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PubMed2000

A novel episomal shuttle vector for transformation of Cryptococcus neoformans with the ccdB gene as a positive selection marker in bacteria.

We report the engineering of a new shuttle vector featuring its episomal maintenance in Cryptococcus neoformans and the lethal Escherichia coli ccdB gene for positive selection in bacteria. Telomere-like sequences from C. neoformans and the STAB fragment confer episomal maintenance to the vector (pPM8) upon transformation in C. neoformans. The vector generated high transformation frequencies and each transformant was estimated to harbor thirty copies of the plasmid. The plasmids recovered in E. coli from the C. neoformans transformants showed no evidence of rearrangement. This construct will be very useful for cloning and studying the regulation of genes in C. neoformans.

Bacterial Proteins↗
PubMed1994

Occurrences, immunoglobulin classes, and biological activities of antibodies in normal human serum that are reactive with Cryptococcus neoformans glucuronoxylomannan.

Serum obtained from normal human subjects contains antibodies reactive in an enzyme-linked immunosorbent assay with the glucuronoxylomannan (GXM) of Cryptococcus neoformans. The frequency of occurrence of class-specific antibodies among normal subjects was 28% for immunoglobulin G (IgG), 98% for IgM, and 3% for IgA. Anti-GXM antibodies with kappa light chains occurred in 98% of normal subjects, while the occurrence of lambda light chains was 28%. Each of five subjects with high levels of anti-GXM IgG antibodies had readily detectable antibodies of the IgG2 isotype; two of the five subjects had readily detectable IgG1 antibody. An examination of sera from human immunodeficiency virus-infected patients showed that human immunodeficiency virus infection was accompanied by a significant decrease in the occurrence of IgM antibodies and anti-GXM antibodies with kappa light chains; these decreases occurred early in infection when CD4 counts were still > or = 500 cells per microliter. A slight but not statistically significant decrease in the occurrence of anti-GXM IgG antibodies was seen only in patients with CD4 levels of < 200 cells per microliter. Sera from normal subjects with high levels of anti-GXM IgG antibodies were examined to identify any contribution of the antibodies to complement activation or to opsonization of the yeast cells. An analysis of the kinetics for activation and binding of C3 to the yeast cell showed no pattern of quantitative or qualitative differences between sera with high or low levels of anti-GXM IgG antibodies. Phagocytosis studies showed that the naturally occurring IgG antibodies did not contribute to opsonization of the yeast cells.

Antibodies, Fungal↗
PubMed2002

Fc-dependent and Fc-independent opsonization of Cryptococcus neoformans by anticapsular monoclonal antibodies: importance of epitope specificity.

Monoclonal antibodies (MAbs) reactive with glucuronoxylomannan (GXM), the major capsular polysaccharide of the yeast Cryptococcus neoformans, produce distinct capsular reactions when viewed by differential interference contrast microscopy. These reactions depend on the epitope specificity of the antibody. Opsonic activities of immunoglobulin G1 (IgG1) MAbs that produce patterns termed rim and puffy were examined. Rim-pattern MAbs are reactive with an epitope shared by GXM serotypes A, B, C, and D. Puffy-pattern MAbs are reactive only with serotypes A and D. In phagocytosis assays, using serotype A cells and resident murine peritoneal macrophages, rim-pattern MAbs were markedly more opsonic than puffy-pattern MAbs. F(ab')(2) fragments of rim-pattern MAbs were synergistic with heat-labile factors in normal human serum for opsonization of the yeast. F(ab')(2) fragments of puffy-pattern MAbs were also synergistic with normal serum in opsonization but at a much lower level than fragments of rim-pattern MAbs. Normal serum alone was not opsonic. F(ab')(2) fragments of rim-pattern MAbs, but not puffy-pattern MAbs, stimulated phagocytosis of encapsulated cryptococci in the absence of serum. This serum-independent opsonic action of F(ab')(2) fragments was abrogated by pretreatment of macrophages with purified GXM, suggesting the involvement of a phagocyte GXM receptor. The results indicate that (i) there are multiple mechanisms by which anticapsular IgG MAbs facilitate phagocytosis of encapsulated cryptococci, (ii) some anti-GXM antibodies are opsonic in an Fc-independent manner, and (iii) opsonic activity correlates with the capsular reaction and occurs in an epitope-specific manner.

Animals↗
PubMed1995

Determination of antigen binding specificities of Cryptococcus neoformans factor sera by enzyme-linked immunosorbent assay.

