Role of Cln1 during melanization of Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that has several well-described virulence determinants. A polysaccharide capsule and the ability to produce melanin are among the most important. Melanization occurs both in vitro, in the presence of catecholamine and indole compounds, and in vivo during the infection. Despite the importance of melanin production for cryptococcal virulence, the component and mechanisms involved in its synthesis have not been fully elucidated. In this work, we describe the role of a G1/S cyclin (Cln1) in the melanization process. Cln1 has evolved specifically with proteins present only in other basidiomycetes. We found that Cln1 is required for the cell wall stability and production of melanin in C. neoformans. Absence of melanization correlated with a defect in the expression of the LAC1 gene. The relation between cell cycle elements and melanization was confirmed by the effect of drugs that cause cell cycle arrest at a specific phase, such as rapamycin. The cln1 mutant was consistently more susceptible to oxidative damage in a medium that induces melanization. Our results strongly suggest a novel and hitherto unrecognized role for C. neoformans Cln1 in the expression of virulence traits.We thank Rajendra Uphadya (Washington University School of Medicine, St. Louis, MI, USA) for providing the sequence of oligonucleotides for 18s gene used in this article. RG-R was supported by a FPI fellowship (reference BES-2009-015913) from the Spanish Ministry of Economics and Competitivity. NT-C is supported by a FPI fellowship (reference BES-2012-051837). OZ is funded by grant SAF2011-25140 and SAF2014-54336 from the Spanish Ministry for Economics and CompetitivityS

Capsule growth in Cryptococcus neoformans is coordinated with cell cycle progression

UNLABELLED: The fungal pathogen Cryptococcus neoformans has several virulence factors, among which the most important is a polysaccharide capsule. The size of the capsule is variable and can increase significantly during infection. In this work, we investigated the relationship between capsular enlargement and the cell cycle. Capsule growth occurred primarily during the G1 phase. Real-time visualization of capsule growth demonstrated that this process occurred before the appearance of the bud and that capsule growth arrested during budding. Benomyl, which arrests the cells in G2/M, inhibited capsule growth, while sirolimus (rapamycin) addition, which induces G1 arrest, resulted in cells with larger capsule. Furthermore, we have characterized a mutant strain that lacks a putative G1/S cyclin. This mutant showed an increased capacity to enlarge the capsule, both in vivo (using Galleria mellonella as the host model) and in vitro. In the absence of Cln1, there was a significant increase in the production of extracellular vesicles. Proteomic assays suggest that in the cln1 mutant strain, there is an upregulation of the glyoxylate acid cycle. Besides, this cyclin mutant is avirulent at 37°C, which correlates with growth defects at this temperature in rich medium. In addition, the cln1 mutant showed lower intracellular replication rates in murine macrophages. We conclude that cell cycle regulatory elements are involved in the modulation of the expression of the main virulence factor in C. neoformans. IMPORTANCE: Cryptococcus neoformans is a pathogenic fungus that has significant incidence worldwide. Its main virulence factor is a polysaccharide capsule that can increase in size during infection. In this work, we demonstrate that this process occurs in a specific phase of the cell cycle, in particular, in G1. In agreement, mutants that have an abnormal longer G1 phase show larger capsule sizes. We believe that our findings are relevant because they provide a link between capsule growth, cell cycle progression, and virulence in C. neoformans that reveals new aspects about the pathogenicity of this fungus. Moreover, our findings indicate that cell cycle elements could be used as antifungal targets in C. neoformans by affecting both the growth of the cells and the expression of the main virulence factor of this pathogenic yeast.O.Z. is funded by grants SAF2008-03761 and SAF2011-25140 from the Spanish Ministry for Economics and Competitivity. R.G.-R. is supported by an FPI fellowship (reference BES-2009-015913) from the Spanish Ministry of Science and Innovation. N.T.-C. is supported by an FPI fellowship (reference BES-2012-051837) from the Spanish Ministry for Economics and Competitivity. A.C. is supported by NIH grants HL059842-3, A1033774, A1052733, and AI033142. R.J.B.C. is supported by T32 AI07506 (NIH/NIAID).S

Quantitative global studies reveal differential translational control by start codon context across the fungal kingdom

