RESUMO
Hybrid AD strains of the human pathogenic Cryptococcus neoformans species complex have been reported from many parts of the world. However, their origin, diversity, and evolution are incompletely understood. In this study, we analyzed 102 AD hybrid strains representing 21 countries on five continents. For each strain, we obtained its mating type and its allelic sequences at each of the seven loci that have been used for genotyping haploid serotypes A and D strains of the species complex by the Cryptococcus research community. Our results showed that most AD hybrids exhibited loss of heterozygosity at one or more of the seven analyzed loci. Phylogenetic and population genetic analyses of the allelic sequences revealed multiple origins of the hybrids within each continent, dating back to one million years ago in Africa and up to the present in other continents. We found evidence for clonal reproduction and long-distance dispersal of these hybrids in nature. Comparisons with the global haploid serotypes A and D strains identified new alleles and new haploid multi-locus genotypes in AD hybrids, consistent with the presence of yet-to-be discovered genetic diversity in haploid populations of this species complex in nature. Together, our results indicate that AD hybrids can be effectively genotyped using the same multi-locus sequencing type approach as that established for serotypes A and D strains. Our comparisons of the AD hybrids among each other as well as with the global haploid serotypes A and D strains revealed novel genetic diversity as well as evidence for multiple origins and dynamic evolution of these hybrids in nature.
Assuntos
Criptococose , Cryptococcus neoformans , Humanos , Cryptococcus neoformans/genética , Tipagem de Sequências Multilocus , Filogenia , GenótipoRESUMO
IMPORTANCE: Cryptococcosis studies often utilize the common C57BL/6J mouse model. Unfortunately, infection in these mice fails to replicate the basic course of human disease, particularly hampering immunological studies. This work demonstrates that SJL/J mice can recapitulate human infection better than other mouse strains. The immunological response to Cryptococcus infection in SJL/J mice was markedly different from C57BL/6J and much more productive in combating this infection. Characterization of infected mice demonstrated strain-specific genetic linkage and differential regulation of multiple important immune-relevant genes in response to Cryptococcus infection. While our results validate many of the previously identified immunological features of cryptococcosis, we also demonstrate limitations from previous mouse models as they may be less translatable to human disease. We concluded that SJL/J mice more faithfully recapitulate human cryptococcosis serving as an exciting new animal model for immunological and genetic studies.
Assuntos
Criptococose , Cryptococcus neoformans , Humanos , Camundongos , Animais , Cryptococcus neoformans/genética , Camundongos Endogâmicos C57BL , Modelos Animais de DoençasRESUMO
Conidial pigment is an important virulence factor in Aspergillus fumigatus, a human fungal pathogen. The biosynthetic gene cluster for 1,8-dihydroxynaphthalene (DHN)-melanin in A. fumigatus consists of six genes, alb1, ayg1, arp1, arp2, abr1 and abr2. In contrast to black DHN-melanin fungi such as Magnaporthe grisea, the polyketide synthase Alb1p in A. fumigatus produces naphthopyrone YWA1 instead of 1,3,6,8-THN (T4HN) and YWA1 is converted to T4HN by Ayg1p. The yeast transformant expressing Alb1p and Arp1p dehydratase produced an unknown compound which was identified to be a novel angular naphthopyrone named YWA3 formed from YWA1. In addition, the amount of YWA3 produced was much more than that of YWA2 formed by non-enzymatic dehydration from YWA1. To further analyse the reaction in vitro, Arp1p was overexpressed in E. coli and purified. Kinetic analysis revealed Km value of Arp1p for YWA1 to be 41 µM which is comparable with that of Ayg1p for YWA1 in conversion to T4HN. The complex structure modelling well explained the mechanism of YWA3 generation by the dehydration of angular YWA1 by Arp1p. These results indicated the possibility that polymerization of angular naphthopyrone YWA3 but not YWA2 could be involved in the characteristic bluish-green conidial pigmentation of A. fumigatus.
