Treatment strategies for cryptococcal infection: challenges, advances and future outlook.

Cryptococcus spp., in particular Cryptococcus neoformans and Cryptococcus gattii, have an enormous impact on human health worldwide. The global burden of cryptococcal meningitis is almost a quarter of a million cases and 181,000 deaths annually, with mortality rates of 100% if infections remain untreated. Despite these alarming statistics, treatment options for cryptococcosis remain limited, with only three major classes of drugs approved for clinical use. Exacerbating the public health burden is the fact that the only new class of antifungal drugs developed in decades, the echinocandins, displays negligible antifungal activity against Cryptococcus spp., and the efficacy of the remaining therapeutics is hampered by host toxicity and pathogen resistance. Here, we describe the current arsenal of antifungal agents and the treatment strategies employed to manage cryptococcal disease. We further elaborate on the recent advances in our understanding of the intrinsic and adaptive resistance mechanisms that are utilized by Cryptococcus spp. to evade therapeutic treatments. Finally, we review potential therapeutic strategies, including combination therapy, the targeting of virulence traits, impairing stress response pathways and modulating host immunity, to effectively treat infections caused by Cryptococcus spp. Overall, understanding of the mechanisms that regulate anti-cryptococcal drug resistance, coupled with advances in genomics technologies and high-throughput screening methodologies, will catalyse innovation and accelerate antifungal drug discovery.

Titan cell formation is unique to Cryptococcus species complex.

Members of the Cryptococcus species complex stand out by unique virulence factors that allowed evolutionary transition to pathogenesis. Among the factors contributing to cryptococcosis is a morphological transformation into giant (Titan) cells. It remains unclear whether species outside of the C. neoformans/C. gattii species complex are capable of titanization. We utilized two recently developed protocols that allow obtaining Titan cells in vitro to test if titanization occurs in non-C. neoformans/C. gattii species. We find that none of the tested strains, representing 10 species of basidiomycetous yeasts and the ascomycetous yeast Saccharomyces cerevisiae, undergo significant titanization under conditions that promote robust Titan cell formation in C. neoformans/C. gattii species complex. C. terreus formed occasional enlarged cells through a mechanism potentially similar to that of titanization. Our findings suggest that titanization is a rare phenomenon among basidiomycetous yeasts that occurs mostly in members of the C. neoformans/C. gattii species complex.

A UBI 31-38 Peptide-coumarin Conjugate: Photophysical Features, Imaging Tracking and Synergism with Amphotericin B Against Cryptococcus.

Cryptococcosis is a fungal disease of global significance for which new effective treatments are needed. The conjugation of the synthetic antimicrobial peptide fragment UBI 31-38 to a coumarin derivative showed to be an effective approach for the design of a novel anticryptococcal agent. In addition to antifungal activity, the conjugate exhibited intense fluorescence, which could be valuable for mechanistic investigations of this molecule. In this work, we studied the photophysical properties of the conjugate and confocal scanning laser microscopy was used to inspect the distribution of the peptide-coumarin conjugate in Cryptococcus cell. The synergism of this compound with amphotericin B or fluconazole against C. gattii and C. neoformans strains was also investigated. The results indicated that the fluorescent conjugate alone as well as its combination with amphotericin B are promising tools against cryptococcosis.

Evaluation of trypan blue stain in the TC20 automated cell counter as a point-of-care for the enumeration of viable cryptococcal cells in cerebrospinal fluid.

Cerebrospinal fluid (CSF) culture can determine a quantitative viability of Cryptococcus yeasts; however, culture has a long turnaround-time. The TC20 automated cell counter (Bio-Rad) is a benchtop instrument used to count cells in 30 seconds. In vitro studies suggest trypan blue staining can distinguish between viable and dead cryptococcal yeasts. We hypothesized that trypan blue staining with automated cell counting may provide rapid quantification of viable CSF Cryptococcus yeasts. In sum, 96 HIV-infected participants with cryptococcal meningitis were enrolled and provided 194 CSF specimens in Kampala, Uganda. Cryptococcosis was diagnosed by CSF cryptococcal antigen (CRAG). CSF was stained with trypan blue and quantified yeasts with the TC20 cell counter. We compared the log10 transformed cell counter readings with gating of 4-10 µm versus log10 quantitative Cryptococcus cultures/ml. TC20 showed more positive results (95.4%) overall than culture (78.4%) with reference to CSF CRAG. TC20 had higher readings compared to culture in most cases with only a 25% level of agreement between the two methods. TC20 had a poor correlation to culture throughout the 14 days of antifungal therapy. The median of log10 transformed counts were 5.22 (IQR = 4.79-5.44) for the TC20 and 3.99 (IQR = 2.59-5.14) for culture. Overall, a linear regression showed no significant relationship between the TC20 and culture (r = -0.0025; P = .92). TC20 automated cell counting with trypan blue staining was poorly predictive of the quantitative CSF culture and could not be used as a substitute for quantitative culture.