International audienceEukaryotic protein synthesis generally initiates at a start codon defined by an AUG and its surrounding Kozak sequence context, but the quantitative importance of this context in different species is unclear. We tested this concept in two pathogenic Crypto-coccus yeast species by genome-wide mapping of translation and of mRNA 5 and 3 ends. We observed thousands of AUG-initiated upstream open reading frames (uORFs) that are a major contributor to translation repression. uORF use depends on the Kozak sequence context of its start codon, and uORFs with strong contexts promote nonsense-mediated mRNA decay. Transcript leaders in Cryptococcus and other fungi are substantially longer and more AUG-dense than in Saccharomyces. Numerous Crypto-coccus mRNAs encode predicted dual-localized proteins , including many aminoacyl-tRNA synthetases, in which a leaky AUG start codon is followed by a strong Kozak context in-frame AUG, separated by mitochondrial-targeting sequence. Analysis of other fungal species shows that such dual-localization is also predicted to be common in the ascomycete mould, Neurospora crassa. Kozak-controlled regulation is correlated with insertions in translational initiation factors in fidelity-determining regions that contact the initiator tRNA. Thus, start codon context is a signal that quantitatively programs both the expression and the structures of proteins in diverse fungi

Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence

UGD1, Encoding the Cryptococcus neoformans UDP-Glucose Dehydrogenase, Is Essential for Growth at 37°C and for Capsule Biosynthesis

We report the identification and disruption of the Cryptococcus neoformans var. grubii UGD1 gene encoding the UDP-glucose dehydrogenase, which catalyzes the conversion of UDP-glucose into UDP-glucuronic acid. Deletion of UGD1 led to modifications in the cell wall, as revealed by changes in the sensitivity of ugd1Δ cells to sodium dodecyl sulfate, NaCl, and sorbitol. Moreover, two of the yeast's major virulence factors—capsule biosynthesis and the ability to grow at 37°C—were impaired in ugd1Δ strains. These results suggest that the UDP-dehydrogenase represents the major, and maybe only, biosynthetic pathway for UDP-glucuronic acid in C. neoformans. Consequently, deletion of UGD1 blocked not only the synthesis of UDP-glucuronic acid but also that of UDP-xylose. To differentiate the phenotype(s) associated with the UDP-glucuronic acid defect alone from those linked to the UDP-xylose defect, ugd1Δ mutants were phenotypically compared to strains from which the gene encoding UDP-xylose synthase (i.e., that required for synthesis of UDP-xylose) had been deleted. Finally, studies of strains from which one of the four CAP genes (CAP10, CAP59, CAP60, or CAP64) had been deleted revealed common cell wall phenotypes associated with the acapsular state

UGE1 and UGE2 Regulate the UDP-Glucose/UDP-Galactose Equilibrium in Cryptococcus neoformans▿ †

The genome of the basidiomycete pathogenic yeast Cryptococcus neoformans carries two UDP-glucose epimerase genes (UGE1 and UGE2). UGE2 maps within a galactose cluster composed of a galactokinase homologue gene and a galactose-1-phosphate uridylyltransferase. This clustered organization of the GAL genes is similar to that in most of the hemiascomycete yeast genomes and in Schizosaccharomyces pombe but is otherwise not generally conserved in the fungal kingdom. UGE1 has been identified as necessary for galactoxylomannan biosynthesis and virulence. Here, we show that UGE2 is necessary for C. neoformans cells to utilize galactose as a carbon source at 30°C but is not required for virulence. In contrast, deletion of UGE1 does not affect cell growth on galactose at this temperature. At 37°C, a uge2Δ mutant grows on galactose in a UGE1-dependent manner. This compensation by UGE1 of UGE2 mutation for growth on galactose at 37°C was not associated with upregulation of UGE1 transcription or with an increase of the affinity of the enzyme for UDP-galactose at this temperature. We studied the subcellular localization of the two enzymes. Whereas at 30°C, Uge1p is at least partially associated with intracellular vesicles and Uge2p is on the plasma membrane, in cells growing on galactose at 37°C, Uge1p colocalizes with Uge2p to the plasma membrane, suggesting that its activity is regulated through subcellular localization

The role of glycosylphosphatidylinositol (gpi) anchored proteins in Cryptococcusneoformans

