Evaluation of Vitek MS for Differentiation of Cryptococcus neoformans and Cryptococcus gattii Genotypes.

Cryptococcus neoformans and Cryptococcus gattii are the main pathogenic species of invasive cryptococcosis among the Cryptococcus species. Taxonomic studies have shown that these two taxa have different genotypes or molecular types with biological and ecoepidemiological peculiarities. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been proposed as an alternative method for labor-intensive methods for C. neoformans and C. gattii genotype differentiation. However, Vitek MS, one of the commercial MALDI-TOF MS instruments, has not been yet been evaluated for this purpose. Thus, we constructed an in-house database with reference strains belonging to the different C. neoformans (VNI, VNII, VNIII, and VNIV) and C. gattii (VGI, VGII, VGIII, and VGIV) major molecular types by using the software Saramis Premium (bioMérieux, Marcy-l'Etoile, France). Then, this new database was evaluated for discrimination of the different genotypes. Our in-house database provided correct identification for all C. neoformans and C. gattii genotypes; however, due to the intergenotypic mass spectral similarities, a careful postanalytic evaluation is necessary to provide correct genotype identification.

The Buoyancy of Cryptococcus neoformans Is Affected by Capsule Size.

Cryptococcus neoformans is an environmental pathogenic fungus with a worldwide geographical distribution that is responsible for hundreds of thousands of human cryptococcosis cases each year. During infection, the yeast undergoes a morphological transformation involving capsular enlargement that increases microbial volume. To understand the factors that play a role in environmental dispersal of C. neoformans and C. gattii, we evaluated the cell density of Cryptococcus using Percoll isopycnic gradients. We found differences in the cell densities of strains belonging to C. neoformans and C. gattii species complexes. The buoyancy of C. neoformans strains varied depending on growth medium. In minimal medium, the cryptococcal capsule made a major contribution to the cell density such that cells with larger capsules had lower density than those with smaller capsules. Removing the capsule, by chemical or mechanical methods, increased the C. neoformans cell density and reduced buoyancy. Melanization of the C. neoformans cell wall, which also contributes to virulence, produced a small but consistent increase in cell density. Encapsulated C. neoformans sedimented much more slowly in seawater as its density approached the density of water. Our results suggest a new function for the capsule whereby it can function as a flotation device to facilitate transport and dispersion in aqueous fluids.IMPORTANCE The buoyancy of a microbial cell is an important physical characteristic that may affect its transportability in fluids and interactions with tissues during infection. The polysaccharide capsule surrounding C. neoformans is required for infection and dissemination in the host. Our results indicate that the capsule has a significant effect on reducing cryptococcal cell density, altering its sedimentation in seawater. Modulation of microbial cell density via encapsulation may facilitate dispersal for other important encapsulated pathogens.

A Predicted Mannoprotein Participates in Cryptococcus gattii Capsular Structure.

The yeast-like pathogen Cryptococcus gattii is an etiological agent of cryptococcosis. The major cryptococcal virulence factor is the polysaccharide capsule, which is composed of glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoproteins (MPs). The GXM and GalXM polysaccharides have been extensively characterized; however, there is little information about the role of mannoproteins in capsule assembly and their participation in yeast pathogenicity. The present study characterized the function of a predicted mannoprotein from C. gattii, designated Krp1. Loss-of-function and gain-of-function mutants were generated, and phenotypes associated with the capsular architecture were evaluated. The null mutant cells were more sensitive to a cell wall stressor that disrupts beta-glucan synthesis. Also, these cells displayed increased GXM release to the culture supernatant than the wild-type strain did. The loss of Krp1 influenced cell-associated cryptococcal polysaccharide thickness and phagocytosis by J774.A1 macrophages in the early hours of interaction, but no difference in virulence in a murine model of cryptococcosis was observed. In addition, recombinant Krp1 was antigenic and differentially recognized by serum from an individual with cryptococcosis, but not with serum from an individual with candidiasis. Taken together, these results indicate that C. gattii Krp1 is important for the cell wall structure, thereby influencing capsule assembly, but is not essential for virulence in vivoIMPORTANCECryptococcus gattii has the ability to escape from the host's immune system through poorly understood mechanisms and can lead to the death of healthy individuals. The role of mannoproteins in C. gattii pathogenicity is not completely understood. The present work characterized a protein, Kpr1, that is essential for the maintenance of C. gattii main virulence factor, the polysaccharide capsule. Our data contribute to the understanding of the role of Kpr1 in capsule structuring, mainly by modulating the distribution of glucans in C. gattii cell wall.

