新生隐球菌的酚氧化酶及用于菌种鉴定的研究

采用4％玉米浸汁咖啡酸琼脂(CACA)培养基。观察了具不同生物学特性的新生隐球菌的酚氧化酶活性,并对临床常见的多种酵母和酵母样真菌作了该酶的检测。结果,受试的3个变种、5种血清型和尿素酶阴性新生隐球菌均呈明确的阳性反应;150株常见酵母和酵母样真菌中43株新生隐球菌全部呈酚氧化酶阳性。107株其它酵母和酵母样真菌全部阴性。具各种不同生物学特性的新生隐球菌均特异性地产生酚氧化酶,用检测该酶的方法作该菌鉴定的特异性和敏感性均为100％,且可于72小时内得到结果。此外,结合尿素酶试验可以准确的鉴定出尿素酶阴性的新生隐球菌。     

Deciphering the model pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is a basidiomycete fungal pathogen of humans that has diverged considerably from other model fungi such as Neurospora crassa, Aspergillus nidulans, Saccharomyces cerevisiae and the common human fungal pathogen Candida albicans. The recent completion of the genome sequences of two related C. neoformans strains and the ongoing genome sequencing of three other divergent Cryptococcus strains with different virulence phenotypes and environmental distributions should improve our understanding of this important pathogen. We discuss the biology of C. neoformans in light of this genomic data, with a special emphasis on the role that evolution and sexual reproduction have in the complex relationships of the fungus with the environment and the host.     

Unique hybrids between the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii

Cryptococcus neoformans and Cryptococcus gattii are yeasts that cause meningoencephalitis, but that differ in host range and geographical distribution. Cryptococcus neoformans occurs world-wide and mostly infects immunocompromised patients, whereas C. gattii occurs mainly in (sub)tropical regions and infects healthy individuals. Anomalous C. neoformans strains were isolated from patients. These strains were found to be monokaryotic, and diploid or aneuploid. Amplified Fragment Length Polymorphism (AFLP) and sequence analyses indicated that AFLP genotypes 2 (C. neoformans) and 4 (C. gattii) were present. The strains were serologically BD. Mating- and serotype-specific PCR reactions showed that the strains were MATa-serotype D/MATalpha-serotype B. This study is the first to describe naturally occurring hybrids between C. neoformans and C. gattii.     

Beta1,2-xylosyltransferase Cxt1p is solely responsible for xylose incorporation into Cryptococcus neoformans glycosphingolipids

The Manalpha1,3(Xylbeta1,2)Manalpha structural motif is common to both capsular polysaccharides of Cryptococcus neoformans and to cryptococcal glycosphingolipids. Comparative analysis of glycosphingolipid structural profiles in wild-type and mutant strains showed that the Xylbeta1,2-transferase (Cxt1p) that participates in capsular polysaccharide biosynthesis is also the sole transferase responsible for adding xylose to C. neoformans glycosphingolipids.     

Medium Containing Trypan Blue and Antibiotics for the Detection of Cryptococcus neoformans in Clinical Samples

A medium containing trypan blue, gentamicin, and chloramphenicol is introduced for the detection of Cryptococcus neoformans and Cryptococcus species from clinical samples. Ten recently isolated strains of Cryptococcus species as well as several clinical isolates of C. neoformans incorporated trypan blue and produced dark blue colonies on this mycological medium, whereas other common yeasts were light blue. The laboratory diagnosis of two cases of cryptococcosis was accomplished by the isolation of C. neoformans on the antibiotic-dye-containing medium. Compared to conventional media supporting large numbers of Pseudomonas aeruginosa and other gram-negative bacilli, the new medium was selective for yeasts. In one instance, the colonization of the respiratory tract by C. neoformans which led to fungemia was traced by the use of the antibiotic-dye medium. The antibiotic mixture, utilized herein, was more effective in suppressing bacteria contained in samples from patients than a medium containing cycloheximide and chloramphenicol.     

利用微卫星标记研究新生隐球菌分子流行病学

目的 研究国内隐球菌临床分离株的遗传多态性和分子流行病学.方法 选择与新生隐球菌遗传相关的9个微卫星标记,分析这9个位点从1993 ～2009年国内分离到的新生隐球菌临床株遗传背景、来源及变异程度.结果 116株被研究的隐球菌临床分离株,主要归属于3个微卫星复合物(MC2,MC3和MC12),其中大部分为MC2(103株).8株菌株属于目前为止未被国内外认识的新复合物(MC12).结论 利用微卫星DNA多态性研究新生隐球菌分子流行病学有较大的应用价值.     

