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PubMed2004

Cryptococcus neoformans methionine synthase: expression analysis and requirement for virulence.

This paper describes (i) the expression profile of the methionine synthase gene (MET6) in the human pathogenic fungus Cryptococcus neoformans and (ii) the phenotypes of a C. neoformans met6 mutant. In contrast to the MET3 gene, which showed no significant change in expression in any environmental condition tested, the MET6 gene showed a substantial induction in response to methionine and a dramatic transcriptional induction in response to homocysteine. Like a met3 mutant, the met6 mutant was a methionine auxotroph. However, relative to a met3 mutant, the met6 mutant grew very slowly and was less heat-shock resistant. In contrast to a met3 mutant, the met6 mutant lost viability when starved of methionine, and it was deficient in capsule formation. Like a met3 mutant, the met6 mutant was avirulent. In contrast to a met3 mutant, the met6 mutant was hypersensitive to fluconazole and to the calcineurin inhibitors FK506 and cyclosporin A. A synergistic fungicidal effect was also found between each of these drugs and met6. The phenotypic differences between the met3 and met6 mutants may be due to the accumulation in met6 mutants of homocysteine, a toxic metabolic intermediate that inhibits sterol biosynthesis.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2003

Contribution of epitope specificity to the binding of monoclonal antibodies to the capsule of Cryptococcus neoformans and the soluble form of its major polysaccharide, glucuronoxylomannan.

Incubation of encapsulated cryptococci with monoclonal antibodies (MAbs) specific for glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, produces two distinct capsular quellung-type reactions termed rim and puffy. The type of capsular reaction that occurs is determined by the epitope specificity of the MAb and the serotype of the yeast cell. Several biological activities, including opsonic activity, complement activation, and protective efficacy, are associated with the type of capsular reaction produced by a MAb. The goal of this study was to examine the reactivities of two families of anti-GXM MAbs with serotype A and D capsular polysaccharides in several immunochemical assays, including agglutination, immunofluorescence, quantitative precipitation, and enzyme-linked immunosorbent assay, in an effort to identify serological assays that are predictive of the capsular quellung reaction. The results showed that the type of capsular reaction (rim versus puffy) is a qualitative assessment of antibody-capsule interaction that cannot be predicted on the basis of a serological assay. The results further showed that antibody reactivity demonstrated in one serological assay is not necessarily predictive of results in another assay, particularly in cases where one assay examines antibody-capsule interactions, e.g., agglutination, and another assay examines interaction of antibody with soluble GXM. Taken together, the results suggest caution in interpretation of immunochemical assays for anti-GXM antibodies and recommend the use of multiple assays formats when studying anticryptococcal antibodies.

Animals↗
PubMed1994

Occurrences, specificities, and functions of ubiquitous antibodies in human serum that are reactive with the Cryptococcus neoformans cell wall.

Previous studies found that normal human serum (NHS) contains an immunoglobulin G (IgG) antibody that mediates initiation of the classical complement pathway by nonencapsulated Cryptococcus neoformans. The present study used an enzyme-linked immunosorbent assay with whole nonencapsulated yeast cells as solid-phase antigens to demonstrate the presence of high levels of IgG antibody in each of 11 sera from normal adult donors. The IgG antibodies were of the IgG2 subclass. The antibody activity was blocked completely by treatment of serum with isolated yeast glucan. Treatment of serum with mannan or chitin had no effect on antibody levels. Antibody activity was adsorbed completely by treatment of serum with zymosan particles. Adsorption with intact cells of Saccharomyces cerevisiae or Candida albicans had no effect, suggesting that the glucan on S. cerevisiae or C. albicans is not surface exposed. Assessment of the opsonic requirements for phagocytosis of nonencapsulated cryptococci by monocyte-derived human macrophages (MO-M phi) showed that high levels of phagocytosis occurred when yeast cells were opsonized with NHS. Removal of anti-glucan antibody by adsorption with whole nonencapsulated cryptococci did not diminish opsonic activity. Heat-inactivated serum or anti-glucan antibody affinity purified from NHS lacked opsonic activity. Taken together, these results indicate that phagocytosis of nonencapsulated cryptococci by monocyte-derived human macrophages has an obligatory requirement for opsonic ligands of the complement system, with no contribution by the anti-glucan IgG that is found in NHS.

