

  9. A potent specific inhibitor of 6-phosphogluconate dehydrogenase of Cryptococcus neoformans and of certain other fungal enzymes.

    PubMed

    Niehaus, W G; Flynn, T

    1993-09-01

    A particular lot of the zwitterionic buffer, 2(N-morpholino) ethane sulfonic acid (MES), contained a contaminant that inhibited a number of fungal NADP-dependent dehydrogenases. Enzymes that were particularly sensitive include 6-phosphogluconate dehydrogenases from Cryptococcus neoformans and Schizophyllum commune and glucose-6-phosphate dehydrogenase from Schizophyllum commune. A number of NADP-dependent dehydrogenases of animal origin were tested and all were completely insensitive to inhibition except for rat liver 6-phosphogluconate dehydrogenase, which was 10-fold less sensitive than the Cryptococcal enzyme. The pattern of inhibition in all cases was linear competitive versus NADP. The inhibitor has been purified and identified as an ethylenesulfonic acid oligomer. This inhibitor holds promise as a model compound for the development of a specific antifungal agent. PMID:8302365

  10. The role of the de novo pyrimidine biosynthetic pathway in Cryptococcus neoformans high temperature growth and virulence

    PubMed Central

    de Gontijo, Fabiano Assis; Pascon, Renata C.; Fernandes, Larissa; Machado, Joel; Alspaugh, J. Andrew; Vallim, Marcelo A

    2015-01-01

    Fungal infections are often difficult to treat due to the inherent similarities between fungal and animal cells and the resulting host toxicity from many antifungal compounds. Cryptococcus neoformans is an opportunistic fungal pathogen of humans that causes life-threatening disease, primarily in immunocompromised patients. Since antifungal therapy for this microorganism is limited, many investigators have explored novel drug targets aim at virulence factors, such as the ability to grow at mammalian physiological temperature (37°C). To address this issue, we used the Agrobacterium tumefaciens gene delivery system to create a random insertion mutagenesis library that was screened for altered growth at elevated temperatures. Among several mutants unable to grow at 37°C, we explored one bearing an interruption in the URA4 gene. This gene encodes dihydroorotase (DHOase) that is involved in the de novo synthesis of pyrimidine ribonucleotides. Loss of the C. neoformans Ura4 protein, by targeted gene interruption, resulted in an expected uracil/uridine auxotrophy and an unexpected high temperature growth defect. In addition, the ura4 mutant displayed phenotypic defects in other prominent virulence factors (melanin, capsule and phospholipase) and reduced stress response compared to wild type and reconstituted strains. Accordingly, this mutant had a decreased survival rate in macrophages and attenuated virulence in a murine model of cryptococcal infection. Quantitative PCR analysis suggests that this biosynthetic pathway is induced during the transition from 30°C to 37°C, and that transcriptional regulation of de novo and salvage pyrimidine pathway are under the control of the Ura4 protein. PMID:25011011

  11. Use of a Suspension Array for Rapid Identification of the Varieties and Genotypes of the Cryptococcus neoformans Species Complex

    PubMed Central

    Diaz, Mara R.; Fell, Jack W.

    2005-01-01

    Cryptococcus neoformans is an encapsulated fungal pathogen known to cause severe disease in immunocompromised patients. The disease, cryptococcosis, is mostly acquired by inhalation and can result in a chronic meningoencephalitis, which can be fatal. Here, we describe a molecular method to identify the varieties and genotypic groups within the C. neoformans species complex from culture-based assays. The method employs a novel flow cytometer with a dual laser system that allows the simultaneous detection of different target sequences in a multiplex and high-throughput format. The assay uses a liquid suspension hybridization format with specific oligonucleotide probes that are covalently bound to the surface of fluorescent color-coded microspheres. Biotinylated target amplicons, which hybridized to their complementary probe sequences, are quantified by the addition of the conjugate, streptavidin R-phycoerythrin. In this study we developed and validated eight probes derived from sequence analysis of the intergenic spacer region of the rRNA gene region. The assay proved to be specific and sensitive, allowed discrimination of a 1-bp mismatch with no apparent cross-reactivity, and detected 101 to 103 genome copies. The described protocol, which can be used directly with yeast cells or isolated DNA, can be undertaken in less than 1 h following PCR amplification and permits identification of species in a multiplex format. In addition to a multiplex capability, the assay allows the simultaneous detection of target sequences in a single reaction. The accuracy, speed, flexibility, and sensitivity of this technology are a few of the advantages that will make this assay useful for the diagnosis of human cryptococcal infections and other pathogenic diseases. PMID:16081894

  12. Sterylglucoside Catabolism in Cryptococcus neoformans with Endoglycoceramidase-related Protein 2 (EGCrP2), the First Steryl-β-glucosidase Identified in Fungi*

    PubMed Central

    Watanabe, Takashi; Ito, Tomoharu; Goda, Hatsumi M.; Ishibashi, Yohei; Miyamoto, Tomofumi; Ikeda, Kazutaka; Taguchi, Ryo; Okino, Nozomu; Ito, Makoto

    2015-01-01

    Cryptococcosis is an infectious disease caused by pathogenic fungi, such as Cryptococcus neoformans and Cryptococcus gattii. The ceramide structure (methyl-d18:2/h18:0) of C. neoformans glucosylceramide (GlcCer) is characteristic and strongly related to its pathogenicity. We recently identified endoglycoceramidase-related protein 1 (EGCrP1) as a glucocerebrosidase in C. neoformans and showed that it was involved in the quality control of GlcCer by eliminating immature GlcCer during the synthesis of GlcCer (Ishibashi, Y., Ikeda, K., Sakaguchi, K., Okino, N., Taguchi, R., and Ito, M. (2012) Quality control of fungus-specific glucosylceramide in Cryptococcus neoformans by endoglycoceramidase-related protein 1 (EGCrP1). J. Biol. Chem. 287, 368–381). We herein identified and characterized EGCrP2, a homologue of EGCrP1, as the enzyme responsible for sterylglucoside catabolism in C. neoformans. In contrast to EGCrP1, which is specific to GlcCer, EGCrP2 hydrolyzed various β-glucosides, including GlcCer, cholesteryl-β-glucoside, ergosteryl-β-glucoside, sitosteryl-β-glucoside, and para-nitrophenyl-β-glucoside, but not α-glucosides or β-galactosides, under acidic conditions. Disruption of the EGCrP2 gene (egcrp2) resulted in the accumulation of a glycolipid, the structure of which was determined following purification to ergosteryl-3β-glucoside, a major sterylglucoside in fungi, by mass spectrometric and two-dimensional nuclear magnetic resonance analyses. This glycolipid accumulated in vacuoles and EGCrP2 was detected in vacuole-enriched fraction. These results indicated that EGCrP2 was involved in the catabolism of ergosteryl-β-glucoside in the vacuoles of C. neoformans. Distinct growth arrest, a dysfunction in cell budding, and an abnormal vacuole morphology were detected in the egcrp2-disrupted mutants, suggesting that EGCrP2 may be a promising target for anti-cryptococcal drugs. EGCrP2, classified into glycohydrolase family 5, is the first steryl

  13. The Cch1-Mid1 High-Affinity Calcium Channel Contributes to the Virulence of Cryptococcus neoformans by Mitigating Oxidative Stress.

    PubMed

    Vu, Kiem; Bautos, Jennifer M; Gelli, Angie

    2015-11-01

    Pathogenic fungi have developed mechanisms to cope with stresses imposed by hosts. For Cryptococcus spp., this implies active defense mechanisms that attenuate and ultimately overcome the onslaught of oxidative stresses in macrophages. Among cellular pathways within Cryptococcus neoformans' arsenal is the plasma membrane high-affinity Cch1-Mid1 calcium (Ca(2+)) channel (CMC). Here we show that CMC has an unexpectedly complex and disparate role in mitigating oxidative stress. Upon inhibiting the Ccp1-mediated oxidative response pathway with antimycin, strains of C. neoformans expressing only Mid1 displayed enhanced growth, but this was significantly attenuated upon H2O2 exposure in the absence of Mid1, suggesting a regulatory role for Mid1 acting through the Ccp1-mediated oxidative stress response. This notion is further supported by the interaction detected between Mid1 and Ccp1 (cytochrome c peroxidase). In contrast, Cch1 appears to have a more general role in promoting cryptococci survival during oxidative stress. A strain lacking Cch1 displayed a growth defect in the presence of H2O2 without BAPTA [(1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, cesium salt] or additional stressors such as antimycin. Consistent with a greater contribution of Cch1 to oxidative stress tolerance, an intracellular growth defect was observed for the cch1Δ strain in the macrophage cell line J774A.1. Interestingly, while the absence of either Mid1 or Cch1 significantly compromises the ability of C. neoformans to tolerate oxidative stress, the absence of both Mid1 and Cch1 has a negligible effect on C. neoformans growth during H2O2 stress, suggesting the existence of a compensatory mechanism that becomes active in the absence of CMC. PMID:26385891

  14. A Homolog of Ste6, the a-Factor Transporter in Saccharomyces cerevisiae, Is Required for Mating but Not for Monokaryotic Fruiting in Cryptococcus neoformans

    PubMed Central

    Hsueh, Yen-Ping; Shen, Wei-Chiang

    2005-01-01

    Fungal pheromones function during the initial recognition stage of the mating process. One type of peptide pheromone identified in ascomycetes and basidiomycetes terminates in a conserved CAAX motif and requires extensive posttranslational modifications to become mature and active. A well-studied representative is the a-factor of Saccharomyces cerevisiae. Unlike the typical secretory pathway utilized by most peptides, an alternative mechanism involving the ATP-binding cassette transporter Ste6 is used for the export of mature a-factor. Cryptococcus neoformans, a bipolar human pathogenic basidiomycete, produces CAAX motif-containing lipopeptide pheromones in both MATa and MATα cells. Virulence studies with a congenic pair of C. neoformans serotype D strains have shown that MATα cells are more virulent than MATa cells. Characterization of the MATα pheromones indicated that an autocrine signaling loop may contribute to the differentiation and virulence of MATα cells. To further address the role of pheromones in the signaling loop, we identified a STE6 homolog in the C. neoformans genome and determined its function by gene disruption. The ste6 mutants in either mating-type background showed partially impaired mating functions, and mating was completely abolished in a bilateral mutant cross. Surprisingly, the MATα ste6 mutant does not exhibit a defect in monokaryotic fruiting, suggesting that the activation of the autocrine signaling loop by the pheromone is via a Ste6-independent mechanism. MFα pheromone itself is essential for this process and could induce the signaling response intracellularly in MATα cells. Our data demonstrate that Ste6 is evolutionarily conserved for mating and is not required for monokaryotic fruiting in C. neoformans. PMID:15643070

  15. Molecular epidemiology and in vitro antifungal susceptibility testing of 108 clinical Cryptococcus neoformans sensu lato and Cryptococcus gattii sensu lato isolates from Denmark.

    PubMed

    Hagen, Ferry; Hare Jensen, Rasmus; Meis, Jacques F; Arendrup, Maiken Cavling

    2016-09-01

    Cryptococcosis is mainly caused by members of the Cryptococcus gattii/Cryptococcus neoformans species complexes. Here, we report the molecular characterisation and in vitro antifungal susceptibility of Danish clinical cryptococcal isolates. Species, genotype, serotype and mating type were determined by amplified fragment length polymorphism (AFLP) fingerprinting and qPCR. EUCAST E.Def 7.2 MICs were determined for amphotericin B, flucytosine, fluconazole, voriconazole and isavuconazole. Most isolates were C. neoformans (serotype A; n = 66) and belonged to genotype AFLP1/VNI (n = 61) or AFLP1B/VNII (n = 5) followed by Cryptococcus deneoformans (serotype D; genotype AFLP2, n = 20), C. neoformans × C. deneoformans hybrids (serotype AD; genotype AFLP3, n = 13) and Cryptococcus curvatus (n = 2). Six isolates were C. gattii sensu lato, and one isolate was a C. deneoformans × C. gattii hybrid (genotype AFLP8). All isolates were amphotericin B susceptible. Flucytosine susceptibility was uniform MIC50 of 4-8 mg l(-1) except for C. curvatus (MICs >32 mg l(-1) ). Cryptococcus gattii sensu lato isolates were somewhat less susceptible to the azoles. MICs of fluconazole (>32 mg l(-1) ), voriconazole (≥0.5 mg l(-1) ) and isavuconazole (0.06 and 0.25 mg l(-1) respectively) were elevated compared to the wild-type population for 1/19 C. deneoformans and 1/2 C. curvatus isolates. Flucytosine MIC was elevated for 1/61 C. neoformans (>32 mg l(-1) ). Antifungal susceptibility revealed species-specific differential susceptibility, but suggested acquired resistance was an infrequent phenomenon. PMID:27061834

  16. Does the Capsule Interfere with Performance of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Cryptococcus neoformans and Cryptococcus gattii?

    PubMed Central

    Grenfell, Rafaella C.; Vidal, Monica S. M.; Giudice, Mauro C.; Del Negro, Gilda M. B.; Juliano, Luiz; Benard, Gil; de Almeida Júnior, João N.

    2015-01-01

    We described the impact of the capsule size for Cryptococcus neoformans and Cryptococcus gattii identification at the species level by Bruker matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). After experimental capsule size modulation, we observed that reducing the capsule size resulted in improved identification by Bruker MALDI-TOF MS across all of the reference strains analyzed. PMID:26659203

  17. Does the Capsule Interfere with Performance of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Cryptococcus neoformans and Cryptococcus gattii?

    PubMed

    Thomaz, Danilo Y; Grenfell, Rafaella C; Vidal, Monica S M; Giudice, Mauro C; Del Negro, Gilda M B; Juliano, Luiz; Benard, Gil; de Almeida Júnior, João N

    2016-02-01

    We described the impact of the capsule size for Cryptococcus neoformans and Cryptococcus gattii identification at the species level by Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). After experimental capsule size modulation, we observed that reducing the capsule size resulted in improved identification by Bruker MALDI-TOF MS across all of the reference strains analyzed. PMID:26659203

  18. The Cch1-Mid1 High-Affinity Calcium Channel Contributes to the Virulence of Cryptococcus neoformans by Mitigating Oxidative Stress

    PubMed Central

    Vu, Kiem; Bautos, Jennifer M.

    2015-01-01

    Pathogenic fungi have developed mechanisms to cope with stresses imposed by hosts. For Cryptococcus spp., this implies active defense mechanisms that attenuate and ultimately overcome the onslaught of oxidative stresses in macrophages. Among cellular pathways within Cryptococcus neoformans' arsenal is the plasma membrane high-affinity Cch1-Mid1 calcium (Ca2+) channel (CMC). Here we show that CMC has an unexpectedly complex and disparate role in mitigating oxidative stress. Upon inhibiting the Ccp1-mediated oxidative response pathway with antimycin, strains of C. neoformans expressing only Mid1 displayed enhanced growth, but this was significantly attenuated upon H2O2 exposure in the absence of Mid1, suggesting a regulatory role for Mid1 acting through the Ccp1-mediated oxidative stress response. This notion is further supported by the interaction detected between Mid1 and Ccp1 (cytochrome c peroxidase). In contrast, Cch1 appears to have a more general role in promoting cryptococci survival during oxidative stress. A strain lacking Cch1 displayed a growth defect in the presence of H2O2 without BAPTA [(1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, cesium salt] or additional stressors such as antimycin. Consistent with a greater contribution of Cch1 to oxidative stress tolerance, an intracellular growth defect was observed for the cch1Δ strain in the macrophage cell line J774A.1. Interestingly, while the absence of either Mid1 or Cch1 significantly compromises the ability of C. neoformans to tolerate oxidative stress, the absence of both Mid1 and Cch1 has a negligible effect on C. neoformans growth during H2O2 stress, suggesting the existence of a compensatory mechanism that becomes active in the absence of CMC. PMID:26385891

  19. Cryptococcus neoformans biofilm formation depends on surface support and carbon source and reduces fungal cell susceptibility to heat, cold, and UV light.

    PubMed

    Martinez, Luis R; Casadevall, Arturo

    2007-07-01

    The fungus Cryptococcus neoformans possesses a polysaccharide capsule and can form biofilms on medical devices. We describe the characteristics of C. neoformans biofilm development using a microtiter plate model, microscopic examinations, and a colorimetric 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay to observe the metabolic activity of cryptococci within a biofilm. A strong correlation between XTT and CFU assays was demonstrated. Chemical analysis of the exopolymeric material revealed sugar composition consisting predominantly of xylose, mannose, and glucose, indicating the presence of other polysaccharides in addition to glucurunoxylomannan. Biofilm formation was affected by surface support differences, conditioning films on the surface, characteristics of the medium, and properties of the microbial cell. A specific antibody to the capsular polysaccharide of this fungus was used to stain the extracellular polysaccharide matrix of the fungal biofilms using light and confocal microscopy. Additionally, the susceptibility of C. neoformans biofilms and planktonic cells to environmental stress was investigated using XTT reduction and CFU assays. Biofilms were less susceptible to heat, cold, and UV light exposition than their planktonic counterparts. Our findings demonstrate that fungal biofilm formation is dependent on support surface characteristics and that growth in the biofilm state makes fungal cells less susceptible to potential environmental stresses. PMID:17513597

  20. 9-O-butyl-13-(4-isopropylbenzyl)berberine, KR-72, Is a Potent Antifungal Agent That Inhibits the Growth of Cryptococcus neoformans by Regulating Gene Expression

    PubMed Central

    Hwang, Hyun Sook; Park, Ki Duk; Kim, Sung Uk; Bahn, Yong-Sun

    2014-01-01

    In this study we explored the mode of action of KR-72, a 9-O-butyl-13-(4-isopropylbenzyl)berberine derivative previously shown to exhibit potent antifungal activity against a variety of human fungal pathogens. The DNA microarray data revealed that KR-72 treatment significantly changed the transcription profiles of C. neoformans, affecting the expression of more than 2,000 genes. Genes involved in translation and transcription were mostly upregulated, whereas those involved in the cytoskeleton, intracellular trafficking, and lipid metabolism were downregulated. KR-72 also exhibited a strong synergistic effect with the antifungal agent FK506. KR-72 treatment regulated the expression of several essential genes, including ECM16, NOP14, HSP10 and MGE1, which are required for C. neoformans growth. The KR-72-mediated induction of MGE1 also likely reduced the viability of C. neoformans by impairing cell cycle or the DNA repair system. In conclusion, KR-72 showed antifungal activity by modulating diverse biological processes through a mode of action distinct from those of clinically available antifungal drugs such as polyene and azole drugs. PMID:25302492

  1. Comparative Transcriptome Analysis Reveals Novel Roles of the Ras and Cyclic AMP Signaling Pathways in Environmental Stress Response and Antifungal Drug Sensitivity in Cryptococcus neoformans ▿ †

    PubMed Central

    Maeng, Shinae; Ko, Young-Joon; Kim, Gyu-Bum; Jung, Kwang-Woo; Floyd, Anna; Heitman, Joseph; Bahn, Yong-Sun

    2010-01-01

    The cyclic AMP (cAMP) pathway plays a central role in the growth, differentiation, and virulence of pathogenic fungi, including Cryptococcus neoformans. Three upstream signaling regulators of adenylyl cyclase (Cac1), Ras, Aca1, and Gpa1, have been demonstrated to control the cAMP pathway in C. neoformans, but their functional relationship remains elusive. We performed a genome-wide transcriptome analysis with a DNA microarray using the ras1Δ, gpa1Δ, cac1Δ, aca1Δ, and pka1Δ pka2Δ mutants. The aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka2Δ mutants displayed similar transcriptome patterns, whereas the ras1Δ mutant exhibited transcriptome patterns distinct from those of the wild type and the cAMP mutants. Interestingly, a number of environmental stress response genes are modulated differentially in the ras1Δ and cAMP mutants. In fact, the Ras signaling pathway was found to be involved in osmotic and genotoxic stress responses and the maintenance of cell wall integrity via the Cdc24-dependent signaling pathway. Notably, the Ras and cAMP mutants exhibited hypersensitivity to a polyene drug, amphotericin B, without showing effects on ergosterol biosynthesis, which suggested a novel method of antifungal combination therapy. Among the cAMP-dependent gene products that we characterized, two small heat shock proteins, Hsp12 and Hsp122, were found to be involved in the polyene antifungal drug susceptibility of C. neoformans. PMID:20097740

  2. The vacuolar-sorting protein Snf7 is required for export of virulence determinants in members of the Cryptococcus neoformans complex.

    PubMed Central

    da C. Godinho, Rodrigo M.; Crestani, Juliana; Kmetzsch, Lívia; de S. Araujo, Glauber; Frases, Susana; Staats, Charley C.; Schrank, Augusto; Vainstein, Marilene H.; Rodrigues, Marcio L.

    2014-01-01

    Fungal pathogenesis requires a number of extracellularly released virulence factors. Recent studies demonstrating that most fungal extracellular molecules lack secretory tags suggest that unconventional secretion mechanisms and fungal virulence are strictly connected. Proteins of the endosomal sorting complex required for transport (ESCRT) have been recently associated with polysaccharide export in the yeast-like human pathogen Cryptococcus neoformans. Snf7 is a key ESCRT operator required for unconventional secretion in Eukaryotes. In this study we generated snf7Δ mutant strains of C. neoformans and its sibling species C. gattii. Lack of Snf7 resulted in important alterations in polysaccharide secretion, capsular formation and pigmentation. This phenotype culminated with loss of virulence in an intranasal model of murine infection in both species. Our data support the notion that Snf7 expression regulates virulence in C. neoformans and C. gattii by ablating polysaccharide and melanin traffic. These results are in agreement with the observation that unconventional secretion is essential for cryptococcal pathogenesis and strongly suggest the occurrence of still obscure mechanisms of exportation of non-protein molecules in Eukaryotes. PMID:25178636

  3. The formation of titan cells in Cryptococcus neoformans depends on the mouse strain and correlates with induction of Th2-type responses.

    PubMed

    García-Barbazán, Irene; Trevijano-Contador, Nuria; Rueda, Cristina; de Andrés, Belén; Pérez-Tavárez, Raquel; Herrero-Fernández, Inés; Gaspar, María Luisa; Zaragoza, Oscar

    2016-01-01

    Cryptococcus neoformans is a pathogenic yeast that can form titan cells in the lungs, which are fungal cells of abnormal enlarged size. Little is known about the factors that trigger titan cells. In particular, it is not known how the host environment influences this transition. In this work, we describe the formation of titan cells in two mouse strains, CD1 and C57BL/6J. We found that the proportion of C. neoformans titan cells was significantly higher in C57BL/6J mice than in CD1. This higher proportion of titan cells was associated with a higher dissemination of the yeasts to the brain. Histology sections demonstrated eosinophilia in infected animals, although it was significantly lower in the CD1 mice which presented infiltration of lymphocytes. Both mouse strains presented infiltration of granulocytes, but the amount of eosinophils was higher in C57BL/6J. CD1 mice showed a significant accumulation of IFN-γ, TNF-α and IL17, while C57BL/BL mice had an increase in the anti-inflammatory cytokine IL-4. IgM antibodies to the polysaccharide capsule and total IgE were more abundant in the sera from C57BL/6J, confirming that these animals present a Th2-type response. We conclude that titan cell formation in C. neoformans depends, not only on microbe factors, but also on the host environment. PMID:26243235

  4. ALL2, a Homologue of ALL1, Has a Distinct Role in Regulating pH Homeostasis in the Pathogen Cryptococcus neoformans

    PubMed Central

    Jain, Neena; Bouklas, Tejas; Gupta, Anjali; Varshney, Avanish K.; Orner, Erika P.

    2015-01-01

    Cryptococcus neoformans is a facultative intracellular fungal pathogen that has a polysaccharide capsule and causes life-threatening meningoencephalitis. Its capsule, as well as its ability to survive in the acidic environment of the phagolysosome, contributes to the pathogen's resilience in the host environment. Previously, we reported that downregulation of allergen 1 (ALL1) results in the secretion of a shorter, more viscous exopolysaccharide with less branching and structural complexity, as well as altered iron homeostasis. Now, we report on a homologous coregulated gene, allergen 2 (ALL2). ALL2's function was characterized by generating null mutants in C. neoformans. In contrast to ALL1, loss of ALL2 attenuated virulence in the pulmonary infection model. The all2Δ mutant shed a less viscous exopolysaccharide and exhibited higher sensitivity to hydrogen peroxide than the wild type, and as a result, the all2Δ mutant was more resistant to macrophage-mediated killing. Transcriptome analysis further supported the distinct function of these two genes. Unlike ALL1's involvement in iron homeostasis, we now present data on ALL2's unique function in maintaining intracellular pH in low-pH conditions. Thus, our data highlight that C. neoformans, a human-pathogenic basidiomycete, has evolved a unique set of virulence-associated genes that contributes to its resilience in the human niche. PMID:26597983

  5. Classification of Cryptococcus neoformans and yeast-like fungus isolates from pigeon droppings by colony phenotyping and ITS genotyping and their seasonal variations in Korea.

    PubMed

    Chae, H S; Jang, G E; Kim, N H; Son, H R; Lee, J H; Kim, S H; Park, G N; Jo, H J; Kim, J T; Chang, K S

    2012-03-01

    Cryptococcus neoformans (C neoformans) is a frequent cause of invasive fungal disease in immunocompromised human hosts. Ninety-eight samples of pigeon droppings were collected from the pigeon shelters in Seoul, and cultured on birdseed agar (BSA) and Sabouraud dextrose agar (SDA). One hundred yeast-like colonies were selected and identified via phenotype characteristics, such as colony morphology and biochemical characteristics. This was then followed with genotyping via sequencing of the internal transcribed spacer (ITS) region. The colonies were classified into four kinds of colony color types: brown type (BrT), beige type (BeT), pink type (PT), and white type (WT). Numbers of isolated BrT, BeT, PT, and WT colonies were 22 (22%), 30 (30%), 19 (19%), and 39 (39%), respectively. All BrT colonies were identified as C neoformans. BeT were identified as 19 isolates of Cryptococcus laurentii, 10 isolates of Malassezia furfur, and 1 isolate of Cryptococcus uniguttulatus. PT was divided into two colony color types: light-PT (l-PT) and deep-PT (d-PT). Eighteen of l-PT and one of d-PT were identified as Rhodotorula glutinis and Rhodotorula mucilaginosa, respectively. WT were identified as 34 isolates of Cryptococcus guilliermondii, 3 isolates of Cryptococcus zeylanoides, 1 isolate of Cryptococcus sake, and 1 isolate of Stephanoascus ciferrii. Most strains were classified identically with the use of either phenotype or genotyping techniques, but C uniguttulatus and C sake classified by phenotyping were Pseudozyma aphidis and Cryptococcus famata by genotyping. This rapid screening technique of pathogenic yeast-like fungi by only colony characteristics is also expected to be very useful for primary yeast screening. Additionally, we investigated the seasonal variations of C neoformans and other yeast-like fungi from 379 pigeon-dropping samples that were collected from February 2011 to March 2011. We isolated 685 yeast-like fungi from the samples. Almost all C neoformans and

  6. Structure and inhibition of the CO2-sensing carbonic anhydrase Can2 from the pathogenic fungus Cryptococcus neoformans.

    PubMed

    Schlicker, Christine; Hall, Rebecca A; Vullo, Daniela; Middelhaufe, Sabine; Gertz, Melanie; Supuran, Claudiu T; Mühlschlegel, Fritz A; Steegborn, Clemens

    2009-01-30

    In the pathogenic fungus Cryptococcus neoformans, a CO(2)-sensing system is essential for survival in the natural environment (approximately 0.03% CO(2)) and mediates the switch to virulent growth in the human host (approximately 5% CO(2)). This system is composed of the carbonic anhydrase (CA) Can2, which catalyzes formation of bicarbonate, and the fungal, bicarbonate-stimulated adenylyl cyclase Cac1. The critical role of these enzymes for fungal metabolism and pathogenesis identifies them as targets for antifungal drugs. Here, we prove functional similarity of Can2 to the CA Nce103 from Candida albicans and describe its biochemical and structural characterization. The crystal structure of Can2 reveals that the enzyme belongs to the "plant-type" beta-CAs but carries a unique N-terminal extension that can interact with the active-site entrance of the dimer. We further tested a panel of compounds, identifying nanomolar Can2 inhibitors, and present the structure of a Can2 complex with the inhibitor and product analog acetate, revealing insights into interactions with physiological ligands and inhibitors. PMID:19071134

  7. Enhanced binding of capsular polysaccharides of Cryptococcus neoformans to polystyrene microtitration plates for enzyme-linked immunosorbent assay.

    PubMed

    Cherniak, R; Cheeseman, M M; Reyes, G H; Reiss, E; Todaro, F

    1988-01-01

    A sensitive enzyme-linked immunosorbent assay (ELISA) to measure antibodies against capsular polysaccharide was developed, based on the enhanced binding of polysaccharide to polystyrene microtitration plates. The wells of the microtitration plate were primed with an adipic acid dihydrazide derivative of bovine serum albumin (AH-BSA) (100 micrograms/mL, 0.01 M NaPO4-0.14 M NaCl, pH 7.2 (PBS]. Capsular polysaccharide, the glucuronoxylomannan of Cryptococcus neoformans serotype A, was oxidized with NaIO4 for 5 min; the reaction was then quenched with ethylene glycol. The partially oxidized polysaccharide was dialyzed vs. PBS, and its concentration was adjusted to 50 micrograms/mL with PBS. This solution (100 microL/well) was covalently bound to the AH-BSA primed microtitration plates through formation of a Schiff base between the hydrazide group on the AH-BSA and the aldehyde groups on the polysaccharide. Antimouse IgG-alkaline phosphatase conjugate was used in an indirect ELISA to measure captured murine monoclonal antibodies directed against glucuronoxylomannan. Mean absorbances, after 15 min, were 0.13 in negative control wells, and greater than 0.7 in test wells. No intermediate steps were required to block nonspecific binding of antibody. PMID:3064947

  8. Enhanced virulence of Histoplasma capsulatum through transfer and surface incorporation of glycans from Cryptococcus neoformans during co-infection

    PubMed Central

    Cordero, Radames J. B.; Liedke, Susie Coutinho; de S. Araújo, Glauber R.; Martinez, Luis R.; Nimrichter, Leonardo; Frases, Susana; Peralta, Jose Mauro; Casadevall, Arturo; Rodrigues, Marcio L.; Nosanchuk, Joshua D.; Guimaraes, Allan J.

    2016-01-01

    Cryptococcus neoformans (Cn) and Histoplasma capsulatum (Hc) co-exist in the environment and occasionally co-infect individuals, which can lead to severe disease/lethal outcomes. We investigated specific interactions between Cn-Hc to determine the impact of synchronous infection in virulence and disease. Co-infected mice had significantly higher mortality than infection with either species or acapsular Cn-Hc. Coating of Hc with cryptococcal glycans (Cn-gly) resulted in higher pulmonary fungal burden in co-infected animals relative to control. Co-cultivation or addition of Cn-gly resulted in enhanced pellicle formation with a hybrid polysaccharide matrix with higher reactivity to GXM mAbs. Transfer and incorporation of Cn polysaccharide onto Hc surface was time and temperature dependent. Cn-gly transfer altered the zeta potential of Hc and was associated with increased resistance to phagocytosis and killing by macrophages. Mice infected with Hc and subsequently injected with purified Cn-gly died significantly more rapidly than Hc alone infected, establishing the precedent that virulence factors from one fungus can enhance the virulence of unrelated species. These findings suggest a new mechanism of microbial interaction involving the transfer of virulence traits that translates into enhanced lethality during mixed fungal infections and highlights the importance of studying heterogeneous microbial populations in the setting of infection. PMID:26908077

  9. Enhanced virulence of Histoplasma capsulatum through transfer and surface incorporation of glycans from Cryptococcus neoformans during co-infection.

    PubMed

    Cordero, Radames J B; Liedke, Susie Coutinho; de S Araújo, Glauber R; Martinez, Luis R; Nimrichter, Leonardo; Frases, Susana; Peralta, Jose Mauro; Casadevall, Arturo; Rodrigues, Marcio L; Nosanchuk, Joshua D; Guimaraes, Allan J

    2016-01-01

    Cryptococcus neoformans (Cn) and Histoplasma capsulatum (Hc) co-exist in the environment and occasionally co-infect individuals, which can lead to severe disease/lethal outcomes. We investigated specific interactions between Cn-Hc to determine the impact of synchronous infection in virulence and disease. Co-infected mice had significantly higher mortality than infection with either species or acapsular Cn-Hc. Coating of Hc with cryptococcal glycans (Cn-gly) resulted in higher pulmonary fungal burden in co-infected animals relative to control. Co-cultivation or addition of Cn-gly resulted in enhanced pellicle formation with a hybrid polysaccharide matrix with higher reactivity to GXM mAbs. Transfer and incorporation of Cn polysaccharide onto Hc surface was time and temperature dependent. Cn-gly transfer altered the zeta potential of Hc and was associated with increased resistance to phagocytosis and killing by macrophages. Mice infected with Hc and subsequently injected with purified Cn-gly died significantly more rapidly than Hc alone infected, establishing the precedent that virulence factors from one fungus can enhance the virulence of unrelated species. These findings suggest a new mechanism of microbial interaction involving the transfer of virulence traits that translates into enhanced lethality during mixed fungal infections and highlights the importance of studying heterogeneous microbial populations in the setting of infection. PMID:26908077

  10. Identification of Genes from the Fungal Pathogen Cryptococcus neoformans Related to Transmigration into the Central Nervous System

    PubMed Central

    Tseng, Hsiang-Kuang; Liu, Chang-Pan; Price, Michael S.; Jong, Ambrose Y.; Chang, Jui-Chih; Toffaletti, Dena L.; Betancourt-Quiroz, Marisol; Frazzitta, Aubrey E.; Cho, Wen-Long; Perfect, John R.

    2012-01-01

    Background A mouse brain transmigration assessment (MBTA) was created to investigate the central nervous system (CNS) pathogenesis of cryptococcal meningoencephalitis. Methodology/Principal Findings Two cryptococcal mutants were identified from a pool of 109 pre-selected mutants that were signature-tagged with the nourseothricin acetyltransferase (NAT) resistance cassette. These two mutants displayed abnormal transmigration into the central nervous system. One mutant displaying decreased transmigration contains a null mutation in the putative FNX1 gene, whereas the other mutant possessing a null mutation in the putative RUB1 gene exhibited increased transmigration into the brain. Two macrophage adhesion-defective mutants in the pool, 12F1 and 3C9, showed reduced phagocytosis by macrophages, but displayed no defects in CNS entry suggesting that transit within macrophages (the “Trojan horse” model of CNS entry) is not the primary mechanism for C. neoformans migration into the CNS in this MBTA. Conclusions/Significance This research design provides a new strategy for genetic impact studies on how Cryptococcus passes through the blood-brain barrier (BBB), and the specific isolated mutants in this assay support a transcellular mechanism of CNS entry. PMID:23028773

  11. TLR9 Signaling Is Required for Generation of the Adaptive Immune Protection in Cryptococcus neoformans-Infected Lungs

    PubMed Central

    Zhang, Yanmei; Wang, Fuyuan; Bhan, Urvashi; Huffnagle, Gary B.; Toews, Galen B.; Standiford, Theodore J.; Olszewski, Michal A.

    2010-01-01

    To determine whether TLR9 signaling contributes to the development of the adaptive immune response to cryptococcal infection, wild-type (TLR9+/+) and TLR9 knockout (TLR9−/−) BALB/c mice were infected intratracheally with 104 C. neoformans 52D. We evaluated 1) organ microbial burdens, 2) pulmonary leukocyte recruitment, 3) pulmonary and systemic cytokine induction, and 4) macrophage activation profiles. TLR9 deletion did not affect pulmonary growth during the innate phase, but profoundly impaired pulmonary clearance during the adaptive phase of the immune response (a 1000-fold difference at week 6). The impaired clearance in TLR9−/− mice was associated with: 1) significantly reduced CD4+, CD8+ T cell, and CD19+ B cell recruitment into the lungs; 2) defects in Th polarization indicated by altered cytokine responses in the lungs, lymphonodes, and spleen; and 3) diminished macrophage accumulation and altered activation profile, including robust up-regulation of Arg1 and FIZZ1 (indicators of alternative activation) and diminished induction of inducible nitric oxide synthase (an indicator of classical activation). Histological analysis revealed defects in granuloma formation and increased numbers of intracellular yeast residing within macrophages in the lungs of TLR9−/− mice. We conclude that TLR9 signaling plays an important role in the development of robust protective immunity, proper recruitment and function of effector cells (lymphocytes and macrophages), and, ultimately, effective cryptococcal clearance from the infected lungs. PMID:20581055

  12. Prostaglandin E2 blockade enhances the pulmonary anti-Cryptococcus neoformans immune reaction via the induction of TLR-4.

    PubMed

    Shen, Liyun; Liu, Ying

    2015-09-01

    The present study aimed to explore whether the inhibition of prostaglandin E2 enhances pulmonary anti-Cryptococcus neoformans immunity. Lung colony forming unit (CFU) assays demonstrated that the cryptococcal infection was dramatically depressed in mice given EP2 and EP4 or single EP antagonist treatment compared to the untreated wild type mice (p<0.05), leading to the increased survival of the infected mice by 8-9 days or 2-4 days, respectively. RT-PCR and flow cytometry assays showed that the expression of IFN-γ, IL-17, IL-22 in M1 macrophages and IL-10 in M2 macrophages increased significantly at 1 week post-infection in mice with either EP2 or EP4 blockade (p<0.05). The polarization of alveolar macrophages showed that, at 1 week post infection, the alveolar macrophages in untreated wild type mice, TLR4(-/-) mice and TLR4(-/-) mice with EP2 and EP4 blockade were strongly M2 polarized, whereas the alveolar macrophages in wild type mice with EP2 and EP4 blockade were M1 polarized. In conclusion, the blockade of EP2 and EP4 promotes mouse survival after cryptococcus infection by promoting the production of cytokines via TLR4, as well as the enhanced M1 polarization of alveolar macrophages. PMID:26122137

  13. Opsonization of Cryptococcus neoformans by a family of isotype-switch variant antibodies specific for the capsular polysaccharide.