The competitive binding specificities of glucuronoxylomannan (GXM) and its derivatives to factor sera of Cryptococcus neoformans were studied by enzyme-linked immunosorbent assay. An effort was made to determine the epitope specificity of each factor serum. Despite the presence of antigenic factor 1 on all serotypes of C. neoformans, variations in inhibition ability were observed with different GXMs. The panspecific component of factor serum 1 (antibody 1) appeared to be due to the presence of more than one antibody component. The activity was dependent on the 6-O-acetyl substituent. GXMs of serotypes A and D inhibited factor serum 2 equally well, indicating a low titer for the antibody 7 component. Serotype B GXM was a poor inhibitor, and serotype C GXM did not inhibit factor serum 2. The activity of factor serum 2 was 6-O-acetyl dependent. GXMs from typical serotype A and serotype D isolates were excellent inhibitors of factor serum 3. GXMs from serotype B were poorly inhibitory and serotype C did not inhibit factor serum 3. The activity of factor serum 3 was 6-O-acetyl dependent. The activity of factor serum 4 was due predominantly to antibody component 6. The activity of factor 4 was directed mainly against serotype C, and it was independent of 6-O-acetyl substitution Factor serum 5 was specific for serotype B GXMs. The inhibitory effect was independent of 6-O-acetyl substitution, but the effect was diminished by reduction of the glucuronic acid. The GXMs with a typical serotype C structure inhibited antibody 6. O deacetylation of the GXMs did not affect their inhibitory activity. However, reduction of glucuronic acid reduced factor serum 6 binding. Factor serum 8 was specific to serotype D; native GXMs of serotype A were slightly inhibitory. O deacetylation of the serotype D GXMs abrogated the inhibitory effect. O deacetylation alone abrogates the activity of antibody components 1, 2, 3, and 8. Reduction of glucuronic acid reduces the inhibitory activity of the GXM to antibody components 4, 5, and 6. Partial GXM structures and methyl glycosides did not effectively inhibit the activity of any of the factor sera.

Animals↗
PubMed1995

Down-regulation of the afferent phase of T cell-mediated pulmonary inflammation and immunity by a high melanin-producing strain of Cryptococcus neoformans.

The interaction(s) between cryptococcal virulence factors and leukocytes involved in generating protective cell-mediated immunity is not well defined. Intratracheal inoculation of Cryptococcus neoformans strain 52 induced a vigorous T cell-mediated pulmonary inflammatory response that controlled the growth of the organism. In contrast, strain 145 induced a pulmonary inflammatory response that was delayed in onset, slower to develop, and ineffective in controlling the infection. In addition, the expansion of cryptococcus-specific lymphocytes in the pulmonary lymph nodes and titer of specific Abs in the serum of strain 145-infected mice were both diminished markedly. Of the known cryptococcal virulence factors, these two strains differed only in melanin production (52-low and 145-high). Heat-killed strain 145 cryptococci (HKC-145) that had been rendered melanin-negative induced TNF-alpha production by alveolar macrophages in vitro and stimulated vigorous cryptococcus-specific lymphoproliferation. In contrast, high melanin-containing HKC-145 inhibited TNF-alpha production and lymphoproliferation. In vivo, mice infected with melanin low strain 52, but not melanin high strain 145, had elevated levels of TNF-alpha in the bronchoalveolar lavage fluid. Mice co-infected with strains 145 and 52 generated a pulmonary inflammatory response resulting in increased long-term survival. Taken together, these studies demonstrate that melanin does not protect cryptococci from being eliminated in vivo by recruited, activated effector cells; but melanin can inhibit the recognition of the organism by host defenses, thereby down-regulating the afferent phase of T cell-mediated immunity, e.g., TNF-alpha production and lymphoproliferation.

Animals↗
PubMed1994

Restriction fragment length polymorphism analysis of Cryptococcus neoformans isolates from environmental (pigeon excreta) and clinical sources in New York City.

Restriction fragment length polymorphism analysis of environmental (pigeon excreta) and clinical Cryptococcus neoformans var. neoformans isolates in a limited geographic area distinguished 6 strains among 8 environmental isolates and 12 strains among 17 clinical isolates. Clusters of patients with three strains types accounted for 47% of clinical isolates. Despite this diversity, two strains were shared by environmental and clinical isolates.

AIDS-Related Opportunistic Infections↗
PubMed1992

Susceptibility testing of Cryptococcus neoformans: a microdilution technique.