Assuntos
Aspergillus fumigatus , Melaninas , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Hidroliases , CinéticaRESUMO
Clinically relevant members of the Scedosporium/Pseudallescheria species complex and Lomentospora prolificans are generally resistant against currently available systemic antifungal agents in vitro, and infection due to these species is difficult to treat. We studied the in vivo efficacy of a new fungicidal agent, olorofim (formerly F901318), against scedosporiosis and lomentosporiosis in neutropenic animals. Cyclophosphamide-immunosuppressed CD-1 mice infected by Scedosporium apiospermum, Pseudallescheria boydii (Scedosporium boydii), and Lomentospora prolificans were treated by intraperitoneal administration of olorofim (15 mg/kg of body weight every 8 h for 9 days). The efficacy of olorofim treatment was assessed by the survival rate at 10 days postinfection, levels of serum (1-3)-ß-d-glucan (BG), histopathology, and fungal burdens of kidneys 3 days postinfection. Olorofim therapy significantly improved survival compared to that of the untreated controls; 80%, 100%, and 100% of treated mice survived infection by Scedosporium apiospermum, Pseudallescheria boydii, and Lomentospora prolificans, respectively, while less than 20% of the control mice (phosphate-buffered saline [PBS] treated) survived at 10 days postinfection. In the olorofim-treated neutropenic CD-1 mice infected with any of the three species, serum BG levels were significantly suppressed and fungal DNA detected in the target organs was significantly lower than in controls. Furthermore, histopathology of kidneys revealed no or only a few lesions with hyphal elements in the olorofim-treated mice, while numerous fungal hyphae were present in control mice. These results indicate olorofim to be a promising therapeutic agent for systemic scedosporiosis/lomentosporiosis, devastating emerging fungal infections that are difficult to treat with currently available antifungals.
Assuntos
Pirimidinas , Scedosporium , Acetamidas , Animais , Antifúngicos/uso terapêutico , Infecções Fúngicas Invasivas , Camundongos , Piperazinas , PirróisRESUMO
The emergence of azole resistance in Aspergillus fumigatus as well as an increasing frequency of multiresistant cryptic Aspergillus spp. necessitates exploration of new classes of antifungals. Olorofim (formerly F901318) is a new fungicidal agent that prevents the growth of ascomycetous mold species via inhibition of de novo pyrimidine biosynthesis, a mechanism of action distinct from that of currently available antifungal drugs. We studied the in vivo efficacy of olorofim intraperitoneal therapy (15 mg/kg of body weight every 8 h for 9 days) against infection with A. fumigatus, A. nidulans, and A. tanneri in both neutropenic CD-1 mice and mice with chronic granulomatous disease (CGD) (gp91-/-phox mice). In the neutropenic mouse model, 80% to 88% of treated mice survived for 10 days, and in the CGD group, 63% to 88% of treated mice survived for 10 days, depending on the infecting species, while less than 10% of the mice in the control groups survived for 10 days. In the olorofim-treated groups, galactomannan levels were significantly suppressed, with lower organ fungal DNA burdens being seen for all three Aspergillus spp. Histopathological slides revealed a limited number of inflammatory foci with or without detectable fungal elements in the kidneys of neutropenic CD-1 mice and in the lungs of CGD mice. Furthermore, the efficacy of olorofim was unrelated to the triazole MICs of the infecting Aspergillus spp. These results show olorofim to be a promising therapeutic agent for invasive aspergillosis.
Assuntos
Acetamidas/farmacologia , Antifúngicos/farmacologia , Aspergilose/tratamento farmacológico , Aspergillus/efeitos dos fármacos , Doença Granulomatosa Crônica/complicações , Neutropenia/complicações , Piperazinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Aspergilose/complicações , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Feminino , Humanos , Masculino , CamundongosRESUMO
Invasive aspergillosis (IA) is the most serious mold infection encountered in patients with iatrogenic immunosuppression. IA is also a major cause of mortality and morbidity in individuals with primary immunodeficiency (PID). Although Aspergillus fumigatus is the most common etiologic agent of IA reported in PID patients, followed by A. nidulans, multiple poorly recognized Aspergillus species such as A. udagawae, A. quadrilineatus, A. pseudoviridinutans, A. tanneri, A. subramanianii, and A. fumisynnematus have been reported almost exclusively from patients with inborn defects in host antifungal defense pathways. Infection in PID patients exhibits patterns of disease progression distinct from those in iatrogenic immunosuppression. Specifically, the disease can be extrapulmonary and chronic with a tendency to disseminate in a contiguous manner across anatomical planes. It is also more refractory to standard antifungal therapy. This synopsis summarizes our understanding of emerging rare Aspergillus species that primarily affect patients with PIDs but not those with acquired immunodeficiencies.