Atypical Morphology and Disparate Speciation in a Case of Feline Cryptococcosis.

A 6-year-old, spayed female cat was presented with acute respiratory signs and pleural effusion. Computed tomography scan revealed a large, lobulated mass effect in the ventral right hemithorax with concurrent sternal lymphadenopathy. A cytologic sample of the mass contained pyogranulomatous inflammation, necrotic material, and abundant yeast structures that lacked a distinct capsule and demonstrated rare pseudohyphal forms. Fungal culture and biochemical testing identified the yeast as Cryptococcus albidus, with susceptibility to all antifungal agents tested. However, subsequent 18S PCR identified 99% homology with a strain of Cryptococcus neoformans and only 92% homology with C. albidus. The patient responded well to fluconazole therapy unlike the only known previous report of C. albidus in a cat. The unusual cytologic morphology in this case underscores the need for ancillary testing apart from microscopy for fungal identification. Though C. albidus should be considered as a potential feline pathogen, confirmation with PCR is recommended when such rare non-neoformans species are encountered.

Fungal genome and mating system transitions facilitated by chromosomal translocations involving intercentromeric recombination.

Species within the human pathogenic Cryptococcus species complex are major threats to public health, causing approximately 1 million annual infections globally. Cryptococcus amylolentus is the most closely known related species of the pathogenic Cryptococcus species complex, and it is non-pathogenic. Additionally, while pathogenic Cryptococcus species have bipolar mating systems with a single large mating type (MAT) locus that represents a derived state in Basidiomycetes, C. amylolentus has a tetrapolar mating system with 2 MAT loci (P/R and HD) located on different chromosomes. Thus, studying C. amylolentus will shed light on the transition from tetrapolar to bipolar mating systems in the pathogenic Cryptococcus species, as well as its possible link with the origin and evolution of pathogenesis. In this study, we sequenced, assembled, and annotated the genomes of 2 C. amylolentus isolates, CBS6039 and CBS6273, which are sexual and interfertile. Genome comparison between the 2 C. amylolentus isolates identified the boundaries and the complete gene contents of the P/R and HD MAT loci. Bioinformatic and chromatin immunoprecipitation sequencing (ChIP-seq) analyses revealed that, similar to those of the pathogenic Cryptococcus species, C. amylolentus has regional centromeres (CENs) that are enriched with species-specific transposable and repetitive DNA elements. Additionally, we found that while neither the P/R nor the HD locus is physically closely linked to its centromere in C. amylolentus, and the regions between the MAT loci and their respective centromeres show overall synteny between the 2 genomes, both MAT loci exhibit genetic linkage to their respective centromere during meiosis, suggesting the presence of recombinational suppressors and/or epistatic gene interactions in the MAT-CEN intervening regions. Furthermore, genomic comparisons between C. amylolentus and related pathogenic Cryptococcus species provide evidence that multiple chromosomal rearrangements mediated by intercentromeric recombination have occurred during descent of the 2 lineages from their common ancestor. Taken together, our findings support a model in which the evolution of the bipolar mating system was initiated by an ectopic recombination event mediated by similar repetitive centromeric DNA elements shared between chromosomes. This translocation brought the P/R and HD loci onto the same chromosome, and further chromosomal rearrangements then resulted in the 2 MAT loci becoming physically linked and eventually fusing to form the single contiguous MAT locus that is now extant in the pathogenic Cryptococcus species.

Acridine orange fluorescent microscopy is more sensitive than India ink light microscopy in the rapid detection of cryptococcosis among CrAg positive HIV patients.

