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PubMed2003

In vitro interaction of flucytosine with conventional and new antifungals against Cryptococcus neoformans clinical isolates.

Combinations of flucytosine with conventional and new antifungals were evaluated in vitro against 30 clinical isolates of Cryptococcus neoformans. Synergy determined by checkerboard analysis was observed with combinations of fluconazole, itraconazole, voriconazole, amphotericin B, and caspofungin with flucytosine against 77, 60, 80, 77, and 67% of the isolates, respectively. Antagonism was never observed. Killing curves showed indifferent interactions between triazoles and flucytosine and synergy between amphotericin B and flucytosine.

Antifungal Agents↗
PubMed2003

G484S amino acid substitution in lanosterol 14-alpha demethylase (ERG11) is related to fluconazole resistance in a recurrent Cryptococcus neoformans clinical isolate.

Five sequential Cryptococcus neoformans isolates recovered from an AIDS patient with recurrent meningitis were analyzed. Four isolates were fluconazole susceptible, while the fifth isolate developed fluconazole resistance. Analysis of the 14-alpha lanosterol demethylase gene (ERG11) showed a point mutation in the resistant strain responsible for the amino acid substitution G484S.

Acquired Immunodeficiency Syndrome↗
PubMed2004

In vitro and in vivo efficacies of the new triazole albaconazole against Cryptococcus neoformans.

The activity of albaconazole (UR-9825; J. Uriach & Cía. S.A., Barcelona, Spain) was compared to that of fluconazole against 12 isolates of Cryptococcus neoformans in vitro and against 1 isolate in vivo in a rabbit model of cryptococcal meningitis. Albaconazole was 100-fold more potent in vitro than fluconazole on a per-weight basis and was fungicidal at potentially relevant concentrations for two isolates. MICs ranged from </=0.0012 to 1.25 micro g/ml, with the MICs for most isolates being between 0.039 and 0.156 micro g/ml. Isolates were from human immunodeficiency virus (HIV)-infected and non-HIV-infected patients and were of serotypes A, B, and C; and the fluconazole MICs for some of the isolates were elevated. Infected rabbits were treated with either fluconazole or albaconazole at dosages ranging from 5 to 80 mg/kg of body weight/day. The peak concentrations of albaconazole in serum and cerebrospinal fluid (CSF) averaged 4.14 and 0.62 micro g/ml, respectively, in animals receiving 80 mg/kg/day. Comparison of the concentrations in serum and CSF suggested a level of CSF penetration of approximately 15%. Despite limited penetration into the subarachnoid space, at all three doses tested albaconazole was as effective as fluconazole for the treatment of cryptococcal meningitis in rabbits.

Animals↗
PubMed2004

Differential gene expression in auristatin PHE-treated Cryptococcus neoformans.

The antifungal pentapeptide auristatin PHE was recently shown to interfere with microtubule dynamics and nuclear and cellular division in the opportunistic pathogen Cryptococcus neoformans. To gain a broader understanding of the cellular response of C. neoformans to auristatin PHE, mRNA differential display (DD) and reverse transcriptase PCR (RT-PCR) were applied. Examination of approximately 60% of the cell transcriptome from cells treated with 1.5 times the MIC (7.89 micro M) of auristatin PHE for 90 min revealed 29 transcript expression differences between control and drug-treated populations. Differential expression of seven of the transcripts was confirmed by RT-PCR, as was drug-dependent modulation of an additional seven transcripts by RT-PCR only. Among genes found to be differentially expressed were those encoding proteins involved in transport, cell cycle regulation, signal transduction, cell stress, DNA repair, nucleotide metabolism, and capsule production. For example, RHO1 and an open reading frame (ORF) encoding a protein with 91% similarity to the Schizophyllum commune 14-3-3 protein, both involved in cell cycle regulation, were down-regulated, as was the gene encoding the multidrug efflux pump Afr1p. An ORF encoding a protein with 57% identity to the heat shock protein HSP104 in Pleurotus sajor-caju was up-regulated. Also, three transcripts of unknown function were responsive to auristatin PHE, which may eventually contribute to the elucidation of the function of their gene products. Further study of these differentially expressed genes and expression of their corresponding proteins are warranted to evaluate how they may be involved in the mechanism of action of auristatin PHE. This information may also contribute to an explanation of the selectivity of auristatin PHE for C. neoformans. This is the first report of drug action using DD in C. neoformans.