RESUMO
Fabrication and efficiency enhancement of tubal field emission lamps (FELs) using multi-walled carbon nanotubes (MWNTs) as the cathode field emitters were studied. The cathode filaments were prepared by eletrolessly plating a nickel (Ni) film on the cathode made of a 304 stainless steel wire dip-coated with MWNTs. The 304 wire was dip-coated with MWNTs and nano-sized Pd catalyst in a solution, and then eletrolessly plated with Ni to form an MWNT-embedded composite film. The MWNTs embedded in Ni not only had better adhesion but also exhibited a higher FE threshold voltage, which is beneficial to our FEL system and can increase the luminous efficiency of the anode phosphor. Our results show that the FE cathode prepared by dipping three times in a solution containing 400 ppm Pd nano-catalysts and 0.2 wt.% MWNTs and then eletrolessly plating a Ni film at a deposition temperature of 60 °C, pH value of 5, and deposition time of 7 min has the best FE uniformity and efficiency. Its emission current can stay as low as 2.5 mA at a high applied voltage of 7 kV, which conforms to the high-voltage-and-low-current requirement of the P22 phosphor and can therefore maximize the luminous efficiency of our FEL. We found that the MWNT cathodes prepared by this approach are suitable for making high-efficiency FELs.
RESUMO
An internal magnetic probe using Hall sensors to measure a current density profile directly with perturbation of less than 10% to the plasma current is successfully operated for the first time in Versatile Experiment Spherical Torus (VEST). An appropriate Hall sensor is chosen to produce sufficient signals for VEST magnetic field while maintaining the small size of 10 mm in outer diameter. Temperature around the Hall sensor in a typical VEST plasma is regulated by blown air of 2 bars. First measurement of 60 kA VEST ohmic discharge shows a reasonable agreement with the total plasma current measured by Rogowski coil in VEST.
RESUMO
Volume-produced negative ion source still requires enhancement of current density with lower input RF (radio-frequency) power in lower operating pressure for various applications. To confirm recent observation of efficient negative ion production with a short cylindrical chamber with smaller effective plasma size, the RF-driven transformer-coupled plasma H(-) ion source at Seoul National University is modified by adopting a newly designed quartz RF window to reduce the chamber length. Experiments with the reduced chamber length show a few times enhancement of H(-) ion beam current compared to that extracted from the previous chamber design, which is consistent with the measured H(-) ion population. Nevertheless, decrease in H(-) ion beam current observed in low pressure regime below â¼5 mTorr owing to insufficient filtering of high energy electrons in the extraction region needs to be resolved to address the usefulness of electron temperature control by the change of geometrical configuration of the discharge chamber. A new discharge chamber with conically converging configuration has been developed, in which the chamber diameter decreases as approaching to the extraction region away from the planar RF antenna such that stronger filter magnetic field can be utilized to prohibit high energy electrons from transporting to the extraction region. First experimental results for the H(-) ion beam extraction with this configuration show that higher magnetic filter field makes peak negative beam currents happen in lower operating pressure. However, overall decrease in H(-) ion beam current due to the change of chamber geometry still requires further study of geometrical effect on particle transport and optimization of magnetic field in this novel configuration.
RESUMO
Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance.