    PubMed Central

    Schlageter, A M; Kozel, T R

    1990-01-01

    A family of immunoglobulin isotype-switch variants was isolated by sib selection from a murine hybridoma which produced an immunoglobulin G subclass 1 (IgG1) antibody specific for the capsular polysaccharide of Cryptococcus neoformans. Antibodies of the IgG1, IgG2a, and IgG2b isotypes had similar serotype specificity patterns in double immunodiffusion assays which used polysaccharides of the four cryptococcal serotypes as antigens. A quantitative difference in the ability of the isotypes to form a precipitate with the polysaccharide was observed in a double immunodiffusion assay and confirmed in a quantitative precipitin assay. The relative precipitating activity of the antibodies was IgG2a greater than IgG1 much greater than IgG2b. Analysis by enzyme-linked immunosorbent assay of the reactivity of the three isotypes with cryptococcal polysaccharide showed identical titers and slopes, suggesting that the variable region of the class-switch antibodies was unaltered. This system allowed us to examine the effect of the Fc portion of the antibody on opsonization of encapsulated cryptococci. Yeast cells were precoated with antibodies of each isotype and incubated with murine macrophages or cultured human monocytes. Antibodies of all three isotypes exhibited a dose-dependent opsonization for phagocytosis by both human and murine phagocytes. The relative opsonic activity of the antibodies was IgG2a greater than IgG1 greater than IgG2b. Images PMID:2187813

  14. Defect of CARD9 leads to impaired accumulation of gamma interferon-producing memory phenotype T cells in lungs and increased susceptibility to pulmonary infection with Cryptococcus neoformans.

    PubMed

    Yamamoto, Hideki; Nakamura, Yuri; Sato, Ko; Takahashi, Yurie; Nomura, Toshiki; Miyasaka, Tomomitsu; Ishii, Keiko; Hara, Hiromitsu; Yamamoto, Natsuo; Kanno, Emi; Iwakura, Yoichiro; Kawakami, Kazuyoshi

    2014-04-01

    Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule signal that is critical for NF-κB activation and is triggered through C-type lectin receptors (CLRs), which are pattern recognition receptors that recognize carbohydrate structures. Previous studies have reported that Cryptococcus neoformans, a fungal pathogen that causes meningoencephalitis in AIDS patients, is recognized through some CLRs, such as mannose receptors or DC-SIGN. However, the role of CARD9 in the host defense against cryptococcal infection remains to be elucidated. In the present study, we analyzed the role of CARD9 in the host defense against pulmonary infection with C. neoformans. CARD9 gene-disrupted (knockout [KO]) mice were highly susceptible to this infection, as shown by the reduced fungal clearance in the infected lungs of CARD9 KO mice, compared to that in wild-type (WT) mice. Gamma interferon (IFN-γ) production was strongly reduced in CARD9 KO mice during the innate-immunity phase of infection. Reduced IFN-γ synthesis was due to impaired accumulation of NK and memory phenotype T cells, which are major sources of IFN-γ innate-immunity-phase production; a reduction in the accumulation of these cells was correlated with reduced CCL4, CCL5, CXCL9, and CXCL10 synthesis. However, differentiation of Th17 cells, but not of Th1 cells, was impaired at the adaptive-immunity phase in CARD9 KO mice compared to WT mice, although there was no significant difference in the infection susceptibility between interleukin 17A (IL-17A) KO and WT mice. These results suggest that CARD9 KO mice are susceptible to C. neoformans infection probably due to the reduced accumulation of IFN-γ-expressing NK and memory phenotype T cells at the early stage of infection. PMID:24470469

  15. The Transcriptional Response of Cryptococcus neoformans to Ingestion by Acanthamoeba castellanii and Macrophages Provides Insights into the Evolutionary Adaptation to the Mammalian Host

    PubMed Central

    Paes, Hugo Costa; Albuquerque, Patrícia; Tavares, Aldo Henrique F. P.; Fernandes, Larissa; Silva-Pereira, Ildinete; Casadevall, Arturo

    2013-01-01

    Virulence of Cryptococcus neoformans for mammals, and in particular its intracellular style, was proposed to emerge from evolutionary pressures on its natural environment by protozoan predation, which promoted the selection of strategies that allow intracellular survival in macrophages. In fact, Acanthamoeba castellanii ingests yeast cells, which then can replicate intracellularly. In addition, most fungal factors needed to establish infection in the mammalian host are also important for survival within the amoeba. To better understand the origin of C. neoformans virulence, we compared the transcriptional profile of yeast cells internalized by amoebae and murine macrophages after 6 h of infection. Our results showed 656 and 293 genes whose expression changed at least 2-fold in response to the intracellular environments of amoebae and macrophages, respectively. Among the genes that were found in both groups, we focused on open reading frame (ORF) CNAG_05662, which was potentially related to sugar transport but had no determined biological function. To characterize its function, we constructed a mutant strain and evaluated its ability to grow on various carbon sources. The results showed that this gene, named PTP1 (polyol transporter protein 1), is involved in the transport of 5- and 6-carbon polyols such as mannitol and sorbitol, but its presence or absence had no effect on cryptococcal virulence for mice or moth larvae. Overall, these results are consistent with the hypothesis that the capacity for mammalian virulence originated from fungus-protozoan interactions in the environment and provide a better understanding of how C. neoformans adapts to the mammalian host. PMID:23524994

  16. Differences between Cryptococcus neoformans and Cryptococcus gattii in the Molecular Mechanisms Governing Utilization of D-Amino Acids as the Sole Nitrogen Source

    PubMed Central

    Chang, Yun C.; Khanal Lamichhane, Ami; Bradley, James; Rodgers, Laura; Ngamskulrungroj, Popchai; Kwon-Chung, Kyung J.

    2015-01-01

    The ability to grow on media containing certain D-amino acids as a sole nitrogen source is widely utilized to differentiate Cryptococcus gattii from C. neoformans. We used the C. neoformans H99 and C. gattii R265 strains to dissect the mechanisms of D-amino acids utilization. We identified three putative D-amino acid oxidase (DAO) genes in both strains and showed that each DAO gene plays different roles in D-amino acid utilization in each strain. Deletion of DAO2 retarded growth of R265 on eleven D-amino acids suggesting its prominent role on D-amino acid assimilation in R265. All three R265 DAO genes contributed to growth on D-Asn and D-Asp. DAO3 was required for growth and detoxification of D-Glu by both R265 and H99. Although growth of H99 on most D-amino acids was poor, deletion of DAO1 or DAO3 further exacerbated it on four D-amino acids. Overexpression of DAO2 or DAO3 enabled H99 to grow robustly on several D-amino acids suggesting that expression levels of the native DAO genes in H99 were insufficient for growth on D-amino acids. Replacing the H99 DAO2 gene with a single copy of the R265 DAO2 gene also enabled its utilization of several D-amino acids. Results of gene and promoter swaps of the DAO2 genes suggested that enzymatic activity of Dao2 in H99 might be lower compared to the R265 strain. A reduction in virulence was only observed when all DAO genes were deleted in R265 but not in H99 indicating a pathobiologically exclusive role of the DAO genes in R265. These results suggest that C. neoformans and C. gattii divergently evolved in D-amino acid utilization influenced by their major ecological niches. PMID:26132227

  17. A Ric8/Synembryn Homolog Promotes Gpa1 and Gpa2 Activation To Respectively Regulate Cyclic AMP and Pheromone Signaling in Cryptococcus neoformans

    PubMed Central

    Gong, Jinjun; Grodsky, Jacob D.; Zhang, Zhengguang

    2014-01-01

    The G protein α subunits Gpa1, Gpa2, and Gpa3 mediate signal transduction and are important in the growth and virulence of Cryptococcus neoformans. To understand how Gpa1 functions without a conventional Gβ subunit, we characterized a resistance to inhibitors of cholinesterase 8 (Ric8) homolog from C. neoformans, which shares amino acid sequence homology with other Ric8 proteins that exhibit guanine nucleotide exchange factor (GEF) activity toward Gα. We found that the ric8 mutant was reduced in capsule size and melanin formation, which could be suppressed by cyclic AMP (cAMP) supplementation or by introducing the activated GPA1Q284L allele. Consistent with the fact that Ric8 participates in cAMP signaling to regulate virulence, the ric8 mutant was attenuated in virulence toward mice. Interestingly, disruption of RIC8 also resulted in opposing effects on pheromone signaling, as the ric8 mutant showed reduced mating but an enhanced ability to induce the pheromone response in the mating partner. To identify Ric8 functional mechanisms, we examined the interactions between Ric8 and the three Gα proteins. Ric8 interacted with Gpa1 and Gpa2, but not Gpa3. The presence of Gpa1Q284L negatively affected its interaction with Ric8, whereas the activated Gpa2Q203L allele abolished the interaction. Collectively, these findings suggest that Ric8 functions as a GEF to facilitate the activation of Gpa1-cAMP signaling and to promote Gpa2, affecting mating efficiency. Our study highlights the distinct and conserved characteristics associated with G protein signaling and contributes to our overall understanding of how G protein α subunits function with or without a canonical Gβ partner in C. neoformans. PMID:25084863

  18. Th2 but Not Th1 Immune Bias Results in Altered Lung Functions in a Murine Model of Pulmonary Cryptococcus neoformans Infection▿

    PubMed Central

    Jain, Aditya V.; Zhang, Yanmei; Fields, W. Bradley; McNamara, David A.; Choe, Mun Y.; Chen, Gwo-hsiao; Erb-Downward, John; Osterholzer, John J.; Toews, Galen B.; Huffnagle, Gary B.; Olszewski, Michal A.

    2009-01-01

    Changes in airway dynamics have been reported in the rat model of pulmonary cryptococcosis. However, it is not known if Cryptococcus neoformans-induced changes in lung functions are related to the immunophenotype that develops in response to cryptococcal infection in the lungs. In this study we performed a parallel analysis of the immunophenotype and airway resistance (standard resistance of the airways [SRAW]) in BALB/c mice infected with highly virulent C. neoformans strain H99 and moderately virulent strain 52D. H99 infection evoked a Th2 response and was associated with increased SRAW, while the SRAW for 52D infection, which resulted in a predominantly Th1-skewed response, did not differ from the SRAW for uninfected mice. We found that an altered SRAW in mice did not positively or negatively correlate with the pulmonary fungal burden, the magnitude of inflammatory response, the numbers of T cells, eosinophils or eosinophil subsets, neutrophils, or monocytes/macrophages, or the levels of cytokines (interleukin-4 [IL-4], IL-10, gamma interferon, or IL-13) produced by lung leukocytes. However, the level of a systemic Th2 marker, serum immunoglobulin E (IgE), correlated significantly with SRAW, indicating that the changes in lung functions were proportional to the level of Th2 skewing in this model. These data also imply that IgE may contribute to the altered SRAW observed in H99-infected mice. Lung histological analysis revealed severe allergic bronchopulmonary mycosis pathology in H99-infected mice and evidence of protective responses in 52D-infected mice with well-marginalized lesions. Taken together, the data show that C. neoformans can significantly affect airflow physiology, particularly in the context of a Th2 immune response with possible involvement of IgE as an important factor. PMID:19752036

  19. Correlation of natural killer cell activity and clearance of Cryptococcus neoformans from mice after adoptive transfer of splenic nylon wool-nonadherent cells.

    PubMed

    Hidore, M R; Murphy, J W

    1986-02-01

    Previous reports demonstrate that natural killer (NK) cells inhibit the growth of Cryptococcus neoformans in vitro, but conclusive evidence supporting the effectiveness of NK cells in host resistance to cryptococci is not available. The objective of these studies was to assess the ability of NK cells to clear C. neoformans from the lungs, livers, and spleens of infected mice. CBA/J mice were depleted of NK cells, as well as other natural effector cells, by an intraperitoneal injection of cyclophosphamide (Cy), 240 mg/kg of body weight. One day later, 7.5 X 10(7) nylon wool-nonadherent (NWN) spleen cells, either untreated or treated with anti-asialo GM1 and complement to remove NK cells, were adoptively transferred to Cy-pretreated mice. On day 2 after Cy treatment, the mice were injected intravenously with 2 X 10(4) cryptococci. At 4 and 6 days after Cy treatment, tissues were assayed for NK reactivity, using a 4-h 51Cr-release assay, and for in vivo clearance of cryptococci as reflected by mean log10 CFU per organ. We observed that Cy treatment depleted NK activity against YAC-1 targets and reduced in vivo clearance of C. neoformans from the tissues of infected mice. Additionally, Cy treatment depleted the total lung and spleen cellularity and the total number of peripheral blood lymphocytes when compared with those in normal untreated control mice. Also, spleen weights were significantly decreased in comparison with those of untreated animals 4 days after Cy treatment. Adoptive transfer of untreated NWN spleen cells into Cy-depressed mice restored the NK cell activity which correlated with enhanced clearance of cryptococci from lungs, livers, and spleens. In contrast, treatment of NWN spleen cells with anti-asialo GM1 and complement before adoptive transfer abrogated the ability of these cells to restore NK activity or reduce the numbers of cryptococci present in tissues of infected mice. Taken together, these data indicate that NK cells are the cells effective

  20. Modulation of Macrophage Inflammatory Nuclear Factor κB (NF-κB) Signaling by Intracellular Cryptococcus neoformans.

    PubMed

    Hayes, James B; Sircy, Linda M; Heusinkveld, Lauren E; Ding, Wandi; Leander, Rachel N; McClelland, Erin E; Nelson, David E

    2016-07-22

    Cryptococcus neoformans (Cn) is a common facultative intracellular pathogen that can cause life-threatening fungal meningitis in immunocompromised individuals. Shortly after infection, Cn is detectable as both extra- and intracellular yeast particles, with Cn being capable of establishing long-lasting latent infections within host macrophages. Although recent studies have shown that shed capsular polysaccharides and intact extracellular Cn can compromise macrophage function through modulation of NF-κB signaling, it is currently unclear whether intracellular Cn also affects NF-κB signaling. Utilizing live cell imaging and computational modeling, we find that extra- and intracellular Cn support distinct modes of NF-κB signaling in cultured murine macrophages. Specifically, in RAW 264.7 murine macrophages treated with extracellular glucuronoxylomannan (GXM), the major Cn capsular polysaccharide, LPS-induced nuclear translocation of p65 is inhibited, whereas in cells with intracellular Cn, LPS-induced nuclear translocation of p65 is both amplified and sustained. Mathematical simulations and quantification of nascent protein expression indicate that this is a possible consequence of Cn-induced "translational interference," impeding IκBα resynthesis. We also show that long term Cn infection induces stable nuclear localization of p65 and IκBα proteins in the absence of additional pro-inflammatory stimuli. In this case, nuclear localization of p65 is not accompanied by TNFα or inducible NOS (iNOS) expression. These results demonstrate that capsular polysaccharides and intact intracellular yeast manipulate NF-κB via multiple distinct mechanisms and provide new insights into how Cn might modulate cellular signaling at different stages of an infection. PMID:27231343

  1. Development of Protective Inflammation and Cell-Mediated Immunity against Cryptococcus neoformans after Exposure to Hyphal Mutants

    PubMed Central

    Zhai, Bing; Wozniak, Karen L.; Masso-Silva, Jorge; Upadhyay, Srijana; Hole, Camaron; Rivera, Amariliz; Wormley, Floyd L.

    2015-01-01

    ABSTRACT Morphological switch is tightly coupled with the pathogenesis of many dimorphic fungal pathogens. Cryptococcus neoformans, the major causative agent of cryptococcal meningitis, mostly presents as the yeast form but is capable of switching to the hyphal form. The filamentous form has long been associated with attenuated virulence, yet the underlying mechanism remains elusive. We previously identified the master regulator Znf2 that controls the yeast-to-hypha transition in Cryptococcus. Activation of Znf2 promotes hyphal formation and abolishes fungal virulence in vivo. Here we demonstrated that the cryptococcal strain overexpressing ZNF2 elicited strong and yet temporally confined proinflammatory responses in the early stage of infection. In contrast, exacerbated inflammation in mice infected with the wild-type (WT) strain showed that they were unable to control the infection. Animals inoculated with this filamentous Cryptococcus strain had fewer pulmonary eosinophils and CD11c+ CD11b+ cells than animals inoculated with WT yeast. Moreover, mice infected with this strain developed protective Th1- or Th17-type T cell responses. These findings suggest that the virulence attenuation of the filamentous form is likely due to its elicitation of protective host responses. The antivirulence effect of Znf2 was independent of two previously identified factors downstream of Znf2. Interestingly, mucosal immunizations with high doses of ZNF2-overexpressing cells, either in the live or heat-killed form, offered 100% protection to the host from a subsequent challenge with the otherwise lethal clinical strain H99. Our results demonstrate that heat-resistant cellular components presented in cryptococcal cells with activated ZNF2 elicit protective host immune responses. These findings could facilitate future research on novel immunological therapies. PMID:26443458

  2. Design, synthesis, and biological evaluation of aminothiazole derivatives against the fungal pathogens Histoplasma capsulatum and Cryptococcus neoformans

    PubMed Central

    Khalil, Ahmed; Edwards, Jessica A.; Rappleye, Chad A.; Tjarks, Werner

    2014-01-01

    Invasive fungal disease constitutes a growing health burden and development of novel antifungal drugs with high potency and selectivity against new fungal molecular targets are urgently needed. Previously, an aminothiazole derivative, designated as 41F5, was identified in our laboratories as highly active against Histoplasma yeast (MIC50 0.4-0.8 µM) through phenotypic high-throughput screening of a commercial library of 3600 purine mimicking compounds (Edwards, JA et al. Antimicrob. Agents Chemother. 2013, 57:4349-5359). Consequently, 68 analogues of 41F5 were designed and synthesized or obtained from commercial sources and their MIC50s of growth inhibition were evaluated in Histoplasma capsulatum to establish a basic Structure-Activity-Relationship (SAR) for this potentially new class of antifungals. The growth inhibiting potentials of smaller subsets of this library were also evaluated in Cryptococcus neoformans and human hepatocyte HepG2 cells, the latter to obtain Selectivity Indices (SIs). The results indicate that a thiazole core structure with a naphth-1-ylmethyl group at the 5-position and cyclohexylamide-, cyclohexylmethylamide-, or cyclohexylethylamide substituents at the 2-position caused the highest growth inhibition of Histoplasma yeast with MIC50s of 0.4 µM. For these analogues, SIs of 92 - >100 indicated generally low host toxicity. Substitution at the 3- and 4-position decreased antifungal activity. Similarities and differences were observed between Histoplasma and Cryptococcus SARs. For Cryptococcus, the naphth-1-ylmethyl substituent at the 5-position and smaller cyclopentylamide or cyclohexylamide groups at the 2-position were important for activity. In contrast, slightly larger cyclohexylmethyl- and cyclohexylethyl substituents markedly decreased activity. PMID:25543205

  3. Virulence Attributes and Hyphal Growth of C. neoformans Are Quantitative Traits and the MATα Allele Enhances Filamentation

    PubMed Central

    Lin, Xiaorong; Huang, Johnny C; Mitchell, Thomas G; Heitman, Joseph

    2006-01-01

    Cryptococcus neoformans is a fungal human pathogen with a bipolar mating system. It undergoes a dimorphic transition from a unicellular yeast to hyphal filamentous growth during mating and monokaryotic fruiting. The traditional sexual cycle that leads to the production of infectious basidiospores involves cells of both α and a mating type. Monokaryotic fruiting is a modified form of sexual reproduction that involves cells of the same mating type, most commonly α, which is the predominant mating type in both the environment and clinical isolates. However, some a isolates can also undergo monokaryotic fruiting. To determine whether mating type and other genetic loci contribute to the differences in fruiting observed between α and a cells, we applied quantitative trait loci (QTL) mapping to an inbred population of F2 progeny. We discovered that variation in hyphal length produced during fruiting is a quantitative trait resulting from the combined effects of multiple genetic loci, including the mating type (MAT) locus. Importantly, the α allele of the MAT locus enhanced hyphal growth compared with the a allele. Other virulence traits, including melanization and growth at 39 °C, also are quantitative traits that share a common QTL with hyphal growth. The Mac1 transcription factor, encoded in this common QTL, regulates copper homeostasis. MAC1 allelic differences contribute to phenotypic variation, and mac1Δ mutants exhibit defects in filamentation, melanin production, and high temperature growth. Further characterization of these QTL regions will reveal additional quantitative trait genes controlling biological processes central to fungal development and pathogenicity. PMID:17112316

  4. Synthesis of a Glucuronic Acid-Containing Thioglycoside Trisaccharide Building Block and Its Use in the Assembly of Cryptococcus Neoformans Capsular Polysaccharide Fragments.

    PubMed

    Guazzelli, Lorenzo; Ulc, Rebecca; Oscarson, Stefan

    2015-12-01

    As part of an ongoing project aimed at identifying protective capsular polysaccharide epitopes for the development of vaccine candidates against the fungal pathogen Cryptococcus neoformans, the synthesis and glycosylation properties of a naphthalenylmethyl (NAP) orthogonally protected trisaccharide thioglycoside, a common building block for construction of serotype B and C capsular polysaccharide structures, were investigated. Ethyl (benzyl 2,3,4-tri-O-benzyl-β-d-glucopyranosyl- uronate)-(1→2)-[2,3,4-tri-O-benzyl-β-d-xylopyranosyl-(1→4)]-6-O-benzyl-3-O-(2-naphthalenylmethyl)-1-thio-α-d-mannopyranoside was prepared and used both as a donor and an acceptor in glycosylation reactions to obtain spacer equipped hexa- and heptasaccharide structures suitable either for continued elongation or for deprotection and printing onto a glycan array or conjugation to a carrier protein. The glycosylation reactions proceeded with high yields and α-selectivity, proving the viability of the building block approach also for construction of 4-O-xylosyl-containing C. neoformans CPS structures. PMID:27308199

  5. Cryptococcus neoformans Yop1, an ER curvature-stabilizing protein, participates with Sey1 in influencing fluconazole-induced disomy formation

    PubMed Central

    Ngamskulrungroj, Popchai; Chang, Yun; Hansen, Bryan; Bugge, Cliff; Fischer, Elizabeth; Kwon-Chung, Kyung J.

    2012-01-01

    Cryptococcus neoformans, an opportunistic fungal pathogen, manifests an intrinsic adaptive mechanism of resistance toward fluconazole (FLC) termed heteroresistance. Heteroresistance is characterized by the emergence of minor resistant subpopulations at levels of FLC that are higher than the strain’s minimum inhibitory concentration. The heteroresistant clones that tolerate high concentrations of FLC often contain disomic chromosome 4 (Chr4). SEY1, GLO3 and GCS2 on Chr4 are responsible for ER integrity and important for Chr4 disomy formation under FLC stress. We sought an evidence of a direct relationship between ER morphology and Chr4 disomy formation. Deletion of the YOP1 gene on Chr7, which encodes an ER curvature-stabilizing protein that interacts with Sey1, perturbed ER morphology without affecting FLC susceptibility or the frequency of FLC-induced disomies. However, deletion of both YOP1 and SEY1, not only perturbed ER morphology more severely than in sey1Δ or yop1Δ strains but also abrogated the FLC-induced disomy. Although the heteroresistance phenotype was retained in the sey1Δyop1Δ strains, tolerance to FLC appeared to have resulted not from chromosome duplication but from gene amplification restricted to the region surrounding ERG11 on Chr1. These data support the importance of ER integrity in C. neoformans for the formation of disomy under FLC stress. PMID:22731401

  6. KRE genes are required for β-1,6-glucan synthesis, maintenance of capsule architecture and cell wall protein anchoring in Cryptococcus neoformans

    PubMed Central

    Gilbert, Nicole M.; Donlin, Maureen J.; Gerik, Kimberly J.; Specht, Charles A.; Djordjevic, Julianne T.; Wilson, Christabel F.; Sorrell, Tania C.; Lodge, Jennifer K.

    2010-01-01

    Summary The polysaccharide β-1,6-glucan is a major component of the cell wall of Cryptococcus neoformans, but its function has not been investigated in this fungal pathogen. We have identified and characterized seven genes, belonging to the KRE family, which are putatively involved in β-1,6-glucan synthesis. The H99 deletion mutants kre5Δ and kre6Δskn1Δ contained less cell wall β-1,6-glucan, grew slowly with an aberrant morphology, were highly sensitive to environmental and chemical stress and were avirulent in a mouse inhalation model of infection. These two mutants displayed alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant. The cell wall content of the GPI-anchored phospholipase B1 (Plb1) enzyme, which is required for cryptococcal cell wall integrity and virulence, was reduced in kre5Δ and kre6Δskn1Δ. Our results indicate that KRE5, KRE6 and SKN1 are involved in β-1,6-glucan synthesis, maintenance of cell wall integrity and retention of mannoproteins and known cryptococcal virulence factors in the cell wall of C. neoformans. This study sets the stage for future investigations into the function of this abundant cell wall polymer. PMID:20384682

  7. The Membrane Phospholipid Binding Protein Annexin A2 Promotes Phagocytosis and Nonlytic Exocytosis of Cryptococcus neoformans and Impacts Survival in Fungal Infection.

    PubMed

    Stukes, Sabriya; Coelho, Carolina; Rivera, Johanna; Jedlicka, Anne E; Hajjar, Katherine A; Casadevall, Arturo

    2016-08-15

    Cryptococcus neoformans is a fungal pathogen with a unique intracellular pathogenic strategy that includes nonlytic exocytosis, a phenomenon whereby fungal cells are expunged from macrophages without lysing the host cell. The exact mechanism and specific proteins involved in this process have yet to be completely defined. Using murine macrophages deficient in the membrane phospholipid binding protein, annexin A2 (ANXA2), we observed a significant decrease in both phagocytosis of yeast cells and the frequency of nonlytic exocytosis. Cryptococcal cells isolated from Anxa2-deficient (Anxa2(-/-)) bone marrow-derived macrophages and lung parenchyma displayed significantly larger capsules than those isolated from wild-type macrophages and tissues. Concomitantly, we observed significant differences in the amount of reactive oxygen species produced between Anxa2(-/-) and Anxa2(+/+) macrophages. Despite comparable fungal burden, Anxa2(-/-) mice died more rapidly than wild-type mice when infected with C. neoformans, and Anxa2(-/-) mice exhibited enhanced inflammatory responses, suggesting that the reduced survival reflected greater immune-mediated damage. Together, these findings suggest a role for ANXA2 in the control of cryptococcal infection, macrophage function, and fungal morphology. PMID:27371724

  8. Analysis of the Protein Kinase A-Regulated Proteome of Cryptococcus neoformans Identifies a Role for the Ubiquitin-Proteasome Pathway in Capsule Formation

    PubMed Central

    Geddes, J. M. H.; Caza, M.; Croll, D.; Stoynov, N.; Foster, L. J.

    2016-01-01

    ABSTRACT The opportunistic fungal pathogen Cryptococcus neoformans causes life-threatening meningitis in immunocompromised individuals. The expression of virulence factors, including capsule and melanin, is in part regulated by the cyclic-AMP/protein kinase A (cAMP/PKA) signal transduction pathway. In this study, we investigated the influence of PKA on the composition of the intracellular proteome to obtain a comprehensive understanding of the regulation that underpins virulence. Through quantitative proteomics, enrichment and bioinformatic analyses, and an interactome study, we uncovered a pattern of PKA regulation for proteins associated with translation, the proteasome, metabolism, amino acid biosynthesis, and virulence-related functions. PKA regulation of the ubiquitin-proteasome pathway in C. neoformans showed a striking parallel with connections between PKA and protein degradation in chronic neurodegenerative disorders and other human diseases. Further investigation of proteasome function with the inhibitor bortezomib revealed an impact on capsule production as well as hypersusceptibility for strains with altered expression or activity of PKA. Parallel studies with tunicamycin also linked endoplasmic reticulum stress with capsule production and PKA. Taken together, the data suggest a model whereby expression of PKA regulatory and catalytic subunits and the activation of PKA influence proteostasis and the function of the endoplasmic reticulum to control the elaboration of the polysaccharide capsule. Overall, this study revealed both broad and conserved influences of the cAMP/PKA pathway on the proteome and identified proteostasis as a potential therapeutic target for the treatment of cryptococcosis. PMID:26758180

  9. Synthesis of a Glucuronic Acid‐Containing Thioglycoside Trisaccharide Building Block and Its Use in the Assembly of Cryptococcus Neoformans Capsular Polysaccharide Fragments†

    PubMed Central

    Guazzelli, Lorenzo; Ulc, Rebecca

    2015-01-01

    Abstract As part of an ongoing project aimed at identifying protective capsular polysaccharide epitopes for the development of vaccine candidates against the fungal pathogen Cryptococcus neoformans, the synthesis and glycosylation properties of a naphthalenylmethyl (NAP) orthogonally protected trisaccharide thioglycoside, a common building block for construction of serotype B and C capsular polysaccharide structures, were investigated. Ethyl (benzyl 2,3,4‐tri‐O‐benzyl‐β‐d‐glucopyranosyl‐ uronate)‐(1→2)‐[2,3,4‐tri‐O‐benzyl‐β‐d‐xylopyranosyl‐(1→4)]‐6‐O‐benzyl‐3‐O‐(2‐naphthalenylmethyl)‐1‐thio‐α‐d‐mannopyranoside was prepared and used both as a donor and an acceptor in glycosylation reactions to obtain spacer equipped hexa‐ and heptasaccharide structures suitable either for continued elongation or for deprotection and printing onto a glycan array or conjugation to a carrier protein. The glycosylation reactions proceeded with high yields and α‐selectivity, proving the viability of the building block approach also for construction of 4‐O‐xylosyl‐containing C. neoformans CPS structures. PMID:27308199

  10. Noncanoncial signal recognition particle RNAs in a major eukaryotic phylum revealed by purification of SRP from the human pathogen Cryptococcus neoformans

    PubMed Central

    Dumesic, Phillip A.; Rosenblad, Magnus A.; Samuelsson, Tore; Nguyen, Tiffany; Moresco, James J.; Yates, John R.; Madhani, Hiten D.

    2015-01-01

    Despite conservation of the signal recognition particle (SRP) from bacteria to man, computational approaches have failed to identify SRP components from genomes of many lower eukaryotes, raising the possibility that they have been lost or altered in those lineages. We report purification and analysis of SRP in the human pathogen Cryptococcus neoformans, providing the first description of SRP in basidiomycetous yeast. The C. neoformans SRP RNA displays a predicted structure in which the universally conserved helix 8 contains an unprecedented stem-loop insertion. Guided by this sequence, we computationally identified 152 SRP RNAs throughout the phylum Basidiomycota. This analysis revealed additional helix 8 alterations including single and double stem-loop insertions as well as loop diminutions affecting RNA structural elements that are otherwise conserved from bacteria to man. Strikingly, these SRP RNA features in Basidiomycota are accompanied by phylum-specific alterations in the RNA-binding domain of Srp54, the SRP protein subunit that directly interacts with helix 8. Our findings reveal unexpected fungal SRP diversity and suggest coevolution of the two most conserved SRP features—SRP RNA helix 8 and Srp54—in basidiomycetes. Because members of this phylum include important human and plant pathogens, these noncanonical features provide new targets for antifungal compound development. PMID:26275773

  11. Isocitrate Dehydrogenase Is Important for Nitrosative Stress Resistance in Cryptococcus neoformans, but Oxidative Stress Resistance Is Not Dependent on Glucose-6-Phosphate Dehydrogenase▿

    PubMed Central

    Brown, Sarah M.; Upadhya, Rajendra; Shoemaker, James D.; Lodge, Jennifer K.

    2010-01-01

    The opportunistic intracellular fungal pathogen Cryptococcus neoformans depends on many antioxidant and denitrosylating proteins and pathways for virulence in the immunocompromised host. These include the glutathione and thioredoxin pathways, thiol peroxidase, cytochrome c peroxidase, and flavohemoglobin denitrosylase. All of these ultimately depend on NADPH for either catalytic activity or maintenance of a reduced, functional form. The need for NADPH during oxidative stress is well established in many systems, but a role in resistance to nitrosative stress has not been as well characterized. In this study we investigated the roles of two sources of NADPH, glucose-6-phosphate dehydrogenase (Zwf1) and NADP+-dependent isocitrate dehydrogenase (Idp1), in production of NADPH and resistance to oxidative and nitrosative stress. Deletion of ZWF1 in C. neoformans did not result in an oxidative stress sensitivity phenotype or changes in the amount of NADPH produced during oxidative stress compared to those for the wild type. Deletion of IDP1 resulted in greater sensitivity to nitrosative stress than to oxidative stress. The amount of NADPH increased 2-fold over that in the wild type during nitrosative stress, and yet the idp1Δ strain accumulated more mitochondrial damage than the wild type during nitrosative stress. This is the first report of the importance of Idp1 and NADPH for nitrosative stress resistance. PMID:20400467

  12. Identification of a major IP5 kinase in Cryptococcus neoformans confirms that PP-IP5/IP7, not IP6, is essential for virulence

    PubMed Central

    Li, Cecilia; Lev, Sophie; Saiardi, Adolfo; Desmarini, Desmarini; Sorrell, Tania C.; Djordjevic, Julianne T.

    2016-01-01

    Fungal inositol polyphosphate (IP) kinases catalyse phosphorylation of IP3 to inositol pyrophosphate, PP-IP5/IP7, which is essential for virulence of Cryptococcus neoformans. Cryptococcal Kcs1 converts IP6 to PP-IP5/IP7, but the kinase converting IP5 to IP6 is unknown. Deletion of a putative IP5 kinase-encoding gene (IPK1) alone (ipk1Δ), and in combination with KCS1 (ipk1Δkcs1Δ), profoundly reduced virulence in mice. However, deletion of KCS1 and IPK1 had a greater impact on virulence attenuation than that of IPK1 alone. ipk1Δkcs1Δ and kcs1Δ lung burdens were also lower than those of ipk1Δ. Unlike ipk1Δ, ipk1Δkcs1Δ and kcs1Δ failed to disseminate to the brain. IP profiling confirmed Ipk1 as the major IP5 kinase in C. neoformans: ipk1Δ produced no IP6 or PP-IP5/IP7 and, in contrast to ipk1Δkcs1Δ, accumulated IP5 and its pyrophosphorylated PP-IP4 derivative. Kcs1 is therefore a dual specificity (IP5 and IP6) kinase producing PP-IP4 and PP-IP5/IP7. All mutants were similarly attenuated in virulence phenotypes including laccase, urease and growth under oxidative/nitrosative stress. Alternative carbon source utilisation was also reduced significantly in all mutants except ipk1Δ, suggesting that PP-IP4 partially compensates for absent PP-IP5/IP7 in ipk1Δ grown under this condition. In conclusion, PP-IP5/IP7, not IP6, is essential for fungal virulence. PMID:27033523

  13. Identification of a major IP5 kinase in Cryptococcus neoformans confirms that PP-IP5/IP7, not IP6, is essential for virulence.

    PubMed

    Li, Cecilia; Lev, Sophie; Saiardi, Adolfo; Desmarini, Desmarini; Sorrell, Tania C; Djordjevic, Julianne T

    2016-01-01

    Fungal inositol polyphosphate (IP) kinases catalyse phosphorylation of IP3 to inositol pyrophosphate, PP-IP5/IP7, which is essential for virulence of Cryptococcus neoformans. Cryptococcal Kcs1 converts IP6 to PP-IP5/IP7, but the kinase converting IP5 to IP6 is unknown. Deletion of a putative IP5 kinase-encoding gene (IPK1) alone (ipk1Δ), and in combination with KCS1 (ipk1Δkcs1Δ), profoundly reduced virulence in mice. However, deletion of KCS1 and IPK1 had a greater impact on virulence attenuation than that of IPK1 alone. ipk1Δkcs1Δ and kcs1Δ lung burdens were also lower than those of ipk1Δ. Unlike ipk1Δ, ipk1Δkcs1Δ and kcs1Δ failed to disseminate to the brain. IP profiling confirmed Ipk1 as the major IP5 kinase in C. neoformans: ipk1Δ produced no IP6 or PP-IP5/IP7 and, in contrast to ipk1Δkcs1Δ, accumulated IP5 and its pyrophosphorylated PP-IP4 derivative. Kcs1 is therefore a dual specificity (IP5 and IP6) kinase producing PP-IP4 and PP-IP5/IP7. All mutants were similarly attenuated in virulence phenotypes including laccase, urease and growth under oxidative/nitrosative stress. Alternative carbon source utilisation was also reduced significantly in all mutants except ipk1Δ, suggesting that PP-IP4 partially compensates for absent PP-IP5/IP7 in ipk1Δ grown under this condition. In conclusion, PP-IP5/IP7, not IP6, is essential for fungal virulence. PMID:27033523

  14. Cryptococcus neoformans Infection in Mice Lacking Type I Interferon Signaling Leads to Increased Fungal Clearance and IL-4-Dependent Mucin Production in the Lungs

    PubMed Central

    Sato, Ko; Yamamoto, Hideki; Nomura, Toshiki; Matsumoto, Ikumi; Miyasaka, Tomomitsu; Zong, Tong; Kanno, Emi; Uno, Kazuko; Ishii, Keiko; Kawakami, Kazuyoshi

    2015-01-01

    Type I interferons (IFNs) are secreted by many cell types upon stimulation via pattern recognition receptors and bind to IFN-α/β receptor (IFNAR), which is composed of IFNAR1 and IFNAR2. Although type I IFNs are well known as anti-viral cytokines, limited information is available on their role during fungal infection. In the present study, we addressed this issue by examining the effect of IFNAR1 defects on the host defense response to Cryptococcus neoformans. In IFNAR1KO mice, the number of live colonies was lower and the host immune response mediated not only by Th1 but also by Th2 and Th17-related cytokines was more accelerated in the infected lungs than in WT mice. In addition, mucin production by bronchoepithelial cells and expression of MUC5AC, a major core protein of mucin in the lungs, were significantly higher in IFNAR1KO mice than in WT mice. This increase in mucin and MUC5AC production was significantly inhibited by treatment with neutralizing anti-IL-4 mAb. In contrast, administration of recombinant IFN-αA/D significantly suppressed the production of IL-4, but not of IFN-γ and IL-17A, in the lungs of WT mice after cryptococcal infection. These results indicate that defects of IFNAR1 led to improved clearance of infection with C. neoformans and enhanced synthesis of IFN-γ and the IL-4-dependent production of mucin. They also suggest that type I IFNs may be involved in the negative regulation of early host defense to this infection. PMID:26384031

  15. Cryptococcal heat shock protein 70 homolog Ssa1 contributes to pulmonary expansion of Cryptococcus neoformans during the afferent phase of the immune response by promoting macrophage M2 polarization.