We studied a series of test conditions in a microtiter system to define the optimal method for determining the susceptibility of Cryptococcus neoformans to antifungal agents. Twenty-one isolates of C. neoformans were grown for 24 or 48 h in four chemically defined media: yeast nitrogen base (BYNB 7); RPMI 1640; synthetic amino acid medium--fungal (SAAMF), buffered at pH 7.0 to select the medium that best supported growth of this fastidious yeast; and yeast nitrogen base, pH 5.4 (YNB 5.4). Maximum growth of C. neoformans, at 35 degrees C, was obtained in YNB 5.4, with the next highest growth levels in BYNB 7, SAAMF, and RPMI. Growth at 24 h was uniformly poor in all media and lacked reproducibility. In contrast, incubation for 48 h gave adequate growth with low standard deviations, and 48 h was selected as the optimal incubation period for this study. Comparison of the relationship between growth kinetics and initial inoculum size for eight cryptococcal isolates showed that 10(4) cells per ml yielded optimal growth in BYNB 7 and YNB 5.4, whereas 10(5) cells per ml was optimal in RPMI and SAAMF. Furthermore, variation of inocula from 10(3) to 10(5) cells per ml showed small but significant inoculum effects in determining MICs of fluconazole, amphotericin B, and flucytosine for C. neoformans. Therefore, 10(4) cells per ml was chosen as the optimal inoculum for susceptibility testing in this study. Mean MICs of fluconazole, amphotericin B, and flucytosine for 21 crytococcal isolates in RPMI and BYNB 7 were low (for example, fluconazole had mean MICs of 1.2 and 1.3 micrograms/ml in RPMI and BYNB 7, respectively) and differed significantly from medium to medium. In contrast, the MICs obtained in SAAMF were significantly higher (e.g., fluconazole had a mean MIC of 2.2 micrograms/ml). Variance in MICs was large with fluconazole and flucytosine but small with amphotericin B, irrespective of the medium used. A microtiter system employing BYNB 7 as the medium, 48 h as the incubation period, and 10(4) cells per ml as the final inoculum is a simple, accurate, and reproducible method for the testing of C. neoformans susceptibility to fluconazole, amphotericin B, and flucytosine.

AIDS-Related Opportunistic Infections↗
PubMed1992

A 34- to 38-kilodalton Cryptococcus neoformans glycoprotein produced as an exoantigen bearing a glycosylated species-specific epitope.

Three monoclonal antibodies (MAbs), all of the immunoglobulin G1 subclass, were raised against Cryptococcus neoformans by using the technique of cyclophosphamide ablation of B-cell responses against shared epitopes of the cross-reactive fungus Trichosporon beigelii. MAb 3C2 was reactive against the encapsulated and nonencapsulated isolates of C. neoformans var. neoformans by enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot), and in addition to a 34- to 38-kDa determinant, it recognized a series of lower-molecular-weight species. 3C2 also reacted strongly with culture supernatant preparations of C. neoformans var. neoformans by ELISA. 3C2 showed no recognition of either T. beigelii or C. neoformans var. gattii antigens. Enzymatic deglycosylation followed by reaction with 3C2 on Western blots revealed that sialic acid was an integral part of the determinant, together with N-acetylglucosaminyl-asparagine and alpha-mannose. Proteolytic digestion showed that the epitope was pepsin sensitive and that it also contained tryptophan and glycine and/or leucine as determinants of recognition by 3C2. The pI of the glycoprotein was 7.1. Affinity chromatography-purified antigen did not exhibit proteolytic activity on sodium dodecyl sulfate-polyacrylamide substrate gels. Indirect fluorescence antibody tests revealed that 3C2 labelling was confined to the cell membrane and cytoplasm of yeasts. The remaining MAbs, 7H4 and 5G5, recognized both capsulated and nonencapsulated strains of C. neoformans var. neoformans by both ELISA and Western Blot, identifying linear determinants with molecular masses of 36 and 30 kDa. They were unreactive against culture supernatant antigen (exoantigen) from either variant of C. neoformans.

Animals↗
PubMed2000

In vitro activities of ravuconazole (BMS-207147) against 541 clinical isolates of Cryptococcus neoformans.

