Interpretation of data comparisons, and subsequent predictions of virulence genes, are heavily dependent on the experimental design, and relate directly to the choice of the time point(s), choice of the reference sample(s) and reliance on data drawn from populations of cells. Single time-point analyses evidently do not provide the resolving power necessary to predict virulence determinants relevant to multistage pathogenetic processes, as evidenced by the requirement for glyoxylate cycle-encoding gene products,

acting at prepenetrative stages of infection, for virulence in M. grisea (Wang et al., 2003) selleck compound and their apparently static levels of transcription (Table 2) in invasive hyphae. For comparative microarray analyses (including the AZD4547 cell line choice of the comparator SAGE tag library in SAGE analytical approaches), the origin of the reference sample profoundly impacts on up- and downregulated genesets. It may, therefore, be naive to expect experiments using reference samples of diverse nutrient compositions (e.g. YPD, RPMI1640 and LIM) to result in similar gene expression profiles. A case in point is provided by a collective

impediment to fungal propagation in plant and animals: the lack of available iron, which is an essential cofactor for many cellular processes. Ustilago maydis, M. grisea and A. fumigatus use siderophores, a class of nonribosomal peptide synthase (NRPS)-dependent secondary metabolites, to scavenge ferric ion selectively through the formation of soluble chelation complexes (Schrettl et al., 2007; Bolker et al., 2008; Hof et al., 2009). Intra- and extracellular siderophores are required for full virulence in a pulmonary murine model of invasive aspergillosis (Schrettl et al., 2007), and accordingly, gene expression at siderophore biosynthetic gene clusters was induced in a similar murine model at 14-h postinfection, indicating that the response to iron limitation in the mammalian host is addressed at a very early stage of infection (McDonagh et al., 2008). Therefore,

concordance between transcriptional data and important Fluorometholone Acetate virulence determinants can be expected from this type of analysis. However, despite the observed similarity of gene expression profiles between A. fumigatus and C. neoformans, iron acquisition was not identified as an important component of the infecting C. neoformans transcriptome. This may, in part, be due to the use of an LIM comparator in the C. neoformans experimentation, which would undoubtedly occlude, at the transcriptional level, this aspect of pathogenic growth. While C. neoformans does not synthesize siderophores, iron acquisition is crucial for C. neoformans virulence (Jung et al., 2009). Thewes and colleagues also found gene expression that reflected iron limitation.