Dsc orthologs are required for hypoxia adaptation, triazole drug responses, and fungal virulence in Aspergillus fumigatus.

Hypoxia is an environmental stress encountered by Aspergillus fumigatus during invasive pulmonary aspergillosis (IPA). The ability of this mold to adapt to hypoxia is important for fungal virulence and genetically regulated in part by the sterol regulatory element binding protein (SREBP) SrbA. SrbA is required for fungal growth in the murine lung and to ultimately cause lethal disease in murine models of IPA. Here we identified and partially characterized four genes (dscA, dscB, dscC, and dscD, here referred to as dscA-D) with previously unknown functions in A. fumigatus that are orthologs of the Schizosaccharomyces pombe genes dsc1, dsc2, dsc3, and dsc4 (dsc1-4), which encode a Golgi E3 ligase complex critical for SREBP activation by proteolytic cleavage. A. fumigatus null dscA-D mutants displayed remarkable defects in hypoxic growth and increased susceptibility to triazole antifungal drugs. Consistent with the confirmed role of these genes in S. pombe, both ΔdscA and ΔdscC resulted in reduced cleavage of the SrbA precursor protein in A. fumigatus. Inoculation of corticosteroid immunosuppressed mice with ΔdscA and ΔdscC strains revealed that these genes are critical for A. fumigatus virulence. Reintroduction of SrbA amino acids 1 to 425, encompassing the N terminus DNA binding domain, into the ΔdscA strain was able to partially restore virulence, further supporting a mechanistic link between DscA and SrbA function. Thus, we have shown for the first time the importance of a previously uncharacterized group of genes in A. fumigatus that mediate hypoxia adaptation, fungal virulence, and triazole drug susceptibility and that are likely linked to regulation of SrbA function.

A microarray screen for direct targets of Zic1 identifies an aquaporin gene, aqp-3b, expressed in the neural folds.

The Zic1 transcription factor plays multiple roles during early development, for example, in patterning the early neural plate and formation of the neural crest, somites, and cerebellum. To identify direct downstream target genes of Zic1, a microarray screen was conducted in Xenopus laevis that identified 85 genes upregulated twofold or more. These include transcription factors, receptors, enzymes, proteins involved in retinoic acid signaling, and an aquaglyceroporin (aqp-3b), but surprisingly no genes known to be involved in cell proliferation. We show that both aqp-3 and aqp-3b were expressed in adult tissues, while during early embryonic development, only aqp-3b was transcribed. During neurula stages, aqp-3b was expressed specifically in the neural folds. This pattern of aqp-3b expression closely resembled that of NF-protocadherin (NFPC), which is involved in cell adhesion and neural tube closure. Aqp-3b may also be involved in neural tube closure, since mammalian Aqp-3 promotes cell migration and proliferation.

Reduced nicotinamide adenine dinucleotide phosphate oxidase-independent resistance to Aspergillus fumigatus in alveolar macrophages.

The fungal pathogen Aspergillus fumigatus is responsible for increasing numbers of fatal infections in immune-compromised humans. Alveolar macrophages (AM) are important in the innate defense against aspergillosis, but little is known about their molecular responses to fungal conidia in vivo. We examined transcriptional changes and superoxide release by AM from C57BL/6 and gp91(phox)(-/-) mice in response to conidia. Following introduction of conidia into the lung, microarray analysis of AM showed the transcripts most strongly up-regulated in vivo to encode chemokines and additional genes that play a critical role in neutrophil and monocyte recruitment, indicating that activation of phagocytes represents a critical early response of AM to fungal conidia. Of the 73 AM genes showing > or = 2-fold changes, 8 were also increased in gp91(phox)(-/-) mice by conidia and in C57BL/6 mice by polystyrene beads, suggesting a common innate response to particulate matter. Ingenuity analysis of the microarray data from C57BL/6 mice revealed immune cell signaling and gene expression as primary mechanisms of this response. Despite the well-established importance of phagocyte NADPH oxidase in resisting aspergillosis, we found no evidence of this mechanism in AM following introduction of conidia into the mouse lung using transcriptional, luminometry, or NBT staining analysis. In support of these findings, we observed that AM from C57BL/6 and gp91(phox)(-/-) mice inhibit conidial germination equally in vitro. Our results indicate that early transcription in mouse AM exposed to conidia in vivo targets neutrophil recruitment, and that NADPH oxidase-independent mechanisms in AM contribute to inhibition of conidial germination.

Early neutrophil recruitment and aggregation in the murine lung inhibit germination of Aspergillus fumigatus Conidia.

Several types of polymorphonuclear neutrophil (PMN) deficiency are a predisposing condition for fatal Aspergillus fumigatus infection. In order to study the defensive role of PMNs in the lungs, with particular reference to PMN recruitment and antimicrobial oxidant activity, responses to pulmonary instillation of A. fumigatus conidia were examined. Responses in BALB/c and C57BL/6 mice were compared with those in CXCR2(-/-) and gp91(phox-/-) mice, which are known to have delayed recruitment of PMN to the lungs in response to inflammatory stimuli and inactive NADPH oxidase, respectively. In BALB/c mice, PMNs were recruited to the lungs and formed oxidase-active aggregates with conidia, which inhibited germination. In C57BL/6, gp91(phox-/-), and CXCR2(-/-) mice, PMN recruitment was slower and there was increased germination compared to that in BALB/c mice at 6 and 12 h. In gp91(phox-/-) mice, germination was extensive in PMN aggregates but negligible in alveolar macrophages (AM). Lung sections taken at 6 and 48 h from BALB/c mice showed PMN accumulation at peribronchiolar sites but no germinating conidia. Those from C57BL/6 and CXCR2(-/-) mice showed germinating conidia at 6 h but not at 48 h and few inflammatory cells. In contrast, those from gp91(phox-/-) mice showed germination at 6 h with more-extensive hyphal proliferation and tissue invasion at 48 h. These results indicate that when the lungs are exposed to large numbers of conidia, in addition to the phagocytic activity of AM, early PMN recruitment and formation of oxidative-active aggregates are essential in preventing germination of A. fumigatus conidia.