Chromosomal Rearrangements between Serotype A and D Strains in Cryptococcus neoformans

Cryptococcus neoformans is a major human pathogenic fungus that can cause meningoencephalitis in immunocompromised hosts. It contains two divergent varieties, var. grubii (serotype A) and var. neoformans (serotype D), as well as hybrids (serotype AD) between these two varieties. In this study, we investigated the extent of chromosomal rearrangements between the two varieties, estimated the effects of chromosomal rearrangements on recombination frequencies, and surveyed the potential polymorphisms of the rearrangements among natural strains of the three serotypes. Through the analyses of two sequenced genomes from strains H99 (representing var. grubii) and JEC21 (representing var. neoformans), we revealed a total of 32 unambiguous chromosome rearrangements, including five translocations, nine simple inversions, and 18 complex rearrangements. Our analyses identified that overall, rearranged regions had recombination frequencies about half of those around syntenic regions. Using a direct PCR screening strategy, we examined the potential polymorphisms of 11 rearrangements among 64 natural C. neoformans strains from five countries. We found no polymorphism within var. neoformans and very limited polymorphism within var. grubii. However, strains of serotype AD showed significant polymorphism, consistent with their hybrid origins coupled with differential loss of heterozygosity. We discuss the implications of these results on the genome structure, ecology, and evolution of C. neoformans.     

Comparison of Genotypes Between Environmental and Clinical Isolates of Cryptococcus neoformans var. grubii Based on Microsatellite Patterns

We applied multilocus microsatellite typing (MLMT) method to investigate the genetic relation between Cryptococcus neoformans var. grubii clinical and environmental isolates in S?o Paulo, Brazil. This MLMT method includes three functional gene sequences of C. neoformans var. grubii, which are dispersed on three chromosomes. In all, 89 strains (36 clinical and 53 environmental isolates) were analyzed. Of 36 clinical strains, 20 belonged to a major type of MLMT-13 (55.6%). They were mainly isolated from clinical specimens. About 52.8% of strains from the environment belong to a major type of MLMT-36, which are indigenous to environments and which were not isolated from clinical samples. Thus, we recognized two genotypes that distinguish majority of clinical and environmental strains. No differences were found in antifungal susceptibility and capsule size between major environmental and clinical MLMT types.     

Uniparental Mitochondrial Transmission in Sexual Crosses in Cryptococcus neoformans

Restriction fragment length polymorphism (RFLP) in the large ribosomal RNA region of the mitochondrial DNA (mtDNA) was developed as a genetic marker for investigating mitochondrial transmission in sexual crosses of the human pathogenic basidiomycetous yeast Cryptococcus neoformans. Strain JEC20 of C. neoformans var. neoformans (mat a) was mated with six strains of C. neoformans var. grubii (mat alpha). Successful mating was indicated by the formation of hyphae and basidiospores. These basidiospores were examined for mtDNA RFLP genotypes. All 570 basidiospores examined from the six crosses showed the mtDNA genotype of strain JEC20. The failure to recover the C. neoformans var. grubii mtDNA in any cross indicates that the C. neoformans var. grubii mtDNA is either selectively eliminated in the newly formed dikaryon or selectively excluded in the immediate dikaryotic hyphae of the newly formed dikaryon. Received: 20 September 1999 / Accepted: 12 November 1999     

DNA fingerprinting pattern and susceptibility to antifungal drugs in Cryptococcus neoformans variety grubii isolates from Barcelona city and rural environmental samples

Cryptococcus neoformans var. grubii (serotype A) was isolated from 12 soil samples mixed with pigeon droppings (16.9%) from 71 soil samples in Barcelona and rural areas of Catalonia. C. neoformans was not isolated from indoor dust and Eucalyptus debris. PCR fingerprinting was performed in 22 representative isolates and all of them corresponded to the VNI pattern. Susceptibility testing for the 22 isolates of C. neoformans var. grubii showed that all of them were susceptible to amphotericin B. Three isolates presented MICs (Minimal Inhibitory Concentrations) ≥ 1 μg/ml to Itraconazole, five MICs ≥ 1 μg/ml to ketoconazole and four were fluconazole resistant, (MICs ≥ 64 μg/ml), while three of them were shown to have MICs ≥ 1 μg/ml to voriconazole. In spite that all isolates presented the same DNA fingerprinting pattern, the susceptibility to antifungals is very variable. The possibility of acquiring cryptococcosis infection with primarily resistant environment strains is feasible.     

