Identification of Aph1, a phosphate-regulated, secreted, and vacuolar acid phosphatase in Cryptococcus neoformans.

UNLABELLED: Cryptococcus neoformans strains isolated from patients with AIDS secrete acid phosphatase, but the identity and role of the enzyme(s) responsible have not been elucidated. By combining a one-dimensional electrophoresis step with mass spectrometry, a canonically secreted acid phosphatase, CNAG_02944 (Aph1), was identified in the secretome of the highly virulent serotype A strain H99. We created an APH1 deletion mutant (Δaph1) and showed that Δaph1-infected Galleria mellonella and mice survived longer than those infected with the wild type (WT), demonstrating that Aph1 contributes to cryptococcal virulence. Phosphate starvation induced APH1 expression and secretion of catalytically active acid phosphatase in the WT, but not in the Δaph1 mutant, indicating that Aph1 is the major extracellular acid phosphatase in C. neoformans and that it is phosphate repressible. DsRed-tagged Aph1 was transported to the fungal cell periphery and vacuoles via endosome-like structures and was enriched in bud necks. A similar pattern of Aph1 localization was observed in cryptococci cocultured with THP-1 monocytes, suggesting that Aph1 is produced during host infection. In contrast to Aph1, but consistent with our previous biochemical data, green fluorescent protein (GFP)-tagged phospholipase B1 (Plb1) was predominantly localized at the cell periphery, with no evidence of endosome-mediated export. Despite use of different intracellular transport routes by Plb1 and Aph1, secretion of both proteins was compromised in a Δsec14-1 mutant. Secretions from the WT, but not from Δaph1, hydrolyzed a range of physiological substrates, including phosphotyrosine, glucose-1-phosphate, ß-glycerol phosphate, AMP, and mannose-6-phosphate, suggesting that the role of Aph1 is to recycle phosphate from macromolecules in cryptococcal vacuoles and to scavenge phosphate from the extracellular environment. IMPORTANCE: Infections with the AIDS-related fungal pathogen Cryptococcus neoformans cause more than 600,000 deaths per year worldwide. Strains of Cryptococcus neoformans isolated from patients with AIDS secrete acid phosphatase; however, the identity and role of the enzyme(s) are unknown. We have analyzed the secretome of the highly virulent serotype A strain H99 and identified Aph1, a canonically secreted acid phosphatase. By creating an APH1 deletion mutant and an Aph1-DsRed-expressing strain, we demonstrate that Aph1 is the major extracellular and vacuolar acid phosphatase in C. neoformans and that it is phosphate repressible. Furthermore, we show that Aph1 is produced in cryptococci during coculture with THP-1 monocytes and contributes to fungal virulence in Galleria mellonella and mouse models of cryptococcosis. Our findings suggest that Aph1 is secreted to the environment to scavenge phosphate from a wide range of physiological substrates and is targeted to vacuoles to recycle phosphate from the expendable macromolecules.

Ccr4 promotes resolution of the endoplasmic reticulum stress response during host temperature adaptation in Cryptococcus neoformans.

Adaptation to host temperature is a prerequisite for any pathogen capable of causing deep infection in humans. Our previous studies demonstrated that a Cryptococcus neoformans ccr4Δ mutant lacking the major deadenylase involved in regulated mRNA decay was defective in host temperature adaptation and therefore virulence. In this study, the ccr4Δ mutant was found to exhibit characteristics of chronic unfolded-protein response (UPR) engagement in both the gene expression profile and phenotype. We demonstrate that host temperature adaptation in C. neoformans is accompanied by transient induction of the endoplasmic reticulum (ER) stress response and that Ccr4-dependent posttranscriptional gene regulation contributes to resolution of ER stress during host temperature adaptation.

KRE genes are required for beta-1,6-glucan synthesis, maintenance of capsule architecture and cell wall protein anchoring in Cryptococcus neoformans.

The polysaccharide beta-1,6-glucan is a major component of the cell wall of Cryptococcus neoformans, but its function has not been investigated in this fungal pathogen. We have identified and characterized seven genes, belonging to the KRE family, which are putatively involved in beta-1,6-glucan synthesis. The H99 deletion mutants kre5Delta and kre6Deltaskn1Delta contained less cell wall beta-1,6-glucan, grew slowly with an aberrant morphology, were highly sensitive to environmental and chemical stress and were avirulent in a mouse inhalation model of infection. These two mutants displayed alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant. The cell wall content of the GPI-anchored phospholipase B1 (Plb1) enzyme, which is required for cryptococcal cell wall integrity and virulence, was reduced in kre5Delta and kre6Deltaskn1Delta. Our results indicate that KRE5, KRE6 and SKN1 are involved in beta-1,6-glucan synthesis, maintenance of cell wall integrity and retention of mannoproteins and known cryptococcal virulence factors in the cell wall of C. neoformans. This study sets the stage for future investigations into the function of this abundant cell wall polymer.