Antibodies, Fungal↗
PubMed2002

Optimal T cell responses to Cryptococcus neoformans mannoprotein are dependent on recognition of conjugated carbohydrates by mannose receptors.

Cryptococcosis is a leading cause of death among individuals with compromised T cell function. Soluble Cryptococcus neoformans mannoproteins (MP) have emerged as promising vaccine candidates due to their capacity to elicit delayed-type hypersensitivity and Th type 1-like cytokines, both critical to the clearance of this pathogenic yeast. In this study, the mechanisms responsible for the potent immunostimulatory properties of MP were explored. Using Chinese hamster ovary cells expressing human macrophage mannose receptor (MMR), we determined that MP is a MMR ligand. Functionally, competitive blockade of multilectin mannose receptors (MR) on APCs diminished MP-dependent stimulation of primary T cells from immunized mice and the MP-reactive CD4(+) T cell hybridoma, P1D6, by 72 and 99%, respectively. Removal of O-linked saccharides from MP by beta-elimination inhibited MP-dependent stimulation of P1D6 and primary T cells by 89 and 90%, respectively. In addition, MP-dependent stimulation of P1D6 was abrogated after digestion with proteinase K, suggesting the protein core of MP contributed the antigenic moiety presented by APC. Stimulation of P1D6 by MP also was abolished using APC obtained from invariant chain-deficient mice, demonstrating Ag presentation was MHC class II restricted. Our data suggest that MP is a ligand for the MMR and that T cell stimulation is functionally inhibited either by competitive blockade of MR or by removal of carbohydrate residues critical for recognition. The demonstration that efficient T cell responses to MP require recognition of terminal mannose groups by MMR provides both a molecular basis for the immunogenicity of cryptococcal MP and support for vaccination strategies that target MR.

Animals↗
PubMed2000

Cryptococcus neoformans neutralizes macrophage and astrocyte derived nitric oxide without interfering with inducible nitric oxide synthase induction or catalytic activity - possible involvement of nitric oxide consumption.

The effect of Cryptococcus neoformans on the accumulation of nitrite, an indicator of nitric oxide (NO) synthesis, was investigated in cytokine (interferon-gamma [IFN-gamma] and interleukin [IL]-1)-stimulated cultures of rat peritoneal macrophages and C6 astrocytoma cells. Cytokine-induced nitrite generation in cultures of both cell types was inhibited in a dose-dependent manner by live C. neoformans, but not by heat-killed cryptococcal cells or conditioned medium from yeast cultures. C. neoformans-mediated reduction of nitrite formation coincided with impairment of NO-dependent macrophage tumoricidal activity. Cytokine-triggered induction of inducible NO synthase (iNOS) was unaffected in C6 cells, and only marginally reduced in macrophages. When cells were pretreated with cytokines for 24 h to induce iNOS, and any further induction was prevented by inhibition of protein synthesis, C. neoformans was still able to reduce nitrite accumulation in cultures of both cell types. Finally, live C. neoformans, but not heat-killed yeast cells or yeast culture supernatant, significantly reduced nitrite production in a culture solution of NO-releasing compound S-nitrosoglutathione (GSNO). Thus, it appears that cryptococcal reduction of nitrite formation in macrophage and C6 cultures was caused by the consumption of NO by some yeast molecule, rather than by the inhibition of cellular NO synthesis.

Animals↗
PubMed1995

CD4 lymphocyte count in HIV-positive persons exposed to Cryptococcus neoformans.