    PubMed

    Eastman, Alison J; He, Xiumiao; Qiu, Yafeng; Davis, Michael J; Vedula, Priya; Lyons, Daniel M; Park, Yoon-Dong; Hardison, Sarah E; Malachowski, Antoni N; Osterholzer, John J; Wormley, Floyd L; Williamson, Peter R; Olszewski, Michal A

    2015-06-15

    Numerous virulence factors expressed by Cryptococcus neoformans modulate host defenses by promoting nonprotective Th2-biased adaptive immune responses. Prior studies demonstrate that the heat shock protein 70 homolog, Ssa1, significantly contributes to serotype D C. neoformans virulence through the induction of laccase, a Th2-skewing and CNS tropic factor. In the present study, we sought to determine whether Ssa1 modulates host defenses in mice infected with a highly virulent serotype A strain of C. neoformans (H99). To investigate this, we assessed pulmonary fungal growth, CNS dissemination, and survival in mice infected with either H99, an SSA1-deleted H99 strain (Δssa1), and a complement strain with restored SSA1 expression (Δssa1::SSA1). Mice infected with the Δssa1 strain displayed substantial reductions in lung fungal burden during the innate phase (days 3 and 7) of the host response, whereas less pronounced reductions were observed during the adaptive phase (day 14) and mouse survival increased only by 5 d. Surprisingly, laccase activity assays revealed that Δssa1 was not laccase deficient, demonstrating that H99 does not require Ssa1 for laccase expression, which explains the CNS tropism we still observed in the Ssa1-deficient strain. Lastly, our immunophenotyping studies showed that Ssa1 directly promotes early M2 skewing of lung mononuclear phagocytes during the innate phase, but not the adaptive phase, of the immune response. We conclude that Ssa1's virulence mechanism in H99 is distinct and laccase-independent. Ssa1 directly interferes with early macrophage polarization, limiting innate control of C. neoformans, but ultimately has no effect on cryptococcal control by adaptive immunity. PMID:25972480

  16. Influence of climatic conditions on the isolation of members of the Cryptococcus neoformans species complex from trees in Colombia from 1992-2004.

    PubMed

    Granados, Diana Paola; Castañeda, Elizabeth

    2006-06-01

    The aim of this retrospective study was to analyze the relationship between occurrence of the serotypes of the Cryptococcus neoformans species complex in tree samples and the climatic conditions registered during samplings in four cities of Colombia, between 1992 and 2004, by means of a logistic regression model and lagged Pearson correlations. During 97 collection dates, 8220 samples from different tree species were taken, of which 2.63% were positive: 56.5% yielded serotype B, 24.7% serotype C and 18.8% serotype A isolates. The prevalence of the serotypes varied among the cities. The results suggest that environmental climatic conditions, mainly humidity, temperature, evaporation and solar radiation, can affect the occurrence of the different serotypes in trees in a differential manner. These different climatic tolerances were reflected in the geographic distribution of the serotypes in Colombia. The climatic conditions for 15 days before the sampling date were correlated with positive or negative isolation of the different serotypes. PMID:16696660

  17. Wsp1 Is Downstream of Cin1 and Regulates Vesicle Transport and Actin Cytoskeleton as an Effector of Cdc42 and Rac1 in Cryptococcus neoformans

    PubMed Central

    Shen, Gui; Zhou, Erxun; Alspaugh, J. Andrew

    2012-01-01

    Human Wiskott-Aldrich syndrome protein (WASP) is a scaffold linking upstream signals to the actin cytoskeleton. In response to intersectin ITSN1 and Rho GTPase Cdc42, WASP activates the Arp2/3 complex to promote actin polymerization. The human pathogen Cryptococcus neoformans contains the ITSN1 homolog Cin1 and the WASP homolog Wsp1, which share more homology with human proteins than those of other fungi. Here we demonstrate that Cin1, Cdc42/Rac1, and Wsp1 function in an effector pathway similar to that of mammalian models. In the cin1 mutant, expression of the autoactivated Wsp1-B-GBD allele partially suppressed the mutant defect in endocytosis, and expression of the constitutively active CDC42Q61L allele restored normal actin cytoskeleton structures. Similar phenotypic suppression can be obtained by the expression of a Cdc42-green fluorescent protein (GFP)-Wsp1 fusion protein. In addition, Rac1, which was found to exhibit a role in early endocytosis, activates Wsp1 to regulate vacuole fusion. Rac1 interacted with Wsp1 and depended on Wsp1 for its vacuolar membrane localization. Expression of the Wsp1-B-GBD allele restored vacuolar membrane fusion in the rac1 mutant. Collectively, our studies suggest novel ways in which this pathogenic fungus has adapted conserved signaling pathways to control vesicle transport and actin organization, likely benefiting survival within infected hosts. PMID:22327008

  18. Monoclonal Antibodies Specific for Immunorecessive Epitopes of Glucuronoxylomannan, the Major Capsular Polysaccharide of Cryptococcus neoformans, Reduce Serotype Bias in an Immunoassay for Cryptococcal Antigen▿

    PubMed Central

    Percival, Ann; Thorkildson, Peter; Kozel, Thomas R.

    2011-01-01

    Immunoassay for detection of glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, is an important tool for diagnosis of cryptococcosis. However, immunoassays that are based solely or in part on detection with polyclonal antibodies may show serotype bias in detection of GXM, particularly limited sensitivity for serotype C. In this study, we describe detection of GXM in an antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) that used a cocktail of two monoclonal antibodies (MAbs). MAb F12D2 was previously produced by immunization with GXM that had been treated to remove O-acetyl groups, a major source of serotype specificity. MAb F12D2 has a high degree of reactivity with GXM of serotypes A, B, C, and D, but the reactivity with serotype D was less than was found with other MAbs. MAb 339 is highly reactive with GXM of serotypes A and D. Use of a combination of the two MAbs produced an immunoassay that had the best properties of both MAbs, including good reactivity with serotype C, which is an emerging threat in sub-Saharan Africa. These results suggest that next-generation immunoassays for diagnosis of cryptococcosis may be formulated by (i) use of immunization and hybridoma screening strategies that are designed to prospectively meet the needs of immunoassay performance and (ii) careful selection of MAbs that span the expected polysaccharide serotypes in the subject patient population. PMID:21697342

  19. X-linked hyper-IgM syndrome associated with Cryptosporidium parvum and Cryptococcus neoformans infections: the first case with molecular diagnosis in Korea.

    PubMed Central

    Jo, Eun Kyeong; Kim, Hyung Seok; Lee, Min Young; Iseki, Motohiro; Lee, Jae Ho; Song, Chang Hwa; Park, Jeong Kyu; Hwang, Tai Ju; Kook, Hoon

    2002-01-01

    X-linked hyper-IgM syndrome (XHIM) is a rare primary immunodeficiency disorder, caused by mutations of the gene encoding CD40 ligand (CD40L; CD154). We report the clinical manifestations and mutational analysis of the CD40L gene observed in a male patient from a XHIM family. Having hypogammaglobulinemia and elevated IgM, the 3-yr-old boy exhibited the characteristic clinical features of XHIM. The patient suffered from frequent respiratory infections, and chronic enteritis caused by Cryptosporidium parvum. In addition, a lymph node biopsy and a culture from this sample revealed C. neoformans infection. Activated lymphocytes from the patient failed to express CD40L on their surface as assessed by flow cytometry and a missence mutation (W140R) was found at the XHIM hotspot in his CD40L cDNA to confirm the diagnosis. Genetic analysis of the mother and sister showed a heterozygote pattern, indicating carrier status. To our knowledge, this is the first report on the molecular diagnosis of an XHIM patient in Korea. PMID:11850600

  20. Prior Exposure to Live Mycobacterium bovis BCG Decreases Cryptococcus neoformans-Induced Lung Eosinophilia in a Gamma Interferon-Dependent Manner

    PubMed Central

    Walzl, Gerhard; Humphreys, Ian R.; Marshall, Ben G.; Edwards, Lorna; Openshaw, Peter J. M.; Shaw, Rory J.; Hussell, Tracy

    2003-01-01

    Some common childhood infections appear to prevent the development of atopy and asthma. In some Mycobacterium bovis BCG-vaccinated populations, strong delayed-type hypersensitivity responses to mycobacterial antigens are associated with a reduced risk of atopy. Although BCG exposure decreases allergen-induced lung eosinophilia in animal models, little attention has been given to the effect of immunity to BCG on responses against live pathogens. We used the murine Cryptococcus neoformans infection model to investigate whether prior BCG infection can alter such responses. The present study shows that persistent pulmonary BCG infection of C57BL/6 mice induced an increase in gamma interferon, a reduction in interleukin-5, and a decrease in lung eosinophilia during subsequent Cryptococcus infection. This effect was long lasting, depended on the presence of live bacteria, and required persistence of mycobacterial infection in the lung. Reduction of eosinophilia was less prominent after infection with a mutant BCG strain (ΔhspR), which was rapidly cleared from the lungs. These observations have important implications for the development of vaccines designed to prevent Th2-mediated disease and indicate that prior lung BCG vaccination can alter the pattern of subsequent host inflammation. PMID:12761122

  1. A Multiplex Real-Time PCR Assay for Identification of Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii in Samples from AIDS Patients with Opportunistic Pneumonia

    PubMed Central

    Gago, Sara; Esteban, Cristina; Valero, Clara; Zaragoza, Óscar; Puig de la Bellacasa, Jorge

    2014-01-01

    A molecular diagnostic technique based on real-time PCR was developed for the simultaneous detection of three of the most frequent causative agents of fungal opportunistic pneumonia in AIDS patients: Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii. This technique was tested in cultured strains and in clinical samples from HIV-positive patients. The methodology used involved species-specific molecular beacon probes targeted to the internal transcribed spacer regions of the rDNA. An internal control was also included in each assay. The multiplex real-time PCR assay was tested in 24 clinical strains and 43 clinical samples from AIDS patients with proven fungal infection. The technique developed showed high reproducibility (r2 of >0.98) and specificity (100%). For H. capsulatum and Cryptococcus spp., the detection limits of the method were 20 and 2 fg of genomic DNA/20 μl reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log10 copies/20 μl reaction mixture. The sensitivity in vitro was 100% for clinical strains and 90.7% for clinical samples. The assay was positive for 92.5% of the patients. For one of the patients with proven histoplasmosis, P. jirovecii was also detected in a bronchoalveolar lavage sample. No PCR inhibition was detected. This multiplex real-time PCR technique is fast, sensitive, and specific and may have clinical applications. PMID:24478409

  2. Chiral N-benzyl-N-methyl-1-(naphthalen-1-yl)ethanamines and their in vitro antifungal activity against Cryptococcus neoformans, Trichophyton mentagrophytes and Trichophyton rubrum.

    PubMed

    Thvedt, Thor H Krane; Kaasa, Kristin; Sundby, Eirik; Charnock, Colin; Hoff, Bård Helge

    2013-10-01

    In the search for new antifungal compounds and to explore structure activity relationships, a series of 24 chiral benzyl amine type antifungals was synthesised and characterised. In vitro testing against the human pathogen Cryptococcus neoformans revealed that several derivatives had MIC50 values similar to that of the commercial drug Butenafine. All of these contained a bulky group in the para position of the benzyl fragment. Eighteen compounds were also tested for activity against the dermatophytes Trichophyton mentagrophytes and Trichophyton rubrum. Of these (R)-N-(4-tert-butylbenzyl)-N-methyl-1-(naphthalen-1-yl)ethanamine (MIC50: 0.06 μg/mL) and a para-benzyloxy substituted derivative (MIC50: 0.125 μg/mL) possessed high activity. Testing of derivatives with a stereocentre at the benzylic carbon, revealed that (S)-stereochemistry was required for potency: a MIC50 value of 1 μg/mL was obtained for (S)-1-(4-tert-butylphenyl)-N-methyl-N-(naphthalen-1-ylmethyl)ethanamine. Preparation of the corresponding fluoromethyl compound was achieved employing lipase B from Candida antarctica as catalyst in the key step. A low antifungal activity was observed for the fluorinated derivative indicating the importance of the amine basicity for the antifungal potency of these compounds. PMID:24051242

  3. Trends in antifungal drug susceptibility of Cryptococcus neoformans isolates obtained through population-based surveillance in South Africa in 2002-2003 and 2007-2008.

    PubMed

    Govender, Nelesh P; Patel, Jaymati; van Wyk, Marelize; Chiller, Tom M; Lockhart, Shawn R

    2011-06-01

    Cryptococcus neoformans is the most common cause of meningitis among adult South Africans with HIV infection/AIDS. Widespread use of fluconazole for treatment of cryptococcal meningitis and other HIV-associated opportunistic fungal infections in South Africa may lead to the emergence of isolates with reduced fluconazole susceptibility. MIC testing using a reference broth microdilution method was used to determine if isolates with reduced susceptibility to fluconazole or amphotericin B had emerged among cases of incident disease. Incident isolates were tested from two surveillance periods (2002-2003 and 2007-2008) when population-based surveillance was conducted in Gauteng Province, South Africa. These isolates were also tested for susceptibility to flucytosine, itraconazole, voriconazole, and posaconazole. Serially collected isolate pairs from cases at several large South African hospitals were also tested for susceptibility to fluconazole. Of the 487 incident isolates tested, only 3 (0.6%) demonstrated a fluconazole MIC of ≥ 16 μg/ml; all of these isolates were from 2002-2003. All incident isolates were inhibited by very low concentrations of amphotericin B and exhibited very low MICs to voriconazole and posaconazole. Of 67 cases with serially collected isolate pairs, only 1 case was detected where the isolate collected more than 30 days later had a fluconazole MIC value significantly higher than the MIC of the corresponding incident isolate. Although routine antifungal susceptibility testing of incident isolates is not currently recommended in clinical settings, it is still clearly important for public health to periodically monitor for the emergence of resistance. PMID:21444707

  4. Reverse Cross Blot Hybridization Assay for Rapid Detection of PCR-Amplified DNA from Candida Species, Cryptococcus neoformans, and Saccharomyces cerevisiae in Clinical Samples

    PubMed Central

    Posteraro, Brunella; Sanguinetti, Maurizio; Masucci, Luca; Romano, Lucio; Morace, Giulia; Fadda, Giovanni

    2000-01-01

    A PCR-based assay was developed to detect and identify medically important yeasts in clinical samples. Using a previously described set of primers (G. Morace et al., J. Clin. Microbiol. 35:667–672, 1997), we amplified a fragment of the ERG11 gene for cytochrome P-450 lanosterol 14α-demethylase, a crucial enzyme in the biosynthesis of ergosterol. The PCR product was analyzed in a reverse cross blot hybridization assay with species-specific probes directed to a target region of the ERG11 gene of Candida albicans (pCal), C. guilliermondii (pGui), C. (Torulopsis) glabrata (pGla), C. kefyr (pKef), C. krusei (pKru), C. parapsilosis (pPar), C. tropicalis (pTro), the newly described species C. dubliniensis (pDub), Saccharomyces cerevisiae (pSce), and Cryptococcus neoformans (pCry). The PCR-reverse cross blot hybridization assay correctly identified multiple isolates of each species tested. No cross-hybridization was detected with any other fungal, bacteria, or human DNAs tested. The method was tested against conventional identification on 140 different clinical samples, including blood and cerebrospinal fluid, from patients with suspected fungal infections. The results agreed with those of culture and phenotyping for all but six specimens (two of which grew yeasts not included in the PCR panel of probes and four in which PCR positivity-culture negativity was justified by clinical findings). Species identification time was reduced from a mean of 4 days with conventional identification to 7 h with the molecular method. The PCR-reverse cross blot hybridization assay is a rapid method for the direct detection and identification of yeasts in clinical samples. PMID:10747151

  5. Trends in Antifungal Drug Susceptibility of Cryptococcus neoformans Isolates Obtained through Population-Based Surveillance in South Africa in 2002-2003 and 2007-2008▿

    PubMed Central

    Govender, Nelesh P.; Patel, Jaymati; van Wyk, Marelize; Chiller, Tom M.; Lockhart, Shawn R.

    2011-01-01

    Cryptococcus neoformans is the most common cause of meningitis among adult South Africans with HIV infection/AIDS. Widespread use of fluconazole for treatment of cryptococcal meningitis and other HIV-associated opportunistic fungal infections in South Africa may lead to the emergence of isolates with reduced fluconazole susceptibility. MIC testing using a reference broth microdilution method was used to determine if isolates with reduced susceptibility to fluconazole or amphotericin B had emerged among cases of incident disease. Incident isolates were tested from two surveillance periods (2002-2003 and 2007-2008) when population-based surveillance was conducted in Gauteng Province, South Africa. These isolates were also tested for susceptibility to flucytosine, itraconazole, voriconazole, and posaconazole. Serially collected isolate pairs from cases at several large South African hospitals were also tested for susceptibility to fluconazole. Of the 487 incident isolates tested, only 3 (0.6%) demonstrated a fluconazole MIC of ≥16 μg/ml; all of these isolates were from 2002-2003. All incident isolates were inhibited by very low concentrations of amphotericin B and exhibited very low MICs to voriconazole and posaconazole. Of 67 cases with serially collected isolate pairs, only 1 case was detected where the isolate collected more than 30 days later had a fluconazole MIC value significantly higher than the MIC of the corresponding incident isolate. Although routine antifungal susceptibility testing of incident isolates is not currently recommended in clinical settings, it is still clearly important for public health to periodically monitor for the emergence of resistance. PMID:21444707

  6. Synergism Effect of the Essential Oil from Ocimum basilicum var. Maria Bonita and Its Major Components with Fluconazole and Its Influence on Ergosterol Biosynthesis.

    PubMed

    Cardoso, Nathalia N R; Alviano, Celuta S; Blank, Arie F; Romanos, Maria Teresa V; Fonseca, Beatriz B; Rozental, Sonia; Rodrigues, Igor A; Alviano, Daniela S

    2016-01-01

    The aim of this study was to evaluate the activity of the EO and its major components of Ocimum basilicum var. Maria Bonita, a genetically improved cultivar, against the fluconazole sensitive and resistant strains of Candida albicans and Cryptococcus neoformans. Geraniol presented better results than the EO, with a low MIC (76 μg/mL against C. neoformans and 152 μg/mL against both Candida strains). The combination of EO, linalool, or geraniol with fluconazole enhanced their antifungal activity, especially against the resistant strain (MIC reduced to 156, 197, and 38 μg/mL, resp.). The ergosterol assay showed that subinhibitory concentrations of the substances were able to reduce the amount of sterol extracted. The substances tested were able to reduce the capsule size which suggests they have an important mechanism of action. Transmission electron microscopy demonstrated cell wall destruction of C. neoformans after treatment with subinhibitory concentrations. In C. albicans ultrastructure alterations such as irregularities in the membrane, presence of vesicles, and cell wall thickening were observed. The biofilm formation was inhibited in both C. albicans strains at MIC and twice MIC. These results provide further support for the use of O. basilicum EO and its major components as a potential source of antifungal agents. PMID:27274752

  7. Synergism Effect of the Essential Oil from Ocimum basilicum var. Maria Bonita and Its Major Components with Fluconazole and Its Influence on Ergosterol Biosynthesis

    PubMed Central

    Cardoso, Nathalia N. R.; Alviano, Celuta S.; Blank, Arie F.; Romanos, Maria Teresa V.; Fonseca, Beatriz B.; Rozental, Sonia; Rodrigues, Igor A.; Alviano, Daniela S.

    2016-01-01

    The aim of this study was to evaluate the activity of the EO and its major components of Ocimum basilicum var. Maria Bonita, a genetically improved cultivar, against the fluconazole sensitive and resistant strains of Candida albicans and Cryptococcus neoformans. Geraniol presented better results than the EO, with a low MIC (76 μg/mL against C. neoformans and 152 μg/mL against both Candida strains). The combination of EO, linalool, or geraniol with fluconazole enhanced their antifungal activity, especially against the resistant strain (MIC reduced to 156, 197, and 38 μg/mL, resp.). The ergosterol assay showed that subinhibitory concentrations of the substances were able to reduce the amount of sterol extracted. The substances tested were able to reduce the capsule size which suggests they have an important mechanism of action. Transmission electron microscopy demonstrated cell wall destruction of C. neoformans after treatment with subinhibitory concentrations. In C. albicans ultrastructure alterations such as irregularities in the membrane, presence of vesicles, and cell wall thickening were observed. The biofilm formation was inhibited in both C. albicans strains at MIC and twice MIC. These results provide further support for the use of O. basilicum EO and its major components as a potential source of antifungal agents. PMID:27274752

  8. Indolizidine, Antiinfective and Antiparasitic Compounds from Prosopis glandulosa Torr. Var. glandulosa

    PubMed Central

    Samoylenko, Volodymyr; Ashfaq, Mohammad K.; Jacob, Melissa R.; Tekwani, Babu L.; Khan, Shabana I.; Manly, Susan P.; Joshi, Vaishali C.; Walker, Larry A.; Muhammad, Ilias

    2013-01-01

    A new potent antiinfective and antiparasitic 2,3-dihydro-1H-indolizinium chloride, (1), was isolated from Prosopis glandulosa Torr. var. glandulosa. Three additional new (2–4) and one known (5), indolizidines were also isolated, and the dihydrochloride salts of 1–3 (compounds 6, 7 and 8) were prepared. Structures were determined by 1D and 2D NMR and mass spectra. Compound 1 showed potent in vitro antifungal activity against Cryptococcus neoformans and Aspergillus fumigatus (IC50 values = 0.4 and 3.0 μg/mL, respectively), and antibacterial activity against methicillin-resistant Staphylococcus aureus and Mycobacterium intracellulare (IC50 values of 0.35 and 0.9 μg/mL, respectively). The remarkable in vitro fungicidal activity of 1–4 against C. neoformans (MFCs = 0.63→1.25 μg/mL) and 2, 3, and 5 against A. fumigatus (MFCs = 0.63→2.5 μg/mL) were similar to amphotericin B, but >2–4-fold more potent than 6–8. Prosopilosidine (1) showed potent in vivo activity at 0.0625 mg/Kg/day/ip for 5 days in a murine model of cryptococcosis by eliminating ~76% of C. neoformans infection from brain tissue compared to ~83% with amphotericin B at 1.5 mg/Kg/day. Compounds 1 and 4 exhibited potent activity and high selectivity index (SI) values against chloroquine sensitive (D6) and chloroquine resistant (W2) strains of Plasmodium falciparum with IC50 values of 39 and 95 ng/mL, and 42 and 120 ng/mL, respectively; (chloroquine, IC50 = 17 and 140 ng/mL). Prosopilosine (1) also showed in vivo antimalarial activity with an ED50 value of ~2 mg/Kg/day/ip against Plasmodium berghei-infected mice after 3 days of treatment. PMID:19105653

  9. Antiparasitic and Antimicrobial Indolizidines from the Leaves of Prosopis glandulosa var. glandulosa†

    PubMed Central

    Rahman, Aziz Abdur; Samoylenko, Volodymyr; Jacob, Melissa R.; Sahu, Rajnish; Jain, Surendra K; Khan, Shabana I.; Tekwani, Babu L.; Muhammad, Ilias

    2013-01-01

    A new indolizidine alkaloid, named Δ1,6-juliprosopine (1), together with previously known indolizidine analogs (2–6), was isolated from the leaves of Prosopis glandulosa var. glandulosa, collected from Nevada, USA; while two other known indolizidines juliprosopine (6) and juliprosine (7) were isolated from P. glandulosa leaves collected in Texas, USA. The structures of compound 1 and 7 were determined using a combination of NMR and MS techniques. Compound 7 exhibited potent antiplasmodial activity against Plasmodium falciparum D6 and W2 strains with IC50 values of 170 and 150 ng/mL, respectively, while 1 was found to be less active (IC50 values 560 and 600 ng/mL). Both the compounds were devoid of VERO cells toxicity up to a concentration of 23800 ng/mL. The antileishmanial activity of indolizidines was evaluated against Leishmania donovani promastigotes, axenic amastigotes and amastigotes in THP1 macrophage cultures. When tested against macrophage cultures, the tertiary bases (1, 3, 6) were found to be more potent than quaternary salts (2, 5, 7), displayed IC50 values between 0.8–1.7 μg/mL and 3.1–6.0 μg/mL, respectively. In addition, compound 7 showed potent antifungal activity against Cryptococcus neoformans and antibacterial activity against Mycobacterium intracellulare, while 1 was potent only against C. neoformans and weakly active against other organisms. PMID:21384317

  10. Clerodane type diterpene as a novel antifungal agent from Polyalthia longifolia var. pendula.

    PubMed

    Bhattacharya, Asish K; Chand, Hemender R; John, Jyothis; Deshpande, Mukund V

    2015-04-13

    Bioactivity-guided chemical examination of methanolic extract of leaves of Polyalthia longifolia var. pendula led to the isolation of the active constituent, a diterpene 1 which was identified as 16α-hydroxycleroda-3,13(14)Z-dien-15,16-olide on the basis of its spectral data. Among the tested strains, diterpene 1 was found to exhibit antifungal activities having MIC90 values of 50.3, 100.6 and 201.2 μM against Candida albicans NCIM3557, Cryptococcus neoformans NCIM3542 (human pathogens) and Neurospora crassa NCIM870 (saprophyte), respectively. Initial, structure-activity-relationship (SAR) data generated by synthesizing some derivatives revealed that the double bond between C3-C4 and the free hydroxyl group at C16 are crucial for the antifungal activity of the diterpene 1. The mode of action of 1 in C. albicans is due to compromised cell membrane permeability and also probably due to disruption of cell wall structures. The red blood cell haemolysis of all the compounds (1-4) did not show any significant haemolysis and was found to be less than 15% for all the compounds when tested at highest concentration, i.e. 1200 μM. Interestingly, all the tested compounds inhibited Y-H transition in dimorphic C. albicans NCIM3557 at much lower concentration than their MIC90 values. Determination of ROS generation by diterpene 1 using DCFH-DA and DHR123 (dihydrorhodamine) staining of C. albicans NCIM3557 indicated production of intracellular ROS as a mechanism of antifungal activity. PMID:25747495

  11. Direct Invasion of the Optic Nerves, Chiasm, and Tracts by Cryptococcus neoformans in an Immunocompetent Host.

    PubMed

    Merkler, Alexander E; Gaines, Nathan; Baradaran, Hediyeh; Schuetz, Audrey N; Lavi, Ehud; Simpson, Sara A; Dinkin, Marc J

    2015-10-01

    Cryptococcus spp is a common fungal infection and frequent cause of meningitis in immunocompromised patients; however, immunocompetent patients are also at risk of infection. Visual loss often occurs via elevated intracranial hypertension but can rarely occur through direct optic nerve, chiasm, or tract invasion. We report a case of a 38-year-old woman who presented with decreased acuity in both eyes. She had generalized visual field constriction in the right eye and temporal hemianopsia in the left eye. Magnetic resonance imaging of the brain and orbits showed multiple areas of ill-defined enhancement in the optic chiasm and tracts as well as in the diaphragmatic sella, prepontine and interpeduncular cisterns, and along cranial nerves VI, VII, and VIII bilaterally. Initial cerebrospinal fluid (CSF) showed 34 white blood cells, hypoglycorrhachia, and negative cryptococcal antigen and bacterial and fungal cultures. A transphenoidal biopsy of the dura and pituitary gland was unremarkable. Empiric steroids resulted in marked improvement in visual acuity in both eyes, but while tapering steroids, she developed rapid visual loss bilaterally. Repeat CSF performed 6 weeks later demonstrated a cryptococcal antigen titer of 1:512. Retroactive staining of the pituitary biopsy was positive for mucicarmine, a component of the polysaccharide capsule of Cryptococcus spp. After induction therapy with amphotericin B and flucytosine and 1 year of fluconazole, her visual acuity was 20/20 in both eyes. In summary, Cryptococcus can affect immunocompetent patients and often presents with insidious, chronic meningitis. Visual loss is common in cryptococcal meningitis but usually results from fulminant papilledema related to elevated intracranial pressure. In rare cases, direct nerve or chiasm infiltration by the fungus results in vision loss. PMID:26425249

  12. Capsule independent uptake of the fungal pathogen Cryptococcus neoformans into brain microvascular endothelial cells.

    PubMed

    Sabiiti, Wilber; May, Robin C

    2012-01-01

    Cryptococcosis is a life-threatening fungal disease with a high rate of mortality among HIV/AIDS patients across the world. The ability to penetrate the blood-brain barrier (BBB) is central to the pathogenesis of cryptococcosis, but the way in which this occurs remains unclear. Here we use both mouse and human brain derived endothelial cells (bEnd3 and hCMEC/D3) to accurately quantify fungal uptake and survival within brain endothelial cells. Our data indicate that the adherence and internalisation of cryptococci by brain microvascular endothelial cells is an infrequent event involving small numbers of cryptococcal yeast cells. Interestingly, this process requires neither active signalling from the fungus nor the presence of the fungal capsule. Thus entry into brain microvascular endothelial cells is most likely a passive event that occurs following 'trapping' within capillary beds of the BBB. PMID:22530025

  13. Pbx Proteins in Cryptococcus neoformans Cell Wall Remodeling and Capsule Assembly

    PubMed Central

    Kumar, Pardeep; Heiss, Christian; Santiago-Tirado, Felipe H.; Black, Ian; Azadi, Parastoo

    2014-01-01

    The cryptococcal capsule is a critical virulence factor of an important pathogen, but little is known about how it is associated with the cell or released into the environment. Two mutants lacking PBX1 and PBX2 were found to shed reduced amounts of the capsule polysaccharide glucuronoxylomannan (GXM). Nuclear magnetic resonance, composition, and physical analyses showed that the shed material was of normal mass but was slightly enriched in xylose. In contrast to previous reports, this material contained no glucose. Notably, the capsule fibers of pbxΔ mutant cells grown under capsule-inducing conditions were present at a lower than usual density and were loosely attached to the cell wall. Mutant cell walls were also defective, as indicated by phenotypes including abnormal cell morphology, reduced mating filamentation, and altered cell integrity. All observed phenotypes were shared between the two mutants and exacerbated in a double mutant. Consistent with a role in surface glycan synthesis, the Pbx proteins localized to detergent-resistant membrane domains. These results, together with the sequence motifs in the Pbx proteins, suggest that Pbx1 and Pbx2 are redundant proteins that act in remodeling the cell wall to maintain normal cell morphology and precursor availability for other glycan synthetic processes. Their absence results in aberrant cell wall growth and metabolic imbalance, which together impact cell wall and capsule synthesis, cell morphology, and capsule association. The surface changes also lead to increased engulfment by host phagocytes, consistent with the lack of virulence of pbx mutants in animal models. PMID:24585882

  14. Role of a VPS41 homologue in starvation response, intracellular survival and virulence of Cryptococcus neoformans.

    PubMed

    Liu, Xiaoguang; Hu, Guowu; Panepinto, John; Williamson, Peter R

    2006-09-01

    Previous studies have demonstrated an important role for the vacuole in the virulence of the fungus Cryptococcus and studies in yeast have implicated the vacuolar protein Vps41 in copper loading of proteins such as iron transporters. However, our studies found that a cryptococcal vps41Delta strain displayed wild-type growth on media containing iron and copper chelators and normal activity of the copper-containing virulence factor laccase as well as almost normal growth at 37 degrees C and wild-type production of the virulence factor capsule. Despite these attributes, the vps41Delta mutant strain showed a dramatic attenuation of virulence in mice and co-incubation of mutant cells with the macrophage cell line, J774.16, resulted in a dramatic loss in viability of the vps41Delta mutant strain at 10 h compared with wild-type and complemented strains. Closer examination revealed that the vps41Delta mutant displayed a dramatic loss in viability after nutrient starvation which was traced to a failure to undergo G2 arrest, but there was no defect in the formation of autophagic or proteolytic vesicles. Our results indicate that VPS41 plays a key role in regulating starvation response in this pathogenic organism and that defects in cell cycle arrest are associated with attenuated pathogenic fitness in mammalian hosts. PMID:16879414

  15. Increased Antifungal Drug Resistance in Clinical Isolates of Cryptococcus neoformans in Uganda

    PubMed Central

    Smith, Kyle D.; Achan, Beatrice; Hullsiek, Kathy Huppler; McDonald, Tami R.; Okagaki, Laura H.; Alhadab, Ali A.; Akampurira, Andrew; Rhein, Joshua R.; Meya, David B.; Boulware, David R.

    2015-01-01

    Cryptococcal antigen screening is recommended among people living with AIDS when entering HIV care with a CD4 count of <100 cells/μl, and preemptive fluconazole monotherapy treatment is recommended for those with subclinical cryptococcal antigenemia. Yet, knowledge is limited of current antimicrobial resistance in Africa. We examined antifungal drug susceptibility in 198 clinical isolates collected from Kampala, Uganda, between 2010 and 2014 using the CLSI broth microdilution assay. In comparison with two previous studies from 1998 to 1999 that reported an MIC50 of 4 μg/ml and an MIC90 of 8 μg/ml prior to widespread human fluconazole and agricultural azole fungicide usage, we report an upward shift in the fluconazole MIC50 to 8 μg/ml and an MIC90 value of 32 μg/ml, with 31% of isolates with a fluconazole MIC of ≥16 μg/ml. We observed an amphotericin B MIC50 of 0.5 μg/ml and an MIC90 of 1 μg/ml, of which 99.5% of isolates (197 of 198 isolates) were still susceptible. No correlation between MIC and clinical outcome was observed in the context of amphotericin B and fluconazole combination induction therapy. We also analyzed Cryptococcus susceptibility to sertraline, with an MIC50 of 4 μg/ml, suggesting that sertraline is a promising oral, low-cost, available, novel medication and a possible alternative to fluconazole. Although the CLSI broth microdilution assay is ideal to standardize results, limit human bias, and increase assay capacity, such assays are often inaccessible in low-income countries. Thus, we also developed and validated an assay that could easily be implemented in a resource-limited setting, with similar susceptibility results (P = 0.52). PMID:26324276

  16. Simultaneous Detection and Identification of Candida, Aspergillus, and Cryptococcus Species by Reverse Line Blot Hybridization

    PubMed Central

    Playford, E. Geoffrey; Kong, Fanrong; Sun, Ying; Wang, Hui; Halliday, Catriona; Sorrell, Tania C.

    2006-01-01

    We report on a reverse line blot (RLB) assay, utilizing fungal species-specific oligonucleotide probes to hybridize with internal transcribed spacer 2 region sequences amplified using a nested panfungal PCR. Reference and clinical strains of 16 Candida species (116 strains), Cryptococcus neoformans (five strains of Cryptococcus neoformans var. neoformans, five strains of Cryptococcus neoformans var. grubii, and six strains of Cryptococcus gatti), and five Aspergillus species (68 strains) were all correctly identified by the RLB assay. Additional fungal species (16 species and 26 strains) not represented on the assay did not exhibit cross-hybridization with the oligonucleotide probes. In simulated clinical specimens, the sensitivity of the assay for Candida spp. and Aspergillus spp. was 100.5 cells/ml and 102 conidia/ml, respectively. This assay allows sensitive and specific simultaneous detection and identification of a broad range of fungal pathogens. PMID:16517870

  17. Cryptoccocal meningitis in Yaoundé (Cameroon) HIV infected patients: Diagnosis, frequency and Cryptococcus neoformans isolates susceptibility study to fluconazole.

    PubMed

    Kammalac Ngouana, T; Dongtsa, J; Kouanfack, C; Tonfack, C; Fomena, S; Mallié, M; Delaporte, E; Boyom, F-Fekam; Bertout, S

    2015-03-01

    Cryptococcal meningitis is a mycosis encountered especially in patients with Acquired Immunodeficiency Syndrome and is fatal in the absence of treatment. Information on epidemiology, diagnosis and susceptibility profile to antifungal drugs, are scarce in Cameroon. Authors evaluated the diagnosis possibilities of the cryptococcal meningitis in Cameroon, and studied the antifungal susceptibility of isolated strains to fluconazole, used as first line treatment of the disease in Cameroon. Between December 2009 and July 2011, 146 cerebrospinal fluids obtained from HIV patients with suspicion of meningitis were analysed. The diagnosis procedure involved macroscopic and cyto-chemical analysis, India ink test, culture on Sabouraud chloramphenicol medium and antigen latex agglutination test. Antifungal susceptibility testing of isolated strains to fluconazole was done by the E-test(®) method. The diagnosis of cryptococcal meningitis gave 28.08% positive cases. Among these patients, 80% were at stages III and IV and 20% at stage I of the HIV infection, according to the WHO previous classification. Cyto-chemical analysis showed current findings in the case of cryptococcal meningitis. India ink test and latex agglutination test exhibited very high sensitivity and specificity (>94%). Fluconazole antifungal susceptibility testing gave MICs lower than 32μg/mL to 92.7% of isolated strains and MICs greater than this value to 7.3% of isolates. These results showed that cryptococcal meningitis remains a real problem among HIV infected patients in Yaoundé. The emergence of fluconazole reduced susceptibility strains is worrying. Nevertheless, efficacy of rapid detection tests is interesting because this will help in rapid diagnosis and treatment of patients. PMID:25467817

  18. Cryptococcus neoformans constitutes an ideal model organism to unravel the contribution of cellular aging to the virulence of chronic infections.