Assuntos
Criptococose/imunologia , Criptococose/microbiologia , Cryptococcus gattii/genética , Variação Genética , Genoma Bacteriano , Animais , Antifúngicos/farmacologia , Cryptococcus gattii/classificação , Cryptococcus gattii/efeitos dos fármacos , Cryptococcus gattii/isolamento & purificação , Surtos de Doenças , Evolução Molecular , Feminino , Genótipo , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , América do Norte/epidemiologia , FilogeniaRESUMO
We describe a case of invasive fungal infection caused by Volvariella volvacea following double umbilical cord blood transplantation (UCBT). Although infections caused by several mushroom species have been documented, we believe this to be the first published report of invasive infection with Volvariella volvacea, an edible mushroom belonging to Agaricales.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical/efeitos adversos , Micoses/diagnóstico , Volvariella/isolamento & purificação , Adulto , Biópsia , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Evolução Fatal , Feminino , Genes de RNAr , Histocitoquímica , Humanos , Imageamento por Ressonância Magnética , Microscopia , Dados de Sequência Molecular , Micoses/microbiologia , RNA Ribossômico 5,8S/genética , Radiografia Torácica , Análise de Sequência de DNA , Tomografia Computadorizada por Raios XRESUMO
We analyzed 71 clinical and environmental Cryptococcus gattii strains that had been isolated before or after the advent of azole antifungals to determine their level of heteroresistance to fluconazole (LHF). All strains of C. gattii manifested heteroresistance, with LHFs that ranged between 4 microg/ml and 32 microg/ml. A considerably higher proportion of the C. gattii strains (86%) than Cryptococcus neoformans strains (46%) exhibited LHFs that were > or =16 microg/ml. No significant correlation was observed between the molecular type or serotypes of strains and their respective LHF. The strains which expressed a higher LHF were also more resistant to xenobiotics than the strains with a low LHF, and the level of resistance to xenobiotics was significantly higher than that reported for C. neoformans. The heteroresistant subpopulation, whose level of drug resistance had been raised in a stepwise manner to 64 microg/ml, reverted to the original LHF upon daily transfers in drug-free medium. Importantly, the strains with high LHFs were significantly more virulent than those with low LHFs. Since all the clinical isolates that had not been exposed to azole drugs as well as the environmental strains manifested heteroresistance to fluconazole, heteroresistance of C. gattii to azoles is an intrinsic mechanism as in C. neoformans and is associated with the strain's virulence.
Assuntos
Antifúngicos/farmacologia , Cryptococcus gattii/efeitos dos fármacos , Fluconazol/farmacologia , Animais , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Cryptococcus gattii/classificação , Cryptococcus gattii/isolamento & purificação , Cryptococcus gattii/patogenicidade , Cryptococcus neoformans/efeitos dos fármacos , Farmacorresistência Fúngica , Microbiologia Ambiental , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Sorotipagem , Especificidade da Espécie , VirulênciaRESUMO
A recent report on several cases of invasive aspergillosis caused by Neosartorya udagawae suggested distinctive patterns of disease progression between N. udagawae and Aspergillus fumigatus. This prompted us to characterize N. udagawae in comparison to A. fumigatus. Our findings showed that both species exist in two mating types at similar ratios and produce gliotoxin. However, the thermotolerance of the two species differs: while A. fumigatus is able to grow at 55 degrees C but not at 10 degrees C, N. udagawae is able to grow at 10 degrees C but fails to grow at >42 degrees C. Furthermore, compared to A. fumigatus, the conidia of N. udagawae require longer incubation periods to germinate at 37 degrees C and are more susceptible to neutrophil attack as well as hydrogen peroxide; N. udagawae is also less virulent in gp91(phox-/-) mice. These findings suggest that growth and susceptibility to the host response might account for the reduced virulence of N. udagawae and the subtle distinction in the progression of the disease caused by the two species.