Role and mechanism of phosphatidylinositol-specific phospholipase C in survival and virulence of Cryptococcus neoformans.

Phospholipase B1 (Plb1) is secreted after release from its glycosylphosphatidylinositol anchor and is implicated in initiation and dissemination of infection of the pathogenic fungus, Cryptococcus neoformans. To investigate the role of phosphatidylinositol-specific phospholipase C (PI-PLC) in Plb1 secretion, we identified two putative PI-PLC-encoding genes in C. neoformans var. grubii (PLC1 and PLC2), and created Deltaplc1 and Deltaplc2 deletion mutants. In Deltaplc1, which expressed less PI-PLC activity than wild type (WT), three major cryptococcal virulence traits, Plb1 secretion, melanin production and growth at host temperature (37 degrees C) were abolished and absence of Plb1 secretion coincided with Plb1 accumulation in plasma membranes. In addition, Deltaplc1 cell walls were defective, as indicated by cell clumping and irregular morphology, slower growth and an inability to activate mitogen-activated protein kinase (MAPK) in the presence of cell wall-perturbing agents. In contrast to Deltaplc2, which was as virulent as WT, Deltaplc1 was avirulent in mice and exhibited attenuated killing of Caenorhabditis elegans at 25 degrees C, demonstrating that mechanism(s) independent of the 37 degrees C growth defect contribute to the virulence composite. We conclude that Plc1 is a central regulator of cryptococcal virulence, acting through the protein kinase C/MAPK pathway, that it regulates release of Plb1 from the plasma membrane and is a candidate antifungal drug target.

Cloning of CnLYSO1, a novel extracellular lysophospholipase of the pathogenic fungus Cryptococcus neoformans.

We cloned a novel lysophospholipase (CnLYSO1) from Cryptococcus neoformans var. grubii by PCR amplification and a cDNA library screen. The open reading frame (ORF) of 1278 nucleotides coded for a predicted 426-amino-acid protein (CnLyso1p) with two highly conserved GXSXG lipase-specific catalytic motifs and a molecular weight of 48.3 kDa. CnLyso1p exhibited 14% and 21% identity to Arabidopsis thaliana and human lysophospholipases, respectively. Immunoprecipitation and Western blot analyses indicated that CnLyso1p was secreted as a high molecular weight protein of 97-140 kDa. CnLYSO1 expressed in a phospholipase B-null mutant of Saccharomyces cerevisiae demonstrated lysophospholipase and lysophospholipase transacylase activities at pH 4.0. Targeted disruption of CnLYSO1 did not affect growth, melanin or capsule production by C. neoformans. Secreted lysophospholipase and transacylase activities (pH 4.0) were 50% of wild type and CnLyso1p was undetectable on Western blots. Phospholipase B activity was reduced at pH 7.0 (P<0.006) and at pH 4.0 (P=NS). The amount of secreted Plb1p (the gene product of PLB1) was also reduced. Deletion of PLB1 abolished all three secreted activities at pH 4.0 and 7.0. These results are best explained by post-translational interaction, most likely the formation of a functional complex between the independently regulated gene products, CnLyso1p and CnPlb1p.

Strain-dependent effects of environmental signals on the production of extracellular phospholipase by Cryptococcus neoformans.

Extracellular phospholipase (PL) activities comprising phospholipase B, lysophospholipase and lysophospholipase transacylase have been identified in culture supernatants of Cryptococcus neoformans and contribute to virulence. We found that PL production was optimal after fungal growth at 30 degrees C and secretion at 37 degrees C for all six C. neoformans isolates studied (four C. neoformans var. neoformans and two C. neoformans var. gattii). No increase in PL activity was found in one strain, NU-2, in low iron or tissue culture media, conditions where upregulation of other virulence factors has been reported. The most virulent strains in an intravenous mouse model of infection were best able to produce PL at growth and secretion temperatures of 37 degrees C, in tissue culture media and under assay conditions of pH 7.0.