    PubMed

    Bouklas, Tejas; Fries, Bettina C

    2013-08-01

    Aging affects all organisms, from unicellular yeasts to multicellular humans. Studies in model organisms demonstrate that the pathways that mediate the two forms of aging, replicative and chronological, are highly conserved. Most studies are focused on the effect of aging on an individual cell rather than a whole population. Complex longevity regulation, however, makes aging a highly adaptive trait that is subject to natural selection. Recent studies have shed light on the potential relevance of aging in fungal pathogens, which undergo replicative aging when they expand in the host environment. Hence, pathogens causing chronic infections can constitute ideal model organisms in unraveling the contribution of selection to aging within a population and help elucidate the contribution of aging itself to the virulence of infections. PMID:23631868

  19. Toward improved anti-cryptococcal drugs: Novel molecules and repurposed drugs.

    PubMed

    Krysan, Damian J

    2015-05-01

    Cryptococcosis is one of the most important fungal infections of humans. It primarily, but not exclusively, afflicts people with compromised immune function. Cryptococcosis is most commonly caused by Cryptococcus neoformans var. grubii with C. neoformans var. neoformans and C. gatti also contributing to the disease. Cryptococcosis is primarily manifested as meningoencephalitis although pneumonia occurs frequently as well. Globally, the burden of disease is highest among those living with HIV/AIDS and is one of the most common causes of death in this patient population. Cryptococcal meningitisis almost invariably fatal if untreated. The current gold standard therapy is amphotericin B combined with 5-flucytosine. Unfortunately, this therapy has significant toxicity and is not widely available in resource-limited regions. Fluconazole, which is associated with poorer outcomes, is frequently as an alternative. Here, I present the characteristics of an ideal anti-cryptococcal agent and review recent progress toward identifying both novel and repurposed drugs as potential new therapies. PMID:25514636

  20. Specific Oligonucleotide Primers for Identification of Cladophialophora carrionii, a Causative Agent of Chromoblastomycosis

    PubMed Central

    Abliz, Paride; Fukushima, Kazutaka; Takizawa, Kayoko; Nishimura, Kazuko

    2004-01-01

    Cladophialophora carrionii is one of the relatively common causative agents of chromoblastomycosis. We have developed the specific oligonucleotide primer set based on the internal transcribed spacer regions of ribosomal DNA for the rapid identification of this pathogen. PCR with this primer set amplified a 362-bp amplicon from C. carrionii strains. From other relevant dematiaceous species, including medically important dematiaceous fungi, such as Fonsecaea pedrosoi, Phialophora verrucosa, and Exophiala dermatitidis, and eight species of medically important yeasts, such as Candida albicans and Cryptococcus neoformans var. neoformans, the primer set did not produce any amplicon. PCR with this primer set may be a useful tool for the identification of C. carrionii. PMID:14715791

  1. Cryptococcal Genotype Influences Immunologic Response and Human Clinical Outcome after Meningitis

    PubMed Central

    Wiesner, Darin L.; Moskalenko, Oleksandr; Corcoran, Jennifer M.; McDonald, Tami; Rolfes, Melissa A.; Meya, David B.; Kajumbula, Henry; Kambugu, Andrew; Bohjanen, Paul R.; Knight, Joseph F.; Boulware, David R.; Nielsen, Kirsten

    2012-01-01

    ABSTRACT In sub-Saharan Africa, cryptococcal meningitis (CM) continues to be a predominant cause of AIDS-related mortality. Understanding virulence and improving clinical treatments remain important. To characterize the role of the fungal strain genotype in clinical disease, we analyzed 140 Cryptococcus isolates from 111 Ugandans with AIDS and CM. Isolates consisted of 107 nonredundant Cryptococcus neoformans var. grubii strains and 8 C. neoformans var. grubii/neoformans hybrid strains. Multilocus sequence typing (MLST) was used to characterize genotypes, yielding 15 sequence types and 4 clonal clusters. The largest clonal cluster consisted of 74 isolates. The results of Burst and phylogenetic analysis suggested that the C. neoformans var. grubii strains could be separated into three nonredundant evolutionary groups (Burst group 1 to group 3). Patient mortality was differentially associated with the different evolutionary groups (P = 0.04), with the highest mortality observed among Burst group 1, Burst group 2, and hybrid strains. Compared to Burst group 3 strains, Burst group 1 strains were associated with higher mortality (P = 0.02), exhibited increased capsule shedding (P = 0.02), and elicited a more pronounced Th2 response during ex vivo cytokine release assays with strain-specific capsule stimulation (P = 0.02). The results of these analyses suggest that cryptococcal strain variation can be an important determinant of human immune responses and mortality. PMID:23015735

  2. Validation and clinical application of a molecular method for the identification of Cryptococcus neoformans/Cryptococcus gattii complex DNA in human clinical specimens.

    PubMed

    Rivera, Vanessa; Gaviria, Marcela; Muñoz-Cadavid, Cesar; Cano, Luz; Naranjo, Tonny

    2015-01-01

    The diagnosis of cryptococcosis is usually performed based on cultures of tissue or body fluids and isolation of the fungus, but this method may require several days. Direct microscopic examination, although rapid, is relatively insensitive. Biochemical and immunodiagnostic rapid tests are also used. However, all of these methods have limitations that may hinder final diagnosis. The increasing incidence of fungal infections has focused attention on tools for rapid and accurate diagnosis using molecular biological techniques. Currently, PCR-based methods, particularly nested, multiplex and real-time PCR, provide both high sensitivity and specificity. In the present study, we evaluated a nested PCR targeting the gene encoding the ITS-1 and ITS-2 regions of rDNA in samples from a cohort of patients diagnosed with cryptococcosis. The results showed that in our hands, this Cryptococcus nested PCR assay has 100% specificity and 100% sensitivity and was able to detect until 2 femtograms of Cryptococcus DNA. PMID:26365230

  3. A Case of Pneumonia Caused by Pneumocystis Jirovecii and Cryptococcus Neoformans in a Patient with HTLV-1 Associated Adult T- Cell Leukemia/Lymphoma: Occam's Razor Blunted.

    PubMed

    Desai, Anish; Fe, Alexander; Desai, Amishi; Ilowite, Jonathan; Cunha, Burke A; Mathew, Joseph P

    2016-02-01

    Adult T-cell leukemia/lymphoma (ATLL) is usually preceded by infection with human T-cell lymphotropic virus I (HTLV-I). Patients with ATLL frequently get opportunistic infections of the lungs, intestines, and central nervous system. Pneumocystis pneumonia is commonly known as an AIDS defining illness. Grocott's methenamine silver stain of bronchoalveolar lavage (BAL) samples obtained via bronchoscopy remain the gold standard for diagnosis. Pulmonary cryptococcosis is seen in patients with T-cell deficiencies and a diagnosis is made by culture of sputum, BAL, or occasionally of pleural fluid. We present the second case of coinfection with these two organisms in a patient with ATLL who was successfully treated with trimethoprim-sulfamethoxazole, corticosteroids, and fluconazole. We illustrate the need for high clinical vigilance for seeking out an additional diagnosis, especially in immunocompromised patients if they are not improving despite receiving appropriate treatment. PMID:27024978

  4. First Description of Oral Cryptococcus neoformans Causing Osteomyelitis of the Mandible, Manubrium and Third Rib with Associated Soft Tissue Abscesses in an Immunocompetent Host

    PubMed Central

    DiNardo, Andrew R.; Schmidt, Davin; Mitchell, Angela; Kaufman, Yoav; Tweardy, David J.

    2016-01-01

    The majority of disseminated cryptococcal infections occur in patients with acquired immunodeficiency syndrome (AIDS), with only 11-14% of cases occurring in patients without AIDS. Most non-AIDS related cases (75%) occur in patients with another immune deficiency. Here, we present the first case of mucocutaneous cryptococcal disease in an immunocompetent host, review the epidemiology of risk factors associated with disseminated cryptococcal disease, and describe a rational workup for a possible acquired immunodeficiency. While rare, 25% of non-AIDS related cryptococcal disease will occur in individuals without an identifiable immunodeficiency and should prompt a work up for cell-mediated immunodeficiency and monitored for closely for progression of other opportunistic infections.

  5. Economics of static VAR compensation

    SciTech Connect

    Alvarado, F.L.; DeMarco, C.; Jung, T.H. . Dept. of Electrical and Computer Engineering)

    1992-09-01

    This project was initiated in anticipation of widened use of static VAR (volt-ampere-reactive) compensation on US bulk-power transmission systems to increase levels of secure power transfer. Project objectives were to deten-nine power system cost savings and reliability benefits resulting from such use. System operating cost and stability probabilities were compared with and without static VAR compensation, applying simulation techniques. For the particular system model studied, there was a 21.4 percent reduction in operating costs taking into account losses added by the static VAR compensator. A procedure was developed to compare instability probabilities for various loadings and static VAR compensator sizes on a power system. For the particular system model studied, the static VAR compensator provided a significant increase in stability but over a narrow range of loading. Static VAR compensation is one of a number of promising FACTS (Flexible AC Transmission System) technologies for handling the demands of increased power transfers on power systems where transmission lines cannot be built or as a short-term altemative to building additional lines.

  6. Cryptococcemia. An analysis of 28 cases with emphasis on the clinical outcome and its etiologic agent.

    PubMed

    Pasqualotto, Alessandro Comarú; Bittencourt Severo, Cecília; de Mattos Oliveira, Flávio; Severo, Luiz Carlos

    2004-09-01

    Clinical protocols of 28 cases of cryptococcemia studied between April 1995 and November 2002 were reviewed. The varieties of Cryptococcus neorformans, the underlying disease, and the severity and outcome of the disease were emphasized. Most patients were immunossupressed (89.3% with AIDS) and Cryptococcus neoformans var. grubii was the main recovered variety (92.8%). Regardless of antifungal treatment, in-hospital mortality was 41% strongly associated with APACHE II score, >14 (p<0.01). PMID:15709789

  7. Ribosomal genes of Histoplasma capsulatum var. duboisii and var. farciminosum.

    PubMed

    Okeke, C N; Kappe, R; Zakikhani, S; Nolte, O; Sonntag, H G

    1998-11-01

    A total of 1704 basepairs of the 18S rDNA of Histoplasma capsulatum var. duboisii (HCD, strain CBS175.57) and H. capsulatum var. farciminosum (HCF, strain CBS478.64) were sequenced (EMBL accession no. Z75306 and no. Z75307). The 18S rDNA of HCD was 100% identical to a published sequence of H. capsulatum var. capsulatum (HCC). The 18S rDNA of HCF showed one transversional point mutation at the nucleotide position 114 (ref. Saccharomyces cerevisiae). Hybridization confirmed that, in the 18S rDNA of two out of five strains of HCF, guanine was substituted for cytosine at the nucleotide position 114. Furthermore, identical group 1C1 introns (403 bp) were found to be inserted after position 1165 in four out of five strains of HCF, including the two strains with point mutations in the 18S rDNA, and a slightly different group 1C1 intron (408 bp) was detected in one strain of HCC without this point mutation. Intraspecific sequence variability in the highly conserved 18S rDNA because of occurrence of introns and mutations as a possible source of error in molecular diagnostics is discussed. In addition, internal transcribed spacer regions between the 18S rDNA and the 5.8S rDNA (ITS1) of three strains of HCF, and one strain each of HCC and HCD showed significant sequence variability between varieties and strains of H. capsulatum. PMID:9916456

  8. TabVar: Tabulated Variables

    SciTech Connect

    Bachan, John

    2015-12-15

    TabVar: A Python library for manipulating datasets in the form of tabulated variables. Tables in tabvar contain many columns representing independent variables, but exactly one distinguished column for the dependent variable. Having a single distinguished column allows a natural lifting of arithmetic operators to tables, much (and in fact fully generalizing) multidimensional array arithmetic. The convenient syntax of whole-table arithmetic, along with the usual operations of filtering and aggregation, and all in the setting of python's interactive REPL allows for rapid exploration of datasets.

  9. TabVar: Tabulated Variables

    Energy Science and Technology Software Center (ESTSC)

    2015-12-15

    TabVar: A Python library for manipulating datasets in the form of tabulated variables. Tables in tabvar contain many columns representing independent variables, but exactly one distinguished column for the dependent variable. Having a single distinguished column allows a natural lifting of arithmetic operators to tables, much (and in fact fully generalizing) multidimensional array arithmetic. The convenient syntax of whole-table arithmetic, along with the usual operations of filtering and aggregation, and all in the setting ofmore » python's interactive REPL allows for rapid exploration of datasets.« less

  10. Primary cutaneous cryptococcosis in an eight-year-old immunocompetent child: how to treat?

    PubMed

    Lenz, D; Held, J; Goerke, S; Wagner, D; Tintelnot, K; Henneke, P; Hufnagel, M

    2015-01-01

    Here we report on a case of primary cryptococcal skin infection in an immunocompetent 8-year-old boy. The infection first manifested itself as a subcutaneous abscess around the proximal joint of his right thumb after a minor injury from contact with a thorny shrub. After surgical incision and drainage was performed, Cryptococcus neoformans var. neoformans was the only pathogen cultured from the lesion. An agglutination test for the capsular antigen in serum displayed negative results and the immunological work-up revealed no underlying immunodeficiency. A "watch and wait" strategy - one without systemic antifungal treatment - was adopted and this resulted in uneventful healing. In summary, primary cryptococcal skin infections in immunocompetent hosts may be managed successfully by surgical treatment in combination with careful clinical follow-up. This approach may help avoid unnecessary antimicrobial treatments. PMID:25565197

  11. "Var Teatre"--A Pioneer Turns 40.

    ERIC Educational Resources Information Center

    Jones, Pamela L.

    1984-01-01

    Describes the Stockholm Municipal Youth and Children's theatre ("Var Teatre"), an institution of 14 theatres and attendant professional staff devoted exclusively to drama activities for children and teenagers. (PD)

  12. Economics of static VAR compensation. Final report

    SciTech Connect

    Alvarado, F.L.; DeMarco, C.; Jung, T.H.

    1992-09-01

    This project was initiated in anticipation of widened use of static VAR (volt-ampere-reactive) compensation on US bulk-power transmission systems to increase levels of secure power transfer. Project objectives were to deten-nine power system cost savings and reliability benefits resulting from such use. System operating cost and stability probabilities were compared with and without static VAR compensation, applying simulation techniques. For the particular system model studied, there was a 21.4 percent reduction in operating costs taking into account losses added by the static VAR compensator. A procedure was developed to compare instability probabilities for various loadings and static VAR compensator sizes on a power system. For the particular system model studied, the static VAR compensator provided a significant increase in stability but over a narrow range of loading. Static VAR compensation is one of a number of promising FACTS (Flexible AC Transmission System) technologies for handling the demands of increased power transfers on power systems where transmission lines cannot be built or as a short-term altemative to building additional lines.

  13. 4D-Var Developement at GMAO

    NASA Technical Reports Server (NTRS)

    Pelc, Joanna S.; Todling, Ricardo; Akkraoui, Amal El

    2014-01-01

    The Global Modeling and Assimilation Offce (GMAO) is currently using an IAU-based 3D-Var data assimilation system. GMAO has been experimenting with a 3D-Var-hybrid version of its data assimilation system (DAS) for over a year now, which will soon become operational and it will rapidly progress toward a 4D-EnVar. Concurrently, the machinery to exercise traditional 4DVar is in place and it is desirable to have a comparison of the traditional 4D approach with the other available options, and evaluate their performance in the Goddard Earth Observing System (GEOS) DAS. This work will also explore the possibility for constructing a reduced order model (ROM) to make traditional 4D-Var computationally attractive for increasing model resolutions. Part of the research on ROM will be to search for a suitably acceptable space to carry on the corresponding reduction. This poster illustrates how the IAU-based 4D-Var assimilation compares with our currently used IAU-based 3D-Var.

  14. Scopafungin, a Crystalline Antibiotic Produced by Streptomyces hygroscopicus var. enhygrus var. nova

    PubMed Central

    Johnson, LeRoy E.; Dietz, Alma

    1971-01-01

    Scopafungin (U-29,479) is a crystalline, nonpolyenic antimicrobial agent obtained from the culture broth of Streptomyces hygroscopicus var. enhygrus var. nova UC-2397. Scopafungin inhibits, in vitro, a variety of pathogenic fungi, yeasts, and gram-positive bacteria. PMID:4940870

  15. Volatiles of Chrysanthemum zawadskii var. latilobum K

    PubMed Central

    Chang, Kyung-Mi; Kim, Gun-Hee

    2012-01-01

    The volatile aroma constituents of Chrysanthemum zawadskii var. latilobum K. were separated by hydro distillation extraction (HDE) method using a Clevenger-type apparatus, and analyzed by gas chromatography-mass spectrometry (GC/MS). The yield of C. zawadskii var. latilobum K. flower essential oil (FEO) was 0.12% (w/w) and the color was light green. Fifty-five volatile chemical components, which make up 88.38% of the total aroma composition, were tentatively characterized. C. zawadskii var. latilobum K. FEOs contained 27 hydrocarbons, 12 alcohols, 7 ketones, 4 esters, 1 aldehyde, 1 amine, and 3 miscellaneous components. The major functional groups were terpene alcohol and ketone. Borneol (12.96), (±)-7-epi-amiteol (12.60), and camphor (10.54%) were the predominant volatiles. These compounds can be used in food and pharmaceutical industries due to their active bio-functional properties. PMID:24471090

  16. Volatiles of Chrysanthemum zawadskii var. latilobum K.

    PubMed

    Chang, Kyung-Mi; Kim, Gun-Hee

    2012-09-01

    The volatile aroma constituents of Chrysanthemum zawadskii var. latilobum K. were separated by hydro distillation extraction (HDE) method using a Clevenger-type apparatus, and analyzed by gas chromatography-mass spectrometry (GC/MS). The yield of C. zawadskii var. latilobum K. flower essential oil (FEO) was 0.12% (w/w) and the color was light green. Fifty-five volatile chemical components, which make up 88.38% of the total aroma composition, were tentatively characterized. C. zawadskii var. latilobum K. FEOs contained 27 hydrocarbons, 12 alcohols, 7 ketones, 4 esters, 1 aldehyde, 1 amine, and 3 miscellaneous components. The major functional groups were terpene alcohol and ketone. Borneol (12.96), (±)-7-epi-amiteol (12.60), and camphor (10.54%) were the predominant volatiles. These compounds can be used in food and pharmaceutical industries due to their active bio-functional properties. PMID:24471090

  17. VarSITI - SCOSTEP's scientific program

    NASA Astrophysics Data System (ADS)

    Georgieva, Katya; Shiokawa, Kazuo

    2016-07-01

    With the aim to promote international scientific activity in the different branches of solar terrestrial physics and its application for the benefit of humanity, SCOSTEP runs long-term (4-5 years) international interdisciplinary scientific programs. The current SCOSTEP's scientific program (2014-2018) is VarSITI - Variability of the Sun and Its Terrestrial Impacts. It has four scientific projects covering solar terrestrial problems all the way from the Sun through the interplanetary space, magnetosphere, ionosphere, and down to the Earth's atmosphere. We will present the VarSITI's projects and activities, and will highlight some of the results so far and plans for the future.

  18. Chemical constituents from Clematis delavayi var. spinescens.

    PubMed

    Li, Yang; Wang, Si-Feng; Zhao, Yan-Li; Liu, Ke-Chun; Wang, Xi-Min; Yang, Yong-Ping; Li, Xiao-Li

    2009-01-01

    A new coumarin, 7-hydroxy-4,6-dimethoxy-5-methylcoumarin (1), was isolated from the aerial parts of Clematis delavayi var. spinescens together with 17 known compounds. Their structures were identified by extensive spectral analysis, especially 2D NMR techniques. Antiangiogenic effects of all compounds were evaluated using a zebrafish model. PMID:19924077

  19. Static var compensators stabilize power voltages

    SciTech Connect

    Burch, R.

    1996-06-01

    This article discusses the operation of a static var compensator as installed by Alabama Power near a steel mill with a large arc furnace load. This is expected to result in a number of benefits, including flicker reduction, dynamic power factor correction, harmonics filtering and a reduction in system losses.

  20. Bacterial endosymbionts of Pyrodinium bahamense var. compressum.

    PubMed

    Azanza, Ma Patricia V; Azanza, Rhodora V; Vargas, Vanessa Mercee D; Hedreyda, Cynthia T

    2006-11-01

    The study presents evidence in support of the bacterial theory associated with the toxicity of Pyrodinium bahamense var. compressum. Bacterial endosymbionts from Philippine P. bahamense var. compressum strain Pbc MZRVA 042595 were isolated and identified via 16S rDNA sequence analysis. Taxonomic diversity of the identified culturable intracellular microbiota associated with Philippine P. bahamense var. compressum was established to be limited to the Phyla Proteobacteria, Actinobacteria, and Firmicutes. Major endosymbionts identified included Moraxella spp., Erythrobacter spp., and Bacillus spp., whereas Pseudomonas putida, Micrococcus spp., and Dietzia maris were identified as minor isolates. All identified strains except D. maris, P. putida, and Micrococcus spp. were shown to contain either saxitoxin or neo saxitoxin or both at levels < or =73 ng/10(7) bacterial cells based on high-performance liquid chromatography analysis. Paralytic shellfish poisoning-like physiologic reactions in test animals used in the mouse assay were recorded for the endosymbionts except for P. putida. The study is the first to elucidate the possible contribution of bacterial endosymbionts in the toxicity of P. bahamense var. compressum isolated in the Philippines. PMID:16944340

  1. VAR Support from Distributed Wind Energy Resources: Preprint

    SciTech Connect

    Romanowitz, H.; Muljadi, E.; Butterfield, C. P.; Yinger, R.

    2004-07-01

    As the size and quantity of wind farms and other distributed generation facilities increase, especially in relation to local grids, the importance of a reactive power compensator or VAR support from these facilities becomes more significant. Poorly done, it can result in cycling or inadequate VAR support, and the local grid could experience excessive voltage regulation and, ultimately, instability. Improved wind turbine and distributed generation power control technologies are creating VAR support capabilities that can be used to enhance the voltage regulation and stability of local grids. Locating VAR support near the point of consumption, reducing step size, and making the control active all improve the performance of the grid. This paper presents and discusses alternatives for improving the integration of VAR support from distributed generation facilities such as wind farms. We also examine the relative effectiveness of distributed VAR support on the local grid and how it can b e integrated with the VAR support of the grid operator.

  2. Glucose kinases from Streptomyces peucetius var. caesius.

    PubMed

    Ruiz-Villafán, Beatriz; Rodríguez-Sanoja, Romina; Aguilar-Osorio, Guillermo; Gosset, Guillermo; Sanchez, Sergio

    2014-07-01

    Glucose kinases (Glks) are enzymes of the glycolytic pathway involved in glucose phosphorylation. These enzymes can use various phosphoryl donors such as ATP, ADP, and polyphosphate. In several streptomycetes, ATP-glucose kinase (ATP-Glk) has been widely studied and regarded as the main glucose phosphorylating enzyme and is likely a regulatory protein in carbon catabolite repression. In cell extracts from the doxorubicin overproducing strain Streptomyces peucetius var. caesius, grown in glucose, a polyphosphate-dependent Glk (Pp-Glk) was detected by zymogram. Maximum activity was observed during the stationary growth phase (48 h) of cells grown in 100 mM glucose. No activity was detected when 20 mM glutamate was used as the only carbon source, supporting a role for glucose in inducing this enzyme. Contrary to wild-type strains of Streptomyces coelicolor, Streptomyces lividans, and Streptomyces thermocarboxydus K-155, S. peucetius var. caesius produced 1.8 times more Pp-Glk than ATP-Glk. In addition, this microorganism produced five and four times more Pp-Glk and anthracyclines, respectively, than its wild-type S. peucetius parent strain, supporting a role for this enzyme in antibiotic production in the overproducer strain. A cloned 726-bp DNA fragment from S. peucetius var. caesius encoded a putative Pp-Glk, with amino acid identities between 83 and 87 % to orthologous sequences from the above-cited streptomycetes. The cloned fragment showed the polyphosphate-binding sequences GXDIGGXXIK, TXGTGIGSA, and KEX(4)SWXXWA. Sequences for the Zn-binding motif were not detected in this fragment, suggesting that Pp-Glk is not related to the Glk ROK family of proteins. PMID:24687748

  3. Chemical diversity of volatiles of Teucrium orientale L. var. orientale, var. puberulens, and var. glabrescens determined by simultaneous GC-FID and GC/MS techniques.

    PubMed

    Ozek, Gulmira; Ozek, Temel; Dinç, Muhittin; Doǧu, Süleyman; Başer, Kemal H C

    2012-06-01

    In the present work, three varieties of Teucrium orientale, var. orientale, var. puberulens, and var. glabrescens, were collected and investigated for chemical composition of the oils. Subsequent gas chromatography (GC-FID) and gas chromatography coupled to mass spectrometry (GC/MS) revealed high abundance of sesquiterpenes in the essential oils analyzed. All the oils contained β-caryophyllene (22.6, 8.5, and 6.3%, resp.) and hexadecanoic acid (7.9, 12.8, and 13.1%). Germacrene D (24.6 and 33.4%) and bicyclogermacrene (6.7 and 8.5%) were found to be the main constituents of var. orientale and var. puberulens, respectively. The high percentages of β-cubebene (26.9%), α-cubebene (9.0%), and α-copaene (7.2%) established the diversity of var. glabrescens. The qualitative difference between the essential oils allowed the differentiation between the varieties in agreement with the morphological observations described in Flora of Turkey for each variety studied. In addition, a cluster analysis of twelve Teucrium taxa based on the essential-oil composition has been carried out. Hovewer, the analysis did not clearly reflect the infrageneric classification of the genus, it largely confirmed the relationships between the infraspecific taxa of Teucrium orientale and T. chamaedrys. PMID:22700232

  4. Cytotoxic diterpenoids from Rabdosia lophanthoides var. gerardianus.

    PubMed

    Lin, Chao-Zhan; Zhao, Wei; Feng, Xiu-Li; Liu, Fang-Le; Zhu, Chen-Chen

    2016-03-01

    Two new abietane diterpenoids, Gerardianin B (1) and Gerardianin C (2), one new lignan glycoside, Gerardianin D (3) and one new lupane-type triterpenoid, Gerardianol A (4), together with seven known abietane diterpenoids were isolated from the aerial parts of Rabdosia lophanthoides var. gerardianus. Their structures were determined by 1D and 2D NMR spectroscopic data. The cytotoxic activities of the nine diterpenoids were evaluated on human cancer cell lines. Compounds 6-11 exhibited significant cytotoxic activities against HepG2 cell lines with IC50 from 4.68 to 9.43μM and HCF-8 cell lines with IC50 from 9.12 to 13.53μM. PMID:26608401

  5. 1D-VAR Retrieval Using Superchannels

    NASA Technical Reports Server (NTRS)

    Liu, Xu; Zhou, Daniel; Larar, Allen; Smith, William L.; Schluessel, Peter; Mango, Stephen; SaintGermain, Karen

    2008-01-01

    Since modern ultra-spectral remote sensors have thousands of channels, it is difficult to include all of them in a 1D-var retrieval system. We will describe a physical inversion algorithm, which includes all available channels for the atmospheric temperature, moisture, cloud, and surface parameter retrievals. Both the forward model and the inversion algorithm compress the channel radiances into super channels. These super channels are obtained by projecting the radiance spectra onto a set of pre-calculated eigenvectors. The forward model provides both super channel properties and jacobian in EOF space directly. For ultra-spectral sensors such as Infrared Atmospheric Sounding Interferometer (IASI) and the NPOESS Airborne Sounder Testbed Interferometer (NAST), a compression ratio of more than 80 can be achieved, leading to a significant reduction in computations involved in an inversion process. Results will be shown applying the algorithm to real IASI and NAST data.

  6. 4-D-Var or ensemble Kalman filter?

    NASA Astrophysics Data System (ADS)

    Kalnay, Eugenia; Li, Hong; Miyoshi, Takemasa; Yang, Shu-Chih; Ballabrera-Poy, Joaquim

    2007-10-01

    We consider the relative advantages of two advanced data assimilation systems, 4-D-Var and ensemble Kalman filter (EnKF), currently in use or under consideration for operational implementation. With the Lorenz model, we explore the impact of tuning assimilation parameters such as the assimilation window length and background error covariance in 4-D-Var, variance inflation in EnKF, and the effect of model errors and reduced observation coverage. For short assimilation windows EnKF gives more accurate analyses. Both systems reach similar levels of accuracy if long windows are used for 4-D-Var. For infrequent observations, when ensemble perturbations grow non-linearly and become non-Gaussian, 4-D-Var attains lower errors than EnKF. If the model is imperfect, the 4-D-Var with long windows requires weak constraint. Similar results are obtained with a quasi-geostrophic channel model. EnKF experiments made with the primitive equations SPEEDY model provide comparisons with 3-D-Var and guidance on model error and `observation localization'. Results obtained using operational models and both simulated and real observations indicate that currently EnKF is becoming competitive with 4-D-Var, and that the experience acquired with each of these methods can be used to improve the other. A table summarizes the pros and cons of the two methods.

  7. 4D-Var or Ensemble Kalman Filter

    NASA Astrophysics Data System (ADS)

    Kalnay, E.; Li, H.; Yang, S.; Miyoshi, T.; Ballabrera, J.

    2007-05-01

    We consider the relative advantages of two advanced data assimilation systems, 4D-Var and ensemble Kalman filter (EnKF), currently in use or considered for operational implementation. We explore the impact of tuning assimilation parameters such as the assimilation window length and background error covariance in 4D-Var, the variance inflation in EnKF, and the effect of model errors and reduced observation coverage in both systems. For short assimilation windows EnKF gives more accurate analyses. Both systems reach similar levels of accuracy if long windows are used for 4D-Var, and for infrequent observations, when ensemble perturbations grow nonlinearly and become non-Gaussian, 4D-Var attains lower errors than EnKF. Results obtained with variations of EnKF using operational models and both simulated and real observations are reviewed. A table summarizes the pros and cons of the two methods.

  8. CHARACTERIZATION OF THE PARASPORAL INCLUSION OF BACILLUS THURINGIENSIS VAR. KYUSHUENSIS

    EPA Science Inventory

    Bacillus thuringiensis var. kyushuensis synthesizes an irregularly shaped parasporal inclusion during sporulation. lectron microscopy revealed that the inclusions are composed of a relatively homogeneous appearing center surrounded by a thick, electron dense coating. urified incl...

  9. Weak bus-oriented optimal multi-objective VAR planning

    SciTech Connect

    Chen, Y.L.

    1996-11-01

    This paper presents a weak bus-oriented criterion to determine the candidate buses for installing new VAR sources in the VAR planning problem. First, an efficient method, using a voltage collapse proximity indicator, is described for identifying weak buses. Then appropriate VAR planning in those weak buses can enhance the system security margin, in particular, to prevent voltage collapse. Next, the goal attainment (GA) method based on the Simulated Annealing (SA) approach is applied to solving general multi-objective VAR planning problems by assuming that the decisionmaker (DM) has goals for each of the objective functions. The presented method can both obtain a better final solution and reduce the solution space. Results of application of the proposed method to the AEP-14 bus system as well as to a large, actual-size system are also presented.

  10. Puccinia jaceae var.solstitialis teliospore priming on yellow starthistle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Following the introduction of Puccinia jaceae var. solstitialis to California for biological control of yellow starthistle (Centaurea solstitialis, Asteraceae), teliospores, pycnia, and multiple urediniospore generations have been observed in the field. Because urediniospores have a relatively short...

  11. Immunoelectrophoretic Analysis of Mycoplasma mycoides var. mycoides

    PubMed Central

    Stone, S. S.; Razin, S.

    1973-01-01

    Acrylamide gel electrophoresis was used to show the similarities and differences in the membrane proteins of two vaccine and two virulent strains of Mycoplasma mycoides var. mycoides. Immunoelectrophoretic (IEP) analysis was also used to partially characterize the associated antigens. Antibody spectra to the antigens of M. mycoides differ in rabbit, pig, and cattle sera. Rabbits produce better precipitating antibody against the anodic migrating protein mycoplasma antigens than cattle and pigs as seen in IEP. However, rabbit anti-M. mycoides serum did not show precipitating antibody against the heat-stable carbohydrate antigen. As judged by IEP, the major carbohydrate antigen extracted from the media, or boiled whole organism, is similar to that present in the sera-infected cattle and knee joints of calves. This carbohydrate antigen has a cathodic migration in IEP at pH 8.6. Periodate oxidation, classically used to destroy carbohydrate, also destroys most of the protein antigens. Heating the antigens to 56 C for 10 min destroys many of the noncarbohydrate antigens and 100 C eliminates all but the carbohydrate antigen. Extraction of M. mycoides with chloroform-methanol, phenol, ethanol, or ethanol-acetone reduced or eliminated most of the protein antigens. Some of the isolated antigenic fractions of M. mycoides were tested to determine their activity in the diagnostic complement fixation test for contagious bovine pleuropneumonia and their inhibitory effect in this test by using bovine anti-M. mycoides antisera having precipitating antibody and circulating antigen. The complement fixation antigen is not the galactan, cannot be extracted by chloroform-methanol, but is stable to boiling at 100 C and may be extracted by phenol and partially precipitated by ethanol-acetone. Images PMID:4577417

  12. Analysis of asthma patients for cryptococcal seroreactivity in an urban German area.

    PubMed

    Grahnert, Andreas; Müller, Uwe; von Buttlar, Heiner; Treudler, Regina; Alber, Gottfried

    2015-08-01

    Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised patients but is also able to asymptomatically infect immunocompetent individuals. C. neoformans is found ubiquitously especially in urban areas where it is spread by pigeons, and fungal exposure may predispose for asthma development already at an early age, as soon as confronted with pigeon droppings. In the study presented here, we investigated the presence of specific immunoglobulin G (IgG) against C. neoformans in sera from patients suffering from asthma in comparison to a healthy control cohort, accrued from the Leipzig Research Centre for Civilization Diseases (LIFE). For serological analysis we developed a flow cytometry (FACS) based assay specific for an acapsular strain of C. neoformans to comprehensively analyze different cryptococcal serotypes. Compared with the non-asthmatic cohort, asthmatics exhibited, as expected, an elevated level of total serum immunoglobulin E (IgE), whereas the IgG seroreactivity against C. neoformans was not significantly different among the two groups (P = .118). Nevertheless, there was a trend toward increased Cryptococcus-specific IgG antibodies in the serum of asthmatics. Additionally, in male asthmatics an increased IgG-mediated seroreactivity compared to female asthmatics was found. This points to a higher prevalence of subclinical C. neoformans infection in male asthmatics and may support the hypothesis of C. neoformans as a risk factor for the development of asthma in urban areas. PMID:26026172

  13. A Method for Evaluating Volt-VAR Optimization Field Demonstrations

    SciTech Connect

    Schneider, Kevin P.; Weaver, T. F.

    2014-08-31

    In a regulated business environment a utility must be able to validate that deployed technologies provide quantifiable benefits to the end-use customers. For traditional technologies there are well established procedures for determining what benefits will be derived from the deployment. But for many emerging technologies procedures for determining benefits are less clear and completely absent in some cases. Volt-VAR Optimization is a technology that is being deployed across the nation, but there are still numerous discussions about potential benefits and how they are achieved. This paper will present a method for the evaluation, and quantification of benefits, for field deployments of Volt-VAR Optimization technologies. In addition to the basic methodology, the paper will present a summary of results, and observations, from two separate Volt-VAR Optimization field evaluations using the proposed method.

  14. Correlation inequalities for two-component hypercubic /var phi//sub 4/ models

    SciTech Connect

    Soria, J.L.

    1988-08-01

    A collection of new and already known correlation inequalities is found for a family of two-component hypercubic /var phi//sub 4/ models, using techniques of duplicated variables, rotated correlation inequalities, and random walk representation. Among the interesting new inequalities are: rotated very special Dunlop-Newman inequality var phi//sub 1x//sup 2/; /var phi//sub 1z//sup 2/ + /var phi//sub 2z//sup 2/ greater than or equal to 0, rotated Griffiths I inequality var phi//sub 1x//var-phi//sub 1y/; /var phi//sub 1z//sup 2/ - /var phi//sub 2z//sup 2/> greater than or equal to 0, and anti-Lebowitz inequality u/sub 4//sup 1111/ greater than or equal to 0.

  15. Crude oil from the var'egansk field. [Siberia

    SciTech Connect

    Driatskaya, Z.V.; Kaminskii, E.K.; Krylova, S.M.; Mkhchiyan, M.A.

    1982-09-01

    This article presents results from an investigation of a representative sample of the crude oil of the BV group (BV/sub 6/, BV/sub 7/, BV/sub 8/, and BV/sub 9/), taken at a central gathering point in the Tyumen Oblast. It indicates that Var'egansk crude is low-sulfur, medium-resin, and medium-wax. The Var'egansk field is a single-bed field, and its deposits are confined to the Jurassic and Cretaceous systems (Valanginian and Hauterivian-Barremian stages).

  16. BZ UMa and Var Her 04: Orphan TOADS

    NASA Astrophysics Data System (ADS)

    Price, A.; Howell, S.

    2005-05-01

    Both BZ UMa and Var Her 04 are cataclysmic variable stars without a home. Neither fit easily into current classification systems so may extend the population distribution of two unique CV types: UGWZ dwarf novae and intermediate polars. New outburst photometry and archival X-Ray data shed some new light on BZ UMa's high energy state and new spectral and IR observations from Spitzer of dust around the newly discovered cataclysmic variable Var Her 04 may help find it a home as well.

  17. New Record of Mariannaea elegans var. elegans in Korea.

    PubMed

    Tang, Longqing; Hyun, Min Woo; Yun, Yeo Hong; Suh, Dong Yeon; Kim, Seong Hwan; Sung, Gi Ho

    2012-03-01

    A Mariannaea fungus was isolated during investigation of an elm tree infested with unidentified beetles. Based on morphological characteristics and molecular analysis of the internal transcribed spacer rDNA sequence, the fungus was identified as Mariannaea elegans var. elegans. Fungal growth was better on malt extract agar than on potato dextrose agar and oatmeal agar. Optimal temperature and pH for growth of the fungus were 30℃ and pH 7.0, respectively. The fungus was found to have the ability to produce extracellular enzymes such as amylase, β-glucosidase, cellulase, and protease. This is first report on M. elegans var. elegans in Korea. PMID:22783129

  18. The VarS/VarA two-component system modulates the activity of the Vibrio cholerae quorum-sensing transcriptional regulator HapR

    PubMed Central

    Tsou, Amy M.; Liu, Zhi; Cai, Tao

    2011-01-01

    The human pathogen Vibrio cholerae uses quorum sensing to regulate the expression of a number of phenotypes, including virulence factor production, in response to changes in cell density. It produces small molecules called autoinducers that increase in concentration as cell density increases, and these autoinducers bind to membrane sensors once they reach a certain threshold. This binding leads to signalling through a downstream phosphorelay pathway to alter the expression of the transcriptional regulator HapR. Previously, it was shown that the VarS/VarA two-component system acts on a component of the phosphorelay pathway upstream of HapR to regulate HapR expression levels. Here, we show that in addition to this mechanism of regulation, VarS and VarA also indirectly modulate HapR protein activity. This modulation is mediated by the small RNA CsrB but is independent of the known quorum-sensing system that links the autoinducers to HapR. Thus, the VarS/VarA two-component system intersects with the quorum-sensing network at two levels. In both cases, the effect of VarS and VarA on quorum sensing is dependent on the Csr small RNAs, which regulate carbon metabolism, suggesting that V. cholerae may integrate nutrient status and cell density sensory inputs to tailor its gene expression profile more precisely to surrounding conditions. PMID:21393367

  19. Cryptococcal cellulitis on the shin of an immunosuppressed patient.

    PubMed

    Zhu, Tian Hao; Rodriguez, Paola G; Behan, James W; Declerck, Brittney; Kim, Gene H

    2016-01-01

    Cryptococcus neoformans is a common fungus found throughout the environment that causes opportunistic disease in immunocompromised individuals. Infection of humans with C neoformans usually manifests as lung disease through inhalation of spores or meningoencephalitis by involvement of the central nervous system. Rarely, dissemination in the form of cutaneous lesions can occur in individuals with long term immunosuppression. We present a patient with C. neoformans manifesting as cellulitis with focal segmental glomerulosclerosis treated with corticosteroids. Because of the mortality associated with disseminated cryptococcosis, early identification, especially of atypical cutaneous presentations is critical from a dermatological perspective. PMID:27617599

  20. [Induction and identification of polyploid of Astragalus membranaceus var. mongholicus].