一种制备新生隐球菌单细胞的方法

【目的】建立流式细胞仪分选新生隐球菌单细胞的方法,确定新生隐球菌单细胞恢复生长的条件和能力。【方法】利用Moflo XDP流式细胞分析分选仪,体外测定不同条件下新生隐球菌恢复生长的比率。【结果】建立了流式细胞仪新生隐球菌单细胞分选流程。确定流式细胞仪分选得到的新生隐球菌单细胞具有恢复生长的能力,恢复生长的能力受营养条件和菌株差异的影响。在营养丰富的条件下,新生隐球菌JEC21和H99单细胞恢复生长比率分别为74%和89%。在寡营养条件下,JEC21和H99单细胞恢复生长比率分别为37%和80%。JEC21生长比率随细胞数的增加而升高,细胞数为100个时,生长比率为55%;细胞数为1000个时,生长比率为97%。【结论】流式细胞仪分选得到新生隐球菌单细胞具有恢复生长的能力,生长能力受营养条件、菌株差异的影响。     

Growth and Mating of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> var. <Emphasis Type="Italic">grubii</Emphasis> on Woody Debris

A total of 36 Cryptococcus neoformans strains originating from South Africa were screened for wood degrading enzymes. All strains tested positive for cellulase activity while none where capable of xylan degradation. Three C. neoformans var. grubii strains, originating from clinical and environmental samples, representing the same genotype (VNI/AFLP1—C. neoformans var. grubii) and MATα, were evaluated for growth on debris of two common tree species in South Africa: Acacia mearnsii and Eucalyptus camaldulensis. The mating capability of all the C. neoformans strains was evaluated on similar debris. Strains grown on A. mearnsii yielded substantially greater yeast populations. A total of 26%, 6%, 46%, and 80% of the 36 C. neoformans strains tested were either able to mate or develop filaments when crossed on A. mearnsii and E. camaldulensis debris, V8 juice, and yeast carbon base (YCB) agar, respectively. Filamentation and monokaryotic fruiting was observed in 3% of strains when C. neoformans was cultured on either A. mearnsii, E. camaldulensis debris, or YCB. The results indicate that this fungus is capable of completing its life cycle and can produce basidiospores on woody debris. In the future, these findings should be considered when studying the epidemiology, microbial ecology, and proposed infection process of this global pathogen.     

Isolation and Characterization of Cryptococcus neoformans Varieties Recovered from Natural Sources in Bogotá, Colombia, and Study of Ecological Conditions in the Area

Cryptococcus neoformans, the etiological agent of cryptococcosis, has been associated with avian droppings and certain trees in different countries, including Colombia. C neoformans environmental isolates were obtained in urban areas in Bogotá, Colombia, and the strains recovered were phenotypically characterized. Attempts to determine the ecological conditions (micro- and macroclimatic) possibly related to their habitat were also undertaken. Four hundred and eighty samples from bark, soil around trunk bases, and detritus inside hollows of 32 trees were collected in three urban areas during a 5-month period, as well as 89 avian droppings samples from different places. Of plant samples, 6.7% collected from nine tree species yielded C. neoformans var. gattii, serotype B strains in 99% of the cases, and C. neoformans var. grubii, serotype A in 1%. The yeast was more frequently recovered from bark than from soil or detritus inside hollows, and from trees with hollows or rotted wood rather than from trees in which birds nest. C. neoformans was present with higher frequency and density in the rainy season than in the dry season; we found that slightly higher temperature and humidity values of the microhabitat, as compared to those of the environment, favored fungal occurrence, but the phenological state of the tree did not. Of dropping samples, 7.9% yielded C. neoformans strains, all of them C. neoformans var. grubii, serotype A. The yeast was obtained more frequently from dry droppings than from moist ones, but neither the sunlight exposure nor the site of collection of samples was correlated with this occurrence. Population density was significantly higher in droppings than in tree samples. Under laboratory conditions, isolates of different serotype showed similar capsular sizes. Water content and pH ranges were wide and did not show any significant difference between positive and negative samples.     

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

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.     

The isolation,characterization and antifungal susceptibilities of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> from bird excreta in Klang Valley,Malaysia

The occurrence of Cryptococcus neoformans in bird excreta in Klang valley, Malaysia was determined in this study. Of 544 samples of bird excreta collected from a local zoo, pet shops and public areas, 20 strains of C. neoformans were isolated. All C. neoformans strains were serotype A and thus identified as C. neoformans variety grubii. All did not produce color changes on canavanine–glycine–bromothymol blue agar. All were of α-mating types, as determined by a pheromone-specific PCR assay. The antifungal susceptibility testing using agar diffusion method Neo-sensitabs showed that all were susceptible to amphotericin B, fluconazole and itraconazole.     

