SUMMARY Aspergillus species are globally ubiquitous saprophytes found in a variety of ecological niches. Almost 200 species of aspergilli have been identified, less than 20 of which are known to cause human disease. Among them, Aspergillus fumigatus is the most prevalent and is largely responsible for the increased incidence of invasive aspergillosis (IA) in the immunocompromised patient population. IA is a devastating illness, with mortality rates in some patient groups reaching as high as 90%. Studies identifying and assessing the roles of specific factors of A. fumigatus that contribute to the pathogenesis of IA have traditionally focused on single-gene deletion and mutant characterization. In combination with recent large-scale approaches analyzing global fungal responses to distinct environmental or host conditions, these studies have identified many factors that contribute to the overall pathogenic potential of A. fumigatus. Here, we provide an overview of the significant findings regarding A. fumigatus pathogenesis as it pertains to invasive disease.

Cryptococcus neoformans was first described as a human fungal pathogen more than a century ago. One aspect of the C. neoformans infectious life cycle that has been the subject of earnest debate is whether the spores are pathogenic. Despite much speculation, no direct evidence has been presented to resolve this outstanding question. We present evidence that C. neoformans spores are pathogenic in a mouse intranasal inhalation model of infection. In addition, we provide mechanistic insights into spore-host interactions. We found that C. neoformans spores were phagocytosed by alveolar macrophages via interactions between fungal ␤-(1,3)-glucan and the host receptors Dectin-1 and CD11b. Moreover, we discovered an important link between spore survival and macrophage activation state: intracellular spores were susceptible to reactive oxygen-nitrogen species. We anticipate these results will serve as the basis for a model to further investigate the pathogenic implications of infections caused by fungal spores.

Histone deacetylases (HDACs) play an important role in regulation of gene expression through histone modifications. Here we show that the Aspergillus fumigatus HDAC HdaA is involved in regulation of secondary metabolite production and is required for normal germination and vegetative growth. Deletion of the hdaA gene increased the production of several secondary metabolites but decreased production of gliotoxin whereas over-expression hdaA increased production of gliotoxin. RT-PCR analysis of 14 non-ribosomal peptide synthases indicated HdaA regulation of up to 9 of them. A mammalian cell toxicity assay indicated increased activity in the over-expression strain. Neither mutant affected virulence of the fungus as measured by macrophage engulfment of conidia or virulence in a neutropenic mouse model.

In most species, chromatin remodeling mediates critical biological processes ranging from development to disease states. In fungi within the genus Aspergillus, chromatin remodeling may regulate expression of metabolic gene clusters, but other processes regulated by chromatin structure remain to be elucidated. In many eukaryotic species, methylation of lysine 9 of histone 3 (H3K9) is a hallmark of heterochromatin formation and subsequent gene silencing. The sole H3K9 methyltransferase in Schizosaccharomyces pombe is Clr4. We report that disruption of the Clr4 homolog in the pathogenic mold Aspergillus fumigatus (ClrD), which is involved in both mono-and trimethylation of H3K9, results in several growth abnormalities. Developmental defects in ⌬AfclrD include reduction in radial growth, reduction in conidial production, and delayed conidiation after developmental competence mediated by delayed expression of brlA, the master regulator of conidiophore development. Sensitivity of ⌬AfclrD to 6-azauracil suggests that ClrD influences transcriptional processing in A. fumigatus. Despite growth abnormalities, macrophage assays suggest ClrD may be dispensable for host interactions.In eukaryotes, gene expression is greatly influenced by chromatin structure. Heterochromatin exists in either a constitutively transcriptionally silent state, as at centromeres and telomeres, or as facultative heterochromatin capable of switching between transcriptionally active and silent states. The expression of developmentally critical genetic loci present in facultative heterochromatin can be regulated by this shift. Classical examples include X-chromosome inactivation in females (9), derepression of mating-type loci in fission yeast (15), and position effect variegation in Drosophila melanogaster (13).The "histone code" influences nucleosome positioning and chromatin compaction in facultative heterochromatin (Fig. 1). This code consists of specific patterns of posttranslational modifications of key amino acids on histone tails (reviewed in references 18, 20, 22, and 44). Modifications may include acetylation, ubiquitination, phosphorylation, and/or methylation. Such modifications recruit regulatory binding proteins that direct processes such as chromatin compaction (which dictates steric accessibility of DNA to transcriptional activators and repressors) or stabilization of transcriptional machinery.There is limited information available about chromatin remodeling proteins and chromatin-level regulation of gene expression in filamentous fungi of the genus Aspergillus. Three histone arginine methyltransferases (RmtA to RmtC) have been analyzed biochemically in the model species A. nidulans; however, detailed analysis of deletion strain phenotypes was not reported (50). An A. nidulans histone deacetylase, HdaA, is required for normal growth under oxidative stress (49) and is involved in regulating telomere-proximal secondary metabolite gene clusters (42). However, there is a near-absence of studies on chromatin regulation in the opportunistic p...

Secondary metabolite (SM) production in filamentous fungi is mechanistically associated with chromatin remodeling of specific SM clusters. One locus recently shown to be involved in SM suppression in Aspergillus nidulans was CclA, a member of the histone 3 lysine 4 methylating COMPASS complex. Here we examine loss of CclA and a putative H3K4 demethylase, HdmA, in the human pathogen Aspergillus fumigatus. Although deletion of hdmA showed no phenotype under the conditions tested, the cclA deletant was deficient in tri- and di-methylation of H3K4 and yielded a slowly growing strain that was rich in the production of several SMs, including gliotoxin. Similar to deletion of other chromatin modifying enzymes, ΔcclA was sensitive to 6-azauracil indicating a defect in transcriptional elongation. Despite the poor growth, the ΔcclA mutant had wild-type pathogenicity in a murine model and the Toll-deficient Drosophila model of invasive aspergillosis. These data indicate that tri- and di-methylation of H3K4 is involved in the regulation of several secondary metabolites in A. fumigatus, however does not contribute to pathogenicity under the conditions tested.