    PubMed

    Wu, Yuxiang; Gao, Jianping; Zhao, Xiaoming

    2003-05-01

    In this article, improved agar painting method, with semi-solid of 0.2% colchicine and 0.1% agar, was adopted to treat apical buds of Astragalus membranaceus var. mongholicus (Bge.) Hsiao seedlings. Obtained plants were proved to be tetraploids by identification of biological characteristics and chromosome numbers. PMID:14535010

  1. [Experiment on polyploid induction of Angelica dahurica var. formosana].

    PubMed

    Peng, F; Zhou, R; Liu, J

    1999-12-01

    Colchicine solution was applied to the primary adventitious buds of Angelica dahurica var. formosana in vitro to induce the polyploid. Compared with non-treated plantlet, the morphology, microhisology, and chromosome number of treated plantlets are varied. It proved that the polyploid induction was effective. PMID:12571900

  2. Cycloartane-Type Saponins from Astragalus tmoleus var. tmoleus.

    PubMed

    Avunduk, Sibel; Mitaine-Offer, Anne-Claire; Miyamoto, Tomofumi; Tanaka, Chiaki; Lacaille-Dubois, Marie-Aleth

    2016-01-01

    Five known cycloartane-type glycosides were isolated from the roots of A. tmoleus Boiss. var. tmoleus. The identification of these compounds was mainly achieved by 1D and 2D NMR spectroscopic techniques and FABMS. The results of our studies confirm that triterpene saponins with the cycloartane-type skeleton might be chemotaxonomically significant for the genus Astragalus. PMID:26996015

  3. Indolizidine, Antiinfective and Antiparasitic Compounds from Prosopis glandulosa var. glandulosa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prosopilosidine, a new potent antiinfective and antiparasitic 2,3-dihydro-1H-indolizinium chloride, (1), was isolated from Prosopis glandulosa Torr. var. glandulosa. Furthermore, three additional new and one known indolizidines, prosopilosine (2), isoprosopilosine (3), isoprosopilosidine (4) and jul...

  4. Fast Responding Voltage Regulator and Dynamic VAR Compensator

    SciTech Connect

    Divan, Deepak; Moghe, Rohit; Tholomier, Damien

    2014-12-31

    The objectives of this project were to develop a dynamic VAR compensator (DVC) for voltage regulation through VAR support to demonstrate the ability to achieve greater levels of voltage control on electricity distribution networks, and faster response compared to existing grid technology. The goal of the project was to develop a prototype Fast Dynamic VAR Compensator (Fast DVC) hardware device, and this was achieved. In addition to developing the dynamic VAR compensator device, Varentec in partnership with researchers at North Carolina State University (NCSU) successfully met the objectives to model the potential positive impact of such DVCs on representative power networks. This modeling activity validated the ability of distributed dynamic VAR compensators to provide fast voltage regulation and reactive power control required to respond to grid disturbances under high penetration of fluctuating and intermittent distributed energy resources (DERs) through extensive simulation studies. Specifically the following tasks were set to be accomplished: 1) Development of dynamic VAR compensator to support dynamic voltage variations on the grid through VAR control 2) Extensive testing of the DVC in the lab environment 3) Present the operational DVC device to the DOE at Varentec’s lab 4) Formulation of a detailed specification sheet, unit assembly document, test setup document, unit bring-up plan, and test plan 5) Extensive simulations of the DVC in a system with high PV penetration. Understanding the operation with many DVC on a single distribution system 6) Creation and submittal of quarterly and final reports conveying the design documents, unit performance data, modeling simulation charts and diagrams, and summary explanations of the satisfaction of program goals. This report details the various efforts that led to the development of the Fast DVC as well as the modeling & simulation results. The report begins with the introduction in Section II which outlines the

  5. Chemopreventive and Anticancer Activities of Allium victorialis var. platyphyllum Extracts

    PubMed Central

    Kim, Hyun-Jeong; Park, Min Jeong; Park, Hee-Juhn; Chung, Won-Yoon; Kim, Ki-Rim; Park, Kwang-Kyun

    2014-01-01

    Background: Allium victorialis var. platyphyllum is an edible perennial herb and has been used as a vegetable or as a Korean traditional medicine. Allium species have received much attention owing to their diverse pharmacological properties, including antioxidative, anti-inflammatory, and anticancer activities. However, A. victorialis var. platyphyllum needs more study. Methods: The chemopreventive potential of A. victorialis var. platyphyllum methanol extracts was examined by measuring 12-O-tetra-decanoylphorbol 13-acetate (TPA)-induced superoxide anion production in the differentiated HL-60 cells, TPA-induced mouse ear edema, and Ames/Salmonella mutagenicity. The apoptosis-inducing capabilities of the extracts were evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, 4’,6-diamidino-2-phenylindole staining, and the DNA fragmentation assay in human colon cancer HT-29 cells. Antimetastatic activities of the extracts were also investigated in an experimental mouse lung metastasis model. Results: The methanol extracts of A. victorialis var. platyphyllum rhizome (AVP-R) and A. victorialis var. platyphyllum stem (AVP-S) dose-dependently inhibited the TPA-induced generation of superoxide anion in HL-60 cells and TPA-induced ear edema in mice, as well as 7,12-dimethylbenz[a]anthracene (DMBA) and tert-butyl hydroperoxide (t-BOOH) -induced bacterial mutagenesis. AVP-R and AVP-S reduced cell viability in a dose-related manner and induced apoptotic morphological changes and internucleosomal DNA fragmentation in HT-29 cells. In the experimental mouse lung metastasis model, the formation of tumor nodules in lung tissue was significantly inhibited by the treatment of the extracts. Conclusions: AVP-R and AVP-S possess antioxidative, anti-inflammatory, antimutagenic, proapoptotic, and antimetastatic activities. Therefore, these extracts can serve as a beneficial supplement for the prevention and treatment of cancer. PMID:25337587

  6. Plasmodium falciparum var gene expression is modified by host immunity

    PubMed Central

    Warimwe, George M.; Keane, Thomas M.; Fegan, Gregory; Musyoki, Jennifer N.; Newton, Charles R. J. C.; Pain, Arnab; Berriman, Matthew; Marsh, Kevin; Bull, Peter C.

    2009-01-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of immune targets, which play a central role in the host–parasite interaction by binding to various host molecules. They are encoded by a diverse family of genes called var, of which there are ≈60 copies in each parasite genome. In sub-Saharan Africa, although P. falciparum infection occurs throughout life, severe malarial disease tends to occur only in childhood. This could potentially be explained if (i) PfEMP1 variants differ in their capacity to support pathogenesis of severe malaria and (ii) this capacity is linked to the likelihood of each molecule being recognized and cleared by naturally acquired antibodies. Here, in a study of 217 Kenyan children with malaria, we show that expression of a group of var genes “cys2,” containing a distinct pattern of cysteine residues, is associated with low host immunity. Expression of cys2 genes was associated with parasites from young children, those with severe malaria, and those with a poorly developed antibody response to parasite-infected erythrocyte surface antigens. Cys-2 var genes form a minor component of all genomic var repertoires analyzed to date. Therefore, the results are compatible with the hypothesis that the genomic var gene repertoire is organized such that PfEMP1 molecules that confer the most virulence to the parasite tend also to be those that are most susceptible to the development of host immunity. This may help the parasite to adapt effectively to the development of host antibodies through modification of the host–parasite relationship. PMID:20018734

  7. C.V. Riley’s lost aphids: Siphonophora fragariae var. immaculata and Aphis rapae var. laevigata (Hemiptera: Aphididae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The syntypes of Siphonophora fragariae var. immaculata Riley were rediscovered in the Aphidoidea collection of the United States of America National Museum of Natural History. Previously, S. fragariae immaculata was largely lost and forgotten. Through examination of the specimens, we hereby establ...

  8. Photosynthetic redox imbalance governs leaf sectoring in the Arabidopsis thaliana variegation mutants immutans, spotty, var1, and var2.

    PubMed

    Rosso, Dominic; Bode, Rainer; Li, Wenze; Krol, Marianna; Saccon, Diego; Wang, Shelly; Schillaci, Lori A; Rodermel, Steven R; Maxwell, Denis P; Hüner, Norman P A

    2009-11-01

    We hypothesized that chloroplast energy imbalance sensed through alterations in the redox state of the photosynthetic electron transport chain, measured as excitation pressure, governs the extent of variegation in the immutans mutant of Arabidopsis thaliana. To test this hypothesis, we developed a nondestructive imaging technique and used it to quantify the extent of variegation in vivo as a function of growth temperature and irradiance. The extent of variegation was positively correlated (R(2) = 0.750) with an increase in excitation pressure irrespective of whether high light, low temperature, or continuous illumination was used to induce increased excitation pressure. Similar trends were observed with the variegated mutants spotty, var1, and var2. Measurements of greening of etiolated wild-type and immutans cotyledons indicated that the absence of IMMUTANS increased excitation pressure twofold during the first 6 to 12 h of greening, which led to impaired biogenesis of thylakoid membranes. In contrast with IMMUTANS, the expression of its mitochondrial analog, AOX1a, was transiently upregulated in the wild type but permanently upregulated in immutans, indicating that the effects of excitation pressure during greening were also detectable in mitochondria. We conclude that mutations involving components of the photosynthetic electron transport chain, such as those present in immutans, spotty, var1, and var2, predispose Arabidopsis chloroplasts to photooxidation under high excitation pressure, resulting in the variegated phenotype. PMID:19897671

  9. Photosynthetic Redox Imbalance Governs Leaf Sectoring in the Arabidopsis thaliana Variegation Mutants immutans, spotty, var1, and var2[W

    PubMed Central

    Rosso, Dominic; Bode, Rainer; Li, Wenze; Krol, Marianna; Saccon, Diego; Wang, Shelly; Schillaci, Lori A.; Rodermel, Steven R.; Maxwell, Denis P.; Hüner, Norman P.A.

    2009-01-01

    We hypothesized that chloroplast energy imbalance sensed through alterations in the redox state of the photosynthetic electron transport chain, measured as excitation pressure, governs the extent of variegation in the immutans mutant of Arabidopsis thaliana. To test this hypothesis, we developed a nondestructive imaging technique and used it to quantify the extent of variegation in vivo as a function of growth temperature and irradiance. The extent of variegation was positively correlated (R2 = 0.750) with an increase in excitation pressure irrespective of whether high light, low temperature, or continuous illumination was used to induce increased excitation pressure. Similar trends were observed with the variegated mutants spotty, var1, and var2. Measurements of greening of etiolated wild-type and immutans cotyledons indicated that the absence of IMMUTANS increased excitation pressure twofold during the first 6 to 12 h of greening, which led to impaired biogenesis of thylakoid membranes. In contrast with IMMUTANS, the expression of its mitochondrial analog, AOX1a, was transiently upregulated in the wild type but permanently upregulated in immutans, indicating that the effects of excitation pressure during greening were also detectable in mitochondria. We conclude that mutations involving components of the photosynthetic electron transport chain, such as those present in immutans, spotty, var1, and var2, predispose Arabidopsis chloroplasts to photooxidation under high excitation pressure, resulting in the variegated phenotype. PMID:19897671

  10. Multilocus Sequence Typing of Serially Collected Isolates of Cryptococcus from HIV-Infected Patients in South Africa

    PubMed Central

    Van Wyk, Marelize; Govender, Nelesh P.; Litvintseva, Anastasia P.

    2014-01-01

    Patients with cryptococcal meningitis in sub-Saharan Africa frequently relapse following treatment. The natural history and etiology of these recurrent episodes warrant investigation. Here, we used multilocus sequence typing (MLST) to compare the molecular genotypes of strains of Cryptococcus neoformans and Cryptococcus gattii isolated from serial episodes of cryptococcal meningitis that were separated by at least 110 days. The most common MLST genotypes among the isolates were the dominant global clinical genotypes (M5 and M4) of molecular type VNI, as well as the VNI genotypes apparently restricted to southern Africa. In addition, there was considerable genetic diversity among these South African isolates, as 15% of the patients had unique genotypes. Eleven percent of the patients were reinfected with a genetically different strain following their initial diagnosis and treatment. However, the majority of serial episodes (89%) were caused by strains with the same genotype as the original strain. These results indicate that serial episodes of cryptococcosis in South Africa are frequently associated with persistence or relapse of the original infection. Using a reference broth microdilution method, we found that the serial isolates of 11% of the patients infected with strains of C. neoformans var. grubii with identical genotypes exhibited ≥4-fold increases in the MICs to fluconazole. Therefore, these recurrent episodes may have been precipitated by inadequate induction or consolidation of antifungal treatment and occasionally may have been due to increased resistance to fluconazole, which may have developed during the chronic infection. PMID:24648562

  11. An in vivo and in vitro model of Plasmodium falciparum rosetting and autoagglutination mediated by varO, a group A var gene encoding a frequent serotype.

    PubMed

    Vigan-Womas, Inès; Guillotte, Micheline; Le Scanf, Cécile; Igonet, Sébastien; Petres, Stéphane; Juillerat, Alexandre; Badaut, Cyril; Nato, Farida; Schneider, Achim; Lavergne, Anne; Contamin, Hugues; Tall, Adama; Baril, Laurence; Bentley, Graham A; Mercereau-Puijalon, Odile

    2008-12-01

    In the Saimiri sciureus monkey, erythrocytes infected with the varO antigenic variant of the Plasmodium falciparum Palo Alto 89F5 clone bind uninfected red blood cells (rosetting), form autoagglutinates, and have a high multiplication rate, three phenotypic characteristics that are associated with severe malaria in human patients. We report here that varO parasites express a var gene having the characteristics of group A var genes, and we show that the varO Duffy binding-like 1alpha(1) (DBL1alpha(1)) domain is implicated in the rosetting of both S. sciureus and human erythrocytes. The soluble varO N-terminal sequence (NTS)-DBL1alpha(1) recombinant domain, produced in a baculovirus-insect cell system, induced high titers of antibodies that reacted with varO-infected red blood cells and disrupted varO rosettes. varO parasites were culture adapted in vitro using human erythrocytes. They formed rosettes and autoagglutinates, and they had the same surface serotype and expressed the same varO gene as the monkey-propagated parasites. To develop an in vitro model with highly homogeneous varO parasites, rosette purification was combined with positive selection by panning with a varO NTS-DBL1alpha(1)-specific mouse monoclonal antibody. The single-variant, clonal parasites were used to analyze seroprevalence for varO at the village level in a setting where malaria is holoendemic (Dielmo, Senegal). We found 93.6% (95% confidence interval, 89.7 to 96.4%) seroprevalence for varO surface-reacting antibodies and 86.7% (95% confidence interval, 82.8 to 91.6%) seroprevalence for the recombinant NTS-DBL1alpha(1) domain, and virtually all permanent residents had seroconverted by the age of 5 years. These data imply that the varO model is a relevant in vivo and in vitro model for rosetting and autoagglutination that can be used for rational development of vaccine candidates and therapeutic strategies aimed at preventing malaria pathology. PMID:18809668

  12. The complete chloroplast genome sequence of Dianthus superbus var. longicalycinus.

    PubMed

    Gurusamy, Raman; Lee, Do-Hyung; Park, SeonJoo

    2016-05-01

    The complete chloroplast genome (cpDNA) sequence of Dianthus superbus var. longicalycinus is an economically important traditional Chinese medicine was reported and characterized. The cpDNA of Dianthus superbus var. longicalycinus is 149,539 bp, with 36.3% GC content. A pair of inverted repeats (IRs) of 24,803 bp is separated by a large single-copy region (LSC, 82,805 bp) and a small single-copy region (SSC, 17,128 bp). It encodes 85 protein-coding genes, 36 tRNA genes and 8 rRNA genes. Of 129 individual genes, 13 genes encoded one intron and three genes have two introns. PMID:25354144

  13. AI approach to optimal var control with fuzzy reactive loads

    SciTech Connect

    Abdul-Rahman, K.H.; Shahidehpour, S.M.; Daneshdoost, M.

    1995-02-01

    This paper presents an artificial intelligence (AI) approach to the optimal reactive power (var) control problem. The method incorporates the reactive load uncertainty in optimizing the overall system performance. The artificial neural network (ANN) enhanced by fuzzy sets is used to determine the memberships of control variables corresponding to the given load values. A power flow solution will determine the corresponding state of the system. Since the resulting system state may not be feasible in real-time, a heuristic method based on the application of sensitivities in expert system is employed to refine the solution with minimum adjustments of control variables. Test cases and numerical results demonstrate the applicability of the proposed approach. Simplicity, processing speed and ability to model load uncertainties make this approach a viable option for on-line var control.

  14. [Photosynthetic parameters and physiological indexes of Paris polyphylla var. yunnanensis influenced by arbuscular mycorrhizal fungi].

    PubMed

    Wei, Zheng-xin; Guo, Dong-qin; Li, Hai-feng; Ding, Bo; Zhang, Jie; Zhou, Nong; Yu, Jie

    2015-10-01

    Through potted inoculation test at room temperature and indoor analysis, the photosynthetic parameters and physiological and biochemical indexes of Paris polyphylla var. yunnanensis were observed after 28 arbuscular mycorrhizal (AM) fungi were injected into the P. polyphylla var. yunnanensis growing in a sterile soil environment. The results showed that AM fungi established a good symbiosis with P. polyphylla var. yunnanensis. The AM fungi influenced the photosynthetic parameters and physiological and biochemical indexes of P. polyphylla var. yunnanensis. And the influences were varied depending on different AM fungi. The application of AM fungi improved photosynthesis intensity of P. polyphylla var. yunnanensis mesophyll cells, the contents of soluble protein and soluble sugar, protective enzyme activity of P. polyphylla var. yunnanensis leaf, which was beneficial to resist the adverse environment and promote the growth of P. polyphylla var. yunnanensis. Otherwise, there was a certain mutual selectivity between P. polyphylla var. yunnanensis and AM fungi. From the comprehensive effect of inoculation, Racocetra coralloidea, Scutellospora calospora, Claroideoglomus claroideum, S. pellucida and Rhizophagus clarus were the most suitable AM fungi to P. polyphylla var. yunnanensis when P. polyphylla var. yunnanensis was planted in the field. PMID:27062807

  15. Differential var gene transcription in Plasmodium falciparum isolates from patients with cerebral malaria compared to hyperparasitaemia

    PubMed Central

    Kyriacou, Helen M.; Stone, Graham N.; Challis, Richard J.; Raza, Ahmed; Lyke, Kirsten E.; Thera, Mahamadou A.; Koné, Abdoulaye K.; Doumbo, Ogobara K.; Plowe, Christopher V.; Rowe, J. Alexandra

    2006-01-01

    The Plasmodium falciparum variant erythrocyte surface antigens known as PfEMP1, encoded by the var gene family, are thought to play a crucial role in malaria pathogenesis because they mediate adhesion to host cells and immuno-modulation. Var genes have been divided into three major groups (A, B and C) and two intermediate groups (B/A and B/C) on the basis of their genomic location and upstream sequence. We analysed expressed sequence tags of the var gene DBLα domain to investigate var gene transcription in relation to disease severity in Malian children. We found that P. falciparum isolates from children with cerebral malaria (unrousable coma) predominantly transcribe var genes with DBLα1-like domains that are characteristic of Group A or B/A var genes. In contrast, isolates from children with equally high parasite burdens but no symptoms or signs of severe malaria (hyperparasitaemia patients) predominantly transcribe var genes with DBLα0-like domains that are characteristic of the B and C-related var gene groups. These results suggest that var genes with DBLα1-like domains (Group A or B/A) may be implicated in the pathogenesis of cerebral malaria, while var genes with DBLα0-like domains promote less virulent malaria infections. PMID:16996149

  16. Dynamic simulation of Static Var Compensators in distribution systems

    SciTech Connect

    Koessler, R.J. )

    1992-08-01

    This paper is a system study guide for the correction of voltage dips due to large motor startups with Static Var Compensators (SVCs). The method utilizes time simulations, which are an important aid in the equipment design and specification. The paper illustrates the process of setting-up a computer model and performing time simulations. The study process is demonstrated through an example, the Shawnee feeder in the Niagara Mohawk Power Corporation service area.

  17. Graft-related endocarditis caused by Neosartorya fischeri var. spinosa.

    PubMed Central

    Summerbell, R C; de Repentigny, L; Chartrand, C; St Germain, G

    1992-01-01

    The first case of endocarditis caused by Neosartorya fischeri var. spinosa is reported. The patient was a child who received a calf pericardium graft after removal of a previously inserted Dacron graft associated with deterioration of adjacent tissue. Copious vegetations removed from the heart were found to be composed of septate hyaline fungal filaments. The fungus was recognized in culture by its bivalved, winged, spiny ascospores, its Aspergillus fischerianus anamorph, and its thermotolerance. Images PMID:1624579

  18. Transient response of a static VAR shunt compensator

    SciTech Connect

    Best, R.A.; Zelaya-De La Parra, H.

    1996-05-01

    A typical static VAR shunt compensator has been analyzed so that the step response and steady-state errors can be identified. The results show that the steady-state error is dependent upon the error in the measurement of the currents` phase alone. They also show that an unstable condition can occur, though it should rarely arise in practice. All the theory was verified on a low power (240 V, 3 A) system.

  19. Biotransformation of patchoulol by Cunninghamella echinulata var. elegans.

    PubMed

    Xu, Fangfang; Liao, Kangsheng; Liu, Yuhong; Zhang, Zhenbiao; Guo, Dean; Su, Ziren; Liu, Bo

    2016-03-01

    Biocatalysis of patchoulol (PA) was performed by the fungus Cunninghamella echinulata var. elegans. Eight metabolites (1-8) including four new compounds were obtained, and their structures were elucidated as (5R,8S)-5,8 dihydroxypatchoulol (1), (5R*,9R*)-5,9 dihydroxypatchoulol (2), (6S*, 9S*)-6,9 dihydroxypatchoulol (3), and (4R*)-4 hydroxypatchoulol (4) by spectroscopic analysis. The absolute configuration of 1 was determined by single crystal X-ray diffraction. PMID:26778089

  20. New clerodane diterpenes from Tinospora sagittata var. yunnanensis.

    PubMed

    Jiang, Zhi-Yong; Li, Wen-Juan; Jiao, Li-Xiang; Guo, Jun-Ming; Tian, Kai; Yang, Chun-Tao; Huang, Xiang-Zhong

    2014-03-01

    Four new clerodane diterpenes, namely sagittatayunnanosides A-D (1-4), were isolated from the roots of Tinospora sagittata var. yunnanensis, together with two known compounds, tinospinoside C (5) and tinospinoside E (6). The structures of the four new compounds were well elucidated by extensive analyses of the MS, IR, and 1D and 2D NMR data. The cytotoxic and antifouling activities of compounds 1-6 were evaluated. PMID:24634023

  1. Cryptococcosis

    MedlinePlus

    ... Stiff neck Tests that may be done include: Blood culture CT scan of the head Sputum culture and ... sample of cerebrospinal fluid (CSF) Cerebrospinal fluid (CSF) ... that the Cryptococcus neoformans fungus can shed into the blood)

  2. New secondary metabolites from bioactive extracts of the fungus Armillaria tabescens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ethyl acetate extracts of Armillaria tabescens (strain JNB-OZ344) mycelium showed significant fungistatic and bacteristatic activities against several major human pathogens including Candida albicans, Cryptococcus neoformans, Escherichia coli and Mycobacterium intracellulare. Chemical analysis of th...

  3. CHARACTERIZATION ADN BIOLOGICAL ACTIVITY OF SECONDARY METABOLITES FROM ARMILLARIA TABESCENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ethyl acetate extracts from liquid cultures of Armillaria tabescens showed good antimicrobial activity against Candida albicans, Cryptococcus neoformans, Escherichia coli and Mycobacterium intracellulare. Chemical analyses of extract constituents led to the isolation and identification of two new co...

  4. HUMAN ALVEOLAR AND PERITONEAL MACROPHAGES MEDIATE FUNGISTASIS INDEPENDENTLY OF L-ARGININE OXIDATION TO NITRITE OR NITRATE

    EPA Science Inventory

    Human alveolar macrophages (HAM) from 28 normal volunteers were found to inhibit replication of Cryptoccous neoformans. onditions under which fungistasis occurred were different than those required for mouse peritoneal macrophage-mediated fungi stasis. nhibition of fungal replica...

  5. New prediction for the direct CP-violating parameter {var_epsilon}{prime}/{var_epsilon} and the {Delta}I=1/2 rule

    SciTech Connect

    Wu, Yue-Liang

    2001-07-01

    The low-energy dynamics of QCD is investigated with special attention paid to the matching between QCD and chiral perturbation theory (ChPT), and also to some useful algebraic chiral operator relations which survive even when we include chiral loop corrections. It then allows us to evaluate the hadronic matrix elements below the energy scale {Lambda}{sub {chi}}{approx_equal}1GeV. Based on the new analyses, we present a consistent prediction for both the direct CP-violating parameter {var_epsilon}{prime}/{var_epsilon} and the {Delta}I=1/2 rule in kaon decays. In the leading 1/N{sub c} approximation, the isospin amplitudes A{sub 0} and A{sub 2} are found to agree well with the data, and the direct CP-violating parameter {var_epsilon}{prime}/{var_epsilon} is predicted to be large, which also confirms our earlier conclusion. Its numerical value is {var_epsilon}{prime}/{var_epsilon}=23.6{sub {minus}7.8}{sup +12.4}{times}10{sup {minus}4}(Im{lambda}{sub t}/1.2{times}10{sup {minus}4}) which is no longer sensitive to the strange quark mass due to the matching conditions. Taking into account a simultaneous consistent analysis on the isospin amplitudes A{sub 0} and A{sub 2}, the ratio {var_epsilon}{prime}/{var_epsilon} is in favor of the values {var_epsilon}{prime}/{var_epsilon}=(20{+-}9){times}10{sup {minus}4}.

  6. Mosquito Passage Dramatically Changes var Gene Expression in Controlled Human Plasmodium falciparum Infections

    PubMed Central

    Bachmann, Anna; Petter, Michaela; Krumkamp, Ralf; Esen, Meral; Held, Jana; Scholz, Judith A. M.; Li, Tao; Sim, B. Kim Lee; Hoffman, Stephen L.; Kremsner, Peter G.; Mordmüller, Benjamin; Duffy, Michael F.; Tannich, Egbert

    2016-01-01

    Virulence of the most deadly malaria parasite Plasmodium falciparum is linked to the variant surface antigen PfEMP1, which is encoded by about 60 var genes per parasite genome. Although the expression of particular variants has been associated with different clinical outcomes, little is known about var gene expression at the onset of infection. By analyzing controlled human malaria infections via quantitative real-time PCR, we show that parasite populations from 18 volunteers expressed virtually identical transcript patterns that were dominated by the subtelomeric var gene group B and, to a lesser extent, group A. Furthermore, major changes in composition and frequency of var gene transcripts were detected between the parental parasite culture that was used to infect mosquitoes and Plasmodia recovered from infected volunteers, suggesting that P. falciparum resets its var gene expression during mosquito passage and starts with the broad expression of a specific subset of var genes when entering the human blood phase. PMID:27070311

  7. Mosquito Passage Dramatically Changes var Gene Expression in Controlled Human Plasmodium falciparum Infections.

    PubMed

    Bachmann, Anna; Petter, Michaela; Krumkamp, Ralf; Esen, Meral; Held, Jana; Scholz, Judith A M; Li, Tao; Sim, B Kim Lee; Hoffman, Stephen L; Kremsner, Peter G; Mordmüller, Benjamin; Duffy, Michael F; Tannich, Egbert

    2016-04-01

    Virulence of the most deadly malaria parasite Plasmodium falciparum is linked to the variant surface antigen PfEMP1, which is encoded by about 60 var genes per parasite genome. Although the expression of particular variants has been associated with different clinical outcomes, little is known about var gene expression at the onset of infection. By analyzing controlled human malaria infections via quantitative real-time PCR, we show that parasite populations from 18 volunteers expressed virtually identical transcript patterns that were dominated by the subtelomeric var gene group B and, to a lesser extent, group A. Furthermore, major changes in composition and frequency of var gene transcripts were detected between the parental parasite culture that was used to infect mosquitoes and Plasmodia recovered from infected volunteers, suggesting that P. falciparum resets its var gene expression during mosquito passage and starts with the broad expression of a specific subset of var genes when entering the human blood phase. PMID:27070311

  8. A new culture medium for recovering the agents of Cryptococcosis from environmental sources

    PubMed Central

    Silva, Dulcilena de Matos Castro e; Santos, Dayane C.S.; Pukinskas, Sandra R.B.S.; Oshida, Julia T.U.; Oliveira, Lidiane; Carvallho, Anderson F.; Melhem, Márcia S.C.

    2015-01-01

    The isolation of Cryptococcosis agents from environmental samples may be difficult due to the presence of groups of fast-growing fungi. We propose a new culture medium based on a modification of Dichloran Rose-Bengal Chloramphenicol Medium (DRBCm) to detect colonies of Cryptococcus neoformans. Our results indicate that DRBCm is superior to the classical Bird Seed Agar in its ability to detect colonies of C. neoformans. PMID:26273249

  9. A new culture medium for recovering the agents of Cryptococcosis from environmental sources.

    PubMed

    Castro e Silva, Dulcilena de Matos; Santos, Dayane C S; Pukinskas, Sandra R B S; Oshida, Julia T U; Oliveira, Lidiane; Carvallho, Anderson F; Melhem, Márcia S C

    2015-06-01

    The isolation of Cryptococcosis agents from environmental samples may be difficult due to the presence of groups of fast-growing fungi. We propose a new culture medium based on a modification of Dichloran Rose-Bengal Chloramphenicol Medium (DRBCm) to detect colonies of Cryptococcus neoformans. Our results indicate that DRBCm is superior to the classical Bird Seed Agar in its ability to detect colonies of C. neoformans. PMID:26273249

  10. Primary cryptococcal prostatitis--rare occurrence.

    PubMed

    Shah, Vinaya B; Patil, Pallavi A; Agrawa, Vipul; Kaswan, Harish K

    2012-05-01

    Cryptococcosis is a well recognized infection in immunocompromised patients. Cryptococcal infection primarily involves the lung and is hematogeneously spread to other organs. Sometimes it might affect the genitourinary tract. The prostate gland is a rare site of primary infection due to cryptococcus neoformans. We report a case of granulomatous inflammation in the prostate as a result of crypyococcus neoformans infection in a 70 year old immunocompetent patient, a non diabetic, which was diagnosed by transrectal ultrasound guided biopsy. PMID:23029729

  11. Differential expression of var gene groups is associated with morbidity caused by Plasmodium falciparum infection in Tanzanian children.

    PubMed

    Rottmann, Matthias; Lavstsen, Thomas; Mugasa, Joseph Paschal; Kaestli, Mirjam; Jensen, Anja T R; Müller, Dania; Theander, Thor; Beck, Hans-Peter

    2006-07-01

    The var gene family of Plasmodium falciparum encodes the variant surface antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 is considered an important pathogenicity factor in P. falciparum infection because it mediates cytoadherence to host cell endothelial receptors. var genes can be grouped into three major groups, A, B, and C, and the conserved var genes, var1-4, according to sequence similarities in coding and noncoding upstream regions. Using real-time quantitative PCR in a study conducted in Tanzania, the var transcript abundances of the different var gene groups were compared among patients with severe, uncomplicated, and asymptomatic malaria. Transcripts of var group A and B genes were more abundant in patients with severe malaria than in patients with uncomplicated malaria. In general, the transcript abundances of var group A and B genes were higher for children with clinical malaria than for children with asymptomatic infections. The var group C and var1-like transcript abundances were similar between the three sample groups. A transcript abundance pattern similar to that for var group A was observed for var2csa and var3-like genes. These results suggest that substantial and systematic differences in var gene expression exist between different clinical presentations. PMID:16790763

  12. Structural insight into epitopes in the pregnancy-associated malaria protein VAR2CSA.

    PubMed

    Andersen, Pernille; Nielsen, Morten A; Resende, Mafalda; Rask, Thomas S; Dahlbäck, Madeleine; Theander, Thor; Lund, Ole; Salanti, Ali

    2008-02-01

    Pregnancy-associated malaria is caused by Plasmodium falciparum malaria parasites binding specifically to chondroitin sulfate A in the placenta. This sequestration of parasites is a major cause of low birth weight in infants and anemia in the mothers. VAR2CSA, a polymorphic multi-domain protein of the PfEMP1 family, is the main parasite ligand for CSA binding, and identification of protective antibody epitopes is essential for VAR2CSA vaccine development. Attempts to determine the crystallographic structures of VAR2CSA or its domains have not been successful yet. In this study, we propose 3D models for each of the VAR2CSA DBL domains and we show that regions in the fold of VAR2CSA inter-domain 2 and a PfEMP1 CIDR domain seem to be homologous to the EBA-175 and Pk alpha-DBL fold. This suggests that ID2 could be a functional domain. We also identify regions of VAR2CSA present on the surface of native VAR2CSA by comparing reactivity of plasma containing anti-VAR2CSA antibodies in peptide array experiments before and after incubation with native VAR2CSA. By this method we identify conserved VAR2CSA regions targeted by antibodies that react with the native molecule expressed on infected erythrocytes. By mapping the data onto the DBL models we present evidence suggesting that the S1+S2 DBL sub-domains are generally surface-exposed in most domains, whereas the S3 sub-domains are less exposed in native VAR2CSA. These results comprise an important step towards understanding the structure of VAR2CSA on the surface of CSA-binding infected erythrocytes. PMID:18282103

  13. VT-1161 Protects Immunosuppressed Mice from Rhizopus arrhizus var. arrhizus Infection

    PubMed Central

    Gebremariam, Teclegiorgis; Wiederhold, Nathan P.; Fothergill, Annette W.; Garvey, Edward P.; Hoekstra, William J.; Schotzinger, Robert J.; Patterson, Thomas F.; Filler, Scott G.

    2015-01-01

    We studied the efficacy of the investigational drug VT-1161 against mucormycosis. VT-1161 had more potent in vitro activity against Rhizopus arrhizus var. arrhizus than against R. arrhizus var. delemar. VT-1161 treatment demonstrated dose-dependent plasma drug levels with prolonged survival time and lowered tissue fungal burden in immunosuppressed mice infected with R. arrhizus var. arrhizus and was as effective as high-dose liposomal amphotericin B treatment. These results support further development of VT-1161 against mucormycosis. PMID:26369977

  14. VT-1161 Protects Immunosuppressed Mice from Rhizopus arrhizus var. arrhizus Infection.

    PubMed

    Gebremariam, Teclegiorgis; Wiederhold, Nathan P; Fothergill, Annette W; Garvey, Edward P; Hoekstra, William J; Schotzinger, Robert J; Patterson, Thomas F; Filler, Scott G; Ibrahim, Ashraf S

    2015-12-01

    We studied the efficacy of the investigational drug VT-1161 against mucormycosis. VT-1161 had more potent in vitro activity against Rhizopus arrhizus var. arrhizus than against R. arrhizus var. delemar. VT-1161 treatment demonstrated dose-dependent plasma drug levels with prolonged survival time and lowered tissue fungal burden in immunosuppressed mice infected with R. arrhizus var. arrhizus and was as effective as high-dose liposomal amphotericin B treatment. These results support further development of VT-1161 against mucormycosis. PMID:26369977

  15. Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum

    PubMed Central

    Amit-Avraham, Inbar; Pozner, Guy; Eshar, Shiri; Fastman, Yair; Kolevzon, Netanel; Yavin, Eylon; Dzikowski, Ron

    2015-01-01

    The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression. PMID:25691743

  16. Observations on the formation of [var epsilon] martensite in an Fe-23. 2%Mn alloy

    SciTech Connect

    Akguen, I.; Durlu, T.N. . Dept. of Physics)

    1994-11-15

    In Fe-Mn binary alloys the formation behavior of [var epsilon] martensite is quite sensitive to the Mn percentage and although both [var epsilon] and [alpha][prime] type martensites are formed in low Mn alloys, mostly [gamma] [yields] [var epsilon] transformation occur as the Mn concentration is increased. The present study was undertaken to examine the formation of thermally induced and also strain-induced [var epsilon] martensites, and their intersections in a Fe-23.2%Mn alloy by using transmission electron microscopy techniques.

  17. Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis

    PubMed Central

    Mor, Visesato; Farnoud, Amir M.; Singh, Ashutosh; Rella, Antonella; Tanno, Hiromasa; Ishii, Keiko; Kawakami, Kazuyoshi; Sato, Toshiya; Del Poeta, Maurizio

    2016-01-01

    Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus. PMID:27082428

  18. Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis.

    PubMed

    Mor, Visesato; Farnoud, Amir M; Singh, Ashutosh; Rella, Antonella; Tanno, Hiromasa; Ishii, Keiko; Kawakami, Kazuyoshi; Sato, Toshiya; Del Poeta, Maurizio

    2016-01-01

    Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus. PMID:27082428

  19. Regulation of Sugar Transport Systems in Fusarium oxysporum var. lini

    PubMed Central

    Brandão, Rogélio L.; Loureiro-Dias, Maria C.

    1990-01-01

    Fusarium oxysporum var. lini (ATCC 10960) formed a facilitated diffusion system for glucose (Ks, about 10 mM) when grown under repressed conditions. Under conditions of derepression, the same system was present together with a high-affinity (Ks, about 40 μM) active system. The maximum velocity of the latter was about 5% of that of the facilitated diffusion system. The high-affinity system was under the control of glucose repression and glucose inactivation. When lactose was the only carbon source in the medium, a facilitated diffusion system for lactose was found (Ks, about 30 mM). PMID:16348256

  20. AmeriFlux US-Var Vaira Ranch- Ione

    SciTech Connect

    Baldocchi, Dennis

    2016-01-01

    This is the AmeriFlux version of the carbon flux data for the site US-Var Vaira Ranch- Ione. Site Description - Located in the lower foothills of the Sierra Nevada Mountains on privately owned land, the Vaira Ranch site is classified as a grassland dominated by C3 annual grasses. Managed by local rancher, Fran Vaira, brush has been periodically removed for cattle grazing. Species include a variety of grasses and herbs, including purple false brome, smooth cat's ear, and rose clover. Growing season is confined to the wet season only, typically from October to early May.

