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PubMed2004

Regulatory roles for the homeodomain and C2H2 zinc finger regions of Cryptococcus neoformans Ste12alphap.

The STE12alpha gene of Cryptococcus neoformans encodes a protein containing both homeodomain and zinc finger regions. As homeodomains and zinc finger regions are important domains for the function of many transcription factors, we used site-specific mutagenesis to delineate the roles of these two domains. The homeodomain and zinc finger regions are each important for the function of Ste12alphap. DNA binding ability, mating frequency, and haploid fruiting capability were reduced in strains with mutations in the homeodomain, whereas virulence and capsule size in the mouse brain were increased. In contrast, mutations in the zinc fingers region resulted in decreased virulence, reduced capsule size in the mouse brain and decreased gene expression of capsule associated genes. In addition, phospholipase activity was increased in the zinc finger mutants. Taken together, most of the phenotypes previously observed in the ste12alpha deletion strains were reproduced in these two types of mutants. However, unlike mutations in the homeodomain/zinc finger region, complete deletion of STE12alpha caused a severe reduction in virulence and a decrease in phospholipase activity. These data suggest that region(s) other than the homeodomain and zinc finger regions of Ste12alphap contribute to the variable influences on the different phenotypes observed in C. neoformans.

Animals↗

PubMed2002

Effect of different K+ concentrations on Cryptococcus neoformans phenoloxidase activity.

Melanin synthesis in Cryptococcus neoformans, catalyzed by phenoloxidase activity, is one of the oldest virulence factors known. However, until now, the relationship between melanin production in C. neoformans and its virulence has been poorly understood. Among different chemical compounds only Fe3+ and Cu2+ cations enhance the phenoloxidase activity in C. neoformans. A few reports in the literature describe the influence of different cations on C. neoformans phenoloxidase activity, excluding iron. In this study, 13 C. neoformans strains isolated from AIDS patients and 7 from bird droppings (B.D.), were examined in order to clarify the effect of different K+ concentrations on phenoloxidase activity. A new solid and liquid caffeic acid minimal synthetic medium (MSM-CAF) containing only caffeic acid and ferric citrate with different potassium concentrations was used to evaluate C. neoformans phenoloxidase activity. In the MSM-CAF solid medium the degree of brown pigmentation on the agar plates was read on days 1, 2 and 3 of incubation, and the pigmentation of the C. neoformans strains was classed into 5 categories. The brown pigment of the liquid MSM-CAF test tubes were checked after 24 hours of incubation by measuring the optical density (O.D.) at 480 nm. Three C. neoformans AIDS and B.D. strains, randomly chosen, were tested for phenoloxidase activity, according to the modified protocols of Polacheck et al., Torres-Guerrero et al. and Rhodes. According to the results obtained, it has been observed that K+ does not activate the phenoloxidase activity in the C. neoformans AIDS and B.D. strains. In particular, with an increase in potassium concentrations in the MSM-CAF solid and liquid medium, there was a corresponding inhibition of the phenoloxidase activity on both the C. neoformans AIDS and B.D. strains.

Acquired Immunodeficiency Syndrome↗

PubMed2006

Antifungal susceptibility of Cryptococcus neoformans to amphotericin B and fluconazole.

Cryptococcus neoformans has emerged as an important opportunistic fungal pathogen in immunocompromised individuals. The therapeutic options of C. neoformans an opportunistic fungal pathogen include flucytosine, amphotericin B, and azole agents. However in the present scenario, emergence of resistance has been reported, hence this study was undertaken to evaluate antifungal susceptibility pattern of C. neoformans isolates from this southern part of India. Ten isolates of C. neoformans were tested against Amp B and fluconazole, of which 7 were susceptible to both and a single isolate of C. neoformans var gatti was resistant to both with MIC of 32mg/ml and 64mg/ml respectively.

Amphotericin B↗

PubMed1978

Carriage of Cryptococcus neoformans in the crops of pigeons.

