Iron-regulated transcription and capsule formation in the fungal pathogen Cryptococcus neoformans.

Cryptococcus neoformans is the leading cause of fungal meningitis in humans. Production of a polysaccharide capsule is a key virulence property for the fungus and capsule synthesis is regulated by iron levels. Given that iron acquisition is an important aspect of virulence for many pathogens, we employed serial analysis of gene expression (SAGE) to examine the transcriptome under iron-limiting and iron-replete conditions. Initially, we demonstrated by SAGE and Northern analysis that iron limitation results in an elevated transcript level for the CAP60 gene that is required for capsule production. We also identified genes encoding putative components for iron transport and homeostasis, including the FTR1 (iron permease) gene, with higher transcript levels in the low-iron condition. An FTR1 disruption mutant grows more slowly than wild-type cells in low-iron medium, and shows delayed growth and altered capsule regulation in iron-replete medium. Iron deprivation also resulted in elevated SAGE tags for putative extracellular mannoproteins and the GPI8 gene encoding a glycosylphosphatidylinositol (GPI) transamidase. The GPI8 gene appears to be essential while disruption of the CIG1 gene encoding a mannoprotein resulted in impaired growth in low-iron medium and altered capsule response to the iron-replete condition. Additionally, we found that iron-replete conditions led to elevated transcripts for genes for iron storage, nitrogen metabolism, glycolysis, mitochondrial function, lipid metabolism and calmodulin-calcineurin signalling. Overall, these studies provide the first view of the C. neoformans transcriptional response to different iron levels.

Differences in sialic acid density in pathogenic and non-pathogenic Aspergillus species.

ASPERGILLUS: fumigatus is a ubiquitous soil fungus that causes invasive lung disease in the immunocompromised host. The structure of the conidial wall has not been well characterized although it is thought that adhesins present on the surface are involved in attachment of the conidia to host lung cells and proteins, which is a prerequisite for the establishment of infection. Negatively charged carbohydrates on the conidial surface have been previously identified as the molecules responsible for attachment of conidia to extracellular matrix proteins. The aim of this research was to identify carbohydrates on the conidial surface that contribute to its negative charge. Direct chemical analysis and indirect binding assays have demonstrated that A. fumigatus possesses sialic acids on the conidial surface. Pre-treatment of A. fumigatus conidia with sialidase decreased binding of a sialic acid-specific lectin, Limax flavus agglutinin (LFA), to the conidial surface and decreased adhesion of conidia to the positively charged polymer poly L-lysine. Two other sialic acid-specific lectins, Maackia amurensis agglutinin and Sambucus nigra agglutinin, exhibited negligible binding to A. fumigatus conidia indicating that 2,3-alpha- and 2,6-alpha-linked sialic acids are not the major structures found on the conidial surface. Mild acid hydrolysis and purification of conidial wall carbohydrates yielded a product that had the same R(F) as the Neu5Ac standard when analysed by high-performance thin-layer chromatography. A density of 6.7 x 10(5) sialic acid residues per conidium was estimated using a colorimetric assay. Conidia grown on a minimal medium lacking sialic acid also reacted with LFA, indicating that sialic acid biosynthesis occurs de novo. Sialic acid biosynthesis was shown to be regulated by nutrient composition: the density of sialic acids on the surface of conidia grown in minimal media was lower than that observed when conidia were grown on rich, complex media. It has previously been shown that pathogenic Aspergillus species adhere to basal lamina proteins to a greater extent than non-pathogenic Aspergillus species. To determine whether the expression of sialic acid on the conidial surface was correlated with adhesion to basal lamina, conidia from other non-pathogenic Aspergillus species were tested for their reactivity towards LFA. Flow cytometric analysis demonstrated that A. fumigatus had a significantly greater sialic acid density than three non-pathogenic Aspergillus species. Sialic acids on the conidial wall may be involved in adhesion to fibronectin, a component of the basal lamina, as binding of A. fumigatus conidia to fibronectin was strongly inhibited in the presence of a sialylated glycoprotein.