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The role of mating type and morphology in Cryptococcus neoformans pathogenesis.

Cryptococcus neoformans is a major fungal pathogen of both humans and animals. The fungus can be divided into two varieties, with each variety being composed of two serotypes. A sexual phase has been identified, which classifies C. neoformans as a bipolar heterothallic fungus with two mating types, MATa and MATalpha. The analysis of mating and mating type in this organism is important for a number of reasons. Both clinical and environmental isolates display a severe bias of the MATalpha mating type over MATa. MATalpha cells are also more virulent than MATalpha cells. Molecular and genetic analyses of the genes that make up the mating pathway have revealed that some of these genes are required for virulence. Finally, although it is well known that infection begins in the lungs after inhalation of infectious particles, it still remains unclear what constitutes the infectious particle. This review will discuss current information about what is known about the role that mating type and morphology play in virulence.

Animals↗

Chronic diarrhoea caused by Cryptococcus neoformans in a non-human immunodeficiency virus-infected patient.

A 43-y-old woman with underlying cervix carcinoma stage IIIB, status postirradiation, and diabetes mellitus with tripathy suffered from chronic diarrhoea for more than 6 months. Stool examination showed few white blood cells and red blood cells. Rectal swab cultures disclosed Cryptococcus neoformans in 2 samples obtained 2 weeks apart. The diarrhoea responded to treatment with oral fluconazole for 4 weeks. Repeated rectal swabs revealed no yeast growth. During follow-up for 4 y, she had no problems with diarrhoea. Chronic diarrhoea caused by C. neoformans is rare and clinical suspicion is needed.

Administration, Oral↗

False-negative cerebrospinal fluid cryptococcal antigen test due to small-colony variants of Cryptococcus neoformans meningitis in a patient with cystopleural shunt.

This is the first report of a small-colony variant Cryptococcus neoformans isolated from the cerebrospinal fluid of a patient with cystopleural shunt associated chronic meningitis. Cryptococcal antigen testing of the cerebrospinal fluid and the serum were both negative. The atypical morphology and the false-negative test may lead to delay of diagnosis and treatment.

Antigens, Fungal↗

Side group addition by xylosyltransferase and glucuronyltransferase in biosynthesis of capsular polysaccharide in Cryptococcus neoformans.

The capsular polysaccharide of Cryptococcus neoformans consists of an O-acetylated mannan backbone with xylosyl and glucuronyl substituents. We have studied two enzymes in the biosynthetic pathway of this polysaccharide. A particulate enzyme preparation contained xylosyltransferase and glucuronyltransferase activities with pH optima of 7.5 and less than 6.5, and optimum incubation temperatures of 25 degrees and 37 degrees C respectively. O-acetylated mannan served as an acceptor for glucuronyl residues but xylomannan did not. Glucuronomannan served as an acceptor for xylosyl residues but acetylated mannan did not. These observations suggest that glucuronate is added before xylose and after acetate. This hypothesis was supported by studies of lipid-linked sugars in which a microsomal preparation was incubated with various combinations of nucleotide sugars. With GDP-mannose, UDP-glucuronate and UDP-[14C]xylose; GDP-mannose, UDP-[14C]glucuronate and UDP-xylose; or GDP-mannose and UDP-[14C]glucuronate, no evidence for a lipid-linked monosaccharide was obtained. However, lipid-linked xylose was detected only if GDP-mannose and UDP-[14C]xylose were provided, a finding consistent with a pool of lipid-linked xylose which is only transferred following glucuronylation. The order of addition inferred is mannosyl, acetyl, glucuronyl and xylosyl.

Carbohydrate Sequence↗

Inhibition of I-A expression in rat peritoneal macrophages due to T-suppressor cells induced by Cryptococcus neoformans.

The expression of I-A antigen in rat peritoneal cells was significantly reduced during infection with Cryptococcus neoformans. When studying the in vitro action of T-suppressor cells induced by the fungus, or a soluble factor from the T-suppressor cells, a significant decrease in I-A expression by the peritoneal cells was observed. This expression was partially restored by indomethacin.

Animals↗

The 16S-like, 5.8S and 23S-like rRNAs of the two varieties of Cryptococcus neoformans: sequence, secondary structure, phylogenetic analysis and restriction fragment polymorphisms.

