MALDI-TOF MS Enables the Rapid Identification of the Major Molecular Types within the Cryptococcus neoformans/C. gattii Species Complex

BACKGROUND: The Cryptococcus neoformans/C. gattii species complex comprises two sibling species that are divided into eight major molecular types, C. neoformans VNI to VNIV and C. gattii VGI to VGIV. These genotypes differ in host range, epidemiology, virulence, antifungal susceptibility and geographic distribution. The currently used phenotypic and molecular identification methods for the species/molecular types are time consuming and expensive. As Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) offers an effective alternative for the rapid identification of microorganisms, the objective of this study was to examine its potential for the identification of C. neoformans and C. gattii strains at the intra- and inter-species level. METHODOLOGY: Protein extracts obtained via the formic acid extraction method of 164 C. neoformans/C. gattii isolates, including four inter-species hybrids, were studied. RESULTS: The obtained mass spectra correctly identified 100% of all studied isolates, grouped each isolate according to the currently recognized species, C. neoformans and C. gattii, and detected potential hybrids. In addition, all isolates were clearly separated according to their major molecular type, generating greater spectral differences among the C. neoformans molecular types than the C. gattii molecular types, most likely reflecting a closer phylogenetic relationship between the latter. The number of colonies used and the incubation length did not affect the results. No spectra were obtained from intact yeast cells. An extended validated spectral library containing spectra of all eight major molecular types was established. CONCLUSIONS: MALDI-TOF MS is a rapid identification tool for the correct recognition of the two currently recognized human pathogenic Cryptococcus species and offers a simple method for the separation of the eight major molecular types and the detection of hybrid strains within this species complex in the clinical laboratory. The obtained mass spectra provide further evidence that the major molecular types warrant variety or even species status

Low diversity Cryptococcus neoformans variety grubii multilocus sequence types from Thailand are consistent with an ancestral African origin.

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Ctr2 Links Copper Homeostasis to Polysaccharide Capsule Formation and Phagocytosis Inhibition in the Human Fungal Pathogen Cryptococcus neoformans

Cryptococcus neoformans is a human opportunistic fungal pathogen responsible for ∼1/3 of HIV/AIDS deaths worldwide. This budding yeast expresses a polysaccharide capsule necessary for virulence. Capsule production inhibits phagocytosis by macrophages. Here we describe results that link copper homeostasis to capsule production and the inhibition of phagocytosis. Specifically, using Agrobacterium-mediated insertional mutagenesis, we identified an insertion in the promoter region of the putative copper transporter-encoding gene CTR2 that results in reduced expression of CTR2 and increased phagocytosis by murine RAW264.7 macrophages. The mutant also displayed sensitivity to copper starvation and defects in polysaccharide capsule production and melanization. These defects were all reversed by genetic correction of the promoter insertion by homologous targeting. Several melanization-defective mutants identified previously, those in the RIM20, RIM101, and VPS25 genes, also display sensitivity to copper starvation, reduced capsule production and increased phagocytosis. Together these results indicate a previously undescribed link between copper homeostasis to polysaccharide capsule production and phagocytosis inhibition in Cryptococcus neoformans

Transcription Factors Mat2 and Znf2 Operate Cellular Circuits Orchestrating Opposite- and Same-Sex Mating in Cryptococcus neoformans

Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from a unicellular yeast to multicellular hyphae during opposite sex (mating) and unisexual reproduction (same-sex mating). Opposite- and same-sex mating are induced by similar environmental conditions and involve many shared components, including the conserved pheromone sensing Cpk1 MAPK signal transduction cascade that governs the dimorphic switch in C. neoformans. However, the homeodomain cell identity proteins Sxi1α/Sxi2a encoded by the mating type locus that are essential for completion of sexual reproduction following cell–cell fusion during opposite-sex mating are dispensable for same-sex mating. Therefore, identification of downstream targets of the Cpk1 MAPK pathway holds the key to understanding molecular mechanisms governing the two distinct developmental fates. Thus far, homology-based approaches failed to identify downstream transcription factors which may therefore be species-specific. Here, we applied insertional mutagenesis via Agrobacterium-mediated transformation and transcription analysis using whole genome microarrays to identify factors involved in C. neoformans differentiation. Two transcription factors, Mat2 and Znf2, were identified as key regulators of hyphal growth during same- and opposite-sex mating. Mat2 is an HMG domain factor, and Znf2 is a zinc finger protein; neither is encoded by the mating type locus. Genetic, phenotypic, and transcriptional analyses of Mat2 and Znf2 provide evidence that Mat2 is a downstream transcription factor of the Cpk1 MAPK pathway whereas Znf2 functions as a more terminal hyphal morphogenesis determinant. Although the components of the MAPK pathway including Mat2 are not required for virulence in animal models, Znf2, as a hyphal morphology determinant, is a negative regulator of virulence. Further characterization of these elements and their target circuits will reveal genes controlling biological processes central to fungal development and virulence