A report is presented on four HIV-positive homosexual men examined after several months of exposure during cleaning of a flat from masses of pigeon droppings heavily colonized by Cryptococcus neoformans. Only one out of the four persons, with a CD4 lymphocyte count of 50/microL, fell sick from systemic cryptococcosis, but not the others, with CD4 lymphocyte counts of 180, 250, and 630/microL, respectively; they remained clinically and mycologically inconspicuous and free from C. neoformans. Open questions in view of the epidemiology of opportunistic pathogens in AIDS are discussed with regard to the CD4 cell count as a parameter indicating a predisposition for cryptococcosis as an airborne AIDS-defining opportunistic infection. This has been confirmed by specific cultural diagnosis of the agent in both the environment and the patient. Already in 1987/88, the probable source of infection had been the subject of epidemiological studies on C. neoformans in Berlin.

AIDS-Related Opportunistic Infections↗
PubMed1996

Persistence, replacement, and microevolution of Cryptococcus neoformans strains in recurrent meningitis in AIDS patients.

Six separate human immunodeficiency virus-positive patients with cryptococcal meningitis were each found to have been infected with a unique strain of Cryptococcus neoformans on the basis of genomic DNA finger-printing analysis with the microsatellite sequence-containing oligonucleotide probe (GGAT)4 and by random amplification of polymorphic DNA. Two patients (A and B) experienced a recurrent episode of infection. Between 12 and 16 single-colony isolates recovered from primary isolation media (> 50% of C. neoformans colonies recovered) from cerebrospinal fluid specimens were fingerprinted from both patients during each episode. The fingerprints of both isolate collections from patient B were very similar, although minor polymorphisms were evident in both sets of profiles. The fingerprints of the isolate collection from the initial episode of infection in patient A were also identical to each other, apart from minor polymorphisms, but they were clearly different from the corresponding profiles of the isolate collection from the recurrent episode, the latter of which were completely identical, apart from minor polymorphisms in a single isolate. Furthermore, prolonged storage and in vitro subculture of the isolates did not alter the fingerprint profiles. These results provided convincing evidence that patients A and B were each infected with a single C. neoformans strain during each episode of infection and that in patient B, the same strain persisted and caused both episodes, while in patient A, a different strain was responsible for each episode. The prevalence of polymorphisms in multiple single-colony isolates from both patients also suggested that C. neoformans populations may undergo microevolution.

AIDS-Related Opportunistic Infections↗
PubMed1986

Antibody-dependent natural killer cell-mediated growth inhibition of Cryptococcus neoformans.

Previous data from this laboratory indicate that normal murine nylon wool nonadherent splenic cells with characteristics of natural killer (NK) cells effectively inhibit in vitro growth of Cryptococcus neoformans, a yeastlike pathogen. Since NK cells have been shown to be involved in antibody-dependent, cell-mediated cytotoxicity against immunoglobulin G (IgG)-coated tumor cells and xenogenic erythrocytes, we were interested in assessing the effects of the IgG fraction of rabbit anticryptococcal serum on NK cell-mediated inhibition of C. neoformans growth. Early in the study it became apparent that the conventional method of determining the numbers of CFU that was used previously for assessment of viable cryptococci at the end of the growth inhibition assay was not reliable for these studies, owing to minor clumping of the organisms in the presence of anticryptococcal antibody. Therefore, the BACTEC radiometric system was evaluated and determined to be a reliable replacement for the CFU count method. Using the BACTEC methodology, we showed that the anticryptococcal antibody significantly augmented the in vitro ability of NK cells to inhibit the growth of C. neoformans compared with normal rabbit serum or tissue culture medium. Furthermore, the antibody alone did not have an adverse effect on the organism, confirming that reduced growth indices obtained from test wells containing antibody, NK cells, and cryptococci were due to the effects of the NK cells. Maximum anticryptococcal activity of the NK cells was observed in the presence of 16 micrograms of IgG per ml; however, significant augmentation of anticryptococcal activity was seen with antibody concentrations as low as 3 micrograms/ml. Using different populations of murine splenic cells which had varying degrees of NK cell activity, we were able to show that NK cell activities, as determined by 51Cr release from YAC-1 targets, directly correlated with antibody-dependent, cell-mediated growth inhibition against cryptococci, suggesting that NK cells were effector cells in the antibody-dependent assays. Furthermore, in every case, the antibody-dependent activity of NK cells against C. neoformans was higher than the spontaneous activity of NK cells against the organism, emphasizing that NK cell activity against cryptococci can be augmented by specific antibody. When NK cell numbers were enriched by Percoll fractionation of nylon wool nonadherent splenic cells, antibody-dependent and spontaneous growth inhibitory activities of the effector cells were concomitantly augmented, confirming that NK cells were the effector cells in antibody-dependent growth inhibition of cryptococci.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Structure of the 13C-enriched O-deacetylated glucuronoxylomannan of Cryptococcus neoformans serotype A determined by NMR spectroscopy.