BACKGROUND: India ink microscopy on cerebrospinal fluid is still utilized in resource limited settings for the diagnosis of cryptococcal meningitis despite its poor sensitivity. We hypothesized that staining fungal nucleic acids with fluorescent dyes instead of the capsule with India ink might improve sensitivity for the diagnosis of cryptococcal meningitis. METHODS: We enrolled 96 HIV-infected participants with cryptococcal meningitis who provided 194 CSF specimens at serial time points in Kampala, Uganda. Cryptococcosis was diagnosed by cerebrospinal fluid (CSF) cryptococcal antigen (CrAg) test and only positive samples were included. We stained CSF with India ink and acridine orange. We cultured the same samples on standard fungal media. We compared acridine orange to CrAg, India ink and CSF culture. RESULTS: Acridine orange was more sensitive (96%) than India ink (79%) with reference to CSF CrAg. Acridine orange and India ink had a statistically significant difference (P<0.001) with a 25% correlation for detection of Cryptococcus yeasts. India ink had more negative results (22%) than acridine orange (4%). The sensitivity for India ink increased (86%) while that of acridine orange did not change (97%) when compared to CSF culture. However, both India ink and acridine orange had poor predictive values with reference to culture. CONCLUSION: Acridine orange is a better alternative to India ink in the rapid detection of cryptococcosis among CrAg positive HIV patients.

Characterization of calcineurin from Cryptococcus humicola and the application of calcineurin in aluminum tolerance.

BACKGROUND: Calcineurin (CaN) is a Ca2+- and calmodulin (CaM)-dependent serine/threonine phosphatase. Previous studies have found that CaN is involved in the regulation of the stress responses. RESULTS: In this study, the growth of Cryptococcus humicola was inhibited by the CaN inhibitor tacrolimus (FK506) under aluminum (Al) stress. The expression of CNA encoding a catalytic subunit A (CNA) and its interaction with CaM were upregulated when the concentration of Al was increased. A CaM-binding domain and key amino acids responsible for interaction with CaM were identified. ∆CNAb with a deletion from S454 to A639 was detected to bind to CaM, while ∆CNAa with a deletion from R436 to A639 showed no binding to CaM. The binding affinities of CNA1 and CNA2, in which I439 or I443 were replaced by Ala, were decreased relative to wild-type CNA. The phosphatase activities of ∆CNAa, CNA1 and CNA2 were lower than the wild-type protein. These results suggest that the region between R436 and S454 is essential for the interaction with CaM and I439, I443 are key amino acids in this region. The ability of the CNA transgenic yeast to develop resistance to Al was significantly higher than that of control yeast. Residual Al in the CNA transgenic yeast culture media was significantly lower than the amount of Al originally added to the media or the residual Al remaining in the control yeast culture media. These findings suggest that CNA confers Al tolerance, and the mechanism of Al tolerance may involve absorption of active Al. CONCLUSIONS: Al stress up-regulated the expression of CNA. CaM-binding domain and key amino acids responsible for interaction with CaM were identified and both are required for phosphatase activities. CNA conferred yeast Al resistance indicating that the gene has a potential to improve Al-tolerance through gene engineering.

Ebselen exerts antifungal activity by regulating glutathione (GSH) and reactive oxygen species (ROS) production in fungal cells.

BACKGROUND: Ebselen, an organoselenium compound and a clinically safe molecule has been reported to possess potent antifungal activity, but its antifungal mechanism of action and in vivo antifungal activity remain unclear. METHODS: The antifungal effect of ebselen was tested against Candida albicans, C. glabrata, C. tropicalis, C. parapsilosis, Cryptococcus neoformans, and C. gattii clinical isolates. Chemogenomic profiling and biochemical assays were employed to identify the antifungal target of ebselen. Ebselen's antifungal activity in vivo was investigated in a Caenorhabditis elegans animal model. RESULTS: Ebselen exhibits potent antifungal activity against both Candida spp. and Cryptococcus spp., at concentrations ranging from 0.5 to 2µg/ml. Ebselen rapidly eradicates a high fungal inoculum within 2h of treatment. Investigation of the drug's antifungal mechanism of action indicates that ebselen depletes intracellular glutathione (GSH) levels, leading to increased production of reactive oxygen species (ROS), and thereby disturbs the redox homeostasis in fungal cells. Examination of ebselen's in vivo antifungal activity in two Caenorhabditis elegans models of infection demonstrate that ebselen is superior to conventional antifungal drugs (fluconazole, flucytosine and amphotericin) in reducing Candida and Cryptococcus fungal load. CONCLUSION: Ebselen possesses potent antifungal activity against clinically relevant isolates of both Candida and Cryptococcus by regulating GSH and ROS production. The potent in vivo antifungal activity of ebselen supports further investigation for repurposing it for use as an antifungal agent. GENERAL SIGNIFICANCE: The present study shows that ebselen targets glutathione and also support that glutathione as a potential target for antifungal drug development.

Converting paper mill sludge into neutral lipids by oleaginous yeast Cryptococcus vishniaccii for biodiesel production.