Cryptococcus neoformans was found in the crops of four (1.3%) of 319 pigeons in the Charity Bird Hospital and the University Campus, Delhi. It was demonstrated repeatedly at the Bird Hospital in old pigeon excreta as well as in the air. The findings substantiate the current view that the pigeon itself is not a reservoir of C. neoformans although it may serve as a carrier of the fungus.

Air Microbiology↗

PubMed2003

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

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.

Colony Count, Microbial↗

PubMed1999

Pathogenicity of Cryptococcus neoformans: virulence factors and immunological mechanisms.

Cryptococcus neoformans is the causative agent of cryptococcosis and cryptococcal meningitis, which are serious pathological conditions affecting up to 10% of patients with AIDS. Mechanisms of pathogenicity of C. neoformans and the host defenses against this fungus are reviewed, incorporating recent data and perspectives.

AIDS-Related Opportunistic Infections↗

PubMed1986

Canavanine resistance in Cryptococcus neoformans.

All of the isolates of Cryptococcus neoformans var. gattii which we tested were resistant to greater than or equal to 3.5 mM canavanine. All of the serotype D isolates and 28% of the serotype A isolates of C. neoformans var. neoformans tested were susceptible to less than or equal to 18 microM canavanine, whereas the remaining 72% of the serotype A isolates were as resistant as the C. neoformans gattii isolates. In the naturally resistant isolates, the mechanism of resistance appeared to be decomposition of canavanine to a nontoxic product. However, in a resistant mutant derived from a naturally susceptible isolate, the mechanism of resistance was an impaired uptake system for canavanine. The toxic effect of canavanine in Cryptococcus results from the incorporation of canavanine into the protein component that is essential for the synthesis of proteins and RNAs.

Arginase↗

PubMed2003

Clonal reproduction and limited dispersal in an environmental population of Cryptococcus neoformans var gattii isolates from Australia.

Cryptococcus neoformans var. gattii is a causative agent of cryptococcosis and is thought to have a specific ecological association with a number of Eucalyptus species in Australia. However, the role that the tree plays in the life cycle of the fungus and the nature of the infectious propagule are not well understood. This study set out to examine whether sexual recombination is occurring in a natural population of C. neoformans var. gattii and whether the fungus disseminates between colonized trees. Thirty C. neoformans var. gattii isolates, consisting of both the alpha and a mating types, were collected from 13 Eucalyptus camaldulensis trees growing along a riverbank in Renmark, South Australia. The genetic diversity within the population was studied by using amplified fragment length polymorphism fingerprinting, and each isolate was assigned a unique multilocus genotype. Population genetic analyses of the multilocus data found no evidence of genetic exchange between members of the population, indicating a clonal population structure. Canonical variate analysis was then used to study the relationship between isolates from different colonized trees. Isolates from individual trees were strongly correlated, and it appeared that dispersal between trees was not occurring to any appreciable extent. These results suggest that the eucalypt may not be the primary niche for C. neoformans var. gattii but that the decaying wood present in hollows on these trees may provide a favorable substrate for extensive clonal propagation of the yeast cells.

Australia↗

PubMedDate not supplied

Feline meningitis due to Cryptococcus neoformans var. neoformans and review of feline cryptococcosis.

Cryptococcus neoformans var. neoformans was ascertained as the prime cause of fatal meningitis in a young-adult female domestic cat who was suspected of rabies. The pathogen was isolated in heavy growth from the infected brain specimen on simplified sunflower seed (Helianthus annuus) medium at 30 degrees C. Periodic acid-Schiff stained impression smear revealed numerous cryptococcal cells. Histologically, pia-arachnoid showed thickening along with many circular yeast cells with and without budding morphologically consistent with Cr. neoformans. The detailed typing of the strain indicated that it belonged to serotype AD and Filobasidiella neoformans var. neoformans mating type 'alpha'. The emphasis is given on early diagnosis by immunological and mycological techniques and prompt chemotherapy to avert the fatal consequences of this enigmatic mycosis.

Animals↗

PubMedDate not supplied

Identification by polymerase chain reaction fingerprinting of Cryptococcus neoformans serotype AD.

