Sterol regulatory element binding proteins (SREBPs) are a class of basic helix-loop-helix transcription factors that regulate diverse cellular responses in eukaryotes. Adding to the recognized importance of SREBPs in human health, SREBPs in the human fungal pathogens Cryptococcus neoformans and Aspergillus fumigatus are required for fungal virulence and susceptibility to triazole antifungal drugs. To date, the exact mechanism(s) behind the role of SREBP in these observed phenotypes is not clear. Here, we report that A. fumigatus SREBP, SrbA, mediates regulation of iron acquisition in response to hypoxia and low iron conditions. To further define SrbA's role in iron acquisition in relation to previously studied fungal regulators of iron metabolism, SreA and HapX, a series of mutants were generated in the ΔsrbA background. These data suggest that SrbA is activated independently of SreA and HapX in response to iron limitation, but that HapX mRNA induction is partially dependent on SrbA. Intriguingly, exogenous addition of high iron or genetic deletion of sreA in the ΔsrbA background was able to partially rescue the hypoxia growth, triazole drug susceptibility, and decrease in ergosterol content phenotypes of ΔsrbA. Thus, we conclude that the fungal SREBP, SrbA, is critical for coordinating genes involved in iron acquisition and ergosterol biosynthesis under hypoxia and low iron conditions found at sites of human fungal infections. These results support a role for SREBP–mediated iron regulation in fungal virulence, and they lay a foundation for further exploration of SREBP's role in iron homeostasis in other eukaryotes.

Several types of polymorphonuclear neutrophil (PMN) deficiency are a predisposing condition for fatalAspergillus 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.Despite life-long exposure to Aspergillus fumigatus, very low morbidity is seen in immunocompetent individuals, indicating that a rapid and effective resistance to this organism has evolved. Individuals with normal immune systems rarely develop invasive pulmonary aspergillosis (IPA) even when exposed to high environmental concentrations of A. fumigatus conidia arising from, for example, disturbance of moldy wood chip piles, sludge, or compost (20) but may develop allergic bronchopulmonary aspergillosis or extrinsic allergic alveolitis (13). However, in recent decades there has been an increasing number of invasive and often fatal A. fumigatus infections in patients with immunosuppressive disorders (5, 16, 39), and A. fumigatus is now recognized as the leading airborne fungal pathogen in immunocompromised individuals (12,35).Resident alveolar macrophages (AM) phagocytose and subsequently destroy inhaled conidia (8, 38). However, the importance of polymorphonuclear neutrophils (PMN) in innate immunity to aspergillosis is indicated by the observation that patients and mice with PMN deficiency due to a variety of causes are susceptible to IPA (9, 30, 34, 37). The current study with mice was undertaken since there is limited information on PMN recruitment in response to inhalation of conidia and the mechanism by which PMN protect against aspergillosis.Our hypothesis was that following exposure of the lung to conidia, ea...

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 ⌬dscA and ⌬dscC resulted in reduced cleavage of the SrbA precursor protein in A. fumigatus. Inoculation of corticosteroid immunosuppressed mice with ⌬dscA and ⌬dscC 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 ⌬dscA 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 s mortality due to invasive fungal infections (IFIs) continues to remain high despite improved diagnostics and prophylactic use of antifungal drugs, research to better understand fungal pathogenesis mechanisms is important. The aim of this research is to uncover new aspects of fungal physiology or the fungus-host interaction that can be manipulated to improve treatment outcomes. Investigating the host microenvironments encountered by human-pathogenic fungi during the initiation, development, and active stages of subsequent infection and how fungi adapt to these dynamic environments is one area where potential therapeutic opportunities exist.Recently, there has been an increased interest in how levels of oxygen at the site of infection impact the outcome of IFIs (18, 22). A major reason for this interest is that fungal strains with a deficient ability to adapt to hypoxia fail to cause lethal disease in murine models. Among the best examples of these fungal strains are the Cryptococcus neoformans and Aspergillus fumigatus null mutants of the sterol regulatory element-binding proteins (SREBP) (Sre1 and SrbA, respectively) (11,12,46). SREBPs were first identified in higher eukaryotes as regulators of cholesterol and lipid metabolism (7,19,20,37,45). Mammalian SREBPs are synthesized as endoplasmic reticulum (ER) membrane-bound precursors and contain...

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 gp91phox−/− 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 ≥2-fold changes, 8 were also increased in gp91phox−/− 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 gp91phox−/− 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.

3 In coronary arteries incubated with [3H]-(±)-isoprenaline (5 jig/ml) for 1 or 10 min, uptake was extraneuronal since it was inhibited by cortisol, 17,B-oestradiol or phenoxybenzamine. 4 Atria incubated with (±)-isoprenaline (5 to 1000 jig/ml) showed non-neuronal, biphasic accumulation of amine. There was a high capacity, low affinity initial uptake process (apparent Km 136 gM) which was resistant to steroidal extraneuronal uptake inhibitors but which could be abolished by phenoxybenzamine. A slower uptake occurred after 2 min which was sensitive to steroidal and other extraneuronal uptake inhibitors. 5 Inhibition of uptake processes did not alter sensitivity of coronary arteries to dilator effects of catecholamines. However, experiments with extraneuronal uptake inhibitors were limited by the relaxant effects of cortisol and 17f3-oestradiol themselves.