Fisetin as a promising antifungal agent against Cryptocococcus neoformans species complex.

AIMS: The aim of this study was to investigate the mechanisms of action of fisetin, a flavonol with antifungal activity previously evaluated against the Cryptococcus neoformans species complex. METHODS AND RESULTS: Ergosterol content and flow cytometry analysis were determined for the C. neoformans species complex in the presence of fisetin and ultrastructural analysis of morphology was performed on Cryptococcus gattii and C. neoformans. Decrease in the total cellular ergosterol content after exposure to fisetin ranged from 25·4% after exposure to 128 µg ml(-1) to 21·6% after exposure to 64 µg ml(-1) of fisetin compared with the control (without fisetin). The fisetin effects obtained with flow cytometry showed metabolic impairment, and alterations in its normal morphology caused by fisetin in C. neoformans cells were verified using scanning electron microscopy. CONCLUSIONS: Fisetin is a compound that acts in the biosynthesis of ergosterol. Flow cytometry showed that fisetin reduced viability of the metabolically active cells of C. gattii, while morphological changes explain the action of fisetin in inhibiting growth of these fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: This study supports the idea that fisetin may represent a good starting point for the development of future therapeutic substances for cryptococcosis.

Comparative Analysis of Extracellular Polymeric Substances from Cryptococcus gattii VGIIa Strain Isolated for the First Time in Japan.

Cryptococcus gattii and C. neoformans are pathogenic yeasts that cause meningoencephalitis. C. gattii has four molecular types: VGI, VGII, VGIII, and VGIV. Furthermore, three genotypes have been reported for VGII, and a high pathogenicity of the VGIIa genotype has been proposed. The VGIIa strain has been isolated from a patient in Japan, but little is known about the characteristics of the polysaccharides in this strain. In this study we examined the induction of interleukin-8（IL-8）transcriptional activation and compared the nuclear magnetic resonance（NMR）spectra of extracellular polymeric substances（EPSs）, mainly polysaccharides, from the VGIIa, VGIIb, and VGIIc genotypes. The induction of IL-8 by C. gattii EPSs was weaker than that by C. neoformans EPSs. The anomeric proton signals in the NMR spectra of EPSs obtained from VGII isolates were similar, and the polysaccharides were mainly mannose, xylose, galactose, and glucuronic acid. These results suggest that the extracellular polysaccharides from the VGIIa strain isolated in Japan are almost the same as those from other VGII strains.

Peroxide Natural Products from Plakortis zyggompha and the Sponge Association Plakortis halichondrioides-Xestospongia deweerdtae: Antifungal Activity against Cryptococcus gattii.

Cryptococcus gattii is a human pathogen and causative agent of a pernicious, sometimes fatal, disseminated fungal disease. Investigation of antifungal extracts of the marine sponge association Plakortis halichondrioides-Xestospongia deweerdtae and the sponge Plakortis zyggompha from the Bahamas led to the discovery and isolation of 6-epi-7,8-dihydroplakortide K (1), plakortide AA (2), and three new plakinic acids, N-P (4-6; unstable 1,2-dioxolanes bearing benzyl-substituted conjugated dienes), along with known plakinic acids L, K, and M.5 Chiroptical comparisons and DFT calculations of (13)C NMR chemical shifts were used to assign the absolute stereostructure of 4. The stereospecific base-promoted rearrangement-saponification of 1 to 10 was briefly investigated and showed tight kinetic control and stereospecific formation of the new C-2 stereocenter with inversion at C-3. Plakinic acid M and plakortides 9 and 11 exhibited antifungal activity against C. gattii (MIC90 = 2.4 to 36 µM), but plakinic acids N-P were inactive under the same conditions.