Seventy-three Cryptococcus neoformans isolates and eight other yeast strains were studied. Fingerprints produced by priming with (GACA)4 differentiated C. neoformans from all other yeasts tested and identified the five C. neoformans serotypes. Four major bands of molecular size 800, 540, 475 and 410 bp were recognized for serotypes A, AD and D. Two of them were specific for serotype A and the other two for serotype D isolates. Serotype AD strains were identified by five different genotypic patterns in which at least one of the two bands specific for serotype A and D were present in different combinations. On repeated and simultaneously performed genotype and serotype testing of nine strains, the genotypic pattern did not change, whereas serotyping was unstable in three cases. PCR-fingerprinting using (GACA)4 as a primer proved more stable than serology in discriminating among C. neoformans serotypes A, D and AD and was able to distinguish among serotype AD strains.

Base Sequence↗

PubMed2001

Serologic evidence for Cryptococcus neoformans infection in early childhood.

OBJECTIVE: Cryptococcus neoformans is an important cause of central nervous system infection in adults with acquired immunodeficiency syndrome (AIDS) but an unusual cause of disease in children with AIDS. The basis for this age-related difference in incidence is not known but may be caused by differences in exposure or immune response. The objective of this study was to determine whether the low prevalence of cryptococcal disease among children is related to a lack of exposure to C neoformans. METHODS: Sera were obtained from 185 immunocompetent individuals ranging in age from 1 week to 21 years who were being evaluated in an urban emergency department. Sera were analyzed for antibodies to C neoformans and Candida albicans proteins by immunoblotting. Immunoblot patterns were compared with those obtained from sera of patients with cryptococcosis (n = 10) and workers in a laboratory devoted to the study of C neoformans. The specificity of our results was confirmed by several approaches, including antibody absorption and blocking studies. Sera were also analyzed for the presence of cryptococcal polysaccharide by both enzyme-linked immunosorbent assay and latex agglutination assays. RESULTS: Sera from children 1.1 to 2 years old demonstrated minimal reactivity to C neoformans proteins. In contrast, the majority of sera from children >2 years old recognized many (>/=6) C neoformans proteins. For children between 2.1 and 5 years old, 56% of sera (n = 25) reacted with many proteins, whereas for children >5 years old (n = 120), 70% of samples reacted with many proteins. Reactivity was decreased by absorbing sera with C neoformans extracts or by preincubating blots with sera from experimentally infected but not from control rats. Reactivity to C neoformans proteins did not correlate with reactivity to C albicans proteins, which was common in sera from children between the ages of 1.1 and 2 years. Cryptococcal polysaccharide was detected at a titer of 1:16 (~10 ng/mL) in the sera of 1 child, a 5.6-year-old boy who presented to the emergency department with vomiting. CONCLUSIONS: Our findings provide both indirect and direct evidence of C neoformans infection in immunocompetent children. Our results indicate that C neoformans infects a majority of children living in the Bronx after 2 years old. These results are consistent with several observations: the ubiquitous nature of C neoformans in the environment, including its association with pigeon excreta; the large number of pigeons in urban areas; and the increased likelihood of environmental exposure for children once they have learned to walk. The signs and symptoms associated with C neoformans infection in immunocompetent children remained to be determined. Primary pulmonary cryptococcosis may be asymptomatic or produce symptoms confused with viral infections and, therefore, not recognized as a fungal infection. Our results suggest that the low incidence of symptomatic cryptococcal disease in children with AIDS is not a result of lack of exposure to C neoformans. These findings have important implications for C neoformans pathogenesis and the development of vaccine strategies.

AIDS-Related Opportunistic Infections↗

PubMed1989

Paradoxical role of capsule in murine bronchoalveolar macrophage-mediated killing of Cryptococcus neoformans.