The in vitro activities of the new triazole, ravuconazole (BMS-207147), were compared to those of fluconazole and itraconazole against 541 clinical isolates of Cryptococcus neoformans. Isolates were obtained from cerebrospinal fluid (396), blood (116), and miscellaneous clinical specimens (29). Overall, ravuconazole (MIC at which 90% of the isolates are inhibited [MIC(90)], 0.25 microg/ml) was more active than either itraconazole (MIC(90), 0.5 microg/ml) or fluconazole (MIC(90), 8 microg/ml). Among the isolates inhibited by > or =16 microg of fluconazole/ml, 90.2% were inhibited by < or =1 microg of ravuconazole/ml. On the basis of our findings and the favorable pharmacokinetic properties of ravuconazole, we suggest that ravuconazole may be useful for the treatment of infectious diseases due to C. neoformans and that further clinical studies to confirm these promising in vitro results are warranted.

Antifungal Agents↗
PubMedDate not supplied

Differences in Cryptococcus neoformans capsular polysaccharide structure influence assembly of alternative complement pathway C3 convertase on fungal surfaces.

Binding of complement component C3 and Factor B to Cryptococcus neoformans serotypes A through D via the alternative complement pathway was measured in a system containing fresh nonimmune human serum. Serotypes B and C (C. neoformans var. gattii) bound approximately half as many molecules of both complement components as serotypes A and D (C. neoformans var. neoformans). In contrast, removal of xylosyl and glucuronyl side chains from the mannan main chain of capsular polysaccharide by the Smith degradation procedure resulted in binding of similar quantities of C3 to each of the four serotypes. We conclude that the relatively high degree of side chain substitution of capsular polysaccharide from C. neoformans variety gattii contributes to inefficient surface assembly of the alternative pathway C3 convertase. Inefficient binding of alternative pathway complement components to serotypes B and C may contribute to the relative difficulty in successfully treating infections caused by these organisms.

Acetylation↗
PubMed2006

Limited contribution of Toll-like receptor 2 and 4 to the host response to a fungal infectious pathogen, Cryptococcus neoformans.

The present study was designed to elucidate the role of Toll-like receptor (TLR) 2 and TLR4 in the host response to Cryptococcus neoformans. Both TLR2 knockout (KO) and TLR4KO mice produced interleukin-1beta (IL-1beta), IL-6, IL-12p40 and tumor necrosis factor-alpha (TNF-alpha) in sera and cleared this fungal pathogen from infected lungs at a comparable level to control littermate (LM) mice. Synthesis of these cytokines was not significantly different in the lungs of these KO mice and LM mice, although IL-1beta, IL-6 and IL-12p40 tended to be lower in TLR2KO, but not TLR4KO, mice than in controls. In addition, there was no significant reduction detected in the synthesis of IL-12 and TNF-alpha by bone marrow-derived dendritic cells from TLR2KO and TLR4KO mice upon stimulation with live yeast cells. Finally, HEK293 cells expressing either TLR2/dectin-1 or TLR4/MD2/CD14 did not respond to C. neoformans in the activation of nuclear factor kappa B (NFkappaB) detected by a luciferase assay. Our results suggest that TLR2 and TLR4 do not or only marginally contribute to the host and cellular response to this pathogen.

Animals↗
PubMed1997

Serotyping of Cryptococcus neoformans isolates from environmental and clinical sources in extreme southern Italy (Calabria and Sicily, central Mediterranean area).

Ninety-seven environmental and four clinical isolates of Cryptococcus neoformans from several localities in extreme southern Italy were serotyped. All proved to be Cr. neoformans var. neoformans serotype A. The homogeneity of the serotypes suggests that geographical climatological conditions may play a role in Cr. neoformans serotype diffusion. It was furthermore revealed that, unlike the data reported for the serotypes of Cr. neoformans in mainland Italy, Cr. neoformans serotype D was not present in the examined sites in extreme southern Italy.

Animals↗
PubMed1991

Intrapulmonary growth and dissemination of an avirulent strain of Cryptococcus neoformans in mice depleted of CD4+ or CD8+ T cells.

The contribution of T lymphocyte subpopulations to intrapulmonary and systemic resistance against an opportunistic strain of Cryptococcus neoformans was examined. It was found that C. neoformans was destroyed when introduced into the lungs of normal mice, but disseminated to the brains of mice treated with an antibody that depleted them of CD4+ T cells. Depletion of either CD8+ or CD4+ T cells impaired the ability of the host to clear the yeast from the lung. These results, together with the observation that CD8+ T cells accumulate in the lungs of CD4+ T cell-deficient mice, suggest that CD8+ T cells play an important role in resistance to C. neoformans infection acquired via the respiratory tract.