Fluconazole alters the polysaccharide capsule of Cryptococcus gattii and leads to distinct behaviors in murine Cryptococcosis.

Cryptococcus gattii is an emergent human pathogen. Fluconazole is commonly used for treatment of cryptococcosis, but the emergence of less susceptible strains to this azole is a global problem and also the data regarding fluconazole-resistant cryptococcosis are scarce. We evaluate the influence of fluconazole on murine cryptococcosis and whether this azole alters the polysaccharide (PS) from cryptococcal cells. L27/01 strain of C. gattii was cultivated in high fluconazole concentrations and developed decreased drug susceptibility. This phenotype was named L27/01F, that was less virulent than L27/01 in mice. The physical, structural and electrophoretic properties of the PS capsule of L27/01F were altered by fluconazole. L27/01F presented lower antiphagocytic properties and reduced survival inside macrophages. The L27/01F did not affect the central nervous system, while the effect in brain caused by L27/01 strain began after only 12 hours. Mice infected with L27/01F presented lower production of the pro-inflammatory cytokines, with increased cellular recruitment in the lungs and severe pulmonary disease. The behavioral alterations were affected by L27/01, but no effects were detected after infection with L27/01F. Our results suggest that stress to fluconazole alters the capsule of C. gattii and influences the clinical manifestations of cryptococcosis.

Pinpointing differentially expressed domains in complex protein mixtures with the cloud service of PatternLab for Proteomics.

Mass-spectrometry-based shotgun proteomics has become a widespread technology for analyzing complex protein mixtures. Here we describe a new module integrated into PatternLab for Proteomics that allows the pinpointing of differentially expressed domains. This is accomplished by inferring functional domains through our cloud service, using HMMER3 and Pfam remotely, and then mapping the quantitation values into domains for downstream analysis. In all, spotting which functional domains are changing when comparing biological states serves as a complementary approach to facilitate the understanding of a system's biology. We exemplify the new module's use by reanalyzing a previously published MudPIT dataset of Cryptococcus gattii cultivated under iron-depleted and replete conditions. We show how the differential analysis of functional domains can facilitate the interpretation of proteomic data by providing further valuable insight.

Immunoproteomics and immunoinformatics analysis of Cryptococcus gattii: novel candidate antigens for diagnosis.

AIM: To identify immunoreactive proteins of Cryptococcus gattii genotype VGII and their B-cell epitopes. MATERIALS & METHODS: We combined 2D gel electrophoresis, immunoblotting and mass spectrometry to identify immunoreactive proteins from four strains of C. gattii genotype VGII (CG01, CG02, CG03 and R265). Next, we screened the identified proteins to map B-cell epitopes. RESULTS: Sixty-eight immunoreactive proteins were identified. The strains and the number of proteins we found were: CG01 (12), CG02 (12), CG03 (18) and R265 (26). In addition, we mapped 374 peptides potentially targeted by B cells. CONCLUSION: Both immunoreactive proteins and B-cell epitopes of C. gattii genotype VGII that were potentially targeted by a host humoral response were identified. Considering the evolutionary relevance of the identified proteins, we may speculate that they could be used as the initial targets for recombinant protein and peptide synthesis aimed at the development of immunodiagnostic tools for cryptococcosis.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry-based method for discrimination between molecular types of Cryptococcus neoformans and Cryptococcus gattii.

We evaluated the usefulness of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for Cryptococcus identification at the species and subspecies levels by using an in-house database of 25 reference cryptococcal spectra. Eighty-one out of the 82 Cryptococcus isolates (72 Cryptococcus neoformans and 10 Cryptococcus gattii) tested were correctly identified with respect to their molecular type designations. We showed that MALDI-TOF MS is a practicable alternative to conventional mycology or DNA-based methods.

Rapid identification of Cryptococcus neoformans and Cryptococcus gattii by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Compared to DNA sequence analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) correctly identified 100% of Cryptococcus species, distinguishing the notable pathogens Cryptococcus neoformans and C. gattii. Identification was greatly enhanced by supplementing a commercial spectral library with additional entries to account for subspecies variability.