Serotypes and mating types of clinical strains ofCryptococcus neoformans isolated in Taiwan

Twenty-one strains ofCryptococcus neoformans isolated from patients in Taiwan were characterized for serotypes and mating types. Slide agglutination test was performed with 8 factor-specific sera (Iatron Company, Japan) to determine the serotypes. Wheat bran agar (WBA) and malt extract agar (MEA, Wickerham) media were used for the mating tests. Twenty of the isolates were of serotype A, and one was serotype B. Except for 2 strains of serotype A, all of the serotype A strains mated withFilobasidiella neoformans var.neoformans, mating type a. The only serotype B strain mated withF. neoformans var.bacillispora mating type a in MEA medium. These data revealed the low prevalence (1/21; 4.8%) ofC. neoformans var.gattii in Taiwan, a subtropically located island.     

Cyclic AMP-dependent protein kinase catalytic subunits have divergent roles in virulence factor production in two varieties of the fungal pathogen Cryptococcus neoformans

Our earlier findings established that cyclic AMP-dependent protein kinase functions in a signaling cascade that regulates mating and virulence of Cryptococcus neoformans var. grubii (serotype A). Mutants lacking the serotype A protein kinase A (PKA) catalytic subunit Pka1 are unable to mate, fail to produce melanin or capsule, and are avirulent in animal models, whereas mutants lacking the PKA regulatory subunit Pkr1 overproduce capsule and are hypervirulent. Because other mutations have been observed to confer different phenotypes in two diverged varieties of C. neoformans (grubii variety [serotype A] and neoformans variety [serotype D]), we analyzed the functions of the PKA genes in the serotype D neoformans variety. Surprisingly, the Pka1 catalytic subunit was not required for mating, haploid fruiting, or melanin or capsule production of serotype D strains. Here we identify a second PKA catalytic subunit gene, PKA2, that is present in both serotype A and D strains of C. neoformans. The divergent Pka2 catalytic subunit was found to regulate mating, haploid fruiting, and virulence factor production in serotype D strains. In contrast, Pka2 has no role in mating, melanin production, or capsule formation in serotype A strains. Our studies illustrate how different components of signaling pathways can be co-opted and functionally specialized during the evolution of related but distinct varieties or subspecies of a human fungal pathogen.     

Cryptococcus neoformàns var. gattii among patients with cryptococcal meningitis in Mexico. First observations

A retrospective study of 20 patients with cryptococcal meningitis and their isolated strains was performed. Cryptococcus neoformans var. neoformans was recovered from 14 (70%) cases, and var. gattii was recovered from six (30%) patients. Twelve patients had AIDS (all carrying var. neoformans), two had other diseases (one with var. neoformans and one var. gattii) and there was no identifiable underlying disease in six (one var. neoformans and five var. gattii). Fourteen patients (11 var. neoformans and three var. gattii) resided in the Mexico City area, where a temperate climate is prevalent, and there were six cases (three var. neoformans and three var. gattii) from states with a tropical/subtropical climate. Although there was no significant statistical difference between the two varieties, the fatal outcome was higher in patients with var. neoformans. The disease caused by var. gattii strains was characterized by a higher opening pressure, more inflamatory changes of CSF and a longer clinical course (delayed clinical and mycological cure). Cryptococcus neoformans var. gattii is a significant cause of cryptococcal meningitis in patients without underlying diseases in Mexico.     

Origin and Inheritance of Group I Introns in 26S rRNA Genes of Gaeumannomyces graminis

Studies of the distribution of the three group I introns (intron A, intron T, and intron AT) in the 26S rDNA of Gaeumannomyces graminis had suggested that they were transferred to a common ancestor of G. graminis var. avenae and var. tritici after it had branched off from var. graminis. Intron AT and intron A exhibited vertical inheritance and coevolved in concert with their hosts. Intron loss could occur after its acquisition. Loss of any one of the three introns could occur in var. tritici whereas only loss of intron T had been found in the majority of var. avenae isolates. The existence of isolates of var. tritici and var. avenae with three introns suggested that intron loss could be reversed by intron acquisition and that the whole process is a dynamic one. This process of intron acquisition and intron loss reached different equilibrium points for different varieties and subgroups, which explained the irregular distribution of these introns in G. graminis. Each of the three group I introns was more closely related to other intron sequences that share the same insertion point in the 26S rDNA than to each other. These introns in distantly related organisms appeared to have a common ancestry. This system had provided a good model for studies on both the lateral transfer and common ancestry of group I introns in the 26S rRNA genes. Received: 17 May 1996 / Accepted: 14 January 1997     