Assuntos
Aspergilose/epidemiologia , Aspergilose/microbiologia , Aspergillus fumigatus/fisiologia , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/microbiologia , Neosartorya/fisiologia , Animais , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/patogenicidade , Aspergillus fumigatus/efeitos da radiação , Modelos Animais de Doenças , Temperatura Alta , Humanos , Peróxido de Hidrogênio/toxicidade , Camundongos , Neosartorya/efeitos dos fármacos , Neosartorya/patogenicidade , Neosartorya/efeitos da radiação , VirulênciaRESUMO
The aim of this investigation was to study the effect of polysaccharide capsule on the gene expression in dendritic cells (DC) during their interaction with Cryptococcus neoformans. To this end, we used an encapsulated virulent strain of C. neoformans and a cap59 gene-disrupted acapsular avirulent strain derived from the same genetic background. DC were exposed to encapsulated and acapsular C. neoformans strains for 4 h and 18 h, and their transcriptional profiles were analyzed using the Affymetrix mouse gene chip U74Av2. A large number of DC genes were up-regulated after treatment with the acapsular strain. In particular, we observed the up-regulation of the genes involved in DC maturation, such as cell surface receptors, cytokines, and chemokines (interleukin-12 [IL-12], IL-2, IL-1alpha, IL-1beta, IL-6, IL-10, tumor necrosis factor alpha, CCR7, CCL17, CCL22, CCL3, CCL4, CCL7, and CXCL10), membrane proteins, and the genes involved in antigen processing and presentation as well as cell cycle or apoptosis. The chemokine gene expression data were confirmed by real-time reverse transcription-PCR, while the expression of cytokine genes was correlated with their secretion. A completely different pattern of gene expression was observed for DC treated with an encapsulated strain of C. neoformans. In particular, no significant induction was observed in the expression of the genes mentioned above. Moreover, a number of genes, such as those coding for chemokines, were down-regulated. These results suggest that the polysaccharide capsule shrouding the cell wall of C. neoformans plays a fundamental role in inducing DC response, highlighting the molecular basis of the true nature of immune silencing exerted by capsular material.
Assuntos
Cryptococcus neoformans/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Animais , Linhagem Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Camundongos , Ligação Proteica , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Increased levels of cerebrospinal fluid (CSF) 14-3-3 proteins have been reported in acute bacterial meningitis. We tested the hypothesis that CSF 14-3-3 protein levels are substantially increased in acute bacterial meningitis and decreased after anti-microbial therapy, and that CSF 14-3-3 protein levels can predict treatment outcomes. METHODS: We examined serial pan-CSF 14-3-3 (14-3-3-P) protein and five major isoform (beta, gamma, epsilon, eta, zeta) levels in 29 adult community-acquired bacterial meningitis (ACABM) patients. The CSF 14-3-3 protein levels were also evaluated in 12 aseptic meningitis patients during the study period. RESULTS: All of the meningitis patients had a positive result on admission. Levels of CSF 14-3-3 protein in ACABM cases were significantly increased initially, and substantially decreased thereafter. Most of those who survived (survivors = 25 and non-survivors = 4) had nearly cleared their 14-3-3 protein from the CSF before discharge. Conversely, patients who died never cleared their CSF 14-3-3 protein. The median value of CSF 14-3-3-P and 14-3-3 gamma, 14-3-3 eta and 14-3-3 epsilon isoforms on admission in the bacterial meningitis group were 173.7, 137.7, 42.2 and 9.1, respectively, which were statistically significant than those of the aseptic meningitis group (48.4, 39.6, 2.5 and 0, respectively). Stepwise logistic regression analysis showed only CSF 14-3-3 gamma isoform on admission was independently associated with outcome (P = 0.05, OR = 0.991). CONCLUSION: Serial 14-3-3 protein gamma isoform actually meets the major requirements for outcome prediction in the treatment of ACABM patients. Assay of the 14-3-3 protein gamma isoform should be added as a neuro-pathologic marker among the panel of conventional CSF parameters.