Antifungal Agents↗
PubMed2004

In vitro antifungal activities of inhibitors of phospholipases from the fungal pathogen Cryptococcus neoformans.

Secreted phospholipase B is a proven virulence factor for the pathogenic fungus Cryptococcus neoformans and exhibits three phospholipase activities in the one protein. These are phospholipase B (PLB), lysophospholipase (LPL), and lysophospholipase transacylase (LPTA). Our aim was to investigate the feasibility of using this enzyme as a target for antifungal therapy. We determined in C. neoformans var. grubii strain H99 that 82% of PLB activity was secreted but that 64% of LPL activity and 70% of LPTA activity were cell associated. Cell-associated activities (cytosolic and membrane) were further characterized, since it is likely that any fungicidal effect would depend on inhibition of these enzymes. Four commercially available compounds with structural similarities to phospholipid substrates were tested as inhibitors. These were alexidine dihydrochloride (compound A), dioctadecyldimethylammonium bromide (compound O), 1,12 bis-(tributylphosphonium)dodecane dibromide (compound P), and decamethonium dibromide (compound D). The best phospholipase inhibitors (compounds A and P) were also the most potent antifungal agents by the standard broth microdilution test. Compound A was highly selective for secreted and cell-associated PLB activities and showed no inhibition of mammalian phospholipase A(2) at 0.25 micro M. Compound O, which was specific for secretory and cytosolic LPL and LPTA and membrane-associated PLB, was not antifungal. We conclude that inhibitors of cryptococcal phospholipases can be selective for fungal enzymes and intrinsically antifungal. They also provide tools for assessing the relative importance of the various enzyme activities in virulence. Our results enable further rational structure-function studies to validate the use of phospholipases as antifungal targets.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2004

Posaconazole and amphotericin B combination therapy against Cryptococcus neoformans infection.

To investigate the effects of posaconazole (POS) and amphotericin B (AMB) combination therapy in cryptococcal infection, we established an experimental model of systemic cryptococcosis in CD1 mice by intravenous injection of three distinct clinical isolates of Cryptococcus neoformans. Therapy was started 24 h after the infection and continued for 10 consecutive days. POS was given at 3 and 10 mg/kg of body weight/day, while AMB was given at 0.3 mg/kg/day. Combination therapy consisted of POS given at a low (combo 3) or at a high (combo 10) dose plus AMB. Survival studies showed that combo 3 was significantly more effective than POS at 3 mg/kg for two isolates tested (P value, < or = 0.001), while combo 10 was significantly more effective than POS at 10 mg/kg for all three isolates (P values ranging from <0.001 to 0.005). However, neither combination regimen was more effective than AMB alone. For two isolates, combination therapy was significantly more effective than each single drug at reducing the fungal burden in the brain (P values ranging from 0.001 to 0.015) but not in the lungs. This study demonstrates that the major impact of POS and AMB combination therapy is on brain fungal burden rather than on survival.

AIDS-Related Opportunistic Infections↗
PubMed2005

Cryptococcus neoformans resistance to echinocandins: (1,3)beta-glucan synthase activity is sensitive to echinocandins.