Isolation of Cryptococcus neoformans from environmental samples in Alicante

A study of the incidence of Cryptococcus neoformans in Alicante was carried out in environmental samples from the cities of Alicante and Santa Pola. The samples were pigeons faeces and Eucalyptus camaldulensis tissues. This study shows that the prevalence of the yeast in faeces from captive pigeons is higher (81.5%) than in the same samples from urban pigeons (16.3%). Regarding the biotype and varieties, a 79.3% of the isolates belonged to C. neoformans, and all of them were C. neoformans var. neoformans. None of the E. camaldulensis samples showed C. neoformans growth, although some Cryptococcus laurentii strains were isolated from flowers.     

Divergence of protein kinase A catalytic subunits in Cryptococcus neoformans and Cryptococcus gattii illustrates evolutionary reconfiguration of a signaling cascade

Gene duplication and divergence via both the loss and gain of gene activities are powerful evolutionary forces underlying the origin of new biological functions. Here a comparative genetics approach was applied to examine the roles of protein kinase A (PKA) catalytic subunits in three closely related varieties or sibling species of the pathogenic fungus genus Cryptococcus. Previous studies revealed that two PKA catalytic subunits, Pka1 and Pka2, control virulence factor production and mating. However, only one of the two plays the predominant physiological role, and this function has been exchanged between Pka1 and Pka2 in strains of the Cryptococcus neoformans var. grubii serotype A lineage compared to divergent C. neoformans var. neoformans serotype D isolates. To understand the basis for this functional plasticity, here the activities of Pka1 and Pka2 were defined in the two varieties and the related sibling species Cryptococcus gattii by gene disruption and characterization, heterologous complementation, and analysis of serotype AD hybrid mutant strains. The findings provide evidence for a shared ancestral role of PKA in governing mating and virulence factor production and indicate that the exchange of catalytic subunit roles is attributable to loss of function. Our studies illustrate the plasticity of signaling networks enabling rapid rewiring during speciation of a clade of common human fungal pathogens.     

In vitro susceptibility to antifungal agents of environmental Cryptococcus spp. isolated in the city of Ribeirão Preto, São Paulo, Brazil

Infections by Cryptococcus strains other than C. neoformans have been detected in immunocompromised patients. Of these strains, three are considered human pathogens: C. albidus, C. laurenttii, and C. uniguttulatus. This study deals with the in vitro susceptibility of Cryptococcus to drugs such as amphotericin B, itraconazole, fluconazole, and 5-fluorocytosine. Environmental Cryptococcus isolates (50) distributed as follows: C. neoformans var. neoformans (16), C. albidus (17), C. laurentii (14), and C. uniguttulatus (3) were evaluated by the micro and macrodilution techniques, according to EUCAST and NCCLS recommendations, respectively. Considering both methodologies the respective minimal inhibitory concentrations (MIC) were 0.125 and 2 microg/ml for amphotericin B, 0.06 and 8 microg/ml for itraconazole, and 0.5 and more than 64 microg/ml for fluconazole and 5-fluorocytosine. Agreement percentages for the two methodologies were 100% for amphotericin B and fluconazole for all the strains tested. For itraconazole, the agreement percentage was 81.3% in the C. neoformans strain and 100% for all the others. All species had a agreement percentage of 94.1 to 100% when susceptibility to 5-fluorocytosine was tested. It is concluded that environmental isolates of C. neoformans var. neoformans, C. albidus, C. laurentii, and C. uniguttulatus may show high MICs against certain drugs, suggesting in vitro primary resistance to the antifungals tested.     

Detection and identification of probable endemic fungal pathogen, Cryptococcus gattii, and worldwide pathogen, Cryptococcus neoformans, by real-time PCR

A real-time PCR method for detection and identification of Cryptococcus neoformans and Cryptococcus gattii was developed and evaluated using DNA from single-colony or koala nasal smears. Two TaqMan minor groove binder probes that distinguished between these species were designed corresponding to the internal sequences of the CAP59 gene for both species. The real-time PCR assay had 100% specificity, as assessed using 13 reference strains and 300 environmental strains. Twelve smear samples from healthy koalas were analyzed by direct real-time PCR. This method successfully detected C. gattii and C. neoformans in one and three koalas, respectively.     