  1. [Study on chemical constituents of Drosera peltata var. multisepala].

    PubMed

    Li, Lin; Huang, Jin; Xu, Xianghua; Zhang, Yao; Cheng, Kejun; Yu, Peizhong

    2012-01-01

    Chemical investigatation of Drosera peltata var. multisepala led to the isolation of eleven compounds using various chromatographic techniques. The structures of these compounds were elucidated as isoshinanolone-4-O-beta-D-glucoside (1), isoshinanolone (2), epi-isoshinanolone (3), plumbagin (4), droserone (5), droserone-5-O-glucoside (6), quercetin (7), kaempferol (8) , gossypetin-8-O-glucoside (9), 3,3'-dimethoxy ellagic acid (10), and ellagic acid (11) by their physicochemical properties and spectral data analysis. Compound 1 was a new compound. Compounds 3, 8, 10, and 11 were isolated from this plant for the first time. PMID:22737855

  2. Effects of ionizing radiation on Capsicum baccatum var. pendulum (Solanaceae).

    PubMed

    Scaldaferro, M A; Prina, A R; Moscone, E A; Kwasniewska, J

    2013-09-01

    Cytogenetic and somatic effects of various x-ray treatments were evaluated in pepper, Capsicum baccatum var. pendulum cv. "Cayenne", with the aim to assess optimal conditions for obtaining viable lines. The cytogenetic effects were quantified by counting chromosome aberrations. The level of DNA fragmentation was estimated with TUNEL test (terminal transferase mediated dUTP-fluorescein nick end labeling). Irradiation to 20 Gy with 16-h presoaking can be a suitable treatment of the selected pepper cultivar for a mutagenesis program. PMID:23747514

  3. A new jacaranone derivative from Senecio scandens var. incisus.

    PubMed

    Wang, Wen-Shu; Lu, Peng; Duan, Chang-Hong; Feng, Jin-Chao

    2010-03-01

    A new jacaranone derivative, 2-(1,6-dihydroxy-4-oxocyclohex-2-enyl) acetic acid (1), has been isolated from the whole plants of Senecio scandens var. incisus, together with three known compounds: 2'-(p-hydroxyl-cinnamoyl)-6'-jacaranone-D-glucopyranoside (2), 2'-caffeoyl-6'-jacaranone-D-glucopyranoside (3) and kampferol-3-rhamnoside (4). Their structures were elucidated on the basis of spectroscopic data analyses and by comparison with the related known compounds. Cytotoxic activities of 1 against three human tumour cell lines have been evaluated by the MTT method. PMID:20221943

  4. Sesquiterpenes and other constituents from Dendranthema zawadskii var. latilobum.

    PubMed

    Shin, Hyun Jung; Lee, So Young; Kim, Ju Sun; Lee, Sanghyun; Choi, Ran Joo; Chung, Ha Sook; Kim, Yeong Shik; Kang, Sam Sik

    2012-01-01

    Six new germacranolides, zawadskinolides A-F (1-6), and a new eudesmane glucoside, chrysantiloboside (7) were isolated from the aerial parts of Dendranthema zawadskii var. latilobum, along with thirteen known constituents. Their structures were elucidated by means of spectroscopic evidence. Bioassay showed that flavonoids such as apigenin (9), (-)-eriodictyol (10) and nepetin (12), as well as the sesquiterpene lactone, zawadskinolide F (6), inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells with IC50 values of 66.15, 132.55, 35.44, and 91.32 μM, respectively. PMID:22382409

  5. Resistance to Southern Root-knot Nematode (Meloidogyne incognita) in Wild Watermelon (Citrullus lanatus var. citroides)

    PubMed Central

    Thies, Judy A.; Ariss, Jennifer J.; Kousik, Chandrasekar S.; Hassell, Richard L.; Levi, Amnon

    2016-01-01

    Southern root-knot nematode (RKN, Meloidogyne incognita) is a serious pest of cultivated watermelon (Citrullus lanatus var. lanatus) in southern regions of the United States and no resistance is known to exist in commercial watermelon cultivars. Wild watermelon relatives (Citrullus lanatus var. citroides) have been shown in greenhouse studies to possess varying degrees of resistance to RKN species. Experiments were conducted over 2 yr to assess resistance of southern RKN in C. lanatus var. citroides accessions from the U.S. Watermelon Plant Introduction Collection in an artificially infested field site at the U.S. Vegetable Laboratory in Charleston, SC. In the first study (2006), 19 accessions of C. lanatus var. citroides were compared with reference entries of Citrullus colocynthis and C. lanatus var. lanatus. Of the wild watermelon accessions, two entries exhibited significantly less galling than all other entries. Five of the best performing C. lanatus var. citroides accessions were evaluated with and without nematicide at the same field site in 2007. Citrullus lanatus var. citroides accessions performed better than C. lanatus var. lanatus and C. colocynthis. Overall, most entries of C. lanatus var. citroides performed similarly with and without nematicide treatment in regard to root galling, visible egg masses, vine vigor, and root mass. In both years of field evaluations, most C. lanatus var. citroides accessions showed lesser degrees of nematode reproduction and higher vigor and root mass than C. colocynthis and C. lanatus var. lanatus. The results of these two field evaluations suggest that wild watermelon populations may be useful sources of resistance to southern RKN. PMID:27168648

  6. Resistance to Southern Root-knot Nematode (Meloidogyne incognita) in Wild Watermelon (Citrullus lanatus var. citroides).

    PubMed

    Thies, Judy A; Ariss, Jennifer J; Kousik, Chandrasekar S; Hassell, Richard L; Levi, Amnon

    2016-03-01

    Southern root-knot nematode (RKN, Meloidogyne incognita) is a serious pest of cultivated watermelon (Citrullus lanatus var. lanatus) in southern regions of the United States and no resistance is known to exist in commercial watermelon cultivars. Wild watermelon relatives (Citrullus lanatus var. citroides) have been shown in greenhouse studies to possess varying degrees of resistance to RKN species. Experiments were conducted over 2 yr to assess resistance of southern RKN in C. lanatus var. citroides accessions from the U.S. Watermelon Plant Introduction Collection in an artificially infested field site at the U.S. Vegetable Laboratory in Charleston, SC. In the first study (2006), 19 accessions of C. lanatus var. citroides were compared with reference entries of Citrullus colocynthis and C. lanatus var. lanatus. Of the wild watermelon accessions, two entries exhibited significantly less galling than all other entries. Five of the best performing C. lanatus var. citroides accessions were evaluated with and without nematicide at the same field site in 2007. Citrullus lanatus var. citroides accessions performed better than C. lanatus var. lanatus and C. colocynthis. Overall, most entries of C. lanatus var. citroides performed similarly with and without nematicide treatment in regard to root galling, visible egg masses, vine vigor, and root mass. In both years of field evaluations, most C. lanatus var. citroides accessions showed lesser degrees of nematode reproduction and higher vigor and root mass than C. colocynthis and C. lanatus var. lanatus. The results of these two field evaluations suggest that wild watermelon populations may be useful sources of resistance to southern RKN. PMID:27168648

  7. Characterization of 12 polymorphic microsatellite loci of Pityopsis graminifolia var. latifolia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pityopsis graminifolia (Michx.) Small var. latifolia (Fern.) Semple is an herbaceous perennial that grows in close proximity to the federally endangered species P. ruthii (Small) Small. Twelve polymorphic microsatellite loci were identified from 87 samples of P. graminifolia var. latifolia and addit...

  8. A well-conserved Plasmodium falciparum var gene shows an unusual stage-specific transcript pattern

    PubMed Central

    Kyes, Sue A.; Christodoulou, Zoe; Raza, Ahmed; Horrocks, Paul; Pinches, Robert; Rowe, J. Alexandra; Newbold, Chris I.

    2010-01-01

    Summary The var multicopy gene family encodes Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variant antigens, which, through their ability to adhere to a variety of host receptors, are thought to be important virulence factors. The predominant expression of a single cytoadherent PfEMP1 type on an infected red blood cell, and the switching between different PfEMP1 types to evade host protective antibody responses, are processes thought to be controlled at the transcriptional level. Contradictory data have been published on the timing of var gene transcription. Reverse transcription-polymerase chain reaction (RT-PCR) data suggested that transcription of the predominant var gene occurs in the later (pigmented trophozoite) stages, whereas Northern blot data indicated such transcripts only in early (ring) stages. We investigated this discrepancy by Northern blot, with probes covering a diverse var gene repertoire. We confirm that almost all var transcript types were detected only in ring stages. However, one type, the well-conserved varCSA transcript, was present constitutively in different laboratory parasites and does not appear to undergo antigenic variation. Although varCSA has been shown to encode a chondroitin sulphate A (CSA)-binding PfEMP1, we find that the presence of full-length varCSA transcripts does not correlate with the CSA-binding phenotype. PMID:12787360

  9. Generation of Antigenic Diversity in Plasmodium falciparum by Structured Rearrangement of Var Genes During Mitosis

    PubMed Central

    Kekre, Mihir; Otto, Thomas D.; Faizullabhoy, Adnan; Rayner, Julian C.; Kwiatkowski, Dominic

    2014-01-01

    The most polymorphic gene family in P. falciparum is the ∼60 var genes distributed across parasite chromosomes, both in the subtelomeres and in internal regions. They encode hypervariable surface proteins known as P. falciparum erythrocyte membrane protein 1 (PfEMP1) that are critical for pathogenesis and immune evasion in Plasmodium falciparum. How var gene sequence diversity is generated is not currently completely understood. To address this, we constructed large clone trees and performed whole genome sequence analysis to study the generation of novel var gene sequences in asexually replicating parasites. While single nucleotide polymorphisms (SNPs) were scattered across the genome, structural variants (deletions, duplications, translocations) were focused in and around var genes, with considerable variation in frequency between strains. Analysis of more than 100 recombination events involving var exon 1 revealed that the average nucleotide sequence identity of two recombining exons was only 63% (range: 52.7–72.4%) yet the crossovers were error-free and occurred in such a way that the resulting sequence was in frame and domain architecture was preserved. Var exon 1, which encodes the immunologically exposed part of the protein, recombined in up to 0.2% of infected erythrocytes in vitro per life cycle. The high rate of var exon 1 recombination indicates that millions of new antigenic structures could potentially be generated each day in a single infected individual. We propose a model whereby var gene sequence polymorphism is mainly generated during the asexual part of the life cycle. PMID:25521112

  10. Refractory and/or Relapsing Cryptococcosis Associated with Acquired Immune Deficiency Syndrome: Clinical Features, Genotype, and Virulence Factors of Cryptococcus spp. Isolates.

    PubMed

    Nascimento, Erika; Vitali, Lucia H; Tonani, Ludmilla; Kress, Marcia R Von Zeska; Takayanagui, Osvaldo M; Martinez, Roberto

    2016-05-01

    Refractory and relapsing crytocococcosis in acquired immune deficiency syndrome (AIDS) patients have a poor prognosis. The risk factors for this complicated infection course were evaluated by comparing refractory and/or relapsing cryptococcosis in human immunodeficiency virus-coinfected patients (cohort 1) with another group of AIDS patients who adequately responded to antifungals (cohort 2). Except for one isolate of Cryptococcus gattii from a cohort 2 case, all other isolates were identified as Cryptococcus neoformans var. grubii, sex type α, genotype VNI, including Cryptococcus reisolated from the relapse or in the refractory state. No differences were observed with respect to Cryptococcus capsule size and in the melanin and phospholipase production. The cohort 1 patients presented higher prevalence of cryptococcemia, cerebral dissemination, chronic liver disease, and leucopenia, and have increased death rate. Apparently, the refractory and/or relapsing cryptococcosis in the AIDS patients were more related to the host and the extent of the infection than to the fungal characteristics. PMID:26928832

  11. Skeletal cryptococcosis from 1977 to 2013

    PubMed Central

    Zhou, Heng-Xing; Lu, Lu; Chu, Tianci; Wang, Tianyi; Cao, Daigui; Li, Fuyuan; Ning, Guangzhi; Feng, Shiqing

    2015-01-01

    Skeletal cryptococcosis, an aspect of disseminated cryptococcal disease or isolated skeletal cryptococcal infection, is a rare but treatable disease. However, limited information is available regarding its clinical features, treatment, and prognosis. This systematic review examined all cases published between April 1977 and May 2013 with regard to the factors associated with this disease, including patient sex, age, and epidemiological history; affected sites; clinical symptoms; underlying diseases; laboratory tests; radiological manifestations; and delays in diagnosis, treatment, follow-up assessments, and outcomes. We found that immune abnormality is a risk factor but does not predict mortality; these observations are due to recent Cryptococcus neoformans var gattii (CNVG) outbreaks (Chaturvedi and Chaturvedi, 2011). Dissemination was irrespective of immune status and required combination therapy, and dissemination carried a worse prognosis. Therefore, a database of skeletal cryptococcosis cases should be created. PMID:25642211

  12. A weak-constraint 4DEnsembleVar

    NASA Astrophysics Data System (ADS)

    Amezcua, Javier; Goodliff, Michael; van Leeuwen, Peter Jan

    2016-04-01

    4DEnsembleVar is a hybrid data assimilation method which, besides making use of flow-dependent ensemble covariance information, avoids the computation of tangent-linear and adjoint models for the evolution and observation operators. In this method, the information from the observation time is communicated to the initial time via 4D cross-time covariances. Large systems require localisation of covariance matrices to suppress long-distance spurious elements. In a 4D covariance, however, using static localisation matrices (as it is done in practice) can eliminate the effect of observations if their location (at observational time) is far from that of the variable they are influencing (at the initial time). In lack of time-dependent localisation functions, in this work we propose a simpler option to ameliorate this problem. We exploit the presence of model error to spread the information of observations to more time steps, introducing a weak-constrained 4DEnsembleVar. The benefits of this method are illustrated in the Korteweg-de-Vries system, the Lorenz 1996 system, and a modified shallow water system with simulated convection.

  13. Baccharis megapotamica var. weirii poisoning in water buffalo (Bubalus bubalis).

    PubMed

    Oliveira-Filho, José C; Carmo, Priscila M S; Lucena, Ricardo B; Pierezan, Felipe; Barros, Claudio S L

    2011-05-01

    An outbreak of an acute disease in buffalo (Bubalus bubalis) caused by the ingestion of Baccharis megapotamica var. weirii occurred in the southern region of Brazil. Ten out of 50 buffalo died 24-48 hr after being introduced into a pasture containing abundant amounts of the plant. Factors influencing the ingestion of the plant and consequent toxicosis included hunger, stress caused by shipment, and unfamiliarity with the plant. Clinical signs included serous ocular discharge, incoordination, mild bloat, and muscle trembling. One buffalo was necropsied. Gross findings included dehydration, abundant liquid in the rumen, reddening of the mucosa of forestomachs, abomasum, and intestine, and edema of the wall of the rumen. The main histologic lesions were superficial to full thickness degeneration and necrosis of the stratified epithelium lining the forestomachs, necrosis of the intestinal mucosa, and widespread lymphoid necrosis. A calf (Bos taurus) was fed a single dose of 5 g/kg/body weight of B. megapotamica var. weirii harvested from the same site where the buffalo died. Twenty hours after the administration of the plant this calf died with clinical signs and lesions similar to those observed in the naturally poisoned buffalo. PMID:21908301

  14. The Development and Application of an Integrated VAR Process Model

    NASA Astrophysics Data System (ADS)

    Ballantyne, A. Stewart

    2016-07-01

    The VAR ingot has been the focus of several modelling efforts over the years with the result that the thermal regime in the ingot can be simulated quite realistically. Such models provide important insight into solidification of the ingot but present some significant challenges to the casual user such as a process engineer. To provide the process engineer with a tool to assist in the development of a melt practice, a comprehensive model of the complete VAR process has been developed. A radiation heat transfer simulation of the arc has been combined with electrode and ingot models to develop a platform which accepts typical operating variables (voltage, current, and gap) together with process parameters (electrode size, crucible size, orientation, water flow, etc.) as input data. The output consists of heat flow distributions and solidification parameters in the form of text, comma-separated value, and visual toolkit files. The resulting model has been used to examine the relationship between the assumed energy distribution in the arc and the actual energy flux which arrives at the ingot top surface. Utilizing heat balance information generated by the model, the effects of electrode-crucible orientation and arc gap have been explored with regard to the formation of ingot segregation defects.

  15. Impact of EnVar hybrid assimilation using EnKF ensembles

    NASA Astrophysics Data System (ADS)

    Prasad, V. S.; Johny, C. J.; Sodhi, Jagdeep Singh; Rajagopal, E. N.

    2016-05-01

    Performance of an EnVar hybrid data assimilation system based on 3D Var NGFS (NCMRWF Global Forecast System) of T574 configuration and Ensemble Kalman Filter is investigated. The experiment is conducted during the Indian monsoon season (June-September) 2015 and compared against operational GSI 3D Var system. Two way coupled dual resolution hybrid system with 80 member ensemble of T254L64 configuration are used and forecasts are done for 10days. In hybrid experiment 75% weight is given to ensemble covariance and 25% for static covariance. The forecast skill of experiments over different spatial domains is compared against observations and respective analysis. The hybrid experiment produced significant improvement in forecasts compared to 3D Var in all fields except lower level temperature over tropical regions. Improvement is also seen in the prediction of extreme rainfall events. The prediction of monsoon onset and track of cyclone Ashobaa with hybrid and 3D var system is discussed.

  16. Use of incremental analysis updates in 4D-Var data assimilation

    NASA Astrophysics Data System (ADS)

    Zhang, Banglin; Tallapragada, Vijay; Weng, Fuzhong; Sippel, Jason; Ma, Zaizhong

    2015-12-01

    The four-dimensional variational (4D-Var) data assimilation systems used in most operational and research centers use initial condition increments as control variables and adjust initial increments to find optimal analysis solutions. This approach may sometimes create discontinuities in analysis fields and produce undesirable spin ups and spin downs. This study explores using incremental analysis updates (IAU) in 4D-Var to reduce the analysis discontinuities. IAU-based 4D-Var has almost the same mathematical formula as conventional 4D-Var if the initial condition increments are replaced with time-integrated increments as control variables. The IAU technique was implemented in the NASA/GSFC 4D-Var prototype and compared against a control run without IAU. The results showed that the initial precipitation spikes were removed and that other discontinuities were also reduced, especially for the analysis of surface temperature.

  17. Empirical analysis on future-cash arbitrage risk with portfolio VaR

    NASA Astrophysics Data System (ADS)

    Chen, Rongda; Li, Cong; Wang, Weijin; Wang, Ze

    2014-03-01

    This paper constructs the positive arbitrage position by alternating the spot index with Chinese Exchange Traded Fund (ETF) portfolio and estimating the arbitrage-free interval of futures with the latest trade data. Then, an improved Delta-normal method was used, which replaces the simple linear correlation coefficient with tail dependence correlation coefficient, to measure VaR (Value-at-risk) of the arbitrage position. Analysis of VaR implies that the risk of future-cash arbitrage is less than that of investing completely in either futures or spot market. Then according to the compositional VaR and the marginal VaR, we should increase the futures position and decrease the spot position appropriately to minimize the VaR, which can minimize risk subject to certain revenues.

  18. Occurrence of Pyrodinium bahamense var. compressum along the southern coast of the Baja California Peninsula.

    PubMed

    Gárate-Lizárraga, Ismael; González-Armas, Rogelio

    2011-03-01

    As part of a continuing toxic microalgae monitoring program, 22 phytoplankton samples were collected from July to November 2010 at several sampling stations along the southern coast of the Baja California Peninsula. For the first time, the toxic dinoflagellate Pyrodinium bahamense var. compressum was found along the southeastern and southwestern coasts of the peninsula. P. bahamense var. bahamense was first observed off San José del Cabo, which is an extension of the range of this variety. Both varieties occur as solitary cells. P. bahamense var. compressum occurred at temperatures ranging between 24.5°C and 31°C, whereas var. P.bahamense occurred at 28.5°C to 29°C, indicating its tropical and subtropical nature. Occurrence of P. bahamense var. compressum along this coastline may be related to El Niño 2009-2010. PMID:21276986

  19. Variability of chemical composition and antioxidant activity of essential oils between Myrtus communis var. Leucocarpa DC and var. Melanocarpa DC.

    PubMed

    Petretto, Giacomo Luigi; Maldini, Mariateresa; Addis, Roberta; Chessa, Mario; Foddai, Marzia; Rourke, Jonathan P; Pintore, Giorgio

    2016-04-15

    Essential oils (EOs) from several individuals of Myrtus communis L. (M. communis) growing in different habitats in Sardinia have been studied. The analyses were focused on four groups of samples, namely cultivated and wild M. communis var. melanocarpa DC, characterized by red/purple berries, and cultivated and wild M. communis var. leucocarpa DC, characterized by white berries. Qualitative and quantitative analyses demonstrated different EO fingerprints among the studied samples: cultivated and wild leucocarpa variety differs mainly from the melanocarpa variety by a high amount of myrtenyl acetate (>200 mg/mL and 0.4 mg/mL in leucocarpa and melanocarpa varieties respectively). Conversely, the wild group is characterized by a higher amount, compared with the cultivated species, of linalool (about 110 mg/mL and 20 mg/mL respectively), linalyl acetate (about 24 mg/mL and about 6 mg/mL respectively) whereas EOs of the cultivated plants were rich in pinocarveol-cis compared with wild plants (about 2 mg/mL and about 0.5 mg/mL respectively). Principal component analysis applied to the chromatographic data confirm a differentiation and classification of EOs from the four groups of M. communis plants. Finally, antioxidant activity of the studied EOs shows differences between the various categories of samples. PMID:26616932

  20. Identification of cultured isolates of clinically important yeast species using fluorescent fragment length analysis of the amplified internally transcribed rRNA spacer 2 region

    PubMed Central

    De Baere, Thierry; Claeys, Geert; Swinne, Danielle; Massonet, Caroline; Verschraegen, Gerda; Muylaert, An; Vaneechoutte, Mario

    2002-01-01

    Background The number of patients with yeast infection has increased during the last years. Also the variety of species of clinical importance has increased. Correct species identification is often important for efficient therapy, but is currently mostly based on phenotypic features and is sometimes time-consuming and depends largely on the expertise of technicians. Therefore, we evaluated the feasibility of PCR-based amplification of the internally transcribed spacer region 2 (ITS2), followed by fragment size analysis on the ABI Prism 310 for the identification of clinically important yeasts. Results A rapid DNA-extraction method, based on simple boiling-freezing was introduced. Of the 26 species tested, 22 could be identified unambiguously by scoring the length of the ITS2-region. No distinction could be made between the species Trichosporon asteroides and T. inkin or between T. mucoides and T. ovoides. The two varieties of Cryptococcus neoformans (var. neoformans and var. gattii) could be differentiated from each other due to a one bp length difference of the ITS2 fragment. The three Cryptococcus laurentii isolates were split into two groups according to their ITS2-fragment lengths, in correspondence with the phylogenetic groups described previously. Since the obtained fragment lengths compare well to those described previously and could be exchanged between two laboratories, an internationally usable library of ITS2 fragment lengths can be constructed. Conclusions The existing ITS2 size based library enables identification of most of the clinically important yeast species within 6 hours starting from a single colony and can be easily updated when new species are described. Data can be exchanged between laboratories. PMID:12139769

  1. The Granuloma Response Controlling Cryptococcosis in Mice Depends on the Sphingosine Kinase 1–Sphingosine 1-Phosphate Pathway

    PubMed Central

    Farnoud, Amir M.; Bryan, Arielle M.; Kechichian, Talar; Luberto, Chiara

    2015-01-01

    Cryptococcus neoformans is a fungal pathogen that causes pulmonary infections, which may progress into life-threatening meningitis. In commonly used mouse models of C. neoformans infections, fungal cells are not contained in the lungs, resulting in dissemination to the brain. We have previously reported the generation of an engineered C. neoformans strain (C. neoformans Δgcs1) which can be contained in lung granulomas in the mouse model and have shown that granuloma formation is dependent upon the enzyme sphingosine kinase 1 (SK1) and its product, sphingosine 1-phosphate (S1P). In this study, we have used four mouse models, CBA/J and C57BL6/J (both immunocompetent), Tgε26 (an isogenic strain of strain CBA/J lacking T and NK cells), and SK−/− (an isogenic strain of strain C57BL6/J lacking SK1), to investigate how the granulomatous response and SK1-S1P pathway are interrelated during C. neoformans infections. S1P and monocyte chemotactic protein-1 (MCP-1) levels were significantly elevated in the bronchoalveolar lavage fluid of all mice infected with C. neoformans Δgcs1 but not in mice infected with the C. neoformans wild type. SK1−/− mice did not show elevated levels of S1P or MCP-1. Primary neutrophils isolated from SK1−/− mice showed impaired antifungal activity that could be restored by the addition of extracellular S1P. In addition, high levels of tumor necrosis factor alpha were found in the mice infected with C. neoformans Δgcs1 in comparison to the levels found in mice infected with the C. neoformans wild type, and their levels were also dependent on the SK1-S1P pathway. Taken together, these results suggest that the SK1-S1P pathway promotes host defense against C. neoformans infections by regulating cytokine levels, promoting extracellular killing by phagocytes, and generating a granulomatous response. PMID:25895971

  2. Microsatellite markers for Senna spectabilis var. excelsa (Caesalpinioideae, Fabaceae)1

    PubMed Central

    López-Roberts, M. Cristina; Barbosa, Ariane R.; Paganucci de Queiroz, Luciano; van den Berg, Cássio

    2016-01-01

    Premise of the study: Senna spectabilis var. excelsa (Fabaceae) is a South and Central American tree of great ecological importance and one of the most common species in several sites of seasonally dry forests. Our goal was to develop microsatellite markers to assess the genetic diversity and structure of this species. Methods and Results: We designed and assessed 53 loci obtained from a microsatellite-enriched library and an intersimple sequence repeat library. Fourteen loci were polymorphic, and they presented a total of 39 alleles in a sample of 61 individuals from six populations. The mean values of observed and expected heterozygosities were 0.355 and 0.479, respectively. Polymorphism information content was 0.390 and the Shannon index was 0.778. Conclusions: Polymorphism information content and Shannon index indicate that at least nine of the 14 microsatellite loci developed are moderate to highly informative, and potentially useful for population genetic studies in this species. PMID:26819856

  3. Controlling pool depth during VAR of Alloy 718

    NASA Astrophysics Data System (ADS)

    Lopez, F.; Beaman, J.; Williamson, R.; Evans, D.

    2016-07-01

    A longtime goal of superalloy producers has been to control the geometry of the liquid pool in solidifying ingots. Accurate pool depth control at appropriate values is expected to result in ingots free of segregation defects. This article describes an industrial VAR experiment in which a 430mm (17 in) diameter Alloy 718 electrode was melted into a 510mm (20 in) ingot. In the experiment, the depth of the liquid pool at the mid-radius was controlled to three different set-points: 137 mm (nominal), 193 mm (deep) and 118 mm (shallow). At each level, the pool depth was marked by a power cutback of several minutes. The ingot was sectioned and longitudinal slices were cut out. Analysis of the photographed ingot revealed that accurate control was obtained for both the nominal and deep pool cases, while the third one was not conclusive.

  4. Deterioration of expanded polystyrene caused by Aureobasidium pullulans var. melanogenum.

    PubMed

    Castiglia, Valeria C; Kuhar, Francisco

    2015-01-01

    An expanded-polystyrene factory located in northern Buenos Aires reported unusual dark spots causing esthetic damage in their production. A fungal strain forming black-olive colonies on extract malt agar medium was isolated from the damaged material and identified as Aureobasidium pullullans var. melanogenum. This fungus is particularly known for its capacity to produce hydrolytic enzymes and a biodegradable extracellular polysaccharide known as pullulan, which is used in the manufacture of packaging material for food and medicine. Laboratory tests were conducted to characterize its growth parameters. It was found that the organism was resistant to a wide range of pHs but did not survive at temperatures over 65°C. The proposed action plan includes drying of the material prior to packaging and disinfection of the machinery used in the manufacturing process and of the silos used for raw material storage. PMID:26165967

  5. Utilization of Leek (Allium ampeloprasum var. porrum) for inulinase production.

    PubMed

    Tasar, Ozden Canli; Erdal, Serkan; Algur, Omer Faruk

    2015-08-18

    Inulinase production by Rhodotorula glutinis was carried out in this study, using leek (Allium ampeloprasum var. porrum) as an alternative carbon source due to its high inulin content and easy availability. Taguchi orthogonal array (OA) design of experiment (DOE) was used to optimize fermentation conditions. For this purpose, five influential factors (leek concentration, pH, incubation temperature, agitation speed, and fermentation time) related to inulinase production were selected at four convenient levels. The results showed that maximum inulinase activity was obtained as 30.89 U/mL, which was close to the predicted result (30.24 U/mL). To validate the obtained results, analysis of variance (ANOVA) was employed. Consequently, leek has a great potential as an effective and economical carbon source for inulinase production, and the use of Taguchi DOE enhanced enzyme activity about 2.87-fold when compared with the unoptimized condition. PMID:25036570

  6. The complete mitochondrial genome sequence of Malus hupehensis var. pinyiensis.

    PubMed

    Duan, Naibin; Sun, Honghe; Wang, Nan; Fei, Zhangjun; Chen, Xuesen

    2016-07-01

    The complete mitochondrial genome sequence of Malus hupehensis var. pinyiensis, a widely used apple rootstock, was determined using the Illumina high-throughput sequencing approach. The genome is 422,555 bp in length and has a GC content of 45.21%. It is separated by a pair of inverted repeats of 32,504 bp, to form a large single copy region of 213,055 bp and a small single copy region of 144,492 bp. The genome contains 38 protein-coding genes, four pseudogenes, 25 tRNA genes, and three rRNA genes. The genome is 25,608 bp longer than that of M. domestica, and several structural variations between these two mitogenomes were detected. PMID:26539696

  7. Antimicrobial phenolic derivatives from Dendranthema zawadskii var. latilobum Kitamura (Asteraceae).

    PubMed

    Rahman, Aziz Abdur; Moon, Surk-Sik

    2007-11-01

    A phytochemical study on the root of Dendranthema zawadskii var. latilobum Kitamura, using a series of silica gel column chromatography and reversed phase C-18 HPLC chromatography, led to the isolation of (1S, 2S)-1, 2, 3-trihydroxy-1-(3, 4-methylenedioxyphenyl)propane (1), 4-methoxycinnamic acid (2), acacetin (3) and caffeic acid methyl ester (4). The structures of these compounds were determined using spectroscopic analyses (UV, IR, HRTOFMS and NMR), with comparison of their spectral data with previously reported values. Compound 1 was isolated for the first time, with compounds 2 and 4 from this plant reported for the first time. The antibacterial and antifungal activities of the isolated compounds were measured using the disc diffusion method. Also, their cytotoxicities against the cancer cell lines, A549, B16F1 and SK-Mel-2, and brine shrimp lethalities were evaluated. PMID:18087803

  8. Complete plastid genome of Astragalus mongholicus var. nakaianus (Fabaceae).

    PubMed

    Choi, In-Su; Kim, Joo-Hwan; Choi, Byoung-Hee

    2016-07-01

    The first complete plastid genome (plastome) of the largest angiosperm genus, Astragalus, was sequenced for the Korean endangered endemic species A. mongholicus var. nakaianus. Its genome is relatively short (123,633 bp) because it lacks an Inverted Repeat (IR) region. It comprises 110 genes, including four unique rRNAs, 30 tRNAs, and 76 protein-coding genes. Similar to other closely related plastomes, rpl22 and rps16 are absent. The putative pseudogene with abnormal stop codons is atpE. This plastome has no additional inversions when compared with highly variable plastomes from IRLC tribes Fabeae and Trifolieae. Our phylogenetic analysis confirms the non-monophyly of Galegeae. PMID:26119117

  9. Anti-adipogenic effects of extracts of Ficus deltoidea var. deltoidea and var. angustifolia on 3T3-L1 adipocytes*

    PubMed Central

    Woon, Shiau Mei; Seng, Yew Wei; Ling, Anna Pick Kiong; Chye, Soi Moi; Koh, Rhun Yian

    2014-01-01

    Objective: This study examined the anti-adipogenic effects of extracts of Ficus deltoidea var. deltoidia and var. angustifolia, a natural slimming aid, on 3T3-L1 adipocytes. Methods: Methanol and water extracts of leaves of the F. deltoidea varieties were analyzed to determine their total flavonoid content (TFC) and total phenolic content (TPC), respectively. The study was initiated by determining the maximum non-toxic dose (MNTD) of the methanol and water extracts for 3T3-L1 preadipocytes. Possible anti-adipogenic effects were then examined by treating 2-d post confluent 3T3-L1 preadipocytes with either methanol extract or water extract at MNTD and half MNTD (½MNTD), after which the preadipocytces were induced to form mature adipocytes. Visualisation and quantification of lipid content in mature adipocytes were carried out through oil red O staining and measurement of optical density (OD) at 520 nm, respectively. Results: The TFCs of the methanol extracts were 1.36 and 1.97 g quercetin equivalents (QE)/100 g dry weight (DW), while the TPCs of the water extracts were 5.61 and 2.73 g gallic acid equivalents (GAE)/100 g DW for var. deltoidea and var. angustilofia, respectively. The MNTDs determined for methanol and water extracts were (300.0±28.3) and (225.0±21.2) μg/ml, respectively, for var. deltoidea, while much lower MNTDs [(60.0±2.0) μg/ml for methanol extracts and (8.0±1.0) μg/ml for water extracts] were recorded for var. angustifolia. Studies revealed that the methanol extracts of both varieties and the water extracts of var. angustifolia at either MNTD or ½MNTD significantly inhibited the maturation of preadipocytes. Conclusions: The inhibition of the formation of mature adipocytes indicated that leaf extracts of F. deltoidea could have potential anti-obesity effects. PMID:24599694

  10. 40 CFR 80.170 - Volumetric additive reconciliation (VAR), equipment calibration, and recordkeeping requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ..., which must restrict to a limited number of specified people those who have the ability to alter or... VAR period, the total volume in gallons of transfers from the facility of unadditized base...

  11. Phytoremediation of cadmium-contaminated farmland soil by the hyperaccumulator Beta vulgaris L. var. cicla.

    PubMed

    Song, Xueying; Hu, Xiaojun; Ji, Puhui; Li, Yushuang; Chi, Guangyu; Song, Yufang

    2012-04-01

    A field study was conducted to evaluate the phytoremediation efficiency of cadmium (Cd) contaminated soil utilizing the Cd hyperaccumulator Beta vulgaris L. var. cicla during one growing season (about 2 months) on farmland in Zhangshi Irrigation Area, the representative wastewater irrigation area in China. Results showed that B. vulgaris L. var. cicla is a promising plant in the phytoremediation of Cd contaminated farmland soil. The maximum of Cd phytoremediation efficiency by B. vulgaris L. var. cicla reached 144.6 mg/ha during one growing season. Planting density had a significant effect on the plant biomass and the overall Cd phytoremediation efficiency (p < 0.05). The amendment of organic manure promoted the biomass increase of B. vulgaris L. var. cicla (p < 0.05) but inhibited the Cd phytoremediation efficiency. PMID:22286610

  12. ClinVar: public archive of relationships among sequence variation and human phenotype

    PubMed Central

    Landrum, Melissa J.; Lee, Jennifer M.; Riley, George R.; Jang, Wonhee; Rubinstein, Wendy S.; Church, Deanna M.; Maglott, Donna R.

    2014-01-01

    ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/) provides a freely available archive of reports of relationships among medically important variants and phenotypes. ClinVar accessions submissions reporting human variation, interpretations of the relationship of that variation to human health and the evidence supporting each interpretation. The database is tightly coupled with dbSNP and dbVar, which maintain information about the location of variation on human assemblies. ClinVar is also based on the phenotypic descriptions maintained in MedGen (http://www.ncbi.nlm.nih.gov/medgen). Each ClinVar record represents the submitter, the variation and the phenotype, i.e. the unit that is assigned an accession of the format SCV000000000.0. The submitter can update the submission at any time, in which case a new version is assigned. To facilitate evaluation of the medical importance of each variant, ClinVar aggregates submissions with the same variation/phenotype combination, adds value from other NCBI databases, assigns a distinct accession of the format RCV000000000.0 and reports if there are conflicting clinical interpretations. Data in ClinVar are available in multiple formats, including html, download as XML, VCF or tab-delimited subsets. Data from ClinVar are provided as annotation tracks on genomic RefSeqs and are used in tools such as Variation Reporter (http://www.ncbi.nlm.nih.gov/variation/tools/reporter), which reports what is known about variation based on user-supplied locations. PMID:24234437

  13. Genome Sequence of Phytophthora fragariae var. fragariae, a Quarantine Plant-Pathogenic Fungus

    PubMed Central

    Gao, Ruifang; Cheng, Yinghui; Wang, Ying; Wang, Ying; Guo, Liyun

    2015-01-01

    Phytophthora fragariae var. fragariae is a serious plant-pathogenic fungus causing red core disease in strawberries, resulting in a larger number of fruit produced, and the fungus has been regulated as a quarantine pest of many countries and regions. Here, we announce the genome sequence of P. fragariae var. fragariae, and this information might provide insight into the mechanism of pathogenicity and host specificity of this pathogen, as well as help us further identify targets for fungicides. PMID:25814589

  14. A Framework to Quantify the Economic Impact from Local VAR Compensation

    SciTech Connect

    Li, Fangxing; Zhang, Wenjuan; Tolbert, Leon M; Kueck, John D; Rizy, D Tom

    2008-01-01

    It is generally accepted that reactive power (or Var) compensation will bring benefits for utilities and industrial customers by providing local voltage and power factor support. However, there is a lack of a systematic approach to quantitatively identify the economic benefit. In addition, with deregulation and restructuring, it is important to indicate the amount of benefit that each market participant may potentially receive given the right price signals. If such information can be easily obtained and presented, it will be more convenient for decision-markers to determine the cost benefit sharing of installing a Var compensator. The vision of this paper is to lay out a possible method for quantitatively evaluating the benefits from local reactive power compensation. The approach is to quantify the benefits into several categories such as reduced losses, shifting reactive power flow to real power flow, and increased transfer. The calculation of these benefits are illustrated with a simple two-bus power system model and then presented with a more complicated model using Optimal Power Flow to calculate the benefits. Simulation on the more complex system is conducted with seven buses in two areas. The simulation results show that the possible economic benefits can be significant, if compared with capacity payments to central generators or payment of power factor penalties applied by utilities. The potential economic value of local Var compensation may give various parties in electricity supply, delivery and end-use consumption a better understanding of the Var benefits to assist their cost-benefit analysis for Var compensation installation. Sensitivity analysis is also provided to illustrate that the benefits may not be monotonically increasing. Also, this paper suggests that the future reactive power market should consider local Var providers or other way to encourage load Var capability, since local Var benefit is significant.