The Mating Type Locus (MAT) and Sexual Reproduction of Cryptococcus heveanensis: Insights into the Evolution of Sex and Sex-Determining Chromosomal Regions in Fungi

Mating in basidiomycetous fungi is often controlled by two unlinked, multiallelic loci encoding homeodomain transcription factors or pheromones/pheromone receptors. In contrast to this tetrapolar organization, Cryptococcus neoformans/Cryptococcus gattii have a bipolar mating system, and a single biallelic locus governs sexual reproduction. The C. neoformans MAT locus is unusually large (>100 kb), contains >20 genes, and enhances virulence. Previous comparative genomic studies provided insights into how this unusual MAT locus might have evolved involving gene acquisitions into two unlinked loci and fusion into one contiguous locus, converting an ancestral tetrapolar system to a bipolar one. Here we tested this model by studying Cryptococcus heveanensis, a sister species to the pathogenic Cryptococcus species complex. An extant sexual cycle was discovered; co-incubating fertile isolates results in the teleomorph (Kwoniella heveanensis) with dikaryotic hyphae, clamp connections, septate basidia, and basidiospores. To characterize the C. heveanensis MAT locus, a fosmid library was screened with C. neoformans/C. gattii MAT genes. Positive fosmids were sequenced and assembled to generate two large probably unlinked MAT gene clusters: one corresponding to the homeodomain locus and the other to the pheromone/receptor locus. Strikingly, two divergent homeodomain genes (SXI1, SXI2) are present, similar to the bE/bW Ustilago maydis paradigm, suggesting one or the other homeodomain gene was recently lost in C. neoformans/C. gattii. Sequencing MAT genes from other C. heveanensis isolates revealed a multiallelic homeodomain locus and at least a biallelic pheromone/receptor locus, similar to known tetrapolar species. Taken together, these studies reveal an extant C. heveanensis sexual cycle, define the structure of its MAT locus consistent with tetrapolar mating, and support the proposed evolutionary model for the bipolar Cryptococcus MAT locus revealing transitions in sexuality concomitant with emergence of a pathogenic clade. These studies provide insight into convergent processes that independently punctuated evolution of sex-determining loci and sex chromosomes in fungi, plants, and animals

Diploids in the Cryptococcus neoformans Serotype A Population Homozygous for the α Mating Type Originate via Unisexual Mating

The ubiquitous environmental human pathogen Cryptococcus neoformans is traditionally considered a haploid fungus with a bipolar mating system. In nature, the α mating type is overwhelmingly predominant over a. How genetic diversity is generated and maintained by this heterothallic fungus in a largely unisexual α population is unclear. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions generating both diploid intermediates and haploid recombinant progeny. Same-sex mating (α-α) also occurs in nature as evidenced by the existence of natural diploid αADα hybrids that arose by fusion between two α cells of different serotypes (A and D). How significantly this novel sexual style contributes to genetic diversity of the Cryptococcus population was unknown. In this study, ∼500 natural C. neoformans isolates were tested for ploidy and close to 8% were found to be diploid by fluorescence flow cytometry analysis. The majority of these diploids were serotype A isolates with two copies of the α MAT locus allele. Among those, several are intra-varietal allodiploid hybrids produced by fusion of two genetically distinct α cells through same-sex mating. The majority, however, are autodiploids that harbor two seemingly identical copies of the genome and arose via either endoreplication or clonal mating. The diploids identified were isolated from different geographic locations and varied genotypically and phenotypically, indicating independent non-clonal origins. The present study demonstrates that unisexual mating produces diploid isolates of C. neoformans in nature, giving rise to populations of hybrids and mixed ploidy. Our findings underscore the importance of same-sex mating in shaping the current population structure of this important human pathogenic fungus, with implications for mechanisms of selfing and inbreeding in other microbial pathogens