(1,3)Beta-D-glucan synthase (EC 2.4.1.34. UDP-glucose: 1,3-beta-D-glucan 3-beta-glucosyltransferase) uses UDP-glucose as substrate and catalyzes the polymerization of glucose ([1,3]-beta-linkages) to form the major carbohydrate component of the fungal cell wall. We have optimized in vitro assay conditions for (1,3)beta-glucan synthase activity from Cryptococcus neoformans. Cells lysed in 50 mM Tris, pH 7.75, containing 20% glycerol, 2 mM NaF, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM MgCl(2), 0.1% protease and phosphatase inhibitor cocktails, and 60 microM GTPgammaS produced maximum specific activity in vitro. We tested in vitro C. neoformans (1,3)beta-glucan synthase activity against the (1,3)beta-glucan synthase inhibitors, caspofungin and cilofungin, and have determined that (1,3)beta-glucan synthase activity is very sensitive (apparent K(i) of 0.17 +/- 0.02 microM and 22 +/- 5.7 microM, respectively) to these echinocandins. Taken together with high MICs for C. neoformans (caspofungin MIC, 16 microg/ml; cilofungin MIC, 64 microg/ml), our results indicate that C. neoformans is resistant to caspofungin and cilofungin by a mechanism(s) unrelated to (1,3)beta-glucan synthase resistance.

Antifungal Agents↗
PubMed2005

Correspondence of in vitro and in vivo fluconazole dose-response curves for Cryptococcus neoformans.

We conducted in vitro experiments to evaluate the susceptibility of a clinical isolate of Cryptococcus neoformans to a wide range of concentrations of fluconazole. In vitro susceptibility was tested using broth macrodilution methods modified to provide a numeric count of viable organisms. The association between the quantitative in vitro response and fluconazole drug concentrations was estimated using local nonparametric regression. Regression analysis was used to assess the correspondence between the in vitro fluconazole concentration-response curve and the murine dose-response curve observed in our previously reported murine model. The regression model was then used to predict the murine response. There was a strong correspondence between in vitro measures of response to fluconazole alone and the previously reported biologic effects seen in the mouse. In vitro antifungal drug susceptibility testing can reliably predict the murine response to fluconazole.

Animals↗
PubMed2006

Susceptibility of Cryptococcus neoformans biofilms to antifungal agents in vitro.

Microbial biofilms contribute to virulence and resistance to antibiotics by shielding microbial cells from host defenses and antimicrobial drugs, respectively. Cryptococcus neoformans was demonstrated to form biofilms in polystyrene microtiter plates. The numbers of CFU of disaggregated biofilms, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide reduction, and light and confocal microscopy were used to measure the fungal mass, the metabolic activity, and the appearance of C. neoformans biofilms, respectively. Biofilm development by C. neoformans followed a standard sequence of events: fungal surface attachment, microcolony formation, and matrix production. The susceptibilities of C. neoformans cells of the biofilm and planktonic phenotypes to four antifungal agents were examined. The exposure of C. neoformans cells or preformed cryptococcal biofilms to fluconazole or voriconazole did not result in yeast growth inhibition and did not affect the metabolic activities of the biofilms, respectively. In contrast, both C. neoformans cells and preformed biofilms were susceptible to amphotericin B and caspofungin. However, C. neoformans biofilms were significantly more resistant to amphotericin B and caspofungin than planktonic cells, and their susceptibilities to these drugs were further reduced if cryptococcal cells contained melanin. A spot enzyme-linked immunosorbent assay and light and confocal microscopy were used to investigate how antifungal drugs affected C. neoformans biofilm formation. The mechanism by which amphotericin B and caspofungin interfered with C. neoformans biofilm formation involved capsular polysaccharide release and adherence. Our results suggest that biofilm formation may diminish the efficacies of some antifungal drugs during cryptococcal infection.

Amphotericin B↗
PubMed2006

Cryptococcus neoformans can utilize the bacterial melanin precursor homogentisic acid for fungal melanogenesis.