The complete assignment of 1H and 13C chemical shifts for 99% uniformly 13C-labeled O-deacetylated glucuronoxylomannan (GXM) of Cryptococcus neoformans serotype A isolate 9759-Mu-1 was accomplished by the analysis of HCCH-TOCSY and HCCH-COSY spectra. The sequence of the glycosyl residues was determined by a GHMBC experiment using 20% uniformly 13C-labeled GXM; GXM was prepared by a novel procedure that insured the virtual exclusion of adjacent 13C-labeled carbon atoms. For each residue in the GXM of 9759-Mu-1 we determined its linkage position, its anomeric configuration, and its position in the repeating sequence as follows: [sequence: see text]

Carbohydrate Conformation↗
PubMed1993

Two-site comparison of broth microdilution and semisolid agar dilution methods for susceptibility testing of Cryptococcus neoformans in three media.

This study evaluated the inter- and intralaboratory agreement between results of the semisolid agar dilution and broth microdilution methods of antifungal susceptibility testing of Cryptococcus neoformans. Three media were tested in two laboratories. The drugs tested were amphotericin B, flucytosine, itraconazole, fluconazole, and Schering 39304. Analysis by kappa statistics revealed good agreement between the laboratories for the two methods. The highest level of inter- and intralaboratory agreement was observed in RPMI 1640 with L-glutamine followed by Eagle's minimum essential medium and yeast nitrogen broth. The broth microdilution method appears more suitable than the semisolid agar dilution method for testing cryptococci because of its ease in performance, cost, and simplicity.

Agar↗
PubMed1994

Monoclonal antibodies to Cryptococcus neoformans capsular polysaccharide modify the course of intravenous infection in mice.

Immunoglobulin G1 (IgG1) monoclonal antibodies (MAbs) to the capsular glucuronoxylomannan (GXM) were studied for their ability to modify the course of intravenous Cryptococcus neoformans infection in mice. A/J mice were given intraperitoneal injection of 1.0 mg of either a GXM-binding IgG1 MAb (2H1 or 2D10 gamma 1) or the irrelevant isotype-matched control MAb 36-65 prior to intravenous infection. Parameters used to study antibody efficacy were lung and brain tissue fungal burden, lung and brain weights, serum GXM levels, and histopathological examination of lung, brain, heart, kidney, and spleen tissues. Mice given GXM-binding MAb had significantly reduced lung tissue fungal burden as measured by CFU. In contrast to the reduction in lung tissue burden, the reduction in brain tissue burden was small and did not achieve statistical significance. Serum GXM levels were reduced in mice receiving GXM-binding MAb. Histopathological examination revealed reduced numbers of granulomas and C. neoformans organisms in the lungs, brains, and kidneys of MAb 2H1-treated mice relative to control mice. The lungs and brains of mice receiving GXM-binding MAb weighed significantly less than those of control animals, consistent with the reduced inflammation noted histologically. Subendocardial inflammation and kidney cortical infarctions were present in control infected mice but not in MAb 2H1-treated mice. Immunocytochemical staining for polysaccharide antigen revealed a marked reduction in the amount of tissue polysaccharide in mice treated with MAb 2H1 relative to control mice. The results support an useful role for passive antibody administration in C. neoformans infections.

Animals↗
PubMed2006

Iron regulation of the major virulence factors in the AIDS-associated pathogen Cryptococcus neoformans.