The nucleotide sequences of the 16S-like, 5.8S and 23S-like rDNAs from the two varieties of Cryptococcus neoformans, C. neoformans var. neoformans and C. neoformans var. gattii, were determined. The rRNA locus has the typical eukaryote organization of 16S-5.8S-23S with the 16S-like and 5.8S rRNA genes separated by a 124-nucleotide spacer and the 5.8S and 23S-like rRNA genes separated by a 187-nucleotide spacer in each strain. The C. neoformans var. neoformans and C. neoformans var. gattii 16S-like, 5.8S and 23S-like rRNAs are, respectively 1802, 158, and 3358 nucleotides in length and share > 99% nucleotide sequence identity, a finding which strongly supports the present taxonomic classification of two varieties within a species. Comparative structure analysis was used to construct secondary-structure models for the deduced 16S-like and 5.8S-23S-like rRNA sequences, which are similar to those of other fungal rRNAs. The C. neoformans 16S-like and 23S-like rRNA sequences were aligned with other eukaryote sequences based on secondary and higher-order structures predicted by comparative structure analysis for phylogenetic analysis. There was good correspondence between the 16S-like and 23S-like derived phylogenetic trees. The closest known fungal relative is Trichosporon beigelii. Southern blot analysis revealed one C. neoformans strain with two types of DNA repeats coding for rRNA which differed in size by about 1000 bp. Restriction fragment length polymorphisms in the rDNA locus provide useful markers for the study of epidemiology and pathogenesis of C. neoformans infections.

Base Sequence↗

The actin gene from Cryptococcus neoformans: structure and phylogenetic analysis.

Using heterologous probing of a genomic library, we have cloned and sequenced the actin gene from the pathogenic yeast Cryptococcus neoformans. The actin gene is 1371 bp in length, and exists as a single copy, as is the case for all fungi studied to date. The locations of the introns in the C. neoformans actin gene are unique among all other known actin genes, and the deduced coding sequence results in a 375 amino acid chain with very high homology to other actins. A phylogenetic tree comprising 31 actin-coding sequences from a wide variety of organisms shows that the C. neoformans actin gene is grouped on a distinct branch together with all other known fungal actin sequences. The availability of the C. neoformans actin gene will aid future phylogenetic and molecular studies of this important human pathogen.

Actins↗

Assessment of a PCR technique for the detection and identification of Cryptococcus neoformans.

The 18S ribosomal RNA gene of Cryptococcus neoformans was amplified by polymerase chain reaction (PCR). The primers CPL1 and CPR4 were tested for their ability to amplify DNA from 30 strains of C. neoformans and 27 specimens of cerebrospinal fluid (CSF) from patients with cryptococcal meningitis. A 343 bp product was obtained and its specificity confirmed by Southern hybridization with an internal sequence (INSR4) probe. The sensitivity was 100 fg by Southern analysis and 1 pg using the PCR. Neither human nor a variety of other fungal and bacterial strains (n = 78) gave an amplified product. This PCR method can detect as few as 5 cells ml-1 of C. neoformans in spiked-CSF following a simple processing procedure. The developed system of PCR was more sensitive than the culture method and revealed a very high specificity. The PCR was easy to perform and needed only 4 h for all processes from receiving the CSF to detection of a specific DNA band after agarose gel electrophoresis. This would provide another rapid laboratory method for the diagnosis of cryptococcal meningitis.

Bacteria↗

Discordant regulation of phenoloxidase and capsular polysaccharide in Cryptococcus neoformans.

We examined the regulation of two fungal virulence factors, phenoloxidase and capsular polysaccharide, in an ex-type strain of the fungal pathogen, Cryptococcus neoformans. Both were made during the stationary phase of cultural growth. Exogenous iron increased phenoloxidase activity three-fold (from 8.7 to 27.7 units mg-1, P < 0.05) but decreased capsular polysaccharide three-fold (from 9.0 to 3.4% packed cell volume, P < 0.01). A temperature shift from 25 to 37 degrees C decreased phenoloxidase activity three-fold (from 60.6 to 23.7 units mg-1, P < 0.01) but not capsular polysaccharide (8.5 to 6.7% packed cell volume, P not significant). Thus, cryptococcal virulence factors are not regulated coordinately. Moreover, although the phenoloxidase synthesizes an antioxidant, melanin, the enzyme is not induced by the oxidant, hydrogen peroxide, or by a combination of hydrogen peroxide and solubilized ferric ion. As cryptococcal melanin is cheaply made from exogenous catechols, perhaps C. neoformans does not need to regulate the phenoloxidase strongly but, rather, can afford to synthesize the phenoloxidase at a moderate rate whenever it finds its growth limited.

Cryptococcus neoformans↗

Lymphoproliferation and cytokine profiles in human peripheral blood mononuclear cells stimulated by Cryptococcus neoformans.