Nuclear genes encoding chloroplast hemoglobins in the unicellular green alga Chlamydomonas eugametos

When the green unicellular alga Chlamydomonas eugametos is grown under light/dark regimes, nuclear genes are periodically activated in response to the changes in light conditions. These genetic responses are dependent upon the activation of genes associated with photosynthesis (LI616 and LI637), nonphotosynthetic photoreceptors (LI410 and LI818) and the biological clock (LI818). We report here that the LI410 and LI637 genes are part of a small gene family encoding hemoglobins (Hbs) related to those from two unicellular eukaryotes, the ciliated protozoa Paramecium caudatum and Tetrahymena pyriformis, and from the cyanobacterium Nostoc commune. Investigations of the intracellular localization of C. eugametos Hbs by means of immunogold electron microscopy indicate that these proteins are predominantly located in the chloroplast, particularly in the pyrenoid and the thylakoid region. To our knowledge, this constitutes the first evidence for the presence of Hbs in chloroplasts. Alignment of the LI637 cDNA nucleotide sequence with its corresponding genomic sequence indicates that the L1637 gene contains three introns, the positions of which are compared with those in the Hb genes of plants, animals and the ciliate P. caudatum. Although the LI637 gene possesses a three-intron/four-exon pattern similar to that of plant leghemoglobin genes, introns are inserted at different positions. Similarly the position of the single intron in the P. caudatum gene differs from the intron sites in the LI637 gene. The latter observations argue against the current view that all eukaryotic Hbs have evolved from a common ancestor having a gene structure identical to that of plant or animal Hbs.     

Ecological niche of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> var. <Emphasis Type="Italic">grubii</Emphasis> and <Emphasis Type="Italic">Cryptococcus gattii</Emphasis> in decaying wood of trunk hollows of living trees in Jabalpur City of Central India

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 varieties never co-occurred in the same hollow. C. gattii and C. n. var. grubii isolates belonged to serotype B and serotype A respectively. 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 varieties (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 × 10³ cells/g and 6 × 10⁵ cells/g. The seasonal variation has been seen in isolation of this yeast. Our result further reinforce the recently emerging evidence that the natural habitat of C. n. var. grubii and C. gattii is more generalized.     

Cryptococcus neoformans and Cryptococcus gattii Isolated from the Excreta of Psittaciformes in a Southern Brazilian Zoological Garden

Cryptococcus neoformans, a major pathogen in immunocompromised patients, is a ubiquitous free-living fungus that can be isolated from soils, avian excreta and plant material. To further study potential saprophytic sources of this yeast in the Southern Brazilian State Rio Grande do Sul, we analyzed fecal samples from 59 species of captive birds kept in cages at a local Zoological Garden, belonging to 12 different orders. Thirty-eight environmental isolates of C. neoformans were obtained only from Psittaciformes (Psittacidae, Cacatuidae and Psittacula). Their variety and serotype were determined, and the genetic structure of the isolates was analyzed by use of the simple repetitive microsatellite specific primer M13 and the minisatellite specific primer (GACA)₄ as single primers in the PCR. The varieties were confirmed by pulsed-field gel electrophoresis (PFGE). Thirty-three isolates (87%) were from the var. grubii, serotype A, molecular type VNI and five (13%) were Cryptococcus gattii, serotype B, molecular type VGI. All the isolates were mating type α. Isolates were screened for some potential virulence factors. Quantitative urease production by the environmental isolates belonging to the C. gattii was similar to the values usually obtained for clinical ones.     

Gene structure of two-domain arginine kinases from Anthopleura japonicus and Pseudocardium sachalinensis

Unusual two-domain arginine kinases (AKs) arose independently at least two times during molecular evolution of phosphagen kinases: AKs from the primitive sea anemone Anthopleura japonicus and from the clam Pseudocardium sachalinensis. To elucidate its unusual evolution, the structures of Anthopleura and Pseudocardium AK genes have been determined. The Anthopleura gene consisted of 4 exons and 3 introns: two domains are linked by a bridge intron, and each domain contains one intron in different positions. On the other hand, the Pseudocardium gene consisted of 10 exons and 9 introns: two domains are also linked by a bridge intron, and domains 1 and 2 contains 3 and 5 introns, respectively, of which 3 introns are located in exactly same positions. Since the two domains of Pseudocardium AK are estimated to have diverged about 290 million years ago, the 3 introns have been conserved at least for this long. Comparison of intron positions in Anthopleura, Pseudocardium and C. elegans AK genes indicates that there is no intron conserved through the three AK lineages, in sharp contrast to relatively conservative intron positions in creatine kinase (CK) gene family.