At the site of microbial infections, the significant influx of immune effector cells and the necrosis of tissue by the invading pathogen generate hypoxic microenvironments in which both the pathogen and host cells must survive. Currently, whether hypoxia adaptation is an important virulence attribute of opportunistic pathogenic molds is unknown. Here we report the characterization of a sterol-regulatory element binding protein, SrbA, in the opportunistic pathogenic mold, Aspergillus fumigatus. Loss of SrbA results in a mutant strain of the fungus that is incapable of growth in a hypoxic environment and consequently incapable of causing disease in two distinct murine models of invasive pulmonary aspergillosis (IPA). Transcriptional profiling revealed 87 genes that are affected by loss of SrbA function. Annotation of these genes implicated SrbA in maintaining sterol biosynthesis and hyphal morphology. Further examination of the SrbA null mutant consequently revealed that SrbA plays a critical role in ergosterol biosynthesis, resistance to the azole class of antifungal drugs, and in maintenance of cell polarity in A. fumigatus. Significantly, the SrbA null mutant was highly susceptible to fluconazole and voriconazole. Thus, these findings present a new function of SREBP proteins in filamentous fungi, and demonstrate for the first time that hypoxia adaptation is likely an important virulence attribute of pathogenic molds.

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...

The phagocyte NADPH-dependent oxidase generates superoxide by reducing molecular oxygen through a transmembrane heterodimer known as flavocytochrome b 558 (flavocytochrome b). We investigated the biosynthesis of flavocytochrome b subunits gp91 phox and p22 phox to elucidate features of flavocytochrome b processing in myeloid cells. Although the gp91 phox precursor, gp65, was processed to gp91phox within 4 -8 h of chase, unassembled gp65 and p22 phox monomers were degraded by the cytosolic proteasome. gp65 associated with p22 phox post-translationally, within 1-4 h of chase, but prior to its modification in the Golgi complex. Moreover, p22 phox coprecipitated with unglycosylated gp91 phox primary translation product made in the presence of tunicamycin, suggesting that heterodimer formation does not require glycosylation. Blocking heme synthesis with succinyl acetone completely inhibited heterodimer formation, although biogenesis of gp65 and p22 phox was unaffected. In succinyl acetone-treated cells, p22 phox and gp65 were degraded completely by 8 h of chase, a process mediated by the cytosolic proteasome. Taken together, these data suggest that the formation of the gp65-p22 phox heterodimer is relatively inefficient and that acquisition of heme by gp65 precedes and is required for its association with p22 phox , a process that requires neither the addition of N-linked oligosaccharides nor modification in the Golgi complex.

During assembly of the phagocyte NADPH oxidase, cytosolic p47-phox translocates to the plasma membrane and binds to flavocytochrome b, and binding domains for p47-phox have been identified on the C-terminal tails of both flavocytochrome b subunits. In the present report, we further examine the interaction of these two oxidase components by using random-sequence peptide phage display library analysis. Screening p47-phox with the peptide libraries identified five potential sites of interaction with flavocytochrome b, including three previously reported regions of interaction and two additional regions of interaction of p47-phox with gp91-phox and p22-phox. The additional sites were mapped to a domain on the first predicted cytosolic loop of gp9l-phox encompassing residues S8TRVRRQL93 and to a domain near the cytosolic C-terminal tail of gp91-phox encompassing residues F450EWFADLL457. The mapping also confirmed a previously reported binding domain on gp91-phox (E-5mSGPRGVHFIF564) and putative Src homology 3 domain binding sites on p22-phox (P156PRPP'60 and G177GPPGGP'83). To demonstrate that the additional regions identified were biologically significant, peptides mimicking the gp9l-phox sequences F77LRGSSACCSTRVRRQL93 and E451WFADLLQLLESQ40were synthesized and assayed for their ability to inhibit NADPH oxidase activity. These peptides had EC50 values of 1 ,LM and 230 ,uM, respectively, and inhibited activation when added prior to assembly but did not affect activity of the preassembled oxidase. Our data demonstrate the usefulness of phage display library analysis for the identification of biologically relevant sites of protein-protein interaction and show that the binding of p47-phox to flavocytochrome b involves multiple binding sites along the C-terminal tails of both gp91-and p22-phox and other regions of gp9l-phox nearer to the N terminus.

Cytochrome b of human neutrophils is the central component of the microbicidal NADPH-oxidase system. However, the folding topology of this integral membrane protein remains undetermined. Two random-sequence bacteriophage peptide libraries were used to map structural features of cytochrome b by determining the epitopes of monoclonal antibodies (mAbs) 44.1 and 54.1, specific for the p22phox and gp91phox cytochrome b chains, respectively. The unique peptides of phage selected by mAb affinity purification were deduced from the phage DNA sequences. Phage selected by mAb 44.1 displayed the consensus peptide sequence GGPQVXPI, which is nearly identical to 181GGPQVNPI18 of p22phox. Phage selected by mAb 54.1 displayed the consensus sequence PKXAVDGP, which resembles 382PKIAVDGP389 of gp91phox. Western blotting demonstrated specific binding of each mAb to the respective cytochrome b subunit and selected phage peptides. In flow cytometric analysis, mAb 44.1 bound only permeabilized neutrophils, while 54.1 did not bind intact or permeabilized cells. However, mAb 54.1 immunosedimented detergent-solubilized cytochrome b in sucrose gradients. These results suggest the 181GGPQVNPI188 segment of p22phox is accessible on its intracellular surface, but the 382PKIAVDGP389 region on gp91phox is not accessible to antibody, and probably not on the protein surface.