Cryptococcus neoformans melanizes in the environment and in mammalian tissues, but the process of melanization in either venue is mysterious given that this microbe produces melanin only from exogenous substrates. Understanding the process of melanization is important because melanization is believed to protect against various stresses in the environment, including UV radiation, and pigment production is associated with virulence. Melanization in C. neoformans requires the availability of diphenolic precursors. In contrast, many bacteria synthesize melanin from homogentisic acid (HGA). We report that C. neoformans strains representing all four serotypes can produce a brown pigment from HGA. The brown pigment was acid resistant and had the electron paramagnetic resonance spectrum of a stable free radical, qualities that identified it as a melanin. Melanin "ghost"-like particles obtained from pigmented C. neoformans cells were hydrophobic, fluorescent under a variety of irradiation wavelengths, negatively charged, insoluble in organic solvents and alcohols, resistant to degradation by strong acids, and vulnerable to bleaching. HGA melanization was laccase dependent and repressed by high concentrations of glucose. The ability of C. neoformans to utilize a bacterial melanin precursor compound suggests a new substrate source for melanization in the environment.

Animals↗
PubMed2003

PCR-restriction fragment length polymorphism analysis of the phospholipase B (PLB1) gene for subtyping of Cryptococcus neoformans isolates.

Cryptococcus neoformans is a pathogenic yeast that is currently divided into three varieties, five serotypes, and eight molecular types. The following report describes the use of PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene (PLB1) as a simple tool to differentiate between C. neoformans subgroups. A PLB1 fragment, 1,970 bp, was amplified and digested with either AvaI or HindIII. Both sets of profiles grouped the isolates into their respective varieties, but only the AvaI profiles allowed for the identification of the eight molecular types via the corresponding RFLP profiles A1 to A8. Digestion of the same fragments with HindIII resulted in RFLP profiles H1 to H5, which distinguished only between serotype A, AD, D, and B/C. Neither enzyme distinguished serotype B from serotype C. The serotype AD profile was a composite of the serotype A and D profiles. Further investigation showed that the serotype AD isolates used in this study are heterozygous, with one allele of PLB1 originating from a serotype A parent and the other from a serotype D parent.

Cryptococcus neoformans↗
PubMed2005

Cutinase-like enzyme from the yeast Cryptococcus sp. strain S-2 hydrolyzes polylactic acid and other biodegradable plastics.

A purified lipase from the yeast Cryptococcus sp. strain S-2 exhibited remote homology to proteins belonging to the cutinase family rather than to lipases. This enzyme could effectively degrade the high-molecular-weight compound polylactic acid, as well as other biodegradable plastics, including polybutylene succinate, poly (epsilon-caprolactone), and poly(3-hydroxybutyrate).

Amino Acid Sequence↗
PubMed2006

Induction by Klebsiella aerogenes of a melanin-like pigment in Cryptococcus neoformans.

While studying the interaction of Cryptococcus neoformans with Dictyostelium discoideum, we noticed that yeast colonies in agar with a feeder lawn of Klebsiella aerogenes were brown. This finding was intriguing because C. neoformans colonies are not pigmented unless they are provided with precursors for melanization. Strains of all C. neoformans serotypes produced brown pigment in response to K. aerogenes at 22, 30, and 37 degrees C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when C. neoformans was exposed to live Escherichia coli or heat-killed K. aerogenes. Analysis of K. aerogenes supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by C. neoformans. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for C. neoformans melanization in the environment.

Animals↗
PubMed1962

Production of a new polysaccharide with Cryptococcus laurentii var. flavescens.

Conditions were investigated for the production of a new gum by the yeast Cryptococcus laurentii var. flavescens NRRL Y-1401 in shaken flasks and 20-liter fermentors. The most suitable medium contained 6% commercial glucose, 0.25% autolyzed brewer's yeast, and 0.001% MnSO(4) and had an initial pH of 6.5. Polysaccharide yields, as measured by the dry weight of the alcohol precipitates, were in the range of 30 to 35% based on initial glucose.

Cryptococcus↗
PubMed1968

Modified Littman oxgall agar to isolate Cryptococcus neoformans.

Littman Oxgall Agar was modified by adding an extract of Guizotia abyssinica seeds with a water diluent for rehydration. In this medium, colonies of Cryptococcus neoformans became brown, but the color failed to develop in eight other yeasts and yeast phases of diphasic organisms. The depression of saprophytic fungi noted on Littman Oxgall Agar was not lost in the modification.

Blastomyces↗