  15. A Framework to Quantify the Economic Benefit from Local VAR Compensation

    SciTech Connect

    Li, Fangxing; Zhang, Wenjuan; Tolbert, Leon M; Kueck, John D; Rizy, D Tom

    2008-01-01

    Abstract It is generally accepted that reactive power (or Var) compensation will bring benefits for utilities and industrial customers by providing local voltage and power factor support. However, there is a lack of a systematic approach to quantitatively identify the economic benefit. In addition, with the deregulation and restructuring, it is important to indicate the amount of benefit that each market participant may potentially receive given the right price signals. If such information can be easily obtained and presented, it will be more convenient for decision-markers to determine the cost benefit sharing of installing a Var compensator. This vision of this paper is to lay out a possible method for quantitatively evaluating the benefits from local reactive power compensation. The approach is to quantify the benefits into several categories such as reduced losses, shifting reactive power flow to real power flow, and increased transfer. The calculation of these benefits are illustrated with a simple two-bus power system model and then presented with a more complicated model using Optimal Power Flow to calculate the benefits. Simulation on the more complex system is conducted with seven buses in two areas. The simulation results show that the possible economic benefits can be significant, if compared with capacity payments to central generators or payment of power factor penalties applied by utilities. The potential economic value of local Var compensation may give various parties in electricity supply, delivery and end-use consumption a better understanding of the Var benefits to assist their cost-benefit analysis for Var compensation installation. Sensitivity analysis is also provided to illustrate that the benefits may not be monotonically increasing. Also, this paper suggests that the future reactive power market should consider local Var providers or other way to encourage load Var capability, since local Var benefit is significant.

  16. Several domains from VAR2CSA can induce Plasmodium falciparum adhesion-blocking antibodies

    PubMed Central

    2010-01-01

    Background Malaria caused by Plasmodium falciparum can result in several different syndromes with severe clinical consequences for the about 200 million individuals infected each year. During pregnancy, women living in endemic areas become susceptible to malaria due to lack of antibodies against a unique P. falciparum membrane protein, named VAR2CSA. This antigen is not expressed in childhood infections, since it binds chondroitin sulphate A (CSA) expressed on the intervillous space in the placenta. A vaccine appears possible because women acquire protective antibodies hindering sequestration in the placenta as a function of parity. A challenge for vaccine development is to design small constructs of this large antigen, which can induce broadly protective antibodies. It has previously been shown that one domain of VAR2CSA, DBL4-FCR3, induces parasite adhesion-blocking antibodies. In this study, it is demonstrated that other domains of VAR2CSA also can induce antibodies with inhibitory activity. Methods All VAR2CSA domains from the 3D7 and HB3 parasites were produced in Baculovirus-transfected insect cells. Groups of three rats per protein were immunized and anti-sera were tested for surface reactivity against infected erythrocytes expressing FCR3 VAR2CSA and for the ability to inhibit FCR3CSA parasite adhesion to CSA. The fine specificity of the immune sera was analysed by VAR2CSA peptide arrays. Results Inhibitory antibodies were induced by immunization with DBL3-HB3 T1 and DBL1-3D7. However, unlike the previously characterised DBL4-FCR3 response the inhibitory response against DBL1-3D7 and DBL3-HB3 T1 was poorly reproduced in the second rounds of immunizations. Conclusion It is possible to induce parasite adhesion-blocking antibodies when immunizing with a number of different VAR2CSA domains. This indicates that the CSA binding site in VAR2CSA is comprised of epitopes from different domains. PMID:20064234

  17. Four-dimensional ensemble variational (4D-En-Var) data assimilation for the HIgh Resolution Limited Area Model (HIRLAM)

    NASA Astrophysics Data System (ADS)

    Gustafsson, N.; Bojarova, J.

    2014-07-01

    A four-dimensional ensemble variational (4D-En-Var) data assimilation has been developed for a limited area model. The integration of tangent linear and adjoint models, as applied in standard 4D-Var, is replaced with the use of an ensemble of non-linear model states to estimate four-dimensional background error covariances over the assimilation time window. The computational costs for 4D-En-Var are therefore significantly reduced in comparison with standard 4D-Var and the scalability of the algorithm is improved. The flow dependency of 4D-En-Var assimilation increments is demonstrated in single simulated observation experiments and compared with corresponding increments from standard 4D-Var and Hybrid 4D-Var ensemble assimilation experiments. Real observation data assimilation experiments carried out over a 6-week period show that 4D-En-Var outperforms standard 4D-Var as well as Hybrid 4D-Var ensemble data assimilation with regard to forecast quality measured by forecast verification scores.

  18. Working alliance inventory applied to virtual and augmented reality (WAI-VAR): psychometrics and therapeutic outcomes.

    PubMed

    Miragall, Marta; Baños, Rosa M; Cebolla, Ausiàs; Botella, Cristina

    2015-01-01

    This study examines the psychometric properties of the Working Alliance Inventory-Short (WAI-S) adaptation to Virtual Reality (VR) and Augmented Reality (AR) therapies (WAI-VAR). The relationship between the therapeutic alliance (TA) with VR and AR and clinically significant change (CSC) is also explored. Seventy-five patients took part in this study (74.7% women, M age = 34.41). Fear of flying and adjustment disorder patients received VR therapy, and cockroach phobia patients received AR therapy. Psychometric properties, CSC, one-way ANOVA, Spearman's Correlations and Multiple Regression were calculated. The WAI-VAR showed a unidimensional structure, high internal consistency and adequate convergent validity. "Not changed" patients scored lower on the WAI-VAR than "improved" and "recovered" patients. Correlation between the WAI-VAR and CSC was moderate. The best fitting model for predicting CSC was a linear combination of the TA with therapist (WAI-S) and the TA with VR and AR (WAI-VAR), due to the latter variable slightly increased the percentage of variability accounted for in CSC. The WAI-VAR is the first validated instrument to measure the TA with VR and AR in research and clinical practice. This study reveals the importance of the quality of the TA with technologies in achieving positive outcomes in the therapy. PMID:26500589

  19. Working alliance inventory applied to virtual and augmented reality (WAI-VAR): psychometrics and therapeutic outcomes

    PubMed Central

    Miragall, Marta; Baños, Rosa M.; Cebolla, Ausiàs; Botella, Cristina

    2015-01-01

    This study examines the psychometric properties of the Working Alliance Inventory-Short (WAI-S) adaptation to Virtual Reality (VR) and Augmented Reality (AR) therapies (WAI-VAR). The relationship between the therapeutic alliance (TA) with VR and AR and clinically significant change (CSC) is also explored. Seventy-five patients took part in this study (74.7% women, Mage = 34.41). Fear of flying and adjustment disorder patients received VR therapy, and cockroach phobia patients received AR therapy. Psychometric properties, CSC, one-way ANOVA, Spearman’s Correlations and Multiple Regression were calculated. The WAI-VAR showed a unidimensional structure, high internal consistency and adequate convergent validity. “Not changed” patients scored lower on the WAI-VAR than “improved” and “recovered” patients. Correlation between the WAI-VAR and CSC was moderate. The best fitting model for predicting CSC was a linear combination of the TA with therapist (WAI-S) and the TA with VR and AR (WAI-VAR), due to the latter variable slightly increased the percentage of variability accounted for in CSC. The WAI-VAR is the first validated instrument to measure the TA with VR and AR in research and clinical practice. This study reveals the importance of the quality of the TA with technologies in achieving positive outcomes in the therapy. PMID:26500589

  20. Semi-nonparametric VaR forecasts for hedge funds during the recent crisis

    NASA Astrophysics Data System (ADS)

    Del Brio, Esther B.; Mora-Valencia, Andrés; Perote, Javier

    2014-05-01

    The need to provide accurate value-at-risk (VaR) forecasting measures has triggered an important literature in econophysics. Although these accurate VaR models and methodologies are particularly demanded for hedge fund managers, there exist few articles specifically devoted to implement new techniques in hedge fund returns VaR forecasting. This article advances in these issues by comparing the performance of risk measures based on parametric distributions (the normal, Student’s t and skewed-t), semi-nonparametric (SNP) methodologies based on Gram-Charlier (GC) series and the extreme value theory (EVT) approach. Our results show that normal-, Student’s t- and Skewed t- based methodologies fail to forecast hedge fund VaR, whilst SNP and EVT approaches accurately success on it. We extend these results to the multivariate framework by providing an explicit formula for the GC copula and its density that encompasses the Gaussian copula and accounts for non-linear dependences. We show that the VaR obtained by the meta GC accurately captures portfolio risk and outperforms regulatory VaR estimates obtained through the meta Gaussian and Student’s t distributions.

  1. Toxicological assessment of nattokinase derived from Bacillus subtilis var. natto.

    PubMed

    Lampe, Bradley J; English, J Caroline

    2016-02-01

    Subtilisin NAT, commonly known as "nattokinase," is a fibrinolytic enzyme produced by the bacterial strain B. subtilis var. natto, which plays a central role in the fermentation of soybeans into the popular Japanese food natto. Recent studies have reported on the potential anticoagulatory and antihypertensive effects of nattokinase administration in humans, with no indication of adverse effects. To evaluate the safety of nattokinase in a more comprehensive manner, several GLP-compliant studies in rodents and human volunteers have been conducted with the enzyme product, NSK-SD (Japan Bio Science Laboratory Co., Ltd., Japan). Nattokinase was non-mutagenic and non-clastogenic in vitro, and no adverse effects were observed in 28-day and 90-day subchronic toxicity studies conducted in Sprague-Dawley rats at doses up to 167 mg/kg-day and 1000 mg/kg-day, respectively. Mice inoculated with 7.55 × 10(8) CFU of the enzyme-producing bacterial strain showed no signs of toxicity or residual tissue concentrations of viable bacteria. Additionally consumption of 10 mg/kg-day nattokinase for 4 weeks was well tolerated in healthy human volunteers. These findings suggest that the oral consumption of nattokinase is of low toxicological concern. The 90-day oral subchronic NOAEL for nattokinase in male and female Sprague-Dawley rats is 1000 mg/kg-day, the highest dose tested. PMID:26740078

  2. In vitro propagation of Dioscorea alata var. purpurae.

    PubMed

    Shah, Heena J; Lele, S S

    2012-07-01

    Dioscorea alata var. purpurae (Indian purple yam) is an important source of diosgenin, a triterpenoid that is used as a raw material in the synthesis of corticosteroid hormones. These drugs are used for the treatment of pharmacological conditions such as arthritis. This paper reports in vitro propagation of explants of various parts of Dioscorea-tuber, leaves, and nodes. Murashige and Skoog media supplemented with hormones and additives was used to get maximum callus initiation and shoot/root induction. All the cultures were maintained at 25 ± 2 °C under cool-white fluorescent tubes with 16-h photoperiod. Callus initiation was observed from 8th to 11th day of inoculation, and subsequent root/shoot was initiated in nodal callus after 21 days. Hormones such as kinetin, indole-3-acetic acid, indole-3-butyric acid, α-naphthalene acetic acid, and thiadizuron did not show significant enhancement. Also, there was no need for supplementing additives (silver nitrate, glutamine, L-: asparagine monohydrate, polyethylene glycol). Combination of 6-benzylaminopurine (0.2 ppm) and 2,4-dichlorophenoxyacetic acid (2 ppm) hormones gave the best results, and all parts of the plants gave similar callus induction. PMID:22476929

  3. Periodicity of Wuchereria bancrofti var. pacifica filariasis in French Polynesia.

    PubMed

    Moulia-Pelat, J P; Glaziou, P; Chanteau, S; Nguyen-Ngoc, L; Marcet, Y; Gardines, R; Martin, P M; Cartel, J L

    1993-06-01

    In 1992, a study on microfilaremia periodicity was carried out on 12 Wuchereria bancrofti carriers in the Marquesas islands. Blood samples were collected simultaneously every 4 hours during a 48 hour period by finger-prick and venipuncture for determination of microfilaremia by both blood film and membrane filtration technique methods, and for determination of antigenemia. The membrane filtration results showed no significant nycthemeral variations between the microfilaria densities at hours 16:00, 20:00, 24:00, 04:00, 08:00 and 12:00. Conversely, the blood film method showed a significant difference between the microfilaria densities: the microfilaremia was higher during the day (12:00-20:00 hours) than during the night (24:00-08:00 hours). As for antigenemia, using Og 4 C3 monoclonal antibody, there was no significant fluctuation during 48 hours. These results confirm that W. bancrofti var. pacifica is subperiodic and diurnal in French Polynesia. In particular, they substantiate the validity of examining venous blood by the membrane filtration technique as the judgment criterion of choice in therapeutic trials and of examining capillary blood during peak hours by the blood film method for evaluating the endemic level in a population. PMID:8367671

  4. Reduced order POD/DEIM 4-D Var data assimilation

    NASA Astrophysics Data System (ADS)

    Navon, Michael; Stefanescu, Razvan

    2014-05-01

    The computational cost of realistic ensemble and hybrid variational/ensemble data assimilation is typically dominated by the cost of ensemble forecasting. The high computational cost of ensemble forecasting limits the number of ensembles, eventually creating a severe rank reduction. Consequently, the efficiency and quality of ensemble-based data assimilation are greatly reduced. With the ever-increasing spatiotemporal resolution and complexity of numerical weather prediction (NWP) models, the room for ensemble forecasting is getting even smaller, creating a paradox: Although the NWP generally benefits from increased resolution and complexity of the models, the quality of their data assimilation is getting worse due to additional computational restrictions. We propose POD model order reduction substantially improving computational efficiency of NWP models. We present recent advances in this domain and the state-of the art of hyper reduction addressing issues of turbulence closure and nonlinearities allowing CPU speed -ups of orders of magnitude, reduced order 4-D VAR and future prospects of implementation to operational NMP models.

  5. De Novo Transcriptome Analysis of Cucumis melo L. var. makuwa

    PubMed Central

    Kim, Hyun A; Shin, Ah-Young; Lee, Min-Seon; Lee, Hee-Jeong; Lee, Heung-Ryul; Ahn, Jongmoon; Nahm, Seokhyeon; Jo, Sung-Hwan; Park, Jeong Mee; Kwon, Suk-Yoon

    2016-01-01

    Oriental melon (Cucumis melo L. var. makuwa) is one of six subspecies of melon and is cultivated widely in East Asia, including China, Japan, and Korea. Although oriental melon is economically valuable in Asia and is genetically distinct from other subspecies, few reports of genome-scale research on oriental melon have been published. We generated 30.5 and 36.8 Gb of raw RNA sequence data from the female and male flowers, leaves, roots, and fruit of two oriental melon varieties, Korean landrace (KM) and Breeding line of NongWoo Bio Co. (NW), respectively. From the raw reads, 64,998 transcripts from KM and 100,234 transcripts from NW were de novo assembled. The assembled transcripts were used to identify molecular markers (e.g., single-nucleotide polymorphisms and simple sequence repeats), detect tissue-specific expressed genes, and construct a genetic linkage map. In total, 234 single-nucleotide polymorphisms and 25 simple sequence repeats were screened from 7,871 and 8,052 candidates, respectively, between the KM and NW varieties and used for construction of a genetic map with 94 F2 population specimens. The genetic linkage map consisted of 12 linkage groups, and 248 markers were assigned. These transcriptome and molecular marker data provide information useful for molecular breeding of oriental melon and further comparative studies of the Cucurbitaceae family. PMID:26743902

  6. De Novo Transcriptome Analysis of Cucumis melo L. var. makuwa.

    PubMed

    Kim, Hyun A; Shin, Ah-Young; Lee, Min-Seon; Lee, Hee-Jeong; Lee, Heung-Ryul; Ahn, Jongmoon; Nahm, Seokhyeon; Jo, Sung-Hwan; Park, Jeong Mee; Kwon, Suk-Yoon

    2016-02-01

    Oriental melon (Cucumis melo L. var. makuwa) is one of six subspecies of melon and is cultivated widely in East Asia, including China, Japan, and Korea. Although oriental melon is economically valuable in Asia and is genetically distinct from other subspecies, few reports of genome-scale research on oriental melon have been published. We generated 30.5 and 36.8 Gb of raw RNA sequence data from the female and male flowers, leaves, roots, and fruit of two oriental melon varieties, Korean landrace (KM) and Breeding line of NongWoo Bio Co. (NW), respectively. From the raw reads, 64,998 transcripts from KM and 100,234 transcripts from NW were de novo assembled. The assembled transcripts were used to identify molecular markers (e.g., single-nucleotide polymorphisms and simple sequence repeats), detect tissue-specific expressed genes, and construct a genetic linkage map. In total, 234 single-nucleotide polymorphisms and 25 simple sequence repeats were screened from 7,871 and 8,052 candidates, respectively, between the KM and NW varieties and used for construction of a genetic map with 94 F2 population specimens. The genetic linkage map consisted of 12 linkage groups, and 248 markers were assigned. These transcriptome and molecular marker data provide information useful for molecular breeding of oriental melon and further comparative studies of the Cucurbitaceae family. PMID:26743902

  7. Genetic dissection of agronomic traits in Capsicum baccatum var. pendulum.

    PubMed

    Moulin, M M; Rodrigues, R; Bento, C S; Gonçalves, L S A; Santos, J O; Sudré, C P; Viana, A P

    2015-01-01

    Genetic mapping is very useful for dissecting complex agronomic traits. Genetic mapping allows for identification of quantitative trait loci (QTL), provide knowledge on a gene position and its adjacent region, and enable prediction of evolutionary mechanisms, in addition to contributing to synteny studies. The aim of this study was to predict genetic values associated with different agronomic traits evaluated in an F2 population of Capsicum baccatum var. pendulum. Previously, a reference genetic map for C. baccatum was constructed, which included 183 markers (42 microsatellite, 85 inter-simple sequence repeat, and 56 random amplification of polymorphic DNA) arranged in 16 linkage groups. The map was used to identify QTL associated with 11 agronomic traits, including plant height, crown diameter, number of days to flowering, days to fruiting, number of fruits per plant, average fruit weight, fruit length, fruit diameter, fruit pulp thickness, soluble solids, and fruit dry weight. QTL mapping was performed by standard interval mapping. The number of small QTL effects ranged from 3-11, with a total of 61 QTL detected in 9 linkage groups. This is the first report involving QTL analysis for C. baccatum species. PMID:25867359

  8. Foam Separation of Pseudomonas fluorescens and Bacillus subtilis var. niger

    PubMed Central

    Grieves, R. B.; Wang, S. L.

    1967-01-01

    An experimental investigation established the effect of the presence of inorganic salts on the foam separation of Pseudomonas fluorescens and of Bacillus subtilis var. niger (B. globigii) from aqueous suspension by use of a cationic surfactant. For P. fluorescens, 5.0 μeq/ml of NaCl, KCl, Na2SO4, K2SO4, CaCl2, CaSO4, MgCl2, or MgSO4 produced increases in the cell concentration in the residual suspension (not carried into the foam) from 2.9 × 105 up to 1.6 × 106 to 2.8 × 107 cells per milliliter (initial suspensions contain from 3.3 × 107 to 4.8 × 107 cells per milliliter). The exceptional influence of magnesium was overcome by bringing the cells into contact first with the surfactant and then the salt. For B. subtilis, the presence of 5.0 μeq/ml of any of the eight salts increased the residual cell concentration by one order of magnitude from 1.2 × 104 to about 4.0 × 105 cells per milliliter. This occurred regardless of the sequence of contact as long as the surfactant contact period was sufficient. The presence of salts increased collapsed foam volumes with P. fluorescens and decreased collapsed foam volumes with B. subtilis. PMID:4961933

  9. Micropropagation of globe artichoke (Cynara cardunculus L. var. scolymus).

    PubMed

    Iapichino, Giovanni

    2013-01-01

    The globe artichoke (Cynara cardunculus L. var. scolymus) is a perennial plant cultivated in the Mediterranean region and the Americas for its edible young flower heads. Although vegetative propagation by offshoots or by "ovoli" (underground dormant axillary buds) has been the primary method of propagation, the potential for the diffusion of diseases and the phenotypic variability can be very high. The propagation of this species by axillary shoot proliferation from in vitro-cultured meristems produces systemic pathogen-free plants and a higher multiplication rate as compared to that obtained by conventional agamic multiplication. Axillary shoot proliferation can be induced from excised shoot apices cultured on Murashige and Skoog agar solidified medium supplemented with various concentrations of cytokinins and auxins, depending on genotype. For the production of virus-free plants, meristems, 0.3-0.8 mm long are excised from shoot apices and surface sterilized. The transfer of artichoke microshoots to a medium lacking cytokinins or with low cytokinin concentration is critical for rooting. Adventitious roots develop within 3-5 weeks after transfer to root induction MS medium containing NAA or IAA at various concentrations. However, in vitro rooting frequency rate is dependent on the genotype and the protocol used. Acclimatization of in vitro microshoots having 3-4 roots is successfully accomplished; plantlets develop new roots in ex vitro conditions and continue to grow. PMID:23179714

  10. Intraspecific Variation in Carotenoids of Brassica oleracea var. sabellica.

    PubMed

    Mageney, Vera; Baldermann, Susanne; Albach, Dirk C

    2016-04-27

    Carotenoids are best known as a source of natural antioxidants. Physiologically, carotenoids are part of the photoprotection in plants as they act as scavengers of reactive oxygen species (ROS). An important source of carotenoids in European food is Brassica oleracea. Focusing on the most abundant carotenoids, we estimated the contents of ß-carotene, (9Z)-neoxanthin, zeaxanthin, and lutein as well as those of chlorophylls a and b to assess their variability in Brassica oleracea var. sabellica. Our analyses included more than 30 cultivars categorized in five distinct sets grouped according to morphological characteristics or geographical origin. Our results demonstrated specific carotenoid patterns characteristic for American, Italian, and red-colored kale cultivars. Moreover, we demonstrated a tendency of high zeaxanthin proportions under traditional harvest conditions, which accord to low-temperature regimes. We also compared the carotenoid patterns of self-generated hybrid lines. Corresponding findings indicated that crossbreeding has a high potential for carotenoid content optimization in kale. PMID:27045759

  11. Antiplasmodial sesquiterpenes from the seeds of Salacia longipes var. camerunensis.

    PubMed

    Mba'ning, Brice M; Lenta, Bruno N; Noungoué, Diderot T; Antheaume, Cyril; Fongang, Yanick F; Ngouela, Silvère A; Boyom, Fabrice F; Rosenthal, Philip J; Tsamo, Etienne; Sewald, Norbert; Laatsch, Hartmut

    2013-12-01

    Phytochemical investigation of the seeds of Salacia longipes var. camerunensis led to the isolation of four sesquiterpenoid derivatives, salaterpene A (1) (1α,2β,8β-triacetoxy-6β,9β-dibenzoyloxy-4β-hydroxy-dihydro-β-agarofuran), salaterpene B (2) (1α,2β,8β-triacetoxy-9β-benzoyloxy-6β-cinnamoyloxy-4β-hydroxy-dihydro-β-agarofuran), salaterpene C (3) (1α,2β-diacetoxy-6β,9β-dibenzoyloxy-4β-hydroxy-dihydro-β-agarofuran) and salaterpene D (4) (2β-acetoxy-1α,6β-dibenzoyloxy-4β-hydroxy-9β-nicotinoyloxy-dihydro-β-agarofuran) together with two known compounds (5 and 6). The structures of the compounds were established by means of NMR spectroscopy. Compounds 1-4 and 6 were tested in vitro for their antiplasmodial activity against Plasmodium falciparum chloroquine-resistant strain W2. All the tested compounds exhibited a moderate potency with IC50 below 2.7 μM. PMID:23863332

  12. Antimicrobial evaluation of clerodane diterpenes from Polyalthia longifolia var. pendula.

    PubMed

    Sashidhara, Koneni V; Singh, Suriya P; Shukla, P K

    2009-03-01

    Phytochemical investigation of the ethanolic extract of leaves of Polyalthia longifolia var. pendula has led to the isolation of seven clerodane diterpenoids and five alkaloids. (-)-14, 15-bisnor-3, 11E-kolavadien-13-one (1), (-)-16-oxocleroda-3,13(14)E-dien-15-oic acid (2), (-)-16alpha-hydroxycleroda-3,13 (14)Z-dien-15,16-olide (3), (+)-(4-->2)-abeo-16(R/S)-2, 13Z-kolavadien-15, 16-olide-3-al (4), (-)-3beta, 16beta-dihydroxycleroda-4(18), 13(14)Z-dien-15,16-olide (5), (-)-3, 12E-kolavadien-15-oic acid-16-al (6), (-)-labd-13E-en-8-ol-15-oic acid (7), liriodenine (8), (-)-anonaine (9), (+)-isoboldine (10), (-)-asimilobine (11) and hordenine (12) have been isolated. This is the first report of 1, 6 and 10 from this plant species while 12 is reported for first time from this genus. Clerodane derivatives 1-7 were evaluated for their antimicrobial activity. Diterpene 3 was found to be most potent agent with MIC value of 6.25 microg/mL against Staphylococcus aureus and Sporothrix schenckii. PMID:19413108

  13. In vitro antioxidant and anticancer effects of solvent fractions from prunella vulgaris var. lilacina

    PubMed Central

    2013-01-01

    Background Recently, considerable attention has been focused on exploring the potential antioxidant properties of plant extracts or isolated products of plant origin. Prunella vulgaris var. lilacina is widely distributed in Korea, Japan, China, and Europe, and it continues to be used to treat inflammation, eye pain, headache, and dizziness. However, reports on the antioxidant activities of P. vulgaris var. lilacina are limited, particularly concerning the relationship between its phenolic content and antioxidant capacity. In this study, we investigated the antioxidant and anticancer activities of an ethanol extract from P. vulgaris var. lilacina and its fractions. Methods Dried powder of P. vulgaris var. lilacina was extracted with ethanol, and the extract was fractionated to produce the hexane fraction, butanol fraction, chloroform fraction and residual water fraction. The phenolic content was assayed using the Folin-Ciocalteu colorimetric method. Subsequently, the antioxidant activities of the ethanol extract and its fractions were analyzed employing various antioxidant assay methods including DPPH, FRAP, ABTS, SOD activity and production of reactive oxygen species. Additionally, the extract and fractions were assayed for their ability to exert cytotoxic activities on various cancer cells using the MTT assay. We also investigated the expression of genes associated with apoptotic cell death by RT-PCR. Results The total phenolic contents of the ethanol extract and water fraction of P. vulgaris var. lilacina were 303.66 and 322.80 mg GAE/g dry weight (or fractions), respectively. The results showed that the ethanol extract and the water fraction of P. vulgaris var. lilacina had higher antioxidant content than other solvent fractions, similar to their total phenolic content. Anticancer activity was also tested using the HepG2, HT29, A549, MKN45 and HeLa cancer cell lines. The results clearly demonstrated that the P. vulgaris var. lilacina ethanol extract induced

  14. Effect of intercropping Panicum maximum var. Ntchisi and Lablab purpureus on the growth, herbage yield and chemical composition of Panicum maximum var. Ntchisi at different harvesting times.

    PubMed

    Ojo, V O A; Dele, P A; Amole, T A; Anele, U Y; Adeoye, S A; Hassan, O A; Olanite, J A; Idowu, O J

    2013-11-15

    The study was conducted to evaluate the effect of intercropping Panicum maximum var. Ntchisi and Lablab purpureus on the growth, herbage yield and chemical composition of P. maximum var. Ntchisi at different harvesting times at the Teaching and Research farm, Federal University of Agriculture, Abeokuta in a randomized complete block design. Samples were collected at different harvesting times (8, 10, 12, 14 weeks after planting). The growth parameters which were plant height, leaf length, leaf number and tiller number measured showed that the intercropping of grass with legume were higher than in the sole plot of P. maximum var. Ntchisi. The plant yield was consistently higher (p < 0.05) in intercropped forages than in sole throughout the harvesting times. The crude protein contents of the forages were also higher for the intercropped across the treatments. The values of the fibre components were significantly different (p < 0.05) at different harvesting times and it was increasing as the harvesting time was increasing. From this study, considering the herbage yield and chemical composition of intecropping Panicum maximum var. Ntchisi and Lablab purpureus, they can be grazed by ruminant animals or harvested at 12 weeks after planting when the quality and quantity will support livestock productivity and can be conserved to be fed to ruminant animals during dry season when feed availability and quality are extremely low. PMID:24511710

  15. Influence of LAR and VAR on Para-Aminopyridine Antimalarials Targetting Haematin in Chloroquine-Resistance

    PubMed Central

    Warhurst, David C.; Craig, John C.

    2016-01-01

    Antimalarial chloroquine (CQ) prevents haematin detoxication when CQ-base concentrates in the acidic digestive vacuole through protonation of its p-aminopyridine (pAP) basic aromatic nitrogen and sidechain diethyl-N. CQ export through the variant vacuolar membrane export channel, PFCRT, causes CQ-resistance in Plasmodium falciparum but 3-methyl CQ (sontochin SC), des-ethyl amodiaquine (DAQ) and bis 4-aminoquinoline piperaquine (PQ) are still active. This is determined by changes in drug accumulation ratios in parasite lipid (LAR) and in vacuolar water (VAR). Higher LAR may facilitate drug binding to and blocking PFCRT and also aid haematin in lipid to bind drug. LAR for CQ is only 8.3; VAR is 143,482. More hydrophobic SC has LAR 143; VAR remains 68,523. Similarly DAQ with a phenol substituent has LAR of 40.8, with VAR 89,366. In PQ, basicity of each pAP is reduced by distal piperazine N, allowing very high LAR of 973,492, retaining VAR of 104,378. In another bis quinoline, dichlorquinazine (DCQ), also active but clinically unsatisfactory, each pAP retains basicity, being insulated by a 2-carbon chain from a proximal nitrogen of the single linking piperazine. While LAR of 15,488 is still high, the lowest estimate of VAR approaches 4.9 million. DCQ may be expected to be very highly lysosomotropic and therefore potentially hepatotoxic. In 11 pAP antimalarials a quadratic relationship between logLAR and logResistance Index (RI) was confirmed, while log (LAR/VAR) vs logRI for 12 was linear. Both might be used to predict the utility of structural modifications. PMID:27483471

  16. Influence of LAR and VAR on Para-Aminopyridine Antimalarials Targetting Haematin in Chloroquine-Resistance.

    PubMed

    Warhurst, David C; Craig, John C; Raheem, K Saki

    2016-01-01

    Antimalarial chloroquine (CQ) prevents haematin detoxication when CQ-base concentrates in the acidic digestive vacuole through protonation of its p-aminopyridine (pAP) basic aromatic nitrogen and sidechain diethyl-N. CQ export through the variant vacuolar membrane export channel, PFCRT, causes CQ-resistance in Plasmodium falciparum but 3-methyl CQ (sontochin SC), des-ethyl amodiaquine (DAQ) and bis 4-aminoquinoline piperaquine (PQ) are still active. This is determined by changes in drug accumulation ratios in parasite lipid (LAR) and in vacuolar water (VAR). Higher LAR may facilitate drug binding to and blocking PFCRT and also aid haematin in lipid to bind drug. LAR for CQ is only 8.3; VAR is 143,482. More hydrophobic SC has LAR 143; VAR remains 68,523. Similarly DAQ with a phenol substituent has LAR of 40.8, with VAR 89,366. In PQ, basicity of each pAP is reduced by distal piperazine N, allowing very high LAR of 973,492, retaining VAR of 104,378. In another bis quinoline, dichlorquinazine (DCQ), also active but clinically unsatisfactory, each pAP retains basicity, being insulated by a 2-carbon chain from a proximal nitrogen of the single linking piperazine. While LAR of 15,488 is still high, the lowest estimate of VAR approaches 4.9 million. DCQ may be expected to be very highly lysosomotropic and therefore potentially hepatotoxic. In 11 pAP antimalarials a quadratic relationship between logLAR and logResistance Index (RI) was confirmed, while log (LAR/VAR) vs logRI for 12 was linear. Both might be used to predict the utility of structural modifications. PMID:27483471

  17. Stabilization of the HCP [var epsilon] phase in an Fe-21%Mn alloy subjected to cathodic hydrogen charging

    SciTech Connect

    Aikawa, T.; Nishino, Y.; Asano, S. )

    1993-07-01

    Cathodic hydrogen charging induces some phase transformation in the surface layer of FCC iron alloys. Such a hydrogen-induced phase transformation has extensively been studied for austenitic stainless steels, where the FCC [gamma] phase transforms partially into an HCP [var epsilon][sub H] phase. The formation of the [var epsilon][sub H] phase has also been found in FCC Fe-Mn alloys and Fe-Ni-Mn alloys. The [var epsilon][sub H] phase is unstable in the absence of hydrogen and decomposes to an [var epsilon] martensite in the metastable FCC alloys. In order to make clear the nature of the [var epsilon][sub H] phase, it is necessary to reveal the stability of the [var epsilon] phase in FCC iron alloys during cathodic hydrogen charging. In the present study, the authors employed an Fe-21%Mn alloy which consisted of a mixture of the [gamma] and [var epsilon] phases in an as-quenched state. For this two-phase alloy, the [var epsilon] [r arrow] [gamma] transformation has so far been examined by a high-pressure equilibration method in a hydrogen-gas atmosphere. The purpose of this study is to compare the stability of the [gamma] phases in the two-phase alloy subjected to cathodic hydrogen charging in an aqueous solution. The influence of hydrogen on the [var epsilon] phase stability will be discussed, including the transformation in a hydrogen-gas atmosphere.

  18. A Novel Saponin Hydrolase from Neocosmospora vasinfecta var. vasinfecta

    PubMed Central

    Watanabe, Manabu; Sumida, Naomi; Yanai, Koji; Murakami, Takeshi

    2004-01-01

    We isolated a soybean saponin hydrolase from Neocosmospora vasinfecta var. vasinfecta PF1225, a filamentous fungus that can degrade soybean saponin and generate soyasapogenol B. This enzyme was found to be a monomer with a molecular mass of about 77 kDa and a glycoprotein. Nucleotide sequence analysis of the corresponding gene (sdn1) indicated that this enzyme consisted of 612 amino acids and had a molecular mass of 65,724 Da, in close agreement with that of the apoenzyme after the removal of carbohydrates. The sdn1 gene was successfully expressed in Trichoderma viride under the control of the cellobiohydrolase I gene promoter. The molecular mass of the recombinant enzyme, about 69 kDa, was smaller than that of the native enzyme due to fewer carbohydrate modifications. Examination of the degradation products obtained by treatment of soyasaponin I with the recombinant enzyme showed that the enzyme hydrolyzed soyasaponin I to soyasapogenol B and triose [α-l-rhamnopyranosyl (1→2)-β-d-galactopyranosyl (1→2)-d-glucuronopyranoside]. Also, when soyasaponin II and soyasaponin V, which are different from soyasaponin I only in constituent saccharides, were treated with the enzyme, the ratio of the reaction velocities for soyasaponin I, soyasaponin II, and soyasaponin V was 2,680:886:1. These results indicate that this enzyme recognizes the fine structure of the carbohydrate moiety of soyasaponin in its catalytic reaction. The amino acid sequence of this enzyme predicted from the DNA sequence shows no clear homology with those of any of the enzymes involved in the hydrolysis of carbohydrates. PMID:14766566

  19. Novel picornavirus in domestic rabbits (Oryctolagus cuniculus var. domestica).

    PubMed

    Pankovics, Péter; Boros, Ákos; Bíró, Hunor; Horváth, Katalin Barbara; Phan, Tung Gia; Delwart, Eric; Reuter, Gábor

    2016-01-01

    Picornaviruses (family Picornaviridae) are small, non-enveloped viruses with positive sense, single-stranded RNA genomes. The numbers of the novel picornavirus species and genera are continuously increasing. Picornaviruses infect numerous vertebrate species from fish to mammals, but have not been identified in a member of the Lagomorpha order (pikas, hares and rabbits). In this study, a novel picornavirus was identified in 16 (28.6%) out of 56 faecal samples collected from clinically healthy rabbits (Oryctolagus cuniculus var. domestica) in two (one commercial and one family farms) of four rabbit farms in Hungary. The 8364 nucleotide (2486 amino acid) long complete genome sequence of strain Rabbit01/2013/HUN (KT325852) has typical picornavirus genome organization with type-V IRES at the 5'UTR, encodes a leader (L) and a single 2A(H-box/NC) proteins, contains a hepatitis-A-virus-like cis-acting replication element (CRE) in the 2A, but it does not contain the sequence forming a "barbell-like" secondary structure in the 3'UTR. Rabbit01/2013/HUN has 52.9%, 52% and 57.2% amino acid identity to corresponding proteins of species Aichivirus A (genus Kobuvirus): to murine Kobuvirus (JF755427) in P1, to canine Kobuvirus (JN387133) in P2 and to feline Kobuvirus (KF831027) in P3, respectively. The sequence and phylogenetic analysis indicated that Rabbit01/2013/HUN represents a novel picornavirus species possibly in genus Kobuvirus. This is the first report of detection of picornavirus in rabbit. Further study is needed to clarify whether this novel picornavirus plays a part in any diseases in domestic or wild rabbits. PMID:26588888

  20. Anti-Granulocyte-Macrophage Colony-Stimulating Factor Autoantibodies Are a Risk Factor for Central Nervous System Infection by Cryptococcus gattii in Otherwise Immunocompetent Patients

    PubMed Central

    Saijo, Tomomi; Chen, Jianghan; Chen, Sharon C.-A.; Rosen, Lindsey B.; Yi, Jin; Sorrell, Tania C.; Bennett, John E.; Holland, Steven M.; Browne, Sarah K.; Kwon-Chung, Kyung J.