Iron overload is known to exacerbate many infectious diseases, and conversely, iron withholding is an important defense strategy for mammalian hosts. Iron is a critical cue for Cryptococcus neoformans because the fungus senses iron to regulate elaboration of the polysaccharide capsule that is the major virulence factor during infection. Excess iron exacerbates experimental cryptococcosis and the prevalence of this disease in Sub-Saharan Africa has been associated with nutritional and genetic aspects of iron loading in the background of the HIV/AIDS epidemic. We demonstrate that the iron-responsive transcription factor Cir1 in Cr. neoformans controls the regulon of genes for iron acquisition such that cir1 mutants are "blind" to changes in external iron levels. Cir1 also controls the known major virulence factors of the pathogen including the capsule, the formation of the anti-oxidant melanin in the cell wall, and the ability to grow at host body temperature. Thus, the fungus is remarkably tuned to perceive iron as part of the disease process, as confirmed by the avirulence of the cir1 mutant; this characteristic of the pathogen may provide opportunities for antifungal treatment.

AIDS-Related Opportunistic Infections↗
PubMed2006

Influence of climatic conditions on the isolation of members of the Cryptococcus neoformans species complex from trees in Colombia from 1992-2004.

The aim of this retrospective study was to analyze the relationship between occurrence of the serotypes of the Cryptococcus neoformans species complex in tree samples and the climatic conditions registered during samplings in four cities of Colombia, between 1992 and 2004, by means of a logistic regression model and lagged Pearson correlations. During 97 collection dates, 8220 samples from different tree species were taken, of which 2.63% were positive: 56.5% yielded serotype B, 24.7% serotype C and 18.8% serotype A isolates. The prevalence of the serotypes varied among the cities. The results suggest that environmental climatic conditions, mainly humidity, temperature, evaporation and solar radiation, can affect the occurrence of the different serotypes in trees in a differential manner. These different climatic tolerances were reflected in the geographic distribution of the serotypes in Colombia. The climatic conditions for 15 days before the sampling date were correlated with positive or negative isolation of the different serotypes.

Chrysobalanaceae↗
PubMed2000

Sequencing and heterologous expression in Saccharomyces cerevisiae of a Cryptococcus neoformans cDNA encoding a plasma membrane H(+)-ATPase.

A cDNA containing an open reading frame encoding a putative plasma membrane H(+)-ATPase in the human pathogenic basidiomycetous yeast Cryptococcus neoformans was cloned and sequenced by means of PCR and cDNA library hybridization. The cloned cDNA is 3475 bp in length, containing a 2994 bp open reading frame encoding a polypeptide of 997 amino acids. As in the case of another basidiomycetous fungus (Uromyces fabae), the deduced amino acid sequence of CnPMA1 was found to be more homologous to those of P-type H(+)-ATPases from higher plants than to those from ascomycetous fungi. In order to prove the sequenced cDNA corresponds to a H(+)-ATPase, it was expressed in Saccharomyces cerevisiae and found to functionally replace its own H(+)-ATPase. Kinetic studies of CnPMA1 compared to ScPMA1 show differences in V(max) values and H(+)-pumping in reconstituted vesicles. The pH optimum and K(m) values are similar in both enzymes.

Amino Acid Sequence↗
PubMed2006

Phospholipase B activity enhances adhesion of Cryptococcus neoformans to a human lung epithelial cell line.

Secreted phospholipase B (PLB1), which contains three enzyme activities in the one protein, is necessary for the initiation of pulmonary infection by Cryptococcus neoformans and for dissemination from the lung via the lymphatics and blood. Adhesion to lung epithelium is the first step in this process, therefore we investigated the role of PLB1 in adhesion to a human lung epithelial cell line, A549, using C. neoformans var. grubii wild-type strain H99, a PLB1 deletion mutant (deltaplb1), and a reconstituted strain (deltaplb1rec). Adhesion of H99 and deltaplb1rec was approximately 69% greater than deltaplb1 at 4 h. Adhesion of deltaplb1 significantly increased after killing by chemicals or heat, and Fourier-transformed analysis by FTIR spectroscopy indicated this was due to changes in capsular and/or cell wall polysaccharides and proteins. Inhibition by specific PLB1 antibodies, or inhibitors of phospholipase B (PLB), but not lysophospholipase (LPL) or lysophospholipase transacylase (LPTA) activities decreased the adhesion of H99 and deltaplb1rec by 33-58%. Growth under conditions of osmotic stress and high glucose concentration increased both PLB secretion and subsequent cryptococcal adhesion. Dose-dependent increases (to 67%) in adhesion of live deltaplb1 were observed in the presence of 0.1-2 mM palmitic acid. We conclude that PLB1 plays a role in the binding of C. neoformans to host lung epithelial cells, possibly due to production of fatty acids from plasma membranes and/or surfactant by PLB activity.