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Identification of Yeasts Is Contingent on Robust Reference Spectra

BACKGROUND: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for yeast identification is limited by the requirement for protein extraction and for robust reference spectra across yeast species in databases. We evaluated its ability to identify a range of yeasts in comparison with phenotypic methods. METHODS: MALDI-TOF MS was performed on 30 reference and 167 clinical isolates followed by prospective examination of 67 clinical strains in parallel with biochemical testing (total n = 264). Discordant/unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene cluster. PRINCIPAL FINDINGS: Twenty (67%; 16 species), and 24 (80%) of 30 reference strains were identified to species, (spectral score ≥2.0) and genus (score ≥1.70)-level, respectively. Of clinical isolates, 140/167 (84%) strains were correctly identified with scores of ≥2.0 and 160/167 (96%) with scores of ≥1.70; amongst Candida spp. (n = 148), correct species assignment at scores of ≥2.0, and ≥1.70 was obtained for 86% and 96% isolates, respectively (vs. 76.4% by biochemical methods). Prospectively, species-level identification was achieved for 79% of isolates, whilst 91% and 94% of strains yielded scores of ≥1.90 and ≥1.70, respectively (100% isolates identified by biochemical methods). All test scores of 1.70-1.90 provided correct species assignment despite being identified to "genus-level". MALDI-TOF MS identified uncommon Candida spp., differentiated Candida parapsilosis from C. orthopsilosis and C. metapsilosis and distinguished between C. glabrata, C. nivariensis and C. bracarensis. Yeasts with scores of <1.70 were rare species such as C. nivariensis (3/10 strains) and C. bracarensis (n = 1) but included 4/12 Cryptococcus neoformans. There were no misidentifications. Four novel species-specific spectra were obtained. Protein extraction was essential for reliable results. CONCLUSIONS: MALDI-TOF MS enabled rapid, reliable identification of clinically-important yeasts. The addition of spectra to databases and reduction in identification scores required for species-level identification may improve its utility

Characterization of an Nmr Homolog That Modulates GATA Factor-Mediated Nitrogen Metabolite Repression in Cryptococcus neoformans

Nitrogen source utilization plays a critical role in fungal development, secondary metabolite production and pathogenesis. In both the Ascomycota and Basidiomycota, GATA transcription factors globally activate the expression of catabolic enzyme-encoding genes required to degrade complex nitrogenous compounds. However, in the presence of preferred nitrogen sources such as ammonium, GATA factor activity is inhibited in some species through interaction with co-repressor Nmr proteins. This regulatory phenomenon, nitrogen metabolite repression, enables preferential utilization of readily assimilated nitrogen sources. In the basidiomycete pathogen Cryptococcus neoformans, the GATA factor Gat1/Are1 has been co-opted into regulating multiple key virulence traits in addition to nitrogen catabolism. Here, we further characterize Gat1/Are1 function and investigate the regulatory role of the predicted Nmr homolog Tar1. While GAT1/ARE1 expression is induced during nitrogen limitation, TAR1 transcription is unaffected by nitrogen availability. Deletion of TAR1 leads to inappropriate derepression of non-preferred nitrogen catabolic pathways in the simultaneous presence of favoured sources. In addition to exhibiting its evolutionary conserved role of inhibiting GATA factor activity under repressing conditions, Tar1 also positively regulates GAT1/ARE1 transcription under non-repressing conditions. The molecular mechanism by which Tar1 modulates nitrogen metabolite repression, however, remains open to speculation. Interaction between Tar1 and Gat1/Are1 was undetectable in a yeast two-hybrid assay, consistent with Tar1 and Gat1/Are1 each lacking the conserved C-terminus regions present in ascomycete Nmr proteins and GATA factors that are known to interact with each other. Importantly, both Tar1 and Gat1/Are1 are suppressors of C. neoformans virulence, reiterating and highlighting the paradigm of nitrogen regulation of pathogenesis