Assuntos
Proteínas 14-3-3/líquido cefalorraquidiano , Meningites Bacterianas/líquido cefalorraquidiano , Adulto , Idoso , Antibacterianos/uso terapêutico , Biomarcadores/líquido cefalorraquidiano , Infecções Comunitárias Adquiridas/líquido cefalorraquidiano , Infecções Comunitárias Adquiridas/tratamento farmacológico , Infecções Comunitárias Adquiridas/mortalidade , Feminino , Humanos , Contagem de Leucócitos , Modelos Logísticos , Masculino , Meningite Asséptica/líquido cefalorraquidiano , Meningite Asséptica/tratamento farmacológico , Meningite Asséptica/mortalidade , Meningites Bacterianas/tratamento farmacológico , Meningites Bacterianas/mortalidade , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Isoformas de Proteínas/líquido cefalorraquidiano , Curva ROC , Taxa de SobrevidaRESUMO
OBJECTIVES: Tuberculous and cryptococcal meningitis are two of the most common types of chronic meningitis. The study was performed to assess whether the occurrence of intracranial arterial stenosis is a risk factor for poor outcome in tuberculous and cryptococcal meningitis. METHODS: The clinical relevance of intracranial arterial stenoses in tuberculous and cryptococcal meningitis was studied using transcranial color-coded sonography (TCCS) and magnetic resonance angiography (MRA) over the period of 1 year (2003). A comparison was made between patients with and those without intracranial arterial stenoses during hospitalization. FINDINGS: There were 12 males and three females. Four patients (27%, 4/15) exhibited a stenosis of at least one cerebral artery, of which three had bilateral middle cerebral artery (MCA) stenosis and one had unilateral MCA stenoses. The stenoses were demonstrable during the whole observation period. After 6 months of follow-up, fatality rate was 50% (2/4) in those with intracranial arterial stenosis and 9% (1/11) in those without. Risk of poor outcome at a 6-month endpoint had an odds ratio of 5.3 for patients with intracranial arterial stenoses than those without (reference group). CONCLUSIONS: Intracranial arterial stenoses imply danger of cerebrovascular complications for the treatment of chronic meningitis during hospitalization. This study demonstrates that the frequency of intracranial stenosis in patients affected by chronic meningitis is high (27%) and it may be associated with poorer outcomes.
Assuntos
Constrição Patológica/complicações , Doenças Arteriais Intracranianas/complicações , Meningite Criptocócica/mortalidade , Tuberculose Meníngea/mortalidade , Adulto , Idoso , Feminino , Humanos , Angiografia por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Resultado do Tratamento , Ultrassonografia Doppler TranscranianaRESUMO
The polysaccharide capsule is known to be the major factor required for the virulence of Cryptococcus neoformans. We have cloned and characterized a gene, designated CPS1, that encodes a protein containing a glycosyltransferase moiety and shares similarity with the type 3 polysaccharide synthase encoded by the cap3B gene of Streptococcus pneumoniae. Cps1p also shares similarity with hyaluronan synthase of higher eukaryotes. Deletion of the CPS1 gene from a serotype D strain of C. neoformans resulted in a slight reduction of the capsule size as observed by using an India ink preparation. The growth at 37 degrees C was impaired, and the ability to associate with human brain endothelial cells in vitro was also significantly reduced by the deletion of CPS1. Using site-specific mutagenesis, we showed that the conserved glycosyltransferase domains are critical for the ability of the strain to grow at elevated temperatures. A hyaluronan enzyme-linked immunosorbent assay method demonstrated that CPS1 is important for the synthesis of hyaluronan or its related polysaccharides in C. neoformans. Comparisons between the wild-type and the cps1Delta strains, using three different transmission electron microscopic methods, indicated that the CPS1 gene product is involved in the composition or maintenance of an electron-dense layer between the outer cell wall and the capsule. These and the virulence studies in a mouse model suggested that the CPS1 gene is important in the pathobiology of C. neoformans.