Urease activity in Cryptococcus neoformans and Cryptococcus gattii

Urease is an enzyme considered one of the main virulence factors in Cryptococcus neoformans. Quantitative differences in urease production between C. neoformans and the new species Cryptococcus gattii have not been so far documented. Using a standardized method, 25 isolates of C. neoformans and 19 of C. gattii were seeded in Christensen urea broth medium for urease activity detection. Approximately, the 50% of activity of one unit of commercial jack beans urease (A550=0.215) was considered as a reference to classified the Cryptococcus in two cathegories, low (A550<0.215) or high (A550=or>0.215) urease producers. After 72 hours of incubation, 76% of C. neoformans and 15.8% of C. gattii strains were high urease producers (p=0.016). Based on these results, the species C. neoformans appeared as the highest urease producer. Other virulence factors should also be investigated to explain C. gattii pathogenicity.     

The isolation and characterization of virulence factors of Cryptococcus spp. from saprophytic sources in the city of Ribeirão Preto, São Paulo, Brazil

Yeasts of the Cryptococcus genus are distributed in nature associated to animal and vegetal organic residues. Occasionally, species other than C. neoformans may be responsible for infectious diseases in human and animals. This study aims to determine the occurrence of Cryptococcus species in the atmosphere and bird droppings in the city of Ribeir?o Preto, S?o Paulo, Brazil, and to evaluate three virulence factors: capsule formation, growth at 37 degrees C and melanin production. We analyzed 86 environmental samples (54 droppings and 32 air). Of the 41 strains isolated, 15 were C. neoformans var. neoformans (12 droppings and 3 air), 15 C. albidus (12 droppings and 3 air), 9 C. laurentii (7 droppings and 2 air) and 2 C. uniguttulatus (from droppings). Capsules were produced by 93.3% of C. neoformans var. neoformans, 66.7% of C. albidus, 88.9% of C. laurentii and 50% (1/2) of C. uniguttulatus. All strains of C. neoformans, 20% of C. albidus and 44.4% of C. laurentii were able to grow at 37 degrees C. The melanin production on DOPA agar was verified in C. neoformans (93.3%), C. albidus (26.7%) and C. laurentii (66.7%). We concluded that different Cryptococcus species coexist in the same ecological niche and they are able to produce virulence factors.     

Chromosome length polymorphism in Cryptococcus neoformans clinical and environmental isolates

A protocol for intact DNA preparation from the basidiomycetous yeast Cryptococcus neoformans has been developed and applied to karyotyping C. neoformans isolates displaying different degrees of capsule formation. A total of 46 strains have been analyzed: 23 (50%) isolated from environmental samples (pigeon droppings), all of them belonging to C. neoformans var. neoformans; and 23 (50%) from clinical samples (human and veterinarian) including 10 isolates of C. neoformans var. neoformans and 13 isolates of C. neoformans var. gattii. Our results showed a global genome size ranging from 14.2 to 20.9 Mb for variety neoformans and from 7.9 to 16.8 Mb for variety gattii. The karyotype diversity was very high for variety neoformans (29 different patterns for the 33 analyzed strains) and lower for variety gattii (six different patterns for 13 strains). No grouping among variety neoformans strains from the same origin was found indicating very high genome diversity for this variety, irrespectively of the origin of the strains.     

Genotypic and Phenotypic Analyses of Two “Isogenic” Strains of the Human Fungal Pathogen Cryptococcus neoformans var. neoformans

Mycopathologia - The Cryptococcus neoformans species complex is a model organism for fungal studies. Many studies have used two strains, JEC20 and JEC21, and their derivatives. These two strains...     

Laccase activity in Cryptococcus gattii strains isolated from goats

Cryptococcosis is a life-threatening infection in humans and animals caused by encapsulated yeasts of the genus Cryptococcus. Cryptococcus neoformans and Cryptococcus gattii are the main agents of this mycosis. Until 2002 C. gattii was classified as a variety of C. neoformans but now is accepted as an independent species. The laccase (phenoloxydase) enzyme produced by these yeasts is considered one of the main pathogenic factors for its ability to induce melanin from dihydroxyphenolic compounds. The vast majority of the studies in laccase and melanin synthesis have been developed using isolates of C. neoformans. The main objective of this study was to evaluate laccase activity in strains of C. gattii, serotype B isolated from immunocompetent goats that died of lung and disseminated cryptococcosis, in several outbreaks occurring in Spain. The laccase activities of these isolates were compared with those of other strains of C. gattii and C. neoformans. After fungal cell rupture, the supernatant of each isolate was analyzed for its laccase activity using as substrate an L-dopa 20 mM solution. The degree of enzymatic activity was assessed according to its absorbance at 450 nm and scored using Enzymatic Units (EU). The maximum values were observed in three strains of C. gattii from goats (EU > 12). The smallest values were observed in one environmental isolate of C. gattii serotype C (EU = 0.7). The highest recorded value for C. neoformans was 6.3 EU in a serotype A isolate from one human case of meningitis. C. gattii serotype B obtained from goats showed different degrees of laccase activity, being the highest in those isolated from severe outbreaks of cryptococcosis. This enzyme appears to represent a major, though nonexclusive, pathogenic factor for Cryptococcus gattii.     