Paper mill sludge (PMS) was assessed as cheap renewable lignocellulosic biomass for lipid production by the oleaginous yeast Cryptococcus vishniaccii (MTCC 232). The sonicated paper mill sludge extract (PMSE) exhibited enhanced lipid yield and lipid content 7.8±0.57g/l, 53.40% in comparison to 5.5±0.8g/l, 40.44% glucose synthetic medium, respectively. The accumulated triglycerides (TAG) inside the lipid droplets (LDs) were converted to biodiesel by transesterification and thoroughly characterized using GC-MS technique. The fatty acid methyl ester (FAME) profile obtained reveals elevated content of oleic acid followed by palmitic acid, linoleic acid and stearic acid with improved oxidative stability related to biodiesel quality.

Global transcriptome analysis of eukaryotic genes affected by gromwell extract.

BACKGROUND: Gromwell is known to have diverse pharmacological, cosmetic and nutritional benefits for humans. Nevertheless, the biological influence of gromwell extract (GE) on the general physiology of eukaryotic cells remains unknown. In this study a global transcriptome analysis was performed to identify genes affected by the addition of GE with Cryptococcus neoformans as the model system. RESULTS: In response to GE treatment, genes involved in signal transduction were immediately regulated, and the evolutionarily conserved sets of genes involved in the core cellular functions, including DNA replication, RNA transcription/processing and protein translation/processing, were generally up-regulated. In contrast, a number of genes involved in carbohydrate metabolism and transport, inorganic ion transport and metabolism, post-translational modification/protein turnover/chaperone functions and signal transduction were down-regulated. Among the GE-responsive genes that are also evolutionarily conserved in the human genome, the expression patterns of YSA1, TPO2, CFO1 and PZF1 were confirmed by northern blot analysis. Based on the functional characterization of some GE-responsive genes, it was found that GE treatment may promote cellular tolerance against a variety of environmental stresses in eukaryotes. CONCLUSIONS: GE treatment affects the expression levels of a significant portion of the Cryptococcus genome, implying that GE significantly affects the general physiology of eukaryotic cells.

Nitrogen source-dependent capsule induction in human-pathogenic cryptococcus species.

Cryptococcus neoformans and C. gattii cause meningoencephalitis and are an increasing human health threat. These pathogenic Cryptococcus species are neurotropic and persist in the cerebrospinal fluid (CSF) of the mammalian host during infection. In order to survive in the host, pathogenic fungi must procure nutrients, such as carbon and nitrogen, from the CSF. To enhance our understanding of nutrient acquisition during central nervous system infection by Cryptococcus species, we examined the utilization of nitrogen sources available in CSF. We screened for the growth and capsule production of 817 global environmental and clinical isolates on various sources of nitrogen. Both environmental and clinical strains grew robustly on uric acid, Casamino Acids, creatinine, and asparagine as sole nitrogen sources. Urea induced the greatest magnitude of capsule induction. This induction was greater in Cryptococcus gattii than in C. neoformans. We confirmed the ability of nonpreferred nitrogen sources to increase capsule production in pathogenic species of Cryptococcus. Since urea is metabolized to ammonia and CO(2) (a known signal for capsule induction), we examined urea metabolism mutants for their transcriptional response to urea regarding capsule production. The transcriptional profile of C. neoformans under urea-supplemented conditions revealed both similar and unique responses to other capsule-inducing conditions, including both intra- and extracellular urea utilization. As one of the most abundant nitrogen sources in the CSF, the ability of Cryptococcus to import urea and induce capsule production may substantially aid this yeast's survival and propagation in the host.

Capsules from pathogenic and non-pathogenic Cryptococcus spp. manifest significant differences in structure and ability to protect against phagocytic cells.

Capsule production is common among bacterial species, but relatively rare in eukaryotic microorganisms. Members of the fungal Cryptococcus genus are known to produce capsules, which are major determinants of virulence in the highly pathogenic species Cryptococcus neoformans and Cryptococcus gattii. Although the lack of virulence of many species of the Cryptococcus genus can be explained solely by the lack of mammalian thermotolerance, it is uncertain whether the capsules from these organisms are comparable to those of the pathogenic cryptococci. In this study, we compared the characteristic of the capsule from the non-pathogenic environmental yeast Cryptococcus liquefaciens with that of C. neoformans. Microscopic observations revealed that C. liquefaciens has a capsule visible in India ink preparations that was also efficiently labeled by three antibodies generated to specific C. neoformans capsular antigens. Capsular polysaccharides of C. liquefaciens were incorporated onto the cell surface of acapsular C. neoformans mutant cells. Polysaccharide composition determinations in combination with confocal microscopy revealed that C. liquefaciens capsule consisted of mannose, xylose, glucose, glucuronic acid, galactose and N-acetylglucosamine. Physical chemical analysis of the C. liquefaciens polysaccharides in comparison with C. neoformans samples revealed significant differences in viscosity, elastic properties and macromolecular structure parameters of polysaccharide solutions such as rigidity, effective diameter, zeta potential and molecular mass, which nevertheless appeared to be characteristics of linear polysaccharides that also comprise capsular polysaccharide of C. neoformans. The environmental yeast, however, showed enhanced susceptibility to the antimicrobial activity of the environmental phagocytes, suggesting that the C. liquefaciens capsular components are insufficient in protecting yeast cells against killing by amoeba. These results suggest that capsular structures in pathogenic Cryptococcus species and environmental species share similar features, but also manifest significant difference that could influence their potential to virulence.