Cell-mediated immunity is critical to host defenses against the fungal infection cryptococcosis. Here, two functions critical to effective cell-mediated immunity (CMI), lymphoproliferation and cytokine release, were studied in Cryptococcus neoformans-stimulated peripheral blood mononuclear cells (PBMC) from seven healthy donors (controls) and two patients with cryptococcosis. PBMC responses to C. neoformans were compared with responses to Candida albicans. Control and patient PBMC had significant lymphoproliferation in response to whole C. neoformans, with peak proliferation seen following 8 days of culture, but only patient PBMC proliferated when stimulated with C. neoformans mannoprotein. C. neoformans-stimulated control PBMC released IL-2, IFN-gamma, and IL-10 into the supernatant with peak or near peak concentrations of these three cytokines generally seen by day 1. Release of IL-4 was low or undetectable. In contrast, C. neoformans-stimulated patient PBMC released IFN-gamma, which peaked on day 7, as well as IL-4, IL-10, and in one of two patients, IL-2. Cytokine release occurred later in patient (compared with control) PBMC. Lymphoproliferation and cytokine release were similar comparing control PBMC stimulated with C. neoformans versus Candida albicans. Thus, the magnitude and kinetics of the lymphoproliferative response to whole C. neoformans is similar comparing PBMC from controls and patients, but the cytokine profiles differ. Moreover, the capacity of patient PBMC to respond to soluble mannoprotein lends support to studies of mannoprotein components as vaccine candidates.

Cells, Cultured↗

Pharmacokinetics and biodistribution of a monoclonal antibody to Cryptococcus neoformans capsular polysaccharide antigen in a rat model of cryptococcal meningitis: implications for passive immunotherapy.

Several investigators have developed monoclonal antibodies against the capsular polysaccharide of Cryptococcus neoformans which have potential therapeutic applications. Using a rat model of C. neoformans meningitis, we studied the biodistribution and pharmacokinetics of a murine anticryptococcal capsular monoclonal antibody (mAb 2H1) after intravenous and intracisternal administration. After intravenous administration of 125I-labelled 2H1 to infected rats, there was no detectable localization of 125I in the brain or cerebrospinal fluid by either gamma-camera imaging of the whole animal or organ scintillation counting. In contrast, direct intracisternal instillation of 2H1 to infected rats resulted in persistent intracranial activity. In addition, the whole body half-life of intravenously administered radio labelled mAb 2H1 was significantly reduced in infected rats compared with uninfected rats. Our observations suggest that if high central nervous system (CNS) levels of mAb are needed to achieve a therapeutic effect in human C. neoformans meningoencephalitis, direct administration of mAb into the cerebrospinal fluid or modification of the mAb to increase penetration into the CNS may be required. Furthermore, higher or more frequent dosing of mAb may be required to maintain therapeutic levels in the presence of infection. This study demonstrates the usefulness of the rat as an experimental system for studying issues related to cryptococcosis.

Animals↗

Cryptococcus neoformans in Papua New Guinea: a common pathogen but an elusive source.

Around Port Moresby, Papua New Guinea (PNG), the annual incidence of cryptococcal meningitis is estimated to be up to 42.8 per million population; Cryptococcus neoformans var. gattii is the predominant causative agent. In Australia and California, environmental isolations have established an ecological association of C. neoformans var. gattii with Eucalyptus camaldulensis, E. tereticornis, and more recently E. rudis and E. gomphcephala. In PNG few E. camaldulensis survive experimental planting, E. tereticornis is endemic and there are no records of planting of the non-endemic E. rudis and E. gomphcephela. Despite extensive sampling of eucalypt-associated and other sources, we were unable to identify the ecological niche of C. neoformans var. gattii and neoformans in this region.

Cryptococcus neoformans↗

Isolation of Cryptococcus neoformans var. neoformans from pigeon droppings collected throughout Turkey.

The six hundred and thirty-four samples of pigeon droppings were collected throughout Turkey, from 54 of 80 provinces. Cryptococcus neoformans was isolated from 29 (4.6%, overall) of 634 samples and 29 isolates were from 18 provinces. Interestingly, 16 (88.9%) of these provinces occur on the three different coastlines of Turkey, therefore the ecological role of a humid climate was speculated. Almost all isolates [28/29] were recovered from samples collected from roofs (n=14) and dovecotes (n=14). All isolates were found to be C. neoformans var. neoformans.

Animals↗

First report on Cryptococcus laurentii associated with feather loss in a glossy starling (Lamprotornis chalybaeus).