Molecular typing and antifungal susceptibility of clinical sequential isolates of Cryptococcus neoformans from Sao Paulo State, Brazil

The antifungal susceptibility profiles and the genetic variability of 83 sequential clinical isolates of Cryptococcus neoformans, including four Cryptococcus gattii isolates, obtained from 38 Sao Paulo AIDS patients with cryptococcal meningitis were assessed by electrophoretic karyotyping and random amplified polymorphic DNA (RAPD) analysis. The majority of the Cryptococcus neoformans isolates were highly susceptible to amphotericin B and fluconazole. Twenty percent of the minimum inhibitory concentration values for amphotericin B varied from 0.5 to 1 micro g mL(-1). For fluconazole, 22% occurred in the range 8-16 mug mL(-1). Sequential isolates from nine patients showed a trend towards lower susceptibility to fluconazole, flucytosine, itraconazole and amphotericin B. The results of molecular typing by electrophoretic karyotyping and RAPD analysis showed the presence of 22 electrophoretic karyotypes (EK) and 15 RAPD profiles that were highly correlated. Our results provided evidence for the occurrence of genetic changes in some strains associated with microevolution during the course of infection. We also observed both microevolution and simultaneous coinfection with two distinct Cryptococcus neoformans strains in one patient. In some patients, we found changed EK- and RAPD patterns in association with increased MIC values.     

Culture of Cryptococcus neoformans in the nonencapsulated state

Forty-one strains of Cryptococcus neoformans were examined after 3 days growth on a fresh and aged medium at p_H 5 & p_H 7 for comparison of capsule formation. Over one-half of the strains did not form visible capsules on aged medium at p_H 5. Serotypes and source of isolation did not correlate with ability or inability to form capsules. Growth of C. neoformans in the nonencapsulated state makes it possible to culture many strains of C. neoformans in the form that more closely simulates the true infectious particles.     

Human defenses against Cryptococcus neoformans: An update

Cryptococcus neoformans var. neoformans is an opportunistic fungal pathogen, especially in AIDS patients, and is found world-wide. On the other hand, Cryptococcus neoformans var. gatti (CN-g) is restricted to an association with two species of Eucalyptus trees. Alveolar macrophages (AM) constitute the first line of defense to Cryptococcus neoformans and offers some resistance. The inflammatory response to Cryptococcus neoformans with an influx of neutrophils and monocytes affords a second line of defense. Secretion of proinflammatory monokines by human AM is now being defined. The inflammatory phagocytes are efficient in killing Cryptococcus neoformans and offer strong resistance. T and B cell responses to infection, a third line of defense, results in production of lymphokines (IFNg, etc.) and specific antibodies. Enhancement of lymphocyte responses by IL-12 and IL-18 to Cryptococcus neoformans infection appears to be critical. Susceptibility of AIDS patients to Cryptococcus neoformans is associated with low CD4+ T cell counts and likely reduced efficacy of the second line of defense. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evidence that the human pathogenic fungus Cryptococcus neoformans var. grubii may have evolved in Africa

Most of the species of fungi that cause disease in mammals, including Cryptococcus neoformans var. grubii (serotype A), are exogenous and non-contagious. Cryptococcus neoformans var. grubii is associated worldwide with avian and arboreal habitats. This airborne, opportunistic pathogen is profoundly neurotropic and the leading cause of fungal meningitis. Patients with HIV/AIDS have been ravaged by cryptococcosis--an estimated one million new cases occur each year, and mortality approaches 50%. Using phylogenetic and population genetic analyses, we present evidence that C. neoformans var. grubii may have evolved from a diverse population in southern Africa. Our ecological studies support the hypothesis that a few of these strains acquired a new environmental reservoir, the excreta of feral pigeons (Columba livia), and were globally dispersed by the migration of birds and humans. This investigation also discovered a novel arboreal reservoir for highly diverse strains of C. neoformans var. grubii that are restricted to southern Africa, the mopane tree (Colophospermum mopane). This finding may have significant public health implications because these primal strains have optimal potential for evolution and because mopane trees contribute to the local economy as a source of timber, folkloric remedies and the edible mopane worm.