It is becoming increasingly obvious that glycophosphatidylinositol (GPI)-anchored proteins (GAPs) play a prominent role in fungi, a full understanding of GAPs is however lacking especially for the human opportunistic fungus Cryptococcus neoformans. Using online GPI prediction tools, GAPs were identified and subsequently a mutant library for these GAP-encoding genes was developed and a publicly available knock out (KO) mutant library was used. In total, 41 overexpression and 34 KO mutants, representing 47 unique genes, were analyzed. From the analysis of the two libraries, two main gene candidates, a mannoprotein 88 (MP88) (CNAG_00776) and an uncharacterized protein (CNAG_00137) were further investigated by constructing additional independent mutant strains. The CNAG_00776 mutant showed an impaired growth upon plasma membrane stress and significant decreased phagocytosis. The CNAG_00137 mutant showed impaired growth during cell wall stress or increased temperature and significant decreased phagocytosis. By performing a large genetic screen of GAPs in the genome of the human fungal pathogen C. neoformans, we identified two candidate GAP genes involved in C. neoformans/host interaction and stress response. Further research into these two genes could potentially result in new targets for antfungals, treatment strategies or vaccines to manage C. neoformans disease

The role of glycosylphosphatidylinositol (gpi) anchored proteins in Cryptococcus neoformans

International audienceIt is becoming increasingly obvious that glycophosphatidylinositol (GPI)-anchored proteins (GAPs) play a prominent role in fungi, a full understanding of GAPs is however lacking especially for the human opportunistic fungus Cryptococcus neoformans. Using online GPI prediction tools, GAPs were identified and subsequently a mutant library for these GAP-encoding genes was developed and a publicly available knock out (KO) mutant library was used. In total, 41 overexpression and 34 KO mutants, representing 47 unique genes, were analyzed. From the analysis of the two libraries, two main gene candidates, a mannoprotein 88 (MP88) (CNAG_00776) and an uncharacterized protein (CNAG_00137) were further investigated by constructing additional independent mutant strains. The CNAG_00776 mutant showed an impaired growth upon plasma membrane stress and significant decreased phagocytosis. The CNAG_00137 mutant showed impaired growth during cell wall stress or increased temperature as well as decreased phagocytosis compared. By performing a large genetic screen of GAPs in the genome of the human fungal pathogen C. neoformans, we identified two candidate GAP genes involved in C. neoformans/host interaction and stress response. Further research into these two genes could potentially result in new targets for antifungals, treatment strategies or vaccines to manage C. neoformans disease

Cas3p Belongs to a Seven-Member Family of Capsule Structure Designer Proteins

The polysaccharide capsule is the main virulence factor of the basidiomycetous yeast Cryptococcus neoformans. Four genes (CAP10, CAP59, CAP60, and CAP64) essential for capsule formation have been previously identified, although their roles in the biosynthetic pathway remain unclear. A genetic and bioinformatics approach allowed the identification of six CAP64-homologous genes, named CAS3, CAS31, CAS32, CAS33, CAS34, and CAS35, in the C. neoformans genome. This gene family is apparently specific in a subclass of the basidiomycete fungi. Single as well as double deletions of these genes in all possible combinations demonstrated that none of the CAP64-homologous genes were essential for capsule formation, although the cas35Δ strains displayed a hypocapsular phenotype. The chemical structure of the glucuronomannan (GXM) produced by the CAS family deletants revealed that these genes determined the position and the linkage of the xylose and/or O-acetyl residues on the mannose backbone. Hence, these genes are all involved in assembly of the GXM structure in C. neoformans

Glycosylphosphatidylinositol Anchors from Galactomannan and GPI-Anchored Protein Are Synthesized by Distinct Pathways in Aspergillus fumigatus

Glycosylphosphatidylinositols (GPIs) are lipid anchors allowing the exposure of proteins at the outer layer of the plasma membrane. In fungi, a number of GPI-anchored proteins (GPI-APs) are involved in the remodeling of the cell wall polymers. GPIs follow a specific biosynthetic pathway in the endoplasmic reticulum. After the transfer of the protein onto the GPI-anchor, a lipid remodeling occurs to substitute the diacylglycerol moiety by a ceramide. In addition to GPI-APs, A. fumigatus produces a GPI-anchored polysaccharide, the galactomannan (GM), that remains unique in the fungal kingdom. To investigate the role of the GPI pathway in the biosynthesis of the GM and cell wall organization, the deletion of PER1—coding for a phospholipase required for the first step of the GPI lipid remodeling—was undertaken. Biochemical characterization of the GPI-anchor isolated from GPI-APs showed that the PER1 deficient mutant produced a lipid anchor with a diacylglycerol. The absence of a ceramide on GPI-anchors in the Δper1 mutant led to a mislocation of GPI-APs and to an alteration of the composition of the cell wall alkali-insoluble fraction. On the other hand, the GM isolated from the Δper1 mutant membranes possesses a ceramide moiety as the parental strain, showing that GPI anchor of the GM follow a distinct unknown biosynthetic pathway