Although Cryptococcus laurentii has been isolated from fresh droppings and cloaca samples from feral pigeons, it has never before been associated with an actual disease condition in birds. This case study deals with the first report on C. laurentii associated with feather loss in a glossy starling (Lamprotornis chalybaeus). The bird exhibited patchy feather loss, especially around the back and beak area, and greyish crusts sticking quite firmly to the underlying skin. The feathers had a greasy appearance and disseminated a musty odour. Treatment was installed with fluconazole in the drinking water. One month following the onset of treatment, the condition of the plumage had markedly improved.

Animals↗

Fatal biphasic brainstem and spinal leptomeningitis with Cryptococcus neoformans in a non-immunocompromised child.

Cryptococcal meningitis is one of the most common life-threatening, invasive fungal infections of the central nervous system in patients with defective T-lymphocyte function. It is, however, unusual in children. We report on a non-immunocompromised 10-y-old boy without evidence of immunological abnormality who developed headache, vomiting, disturbances of consciousness and areflexia. Magnetic resonance imaging of the brain and the spinal cord revealed enlargement of the ventricles and high signal lesions in the leptomeninges at the level of the cerebral peduncles and the cervical and thoracic cord. Cerebrospinal fluid analysis was positive for Cryptococcus neoformans. He was treated with amphotericin B and was symptom-free within 1 wk. Despite an extended course of therapy his symptoms suddenly relapsed and he succumbed to the medical complications of cardiac and respiratory failure. Central nervous system appearances at postmortem were those of cryptococcal leptomeningitis.

Arachnoid↗

Immunomodulator tuftsin increases the susceptibility of Cryptococcus neoformans to liposomal amphotericin B in immunocompetent BALB/c mice.

The co-administration of immunomodulators and antibiotics has been proved very successful for treatment of opportunistic infectious diseases. In the present study, we evaluated the combination of liposomal amphotericin B (lip-Amp B) and immunomodulator tuftsin to cure Cryptococcus neoformans infection in BALB/c mice. Mice infected with C. neoformans were treated with Amp B deoxycholate and tuftsin free or tuftsin-loaded Amp B liposomes. The results of the present study demonstrated higher efficacy of tuftsin-loaded Amp B liposomes against experimental murine cryptococcosis, in terms of enhanced survival rate and reduced fungal burden in organs (lungs and brain) of the treated mice. Interestingly, pre-treatment of mice with liposomal tuftsin before challenging them with the C. neoformans infection resulted in 100% survival of the treated animals followed by treatment with lip-Amp B. Immunomodulator-based therapy seems likely to be more beneficial for treatment of fungal infectious diseases.

Amphotericin B↗

Comparison of broth dilution and semisolid agar dilution for in vitro susceptibility testing of Cryptococcus neoformans.

We compared the in vitro activity of amphotericin B, flucytosine, itraconazole, fluconazole, ketoconazole and miconazole against 18 strains of Cryptococcus neoformans by using two methods: microbroth dilution and semisolid agar dilution. By both of the methods minimum inhibitory concentrations (MICs) showed a wide range for all antifungal agents but not for amphotericin B. Statistically significant differences between the two methods were observed only with amphotericin B and flucytosine, p = 0.048 and p = 0.045 respectively. Our study suggests that azole susceptibility testing for C. neoformans may be performed by the broth microdilution as well as the semisolid agar test. The choice of the method when testing amphotericin B and flucytosine is more problematic.

Amphotericin B↗

Genetic characterization of environmental isolates of the Cryptococcus neoformans species complex from Brazil.

The genetic affiliation of a large number of isolates of the Cryptococcus neoformans species complex from environmental sources in Brazil has been investigated using amplified fragment length polymorphism (AFLP). The strains of C. neoformans isolated from a single tree, as well as from neighbouring trees, showed high similarity values (> 95%) of their AFLP patterns, thus suggesting considerable genetic homogeneity. The majority of isolates of C. neoformans belonged to AFLP genotype 1, and had serotype A and mating type alpha (= C. neoformans var. grubii). Three isolates belonged to AFLP genotype 2, with serotype D and mating type alpha (= C. neoformans var. neoformans). One isolate, obtained from a building in Rio de Janeiro inhabited by pigeons, belonged to the AD hybrid AFLP genotype 3. All isolates from trees of C. neoformans var. gattii (= C. gattii) belonged to AFLP genotype 6, and their banding patterns showed relatively low genetic homogeneity with a similarity value of about 76%. Isolates of this genotype occupy an environmental niche in the Americas, and they may cause disease in non-AIDS and AIDS patients as well.

Air Microbiology↗