Cryptococcuria as manifestation of disseminated cryptococcosis: Staib agar as a selective identification medium.

We conducted a retrospective study of 58 cases of cryptococcosis (1986-2008) with urine test positive for Cryptococcus sp, in Mycology Laboratory, Santa Casa-Hospital Complex, Porto Alegre, RS, Brazil. The diagnosis of cryptococcuria was based on microscopic examination and culture of urinary sediment. Cryptococcus was isolated from other clinical specimens such as blood, cerebrospinal fluid, ascitic and pleural fluids, respiratory secretions, biopsies of skin, nasal and bone marrow. Cryptocccus neoformans was present in 55 cases and Cryptocccus gattii in three cases. Males predominated (79.3%); age ranged from 12 to 86 years. Acquired Immune Deficiency Syndrome (AIDS) were present in 60.3%, 31.1% did not have AIDS and 5.2% were apparently immunocompetent patients. The most frequent signs and symptoms were headache (53.4%) and fever (51.7%). The most widely used medication was the amphotericin B (43 patients). The mortality rate was 45%. We conclude that the mycological examination of the urine can be an alternative simple, non-invasive and useful in diagnosis of disseminated cryptococcosis, especially when used in conjunction with techniques for demonstration of the capsule (nigrosine) and/or production of melanin in special culture media (Staib agar).

Expanding fungal pathogenesis: Cryptococcus breaks out of the opportunistic box.

Cryptococcus neoformans is generally considered to be an opportunistic fungal pathogen because of its tendency to infect immunocompromised individuals, particularly those infected with HIV. However, this view has been challenged by the recent discovery of specialized interactions between the fungus and its mammalian hosts, and by the emergence of the related species Cryptococcus gattii as a primary pathogen of immunocompetent populations. In this Review, we highlight features of cryptococcal pathogens that reveal their adaptation to the mammalian environment. These features include not only remarkably sophisticated interactions with phagocytic cells to promote intracellular survival, dissemination to the central nervous system and escape, but also surprising morphological and genomic adaptations such as the formation of polyploid giant cells in the lung.

Polymicrobial lung infection in postrenal transplant recipient diagnosed by fine-needle aspiration cytology.

Tuberculous and fungal infections are among the non-neoplastic lesions of the lung, in which fine-needle aspiration cytology (FNAC) has proven to be a useful technique in both immunocompromised and immunocompetent patients. The presence of polymicrobial infection in a renal transplant recipient is documented in the literature, but has rarely been diagnosed on cytology. We report a case of concomitant pulmonary cryptococcosis, aspergillosis, and tuberculosis in a renal transplant recipient diagnosed on FNAC.A 50-year-old renal transplant recipient, asymptomatic for 3 year, presented with intermittent low-grade fever associated with cough, expectoration, and a newly developed cavitatory lesion in the left lung on chest X-ray. Computed tomography-guided FNAC performed on the lung lesion showed fungal profiles with septate hyphae and acute-angled branching consistent with morphology of Aspergillus. In addition, numerous yeast forms of cryptococcus and a few acid-fast mycobacterial tubercle bacilli were seen.Guided FNAC is a useful and reliable technique for the diagnosis of pulmonary infection. One should always keep in mind the possibility of polymicrobial infections especially inimmunocompromised patients.

The influence of N-glycosylation on biochemical properties of Amy1, an alpha-amylase from the yeast Cryptococcus flavus.

The yeast Cryptococcus flavus secretes a glycosylated alpha-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 microg mL(-1) affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 microg mL(-1) TM. SDS-PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The K(M) values of native and non-glycosylated alpha-amylases were 0.052 and 0.098 mg mL(-1), and V(max) values were 0.038 and 0.047 mg min(-1), respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg(2+) and Cu(2+) suggesting that N-glycosylation is involved in the folding of Amy1.