Assuntos
Cápsulas Bacterianas/fisiologia , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/genética , Proteínas Fúngicas/genética , Glicosiltransferases/fisiologia , Streptococcus pneumoniae/enzimologia , Animais , Cápsulas Bacterianas/química , Encéfalo/irrigação sanguínea , Encéfalo/enzimologia , Encéfalo/microbiologia , Células Cultivadas , Cryptococcus neoformans/patogenicidade , Cryptococcus neoformans/ultraestrutura , Modelos Animais de Doenças , Endotélio Vascular/enzimologia , Endotélio Vascular/microbiologia , Feminino , Proteínas Fúngicas/química , Proteínas Fúngicas/fisiologia , Glicosiltransferases/química , Glicosiltransferases/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microcirculação/enzimologia , Microcirculação/microbiologia , Dados de Sequência Molecular , Sepse/microbiologia , Sepse/patologia , Streptococcus pneumoniae/genética , VirulênciaRESUMO
Cryptococcus neoformans and Cryptococcus gattii are the caus-ative agents of cryptococcal meningoencephalitis and are amenable to genetic manipulations, making them important models of pathogenic fungi. To improve the efficiency of Agrobacterium tumefaciens mediated transformation (ATMT) in C. neoformans, we optimized various co-cultivation conditions including incubation time and temperature, and bacteria to yeast ratio. ATMT was also applied to both serotypes (B and C) of C. gattii. Transformation efficiency by ATMT in C. neoformans was comparable to either electroporation or biolistic transformation and gave superior efficiencies in serotypes B and C, but unlike Saccharomyces cerevisiae, adenine auxotrophy did not increase ATMT efficiency in C. neoformans or C. gattii. All transformants tested were stable, with a majority containing only a single T-DNA insertion; however, homologous recombination was not observed. Additionally, we isolated adenine auxotrophs containing a single T-DNA insertion in the ADE2 gene for representative serotype B and C strains.
Assuntos
Agrobacterium tumefaciens/genética , Cryptococcus neoformans/genética , Transformação Bacteriana/genética , Transformação Genética , Agrobacterium tumefaciens/fisiologia , Cryptococcus/classificação , Cryptococcus/genética , Cryptococcus neoformans/classificação , Plasmídeos/genéticaRESUMO
Agrobacterium tumefaciens was used to transform Aspergillus fumigatus by either random or site-directed integration of transforming DNA (T-DNA). Random mutagenesis via Agrobacterium tumefaciens-mediated transformation (ATMT) was accomplished with T-DNA containing a hygromycin resistance cassette. Cocultivation of A. fumigatus conidia and Agrobacterium (1:10 ratio) for 48 h at 24 degrees C resulted in high frequencies of transformation (> 100 transformants/10(7) conidia). The majority of transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable. We chose alb1, a polyketide synthase gene, as the target gene for homologous integration because of the clear phenotype difference between the white colonies of Deltaalb1 mutant strains and the bluish-green colonies of wild-type strains. ATMT with a T-DNA-containing alb1 disruption construct resulted in 66% albino transformants. Southern analysis revealed that 19 of the 20 randomly chosen albino transformants (95%) were disrupted by homologous recombination. These results suggest that ATMT is an efficient tool for transformation, random insertional mutagenesis, and gene disruption in A. fumigatus.
Assuntos
Agrobacterium tumefaciens/genética , Aspergillus fumigatus/genética , Transformação Bacteriana , Deleção de Genes , Humanos , Mutagênese Insercional , Policetídeo Sintases/genéticaRESUMO
Cryptococcus neoformans exists in two mating types MATa and MATalpha. Although the morphology, growth characteristics and genetic segregation patterns among MATa and MATalpha strains are indistinguishable in the laboratory, the predominance of MATalpha strains in nature suggests that MATalpha strains are better suited for survival in nature. We disrupted the TUP1 gene, a global repressor, to find the possible biological differences in congenic MATalpha and MATa cells of C. neoformans. Disruption of TUP1 affected neither the yeast nor the hyphal cell morphology but resulted in a similar reduction of mating frequencies in both MATalpha and MATa cells. Disruption of TUP1, however, functionally manifested itself in several mating type-dependent phenotypes: (i) MATalpha cells became more sensitive to 0.8 M KCl while MATa cells showed no change in sensitivity, (ii) a temperature-dependent growth reduction was exhibited at both 30 degrees C and 25 degrees C in MATa but a similar growth reduction was not observed in MATalpha cells until the temperature was lowered to 25 degrees C and (iii) the transcriptional level of genes in several different biological pathways was markedly altered in a mating type-dependent manner. This work is the first case in which non-mating-related biological differences are observed between two congenic mating partners in yeast.