Infections with the encapsulated fungus Cryptococcus neoformans are usually acquired via inhalation, and the presence of a capsule has been identified as a virulence factor. Therefore, we studied murine bronchoalveolar macrophage (BAM)-mediated killing and phagocytosis of encapsulated and acapsular strains of C. neoformans. After 2 h, BAM killed encapsulated strains CN52 and MP415 more readily than acapsular strains CN602 and CAP67 (54.9 and 36.2% vs 26.1 and 6.7%, respectively, p less than 0.001). Pre-incubating CN602 with purified capsular polysaccharide increased killing to 42.7% (p = 0.04). Significantly greater killing of the encapsulated strains also occurred in vivo. BAM-mediated killing of CN52 appeared to proceed by non-oxidative mechanisms, as BAM released minimal amounts of H2O2 after stimulation with CN52, and killing was not reduced by inhibitors or scavengers of the respiratory burst. The association between encapsulation and susceptibility to BAM fungicidal effects was not attributable to differences in yeast ingestion. Using the same low ratio of organisms to BAM as in the killing assay, greater than 95% of both CN52 and CN602 were phagocytosed. However, BAM phagocytosed significantly greater numbers of acapsular CN602 when incubated with a higher inoculum. Phagocytosis and killing of CN52 and CN602 required fresh serum as a source of C. Phagocytosis of CN52, but not CN602, was profoundly inhibited if BAM were plated on surfaces coated with mAb against the C3bR (CR1). mAb against the iC3b receptor (CR3) did not affect phagocytosis of either strain. These data demonstrate the innate ability of BAM to preferentially kill, by apparently non-oxidative mechanisms, an encapsulated as opposed to acapsular organism. Inasmuch as different receptors appear involved in phagocytosis of encapsulated versus acapsular C. neoformans, the disparity in killing may result from the greater ability of receptors mediating uptake of encapsulated organisms to trigger the antimicrobial armamentarium of the BAM.

Administration, Intranasal↗

PubMed2001

Origin of Cryptococcus neoformans var. neoformans diploid strains.

The basidiomycetous yeast Cryptococcus neoformans is an important human fungal pathogen. Two varieties, C. neoformans var. neoformans and C. neoformans var. gattii, have been identified. Both are heterothallic with two mating types, MATa and MATalpha. Some rare isolates are self-fertile and are considered occasional diploid or aneuploid strains. In the present study, 133 isolates, mostly from Italian patients, were investigated to detect the presence of diploid strains in the Igiene Università Milano culture collection. All of the diploid isolates were further investigated by different methods to elucidate their origins. Forty-nine diploid strains were identified by flow cytometry. PCR fingerprinting using the (GACA)(4) primer showed that the diploid state was associated with two specific genotypes identified as VN3 and VN4. Determination of mating type on V8 juice medium confirmed that the majority of the strains were sterile. PCR and dot blotting using the two pheromone genes (MFa and MFalpha) as probes identified 36 of the 49 diploid isolates as MATa/alpha. The results of pheromone gene sequencing showed that two allelic MFalpha genes exist and are distinct for serotypes A and D. In contrast, the MFa gene sequence was conserved in both serotype alleles. Amplification of serotype-specific STE20 alleles demonstrated that the diploid strains contained one mating locus inherited from a serotype A parent and one inherited from a serotype D parent. The present results suggest that diploid isolates may be common among the C. neoformans population and that in Italy and other European countries serotype A and D populations are not genetically isolated but are able to recombine by sexual reproduction.

Cryptococcosis↗

PubMed2001

A cryptococcal capsular polysaccharide mimotope prolongs the survival of mice with Cryptococcus neoformans infection.

Defined Abs to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been shown to be protective against experimental cryptococcosis. This suggests that if a vaccine could induce similar Abs it might protect against infection. However, the potential use of a GXM-based vaccine has been limited by evidence that GXM is a poor immunogen that can induce nonprotective and deleterious, as well as protective, Abs, and that the nature of GXM oligosaccharide epitopes that can elicit a protective response is unknown. In this study, we investigated whether a peptide surrogate for a GXM epitope could induce an Ab response to GXM in mice. The immunogenicity of peptide-protein conjugates produced by linking a peptide mimetic of GXM, P13, to either BSA, P13-BSA, or tetanus toxoid, P13-tetanus toxoid, was examined in BALB/c and CBA/n mice that received four s.c. injections of the conjugates at 14- to 30-day intervals. All mice immunized with conjugate produced IgM and IgG to P13 and GXM. Challenge of conjugate-immunized mice with C. neoformans revealed longer survival and lower serum GXM levels than control mice. These results indicate that 1) P13 is a GXM mimotope and 2) that it induced a protective response against C. neoformans in mice. P13 is the first reported mimotope of a C. neoformans Ag. Therefore, the P13 conjugates are vaccine candidates for C. neoformans and their efficacy in this study suggests that peptide mimotopes selected by protective Abs deserve further consideration as vaccine candidates for encapsulated pathogens.