    2014-01-01

    ABSTRACT Cryptococcosis is caused by either Cryptococcus neoformans or C. gattii. While cryptococcal meningoencephalitis is caused mostly by C. neoformans in immunocompromised patients, the risk factors remain unclear for patients with no known immune defect. Recently, anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies were detected in the plasma of seven “immunocompetent” cryptococcosis patients, and the cryptococcal strains from these patients were reported as C. neoformans (three strains), C. gattii (one strain), and Cryptococcus (three strains not identified to the species level). We identified all three strains that had not been identified to the species level as C. gattii. Notably, the three strains that were reported as C. neoformans but were unavailable for species confirmation originated from Sothern California and Thailand where C. gattii is endemic. Most clinical laboratories designate C. neoformans without distinguishing between the two species; hence, these three strains could have been C. gattii. Since C. gattii infects more immunocompetent patients than C. neoformans, we pursued the possibility that this antibody may be more prevalent in patients infected with C. gattii than in those infected with C. neoformans. We screened the plasma of 20 healthy controls and 30 “immunocompetent” patients with cryptococcal meningoencephalitis from China and Australia (multiple ethnicities). Anti-GM-CSF autoantibodies were detected only in the plasma of seven patients infected by C. gattii and one healthy volunteer and in none infected by C. neoformans. While plasma from these C. gattii patients completely prevented GM-CSF-induced p-STAT5 in normal human peripheral blood mononuclear cells (PBMCs), plasma from one healthy volunteer positive for anti-GM-CSF autoantibodies caused only partial blockage. Our results suggest that anti-GM-CSF autoantibodies may predispose otherwise immunocompetent individuals to

  1. VarR controls colonization and virulence in the marine macroalgal pathogen Nautella italica R11

    PubMed Central

    Gardiner, Melissa; Fernandes, Neil D.; Nowakowski, Dennis; Raftery, Mark; Kjelleberg, Staffan; Zhong, Ling; Thomas, Torsten; Egan, Suhelen

    2015-01-01

    There is increasing evidence to suggest that macroalgae (seaweeds) are susceptible to infectious disease. However, to date, little is known about the mechanisms that facilitate the colonization and virulence of microbial seaweed pathogens. One well-described example of a seaweed disease is the bleaching of the red alga Delisea pulchra, which can be caused by the bacterium Nautella italica R11, a member of the Roseobacter clade. This pathogen contains a unique luxR-type gene, varR, which we hypothesize controls its colonization and virulence. We show here that a varR knock-out strain is deficient in its ability to cause disease in D. pulchra and is defective in biofilm formation and attachment to a common algal polysaccharide. Moreover complementation of the varR gene in trans can restore these functions to the wild type levels. Proteomic analysis of bacterial cells in planktonic and biofilm growth highlight the potential importance of nitrogen scavenging, mobilization of energy reserves, and stress resistance in the biofilm lifestyle of N. italica R11. Moreover, we show that VarR regulates the expression of a specific subset of biofilm-associated proteins. Taken together these data suggest that VarR controls colonization and persistence of N. italica R11 on the surface of a macroalgal host and that it is an important regulator of virulence. PMID:26528274

  2. Characteristic odorants from bailingu oyster mushroom (Pleurotus eryngii var. tuoliensis) and summer oyster mushroom (Pleurotus cystidiosus).

    PubMed

    Usami, Atsushi; Motooka, Ryota; Nakahashi, Hiroshi; Okuno, Yoshiharu; Miyazawa, Mitsuo

    2014-01-01

    In this study, the characteristic odorants of the volatile oils from Pleurotus species (P. eryngii var. tuoliensis and P. cystidiosus) were extracted by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS), gas chromatography-olfactometry (GC-O), and aroma extract dilution analysis (AEDA). A total of 52 and 54 components (P. eryngii var. tuoliensis and P. cystidiosus, respectively) were identified, representing about 98.8% and 85.1% of the volatile oils, respectively. The main components of the P. eryngii var. tuoliensis oil were palmitic acid (82, 38.0%), oleic acid (86, 25.0%) and linoleic acid (85, 9.7%). The main components of the P. cystidiosus oil, palmitic acid (82, 25.8%), indole (54, 9.1%) and myristic acid (77, 5.3%). Regarding the aroma components, 16 and 13 components were identified in the P. eryngii var. tuoliensis and P. cystidiosus oils respectively, by the GC-O analyses. The results of the sniffing test, odor activity value (OAV) and flavor dilution (FD) factor indicate that methional, 1-octen-3-ol and nonanal are the main aroma-active components of P. eryngii var. tuoliensis oil. On the other hands, dimethyl trisulfide and 1-octen-3-ol were estimated as the main aroma-active components of the P. cystidiosus oil. PMID:24919476

  3. Subcellular distribution and chemical forms of thorium in Brassica juncea var. foliosa.

    PubMed

    Zhou, Sai; Kai, Hailu; Zha, Zhongyong; Fang, Zhendong; Wang, Dingna; Du, Liang; Zhang, Dong; Feng, Xiaojie; Jin, Yongdong; Xia, Chuanqin

    2016-06-01

    Brassica juncea var. foliosa (B. juncea var. foliosa) is a promising species for thorium (Th) phytoextraction due to its large biomass, fast growth rate and high tolerance toward Th. To further understand the mechanisms of Th tolerance, the present study investigated the subcellular distribution and chemical forms of Th found in B. juncea var. foliosa Our results indicated that in both roots and leaves, Th contents in different parts of the cells follow the order of cell wall > membranes and soluble fraction > organelles. In particular, Transmission Electron Microscope (TEM) analysis showed that Th was abundantly located in cell walls of the roots. Additionally, when plants were exposed to different concentrations of Th, we have found that Th existed in B. juncea var. foliosa with different chemical forms. Much of the Th extracted by 2% acetic acid (HAc), 1 M NaCl and HCl in roots with the percentage distribution varied from 47.2% to 62.5%, while in leaves, most of the Th was in the form of residue and the subdominant amount of Th was extracted by HCl, followed by 2% HAc. This suggested that Th compartmentation in cytosol and integration with phosphate or proteins in cell wall might be responsible for the tolerance of B. juncea var. foliosa to the stress of Th. PMID:27010411

  4. VarMod: modelling the functional effects of non-synonymous variants.

    PubMed

    Pappalardo, Morena; Wass, Mark N

    2014-07-01

    Unravelling the genotype-phenotype relationship in humans remains a challenging task in genomics studies. Recent advances in sequencing technologies mean there are now thousands of sequenced human genomes, revealing millions of single nucleotide variants (SNVs). For non-synonymous SNVs present in proteins the difficulties of the problem lie in first identifying those nsSNVs that result in a functional change in the protein among the many non-functional variants and in turn linking this functional change to phenotype. Here we present VarMod (Variant Modeller) a method that utilises both protein sequence and structural features to predict nsSNVs that alter protein function. VarMod develops recent observations that functional nsSNVs are enriched at protein-protein interfaces and protein-ligand binding sites and uses these characteristics to make predictions. In benchmarking on a set of nearly 3000 nsSNVs VarMod performance is comparable to an existing state of the art method. The VarMod web server provides extensive resources to investigate the sequence and structural features associated with the predictions including visualisation of protein models and complexes via an interactive JSmol molecular viewer. VarMod is available for use at http://www.wasslab.org/varmod. PMID:24906884

  5. Allowing for model error in strong constraint 4D-Var

    NASA Astrophysics Data System (ADS)

    Howes, Katherine; Lawless, Amos; Fowler, Alison

    2016-04-01

    Four dimensional variational data assimilation (4D-Var) can be used to obtain the best estimate of the initial conditions of an environmental forecasting model, namely the analysis. In practice, when the forecasting model contains errors, the analysis from the 4D-Var algorithm will be degraded to allow for errors later in the forecast window. This work focusses on improving the analysis at the initial time by allowing for the fact that the model contains error, within the context of strong constraint 4D-Var. The 4D-Var method developed acknowledges the presence of random error in the model at each time step by replacing the observation error covariance matrix with an error covariance matrix that includes both observation error and model error statistics. It is shown that this new matrix represents the correct error statistics of the innovations in the presence of model error. A method for estimating this matrix using innovation statistics, without requiring prior knowledge of the model error statistics, is presented. The method is demonstrated numerically using a non-linear chaotic system with erroneous parameter values. We show that that the new method works to reduce the analysis error covariance when compared with a standard strong constraint 4D-Var scheme. We discuss the fact that an improved analysis will not necessarily provide a better forecast.

  6. Genetic diversity in wild diploid wheats Triticum monococcum var. boeoticum and T. urartu (Poaceae).

    PubMed

    Smith-Huerta, N L; Huerta, A J; Barnhart, D; Waines, J G

    1989-08-01

    The genetic diversity of two wild diploid wheat species, Triticum monococcum var. boeoticum and T. urartu, was assessed using starch gel electrophoresis. Genetic diversity is uniformly low in both species. Number of alleles per locus was very low with a mean of 1.22 for T. monococcum var. boeoticum and 1.19 in T. urartu. Percentage of polymorphic loci was also low, with a mean of 19.71 for T. monococcum var. boeoticum and a mean of 18.35 for T. urartu. Mean gene diversity was low with a mean of 0.052 in populations of T. monococcum var. boeoticum and a mean of 0.040 in populations of T. urartu. Genetic affinities of the species and of populations were computed using Nei's identity index (NI). Overall genetic affinities of the two species are NI=0.697. The genetic affinities of different populations of a species are uniformly high with NIs ranging from 0.894 to 1.000 in T. monococcum var. boeoticum and from 0.898 to 1.000 in T. urartu. PMID:24227153

  7. A new approach for optimal VAR sources planning in large scale electric power systems

    SciTech Connect

    Yingtung Hsiao; Chunchang Liu; Yuanlin Chen . Dept. of Electrical Engineering); Hsiodong Chiang )

    1993-08-01

    This paper presents a new approach for contingency-constrained optimal reactive volt amper (VAR) sources planning in large-scale power systems. Features distinguishing the proposed approach from many of the existing methods include that it allows a more realistic problem formulation, and that it can find the (global) optimal solution. The new problem formulation takes into consideration practical aspects of VAR sources; the load constraints and operational constraints at different load levels. This solution methodology based on simulated annealing determines (1) the location to install VAR sources; (2) the types and sizes of VAR sources to be installed; and (3) the settings of VAR sources at different loading conditions. In order to speed up the solution algorithm, this paper makes a slight modification of the fast decoupled load flow and incorporates it into the solution algorithm. This method is suitable for large-scale power systems and has been tested on several power systems with promising results. Simulation results on the IEEE 30 buses system and the Tai-power 358 buses system are presented.

  8. [Diversity and community structure of endophytic fungi from Taxus chinensis var. mairei].

    PubMed

    2014-07-01

    A total of 628 endophytic fungi were isolated from 480 tissue segments of needles and branches of Taxus chinensis var. mairei. According to morphological characteristics and ITS sequences, they represented 43 taxa in 28 genera, of which 10 Hyphomycetes, 20 Coelomycetes, 12 Ascomycetes and 1 unknown fungus. Phomopsis mali was confirmed as the dominant species. In accordance with relative frequency, Alternaria alternata, Aureobasidium pullulans, Colletotrichum boninense, C. gloeosporioides, Epicoccum nigrum , Fungal sp., Fusarium lateritium, Glomerella cingulata, Magnaporthales sp. , Nigrospora oryzae, Pestalotiopsis maculiformans, P. microspora, Peyronellaea glomerata and Xylaria sp. 1 were more common in T. chinensis var. mairei. T. chinensis var. mairei were severely infected by endophytic fungi. Endophytic fungi were found in 81 percent of plant tissues with a high diversity. Distribution ranges of endophytic fungi were influenced by tissue properties. The colonization rate, richness, diversity of endophytic fungi in needles were obviously lower than in branches, and kinds of endophytic fungi between branches were more similar than those in needles, thus endophytic fungi had tissue preference. In addition, tissue age influenced the community structure of endophytic fungi. The elder branch tissues were, the higher colonization rate, richness, diversity of endophytic fungi were. Systematic studying the diversity and community structure of endophytic fungi in T. chinensis var. mairei and clarifying their distribution regularity in plant tissues would offer basic data and scientific basis for their development and utilization. Discussing the presence of fungal pathogens in healthy plant tissues would be of positive significance for source protection of T. chinensis var. mairei. PMID:25345060

  9. The Cryptococcal Enzyme Inositol Phosphosphingolipid-Phospholipase C Confers Resistance to the Antifungal Effects of Macrophages and Promotes Fungal Dissemination to the Central Nervous System†

    PubMed Central

    Shea, John M.; Kechichian, Talar B.; Luberto, Chiara; Del Poeta, Maurizio

    2006-01-01

    In recent years, sphingolipids have emerged as critical molecules in the regulation of microbial pathogenesis. In fungi, the synthesis of complex sphingolipids is important for the regulation of pathogenicity, but the role of sphingolipid degradation in fungal virulence is not known. Here, we isolated and characterized the inositol phosphosphingolipid-phospholipase C1 (ISC1) gene from the fungal pathogen Cryptococcus neoformans and showed that it encodes an enzyme that metabolizes fungal inositol sphingolipids. Isc1 protects C. neoformans from acidic, oxidative, and nitrosative stresses, which are encountered by the fungus in the phagolysosomes of activated macrophages, through a Pma1-dependent mechanism(s). In an immunocompetent mouse model, the C. neoformans Δisc1 mutant strain is almost exclusively found extracellularly and in a hyperencapsulated form, and its dissemination to the brain is remarkably reduced compared to that of control strains. Interestingly, the dissemination of the C. neoformans Δisc1 strain to the brain is promptly restored in these mice when alveolar macrophages are pharmacologically depleted or when infecting an immunodeficient mouse in which macrophages are not efficiently activated. These studies suggest that Isc1 plays a key role in protecting C. neoformans from the intracellular environment of macrophages, whose activation is important for preventing fungal dissemination of the Δisc1 strain to the central nervous system and the development of meningoencephalitis. PMID:16988277

  10. RBF neural network prediction on weak electrical signals in Aloe vera var. chinensis

    NASA Astrophysics Data System (ADS)

    Wang, Lanzhou; Zhao, Jiayin; Wang, Miao

    2008-10-01

    A Gaussian radial base function (RBF) neural network forecast on signals in the Aloe vera var. chinensis by the wavelet soft-threshold denoised as the time series and using the delayed input window chosen at 50, is set up to forecast backward. There was the maximum amplitude at 310.45μV, minimum -75.15μV, average value -2.69μV and <1.5Hz at frequency in Aloe vera var. chinensis respectively. The electrical signal in Aloe vera var. chinensis is a sort of weak, unstable and low frequency signals. A result showed that it is feasible to forecast plant electrical signals for the timing by the RBF. The forecast data can be used as the preferences for the intelligent autocontrol system based on the adaptive characteristic of plants to achieve the energy saving on the agricultural production in the plastic lookum or greenhouse.

  11. Molecular and morphological evidence for Penstemon luculentus (Plantaginaceae): a replacement name for Penstemon fremontii var. glabrescens.

    PubMed

    Johnson, Robert L; Stevens, Mikel R; Johnson, Leigh A; Robbins, Matthew D; Anderson, Chris D; Ricks, Nathan J; Farley, Kevin M

    2016-01-01

    Penstemon luculentus R.L.Johnson & M.R.Stevens, nom. nov. replaces Penstemon fremontii var. glabrescens Dorn & Lichvar. The varietal name glabrescens was not elevated because it was already occupied by Penstemon glabrescens Pennell, a different species. This new arrangement is supported by molecular and morphological evidence. An analysis of genetic diversity in populations of both varieties of Penstemon fremontii Torr. & A. Gray (glabrescens and fremontii) from the Piceance Basin, Colorado, using SSR (simple sequences repeats) or microsatellites markers, revealed significant genetic differentiation between the two. Penstemon fremontii var. glabrescens was also genetically different from Penstemon gibbensii Dorn and Penstemon scariosus var. garrettii (Pennell) N.H. Holmgren. The combination of hirtellous stems, glabrous leaves, non-glandular inflorescence, and long anther hairs distinguish Penstemon luculentus from other morphologically similar species. PMID:27489478

  12. Molecular and morphological evidence for Penstemon luculentus (Plantaginaceae): a replacement name for Penstemon fremontii var. glabrescens

    PubMed Central

    Johnson, Robert L.; Stevens, Mikel R.; Johnson, Leigh A.; Robbins, Matthew D.; Anderson, Chris D.; Ricks, Nathan J.; Farley, Kevin M.

    2016-01-01

    Abstract Penstemon luculentus R.L.Johnson & M.R.Stevens, nom. nov. replaces Penstemon fremontii var. glabrescens Dorn & Lichvar. The varietal name glabrescens was not elevated because it was already occupied by Penstemon glabrescens Pennell, a different species. This new arrangement is supported by molecular and morphological evidence. An analysis of genetic diversity in populations of both varieties of Penstemon fremontii Torr. & A. Gray (glabrescens and fremontii) from the Piceance Basin, Colorado, using SSR (simple sequences repeats) or microsatellites markers, revealed significant genetic differentiation between the two. Penstemon fremontii var. glabrescens was also genetically different from Penstemon gibbensii Dorn and Penstemon scariosus var. garrettii (Pennell) N.H. Holmgren. The combination of hirtellous stems, glabrous leaves, non-glandular inflorescence, and long anther hairs distinguish Penstemon luculentus from other morphologically similar species. PMID:27489478

  13. Dynamic performance and control of a static var generator using cascade multilevel inverters

    SciTech Connect

    Peng, Fang Zheng; Lai, Jih-Sheng

    1996-10-01

    A cascade multilevel inverter is proposed for static VAR shifting, compensation/generation applications. The new cascade M-level inverter consists of (M-1)/2 single-phase full bridges in which each bridge has its own separate dc source. This inverter can generate almost sinusoidal waveform voltage with only one time switching per cycle. It can eliminate the need for transformers in multipulse inverters. A prototype static VAR generator (SVG) system using 11- level cascade inverter (21-level line-to-line voltage waveform) has been built. The output voltage waveform is equivalent to that of a 60- pulse inverter. This paper focuses on dynamic performance of the cascade inverter based SVG system. Control schemes are proposed to achieve a fast response which is impossible for a conventional static VAR compensator (SVC). Analytical, simulated and experimental results show the superiority of the proposed SVG system.

  14. Volt-VAR Optimization on American Electric Power Feeders in Northeast Columbus

    SciTech Connect

    Schneider, Kevin P.; Weaver, T. F.

    2012-05-10

    In 2007 American Electric Power launched the gridSMART® initiative with the goals of increasing efficiency of the electricity delivery system and improving service to the end-use customers. As part of the initiative, a coordinated Volt-VAR system was deployed on eleven distribution feeders at five substations in the Northeast Columbus Ohio Area. The goal of the coordinated Volt-VAR system was to decrease the amount of energy necessary to provide end-use customers with the same quality of service. The evaluation of the Volt-VAR system performance was conducted in two stages. The first stage was composed of simulation, analysis, and estimation, while the second stage was composed of analyzing collected field data. This panel paper will examine the analysis conducted in both stages and present the estimated improvements in system efficiency.

  15. A novel, high-frequency, per-phase static var compensator

    SciTech Connect

    Mohan, N.; Kamath, G.R.

    1995-12-31

    Static var compensators are being used to provide voltage control, var compensation, damping of oscillations, and improved transient stability in power systems. Conventional SVCs which consist of thyristor-switched capacitors and thyristor controlled inductors suffer from disadvantages of size and slow dynamic response. This makes it unsuitable for applications such as controlling the voltage flicker due to arc furnace loads. Advanced SVCs (STATCON or STATCOM) eliminate the above disadvantages. However, they have a disadvantage due to the presence of a large line-frequency transformer. In this paper, a novel, high-frequency, per-phase static var compensator is proposed, which has all the advantages of the advanced SVC. Various related topologies and control strategies are also enumerated and their advantages and disadvantages are discussed.

  16. FlyVar: a database for genetic variation in Drosophila melanogaster

    PubMed Central

    Wang, Fei; Jiang, Lichun; Chen, Yong; Haelterman, Nele A.; Bellen, Hugo J.; Chen, Rui

    2015-01-01

    FlyVar is a publicly and freely available platform that addresses the increasing need of next generation sequencing data analysis in the Drosophila research community. It is composed of three parts. First, a database that contains 5.94 million DNA polymorphisms found in Drosophila melanogaster derived from whole genome shotgun sequencing of 612 genomes of D. melanogaster. In addition, a list of 1094 dispensable genes has been identified. Second, a graphical user interface (GUI) has been implemented to allow easy and flexible queries of the database. Third, a set of interactive online tools enables filtering and annotation of genomic sequences obtained from individual D. melanogaster strains to identify candidate mutations. FlyVar permits the analysis of next generation sequencing data without the need of extensive computational training or resources. Database URL: www.iipl.fudan.edu.cn/FlyVar. PMID:26289428

  17. An optimization approach for online identification of harmonic resonance due to pending Volt/VAr operation

    NASA Astrophysics Data System (ADS)

    McBee, Kerry D.

    The emphasis on creating a more efficient distribution system has led many utility companies to employ dynamic voltage and VAr compensation (Volt/VAr) applications that reduce energy demand, generation, and losses associated with the transmission and distribution of energy. To achieve these benefits, Volt/VAr applications rely upon algorithms to control voltage support equipment, such as transformer load tap changers, voltage regulators, and capacitor banks. The majority of these algorithms utilize metaheuristic programming methods to determine the Volt/VAr scheme that produces the most energy efficient operating conditions. It has been well documented that the interaction between capacitor bank reactance and the inductive reactance of a distribution system can produce parallel harmonic resonance that can damage utility and customer equipment. The Volt/VAr controlling algorithms that account for harmonics do so in an indirect manner that can mask harmonic resonance conditions. Unlike previous research endeavors, the primary focus of the method described within this dissertation is to identify Volt/VAr schemes that prevent harmonic resonance due to capacitor bank operation. Instead of a metaheuristic approach, the harmonic resonance identification algorithm relies upon constrained mixed integer nonlinear programming (MINLP), which is more suited for analyzing impedance characteristics created by the energized states of a system of capacitor banks. Utilizing a numerical approach improves the accuracy of identifying harmonic resonance conditions, while also reducing the complexity of the process by exclusively relying upon the system's admittance characteristics. The novel harmonic resonance identification method is applicable to distribution systems that are dynamically reconfigured, which can result in a number of unknown harmonic resonance producing conditions, a feature unavailable with existing controlling algorithms. The ability to identify all harmonic

  18. A computer package for optimal multi-objective VAR planning in large scale power systems

    SciTech Connect

    Chiang, H.D. . School of Electrical Engineering); Liu, C.C.; Chen, Y.L. . Dept. of Electrical Engineering); Hsiao, Y.T.

    1994-05-01

    This paper presents a simulated annealing based computer package for multi-objective, VAR planning in large scale power systems - SAMVAR. This computer package has three distinct features. First, the optimal VAR planning is reformulated as a constrained, multi-objective, non-differentiable optimization problem. The new formulation considers four different objective functions related to system investment, system operational efficiency, system security and system service quality. The new formulation also takes into consideration load, operation and contingency constraints. Second, it allows both the objective functions and equality and inequality constraints to be non-differentiable; making the problem formulation more realistic. Third, the package employs a two-stage solution algorithm based on an extended simulated annealing technique and the [var epsilon]-constraint method. The first-stage of the solution algorithm uses an extended simulated annealing technique to find a global, non-inferior solution. The results obtained from the first stage provide a basis for planners to prioritize the objective functions such that a primary objective function is chosen and tradeoff tolerances for the other objective functions are set. The primary objective function and the trade-off tolerances are then used to transform the constrained multi-objective optimization problem into a single-objective optimization problem with more constraints by employing the [var epsilon]-constraint method. The second-stage uses the simulated annealing technique to find the global optimal solution. A salient feature of SAMVAR is that it allows planners to find an acceptable, global non-inferior solution for the VAR problem. Simulation results indicate that SAMVAR has the ability to handle the multi-objective VAR planning problem and meet with the planner's requirements.

  19. Genetic Variability and Population Structure of the Mushroom Pleurotus eryngii var. tuoliensis

    PubMed Central

    Zhao, Mengran; Huang, Chenyang; Chen, Qiang; Wu, Xiangli; Qu, Jibin; Zhang, Jinxia

    2013-01-01

    The genetic diversity of 123 wild strains of Pleurotus eryngii var. tuoliensis, which were collected from nine geographical locations in Yumin, Tuoli, and Qinghe counties in the Xinjiang Autonomous Region of China, was analysed using two molecular marker systems (inter-simple sequence repeat and start codon targeted). At the variety level, the percentage of polymorphic loci and Nei’s gene diversity index for P. eryngii var. tuoliensis was 96.32% and 0.238, respectively. At the population level, Nei’s gene diversity index ranged from 0.149 to 0.218 with an average of 0.186, and Shannon's information index ranged from 0.213 to 0.339 with an average of 0.284. These results revealed the abundant genetic variability in the wild resources of P. eryngii var. tuoliensis. Nei’s gene diversity analysis indicated that the genetic variance was mainly found within individual geographical populations, and the analysis of molecular variance revealed low but significant genetic differentiation among local and regional populations. The limited gene flow (Nm = 1.794) was inferred as a major reason for the extent of genetic differentiation of P. eryngii var. tuoliensis. The results of Mantel tests showed that the genetic distance among geographical populations of P. eryngii var. tuoliensis was positively correlated with the geographical distance and the longitudinal distances (rGo = 0.789 and rLn = 0.873, respectively), which indicates that geographical isolation is an important factor for the observed genetic differentiation. Nine geographical populations of P. eryngii var. tuoliensis were divided into three groups according to their geographical origins, which revealed that the genetic diversity was closely related to the geographical distribution of this wild fungus. PMID:24349475

  20. Resistance to Southern Root-knot Nematode (Meloidogyne incognita) in Wild Watermelon (Citrullus lanatus var. citroides) Populations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Southern root-knot nematode (Meloidogyne incognita) is a serious pest of cultivated watermelon (Citrullus lanatus var. lanatus) in southern regions of the US and no resistance is known to exist in commercial watermelon cultivars. Wild watermelon relatives (C. lanatus var. citroides) have been shown...

  1. 40 CFR 180.1243 - Bacillus subtilis var. amyloliquefaciens strain FZB24; exemption from the requirement of a...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Bacillus subtilis var... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1243 Bacillus subtilis... the requirement of a tolerance for residues of the Bacillus subtilis var. amyloliquefaciens...

  2. 40 CFR 180.1243 - Bacillus subtilis var. amyloliquefaciens strain FZB24; exemption from the requirement of a...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Bacillus subtilis var... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1243 Bacillus subtilis... the requirement of a tolerance for residues of the Bacillus subtilis var. amyloliquefaciens...

  3. Erasing the Epigenetic Memory and Beginning to Switch—The Onset of Antigenic Switching of var Genes in Plasmodium falciparum

    PubMed Central

    Fastman, Yair; Noble, Robert; Recker, Mario; Dzikowski, Ron

    2012-01-01

    Antigenic variation in Plasmodium falciparum is regulated by transcriptional switches among members of the var gene family, each expressed in a mutually exclusive manner and encoding a different variant of the surface antigens collectively named PfEMP1. Antigenic switching starts when the first merozoites egress from the liver and begin their asexual proliferation within red blood cells. By erasing the epigenetic memory we created parasites with no var background, similar to merozoites that egress from the liver where no var gene is expressed. Creating a null-var background enabled us to investigate the onset of antigenic switches at the early phase of infection. At the onset of switching, var transcription pattern is heterogeneous with numerous genes transcribed at low levels including upsA vars, a subtype that was implicated in severe malaria, which are rarely activated in growing cultures. Analysis of subsequent in vitro switches shows that the probability of a gene to turn on or off is not associated with its chromosomal position or promoter type per se but on intrinsic properties of each gene. We concluded that var switching is determined by gene specific associated switch rates rather than general promoter type or locus associated switch rates. In addition, we show that fine tuned reduction in var transcription increases their switch rate, indicating that transcriptional perturbation can alter antigenic switching. PMID:22461905

  4. Accessions of Citrullus lanatus var. Citroides are Valuable Rootstocks for Grafted Watermelon in Fields Infested with Root-Knot Nematodes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wild watermelon (Citrullus lanatus var. citroides) RKVL rootstock lines developed at the U.S. Vegetable Laboratory, USDA, ARS in Charleston, South Carolina, were compared to wild tinda and commercial cucurbit rootstock cultivars for grafting of seedless watermelon ‘Tri-X 313’ (C. lanatus var. lanatu...

  5. Genetic interactions reveal that specific defects of chloroplast translation are associated with the suppression of var2-mediated leaf variegation.

    PubMed

    Liu, Xiayan; Zheng, Mengdi; Wang, Rui; Wang, Ruijuan; An, Lijun; Rodermel, Steve R; Yu, Fei

    2013-10-01

    Arabidopsis thaliana L. yellow variegated (var2) mutant is defective in a chloroplast FtsH family metalloprotease, AtFtsH2/VAR2, and displays an intriguing green and white leaf variegation. This unique var2-mediated leaf variegation offers a simple yet powerful tool for dissecting the genetic regulation of chloroplast development. Here, we report the isolation and characterization of a new var2 suppressor gene, SUPPRESSOR OF VARIEGATION8 (SVR8), which encodes a putative chloroplast ribosomal large subunit protein, L24. Mutations in SVR8 suppress var2 leaf variegation at ambient temperature and partially suppress the cold-induced chlorosis phenotype of var2. Loss of SVR8 causes unique chloroplast rRNA processing defects, particularly the 23S-4.5S dicistronic precursor. The recovery of the major abnormal processing site in svr8 23S-4.5S precursor indicate that it does not lie in the same position where SVR8/L24 binds on the ribosome. Surprisingly, we found that the loss of a chloroplast ribosomal small subunit protein, S21, results in aberrant chloroplast rRNA processing but not suppression of var2 variegation. These findings suggest that the disruption of specific aspects of chloroplast translation, rather than a general impairment in chloroplast translation, suppress var2 variegation and the existence of complex genetic interactions in chloroplast development. PMID:23721655

  6. Hierarchical control design challenge problems for optimal VAR planning and real time voltage control in electric power systems

    SciTech Connect

    Mehra, R.K.

    1994-12-31

    To optimize VAR sources on a power system, system planners must identify the location, size, and type of all available reactive power devices. The Control Design Challenge problem is to determine the least-costly VAR expansion pattern that will enable large power system to maintain acceptable voltage levels in all conditions.

  7. The Chondroitin Sulfate A-binding Site of the VAR2CSA Protein Involves Multiple N-terminal Domains*

    PubMed Central

    Dahlbäck, Madeleine; Jørgensen, Lars M.; Nielsen, Morten A.; Clausen, Thomas M.; Ditlev, Sisse B.; Resende, Mafalda; Pinto, Vera V.; Arnot, David E.; Theander, Thor G.; Salanti, Ali

    2011-01-01

    Malaria during pregnancy is a major health problem for African women. The disease is caused by Plasmodium falciparum malaria parasites, which accumulate in the placenta by adhering to chondroitin sulfate A (CSA). The interaction between infected erythrocytes and the placental receptor is mediated by a parasite expressed protein named VAR2CSA. A vaccine protecting pregnant women against placental malaria should induce antibodies inhibiting the interaction between VAR2CSA and CSA. Much effort has been put into defining the part of the 350 kDa VAR2CSA protein that is responsible for binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with high affinity, however to date no sub-fragment of VAR2CSA has been shown to interact with CSA with similar affinity or specificity. In this study, we used a biosensor technology to examine the binding properties of a panel of truncated VAR2CSA proteins. The experiments indicate that the core of the CSA-binding site is situated in three domains, DBL2X-CIDRPAM and a flanking domain, located in the N-terminal part of VAR2CSA. Furthermore, recombinant VAR2CSA subfragments containing this region elicit antibodies with high parasite adhesion blocking activity in animal immunization experiments. PMID:21398524

  8. 40 CFR 180.1243 - Bacillus subtilis var. amyloliquefaciens strain FZB24; exemption from the requirement of a...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Bacillus subtilis var... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1243 Bacillus subtilis... the requirement of a tolerance for residues of the Bacillus subtilis var. amyloliquefaciens...

  9. 40 CFR 180.1243 - Bacillus subtilis var. amyloliquefaciens strain FZB24; exemption from the requirement of a...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Bacillus subtilis var... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1243 Bacillus subtilis... the requirement of a tolerance for residues of the Bacillus subtilis var. amyloliquefaciens...

  10. 40 CFR 180.1243 - Bacillus subtilis var. amyloliquefaciens strain FZB24; exemption from the requirement of a...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Bacillus subtilis var... EXEMPTIONS FOR PESTICIDE CHEMICAL RESIDUES IN FOOD Exemptions From Tolerances § 180.1243 Bacillus subtilis... the requirement of a tolerance for residues of the Bacillus subtilis var. amyloliquefaciens...

  11. Two New Highly Oxygenated Spirostanol Saponins from Paris polyphylla var. stenophylla.

    PubMed

    Jin, Ling-Yu; Lu, Ting-Xiang; Qin, Xu-Jie; Ni, Wei; Yan, Huan; Chen, Yu; Liu, Hui; He, Hong-Ping; Liu, Hai-Yang

    2016-08-01

    Phytochemical investigation of the rhizomes of Paris polyphylla var. stenophylla led to the isolation of two new highly oxygenated spirostanol saponins, named paristenosides A (1) and B (2), together with seven known compounds. Their structures were established mainly on the base of NMR spectroscopic techniques and mass spectrometry, as well as chemical methods. In addition, the cytotoxicity of the two new saponins was tested. Two new highly oxygenated spirostanol saponins, paristenosides A (1) and B (2), were isolated from the rhizomes of Paris polyphylla var. stenophylla. Their structures were established mainly based on NMR spectroscopic techniques and mass spectrometry, as well as chemical methods. PMID:27255683

  12. Development of polymorphic microsatellite loci in the perennial herb Hepatica nobilis var. japonica (Ranunculaceae)1

    PubMed Central

    Kameoka, Shinichiro; Higashi, Hiroyuki; Setoguchi, Hiroaki

    2015-01-01

    Premise of the study: Microsatellite markers were developed and characterized in the vulnerable plant Hepatica nobilis var. japonica (Ranunculaceae) to investigate its genetic diversity, population structure, and gene flow. Methods and Results: Fourteen microsatellite markers were developed. The number of alleles per locus ranged from one to 12, and the expected heterozygosity per locus ranged from 0.043 to 0.855. Eleven markers were successfully amplified in the cultivar ‘Mego’ from Japan. Conclusions: These microsatellite markers can be used to investigate the genetic diversity, population structure, and gene flow of H. nobilis var. japonica. PMID:25798342

  13. [Finding of dairy yeasts Kluyveromyces lactis var. lactis in natural habitats].

    PubMed

    Naumov, G I; Naumova, E S; Glushakova, A M; Kachalkin, A V; Chernov, I Y

    2014-01-01

    Well-known yeasts Kluyveromyces lactis var. lactis, which are usually associated with dairy prod- ucts, were discovered in nature (in woodland park soil under Impatiens glandulifera Royle plants). Reliable identification of the yeasts was carried out using physiological criteria (lactose and maltose utilization) and molecular markers (nucleotide sequence of the 5.8S-ITS rDNA fragment, pulsed-field electrophoresis, and Southern hybridization of chromosomal DNA with the LAC4 probe). Ecology of KI. lactis var. lactis is discussed. PMID:25941717

  14. Genetic and phenological variation of tocochromanol (vitamin E) content in wild (Daucus carota L. var. carota) and domesticated carrot (D. carota L. var. sativa)

    PubMed Central

    Luby, Claire H; Maeda, Hiroshi A; Goldman, Irwin L

    2014-01-01

    Carrot roots (Daucus carota L. var. sativa) produce tocochromanol compounds, collectively known as vitamin E. However, little is known about their types and amounts. Here we determined the range and variation in types and amounts of tocochromanols in a variety of cultivated carrot accessions throughout carrot postharvest storage and reproductive stages and in wild-type roots (Daucus carota L. var. carota). Of eight possible tocochromanol compounds, we detected and quantified α-, and the combined peak for β- and γ- forms of tocopherols and tocotrienols. Significant variation in amounts of tocochromanol compounds was observed across accessions and over time. Large increases in α-tocopherol were noted during both reproductive growth and the postharvest stages. The variation of tocochromanols in carrot root tissue provides useful information for future research seeking to understand the role of these compounds in carrot root tissue or to breed varieties with increased levels of these compounds. PMID:26504534

  15. Murine Model for Preclinical Studies of Var2CSA-Mediated Pathology Associated with Malaria in Pregnancy.

    PubMed

    de Moraes, Luciana V; Dechavanne, Sebastien; Sousa, Patrícia M; Barateiro, André; Cunha, Sónia F; Nunes-Silva, Sofia; Lima, Flávia A; Murillo, Oscar; Marinho, Claudio R F; Gangnard, Stephane; Srivastava, Anand; Braks, Joanna A; Janse, Chris J; Gamain, Benoit; Franke-Fayard, Blandine; Penha-Gonçalves, Carlos

    2016-06-01

    Plasmodium falciparum infection during pregnancy leads to abortions, stillbirth, low birth weight, and maternal mortality. Infected erythrocytes (IEs) accumulate in the placenta by adhering to chondroitin sulfate A (CSA) via var2CSA protein exposed on the P. falciparum IE membrane. Plasmodium berghei IE infection in pregnant BALB/c mice is a model for severe placental malaria (PM). Here, we describe a transgenic P. berghei parasite expressing the full-length var2CSA extracellular region (domains DBL1X to DBL6ε) fused to a P. berghei exported protein (EMAP1) and characterize a var2CSA-based mouse model of PM. BALB/c mice were infected at midgestation with different doses of P. berghei-var2CSA (P. berghei-VAR) or P. berghei wild-type IEs. Infection with 10(4) P. berghei-VAR IEs induced a higher incidence of stillbirth and lower fetal weight than P. berghei At doses of 10(5) and 10(6) IEs, P. berghei-VAR-infected mice showed increased maternal mortality during pregnancy and fetal loss, respectively. Parasite loads in infected placentas were similar between parasite lines despite differences in maternal outcomes. Fetal weight loss normalized for parasitemia was higher in P. berghei-VAR-infected mice than in P. berghei-infected mice. In vitro assays showed that higher numbers of P. berghei-VAR IEs than P. berghei IEs adhered to placental tissue. Immunization of mice with P. berghei-VAR elicited IgG antibodies reactive to DBL1-6 recombinant protein, indicating that the topology of immunogenic epitopes is maintained between DBL1-6-EMAP1 on P. berghei-VAR and recombinant DBL1-6 (recDBL1-6). Our data suggested that impairments in pregnancy caused by P. berghei-VAR infection were attributable to var2CSA expression. This model provides a tool for preclinical evaluation of protection against PM induced by approaches that target var2CSA. PMID:27045035

  16. Toxicity of "Bacillus thuringiensis var. Kurstaki" to the Painted Lady Butterfly, Vanessa cardui.

    ERIC Educational Resources Information Center

    Stalter, Richard; Nadal, Gerard; Kincaid, Dwight

    2000-01-01

    Reports the effects of Bacillus thuringiensis var. Kurstaki (BT), which is highly toxic, to a