Animals↗
PubMed1996

Asymptomatic solitary pulmonary nodules due to Cryptococcus neoformans in patients infected with human immunodeficiency virus.

We report the cases of three HIV-positive patients with solitary pulmonary nodules caused by Cryptococcus neoformans. Although human infection with C. neoformans occurs via the respiratory tract, isolated pulmonary infection in HIV-positive patients, in contrast with HIV-negative patients, has been thought to be relatively rare. When isolated pulmonary disease in HIV-infected patients, has been described, most of the patients have been symptomatic (symptoms have included fever, cough, and dyspnea). In addition, these patients have had diffuse interstitial infiltrates, alveolar infiltrates, or nodular infiltrates that have often been associated with hilar adenopathy and occasionally with pleural effusions. None of the patients in the previously reported series have had lesions described as small, asymptomatic, isolated pulmonary nodules.

Adult↗
PubMed2004

Serotypes of clinical cerebrospinal fluid Cryptococcus neoformans isolates from southern Taiwan and their in vitro susceptibilities to amphotericin B, fluconazole, and voriconazole.

In this study, 34 clinical cerebrospinal fluid isolates of Cryptococcus neoformans were serotyped, and their in vitro susceptibilities to amphotericin B, fluconazole, and voriconazole were analyzed. Of these 34 isolates, serotype A was found in 29 isolates and serotype B in the other five. The voriconazole geometric mean MIC was significantly lower than the amphotericin B/antibiotic medium 3 geometric mean MIC (P < 0.0001 at both 48 and 72 h), as well as the fluconazole geometric mean MIC (P < 0.0001 at both 48 and 72 h). Of the three antifungal agents, only fluconazole, with geometric mean MICs at both 48 and 72 h, showed significant difference between the serotypes A and B of C. neoformans.

Amphotericin B↗
PubMed2000

In vitro susceptibility of environmental Cryptococcus neoformans variety neoformans isolates from Turkey to six antifungal agents, including SCH56592 and voriconazole.

The in vitro antifungal susceptibility of 27 environmental (pigeon droppings) isolates of Cryptococcus neoformans var. neoformans, isolated from throughout Turkey, to six antifungal agents (amphotericin B, flucytosine, fluconazole, voriconazole, itraconazole, and SCH56592) was studied. Voriconazole, itraconazole, and SCH56592 all showed comparable activity and were more active than the remaining three antifungal agents tested. Overall, SCH56592 was the most active agent (MIC90, 0.015 microg/ml, at both 48 and 72 h), followed by itraconazole (MIC90, 0.03 microg/ml, at both 48 and 72 h) and voriconazole (MIC90, 0.25 microg/ml, at both 48 and 72 h), respectively. Antifungal susceptibility data for environmental isolates may reflect patterns for the clinical isolates recovered from patients from the same geographic area.

Animals↗
PubMed1995

Comparison of multilocus enzyme electrophoresis and random amplified polymorphic DNA analysis for molecular subtyping of Cryptococcus neoformans. The Cryplococcal Disease Active Surveillance Group.

We evaluated multilocus enzyme electrophoresis (MEE) and random amplified polymorphic DNA (RAPD) for their usefulness in subtyping 344 Cryptococcus neoformans clinical isolates obtained from four U.S. metropolitan areas in 1992 to 1994. MEE and RAPD with five primers both discriminated between the two varieties of C. neofromans. MEE divided C. neoformans var. neoformans isolates into 15 enzyme electrophoretic subtypes (ETs) arranged in three complexes. The predominant ET 1 complex contained 10 ETs, with isolates from 70% of patients in 1 ET. RAPD with five primers further sorted this predominant ET into 19 subtypes, with 60% of isolates sorting into three RAPD types. The ET 8 MEE complex, containing three ETs, could not be divided further by RAPD. The ET 7 complex (two ETs) included isolates from all serotype AD patients. Although both MEE and RAPD identified isolates of C. neoformans var. gattii, neither distinguished between serotypes B and C. These results showed that the two C. neoformans varieties could be identified by MEE or RAPD profile as well as by biochemical methods. RAPD improved the discriminatory power of MEE for isolates within the ET 1 complex but with other ETs offered little additional sensitivity over MEE and was less sensitive than MEE with isolates of C. neoformans var. gattii. This information will be useful in identifying particular environmental sources of disease-causing exposures, in seeking clusters of cases, and in determining whether an infecting strain changes over time.

Cryptococcus neoformans↗