Animals↗

PubMed1992

The ecology of Cryptococcus neoformans.

Environmental isolations have established that Cryptococcus neoformans var. gattii serotype B appears to have a specific ecological association with Eucalyptus camaldulensis. The global distribution of the tree appears to correspond to the epidemiologic distribution of cryptococcosis caused by C. neoformans var. gattii. The epidemiology of cryptococcosis can primarily be explained by exposure to an infective aerosolized inoculum, such as basidiospores released from specific host plants and/or desiccated blastoconidia (yeast cells) disseminated from accumulations of dried pigeon dung. The ecology of C. neoformans still remains largely unresolved, studies on the host-parasite interaction between serotype B and E. camaldulensis are still in progress, and extensive environmental searches are now underway to determine the natural habitats of serotypes A, C and D.

Animals↗

PubMed2001

Cryptococcus neoformans var. neoformans (serotype D) strains are more susceptible to heat than C. neoformans var. grubii (serotype A) strains.

Cryptococcus neoformans var. neoformans (serotype D) and C. neoformans var. grubii (serotype A) differ in geographic prevalence and dermatotropism, with C. neoformans var. neoformans strains being more prevalent among isolates from temperate countries as well as from skin infections. Analysis of 19 strains from each serotype revealed wide variation in thermal susceptibility, with C. neoformans var. neoformans strains being more susceptible, on average, to heat killing. The results suggest a consistent explanation for the geographic differences between serotype A and D strains and for the dermatotropism of serotype D strains.

Colony Count, Microbial↗

PubMed1993

Serotyping of Cryptococcus neoformans by using a monoclonal antibody specific for capsular polysaccharide.

The importance of epidemiological studies of cryptococcosis has increased since the beginning of the AIDS epidemic. Cryptococcus neoformans exists in two varieties defining four serotypes, Cryptococcus neoformans var. neoformans (serotypes A and D) and Cryptococcus neoformans var. gattii (serotypes B and C). The varieties are easy to distinguish by their differences of growth on diagnostic media. We propose here an easy serotyping method combining diagnostic media and a direct immunofluorescence assay with one monoclonal antibody (E1) specific for cryptococcal polysaccharide. The method was validated by the blinded testing of four to five reference strains of each serotype. Immunofluorescence patterns were characteristic of a given serotype provided that the variety of the strain had been defined before. For C. neoformans var. neoformans, a bright, homogeneous staining with several cell aggregates was characteristics of serotype A, whereas only a few serotype D cells were positive. For C. neoformans var. gattii, a completely negative isolate was serotype C, whereas the population of serotype B included a majority of negative cells but also included positive cells with a speckled pattern. The method was then used to serotype 156 clinical isolates from France and isolates from areas where C. neoformans var. gattii was endemic before the AIDS epidemic (13 strains from Rwanda and Zaire and 5 strains from Australia). The specificity of E1 was defined by its reactivity with various Cryptococcus spp. and analyzed according to the described cryptococcal antigenic factors. We conclude from this study that E1 provides a rapid and reliable means to serotype multiple isolates of C. neoformans.

AIDS-Related Opportunistic Infections↗

PubMed1993

Cryptococcosis produced by a urease negative strain of Cryptococcus neoformans.

A urease negative strain of Cryptococcus neoformans was isolated form a patient with AIDS. The identification of the yeast was confirmed by physiological and pathogenicity tests. Clinically, the disseminated cryptococcal infection in our patient was identical to those reported in other patients with AIDS.

AIDS-Related Opportunistic Infections↗