Bacterial Adhesion↗
PubMed1996

Molecular subtype distribution of Cryptococcus neoformans in four areas of the United States. Cryptococcal Disease Active Surveillance Group.

To improve understanding of the epidemiology of cryptococcal disease, we analyzed the multilocus genotype distribution of 358 Cryptococcus neoformans isolates obtained from 251 patients through active surveillance in four U.S. geographic areas from 1992 through 1994. Isolates of the predominant enzyme electrophoretic type (ET), ET-1, were recovered in significantly greater proportion from Atlanta, Ga., Houston, Tex., and all major metropolitan areas of Alabama than from San Francisco, Calif. ET-2 and ET-7 complex (serotype AD) isolates were recovered predominantly from San Francisco. ET-3 was recovered less frequently from San Francisco than from the three other locations. These findings may reflect geographic differences in exposure to environmental strains or the identification of previously unrecognized C. neoformans clusters. Analysis by random amplified polymorphic DNA-PCR subtyping further divided 67 ET-1 isolates into 19 additional subtypes, none of which could be associated with a particular geographic region. Multiple isolates from the same patient always revealed the same multilocus enzyme electrophoresis and random amplified polymorphic DNA subtypes. No differences in subtype distribution were found when isolates from AIDS patients were compared with those from persons without or with another underlying disease, although one C. noeformans var. gattii isolate was obtained from an AIDS patient. When body site distribution was analyzed, ET-4 was disproportionately recovered from skin or surface body sites. Evidence for linkage disequilibrium in this fungal population suggests that virulent C. neoformans possesses a clonal population structure. Continued application of molecular subtyping methods will be useful in tracking the source, transmission, and relative virulence of different C. neoformans strains.

AIDS-Related Opportunistic Infections↗
PubMed1995

Role of tumor necrosis factor and gamma interferon in acquired resistance to Cryptococcus neoformans in the central nervous system of mice.

Although naive C.B-17 and BALB/cBy mice die of meningoencephalitis within 5 weeks of intravenous infection with an opportunistic strain of Cryptococcus neoformans, immunized mice express an acquired, CD4+ T-cell-dependent immunity and survive an intravenous infection. Infusion of lymphocytes from immune mice into severe combined immunodeficiency (SCID) mice renders these mice more resistant to cryptococcal brain infection than uninfused controls. We have investigated the role of gamma interferon (IFN-gamma) and tumor necrosis factor (TNF) in acquired resistance to C. neoformans. Neutralization of either IFN-gamma or TNF impaired resistance of immune BALB/cBy or C.B-17 mice to cryptococci. At 10 days postinfection, there were approximately 10 times as many yeast cells in the brains of mice treated with either anticytokine antibody as in the brains of mice treated with control antibody. Simultaneous neutralization of IFN-gamma and TNF further exacerbated infection. Neutralization of IFN-gamma or TNF also impaired resistance in immune lymphocyte-infused SCID mice, resulting in significantly higher yeast burdens in brains of cytokine-neutralized mice than in brains of controls. Concurrent neutralization of IFN-gamma and TNF rendered SCID recipients of immune cells equivalent to uninfused SCID mice with respect both to brain yeast burdens at 10 days and to survival. Anti-TNF treatment alone also curtailed survival. Histological examination of the brains of cytokine-neutralized mice revealed deficiencies in ability to focus inflammatory cells at brain lesions. These data demonstrate that both IFN-gamma and TNF are important mediators of acquired resistance to cryptococcal meningoencephalitis.

Animals↗