HacA-independent functions of the ER stress sensor IreA synergize with the canonical UPR to influence virulence traits in Aspergillus fumigatus.

Endoplasmic reticulum (ER) stress is a condition in which the protein folding capacity of the ER becomes overwhelmed by an increased demand for secretion or by exposure to compounds that disrupt ER homeostasis. In yeast and other fungi, the accumulation of unfolded proteins is detected by the ER-transmembrane sensor IreA/Ire1, which responds by cleaving an intron from the downstream cytoplasmic mRNA HacA/Hac1, allowing for the translation of a transcription factor that coordinates a series of adaptive responses that are collectively known as the unfolded protein response (UPR). Here, we examined the contribution of IreA to growth and virulence in the human fungal pathogen Aspergillus fumigatus. Gene expression profiling revealed that A. fumigatus IreA signals predominantly through the canonical IreA-HacA pathway under conditions of severe ER stress. However, in the absence of ER stress IreA controls dual signaling circuits that are both HacA-dependent and HacA-independent. We found that a ΔireA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasts the partial virulence of a ΔhacA mutant, suggesting that IreA contributes to pathogenesis independently of HacA. In support of this conclusion, we found that the ΔireA mutant had more severe defects in the expression of multiple virulence-related traits relative to ΔhacA, including reduced thermotolerance, decreased nutritional versatility, impaired growth under hypoxia, altered cell wall and membrane composition, and increased susceptibility to azole antifungals. In addition, full or partial virulence could be restored to the ΔireA mutant by complementation with either the induced form of the hacA mRNA, hacA(i), or an ireA deletion mutant that was incapable of processing the hacA mRNA, ireA(Δ10). Together, these findings demonstrate that IreA has both HacA-dependent and HacA-independent functions that contribute to the expression of traits that are essential for virulence in A. fumigatus.

Histoplasma capsulatum utilizes siderophores for intracellular iron acquisition in macrophages.

Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (MΦ). Studies in human and murine MΦ demonstrate that the intracellular growth of H. capsulatum yeasts is exquisitely sensitive to the availability of iron. As H. capsulatum produces hydroxamate siderophores, we sought to determine if siderophores were required for intracellular survival in MΦ, and in a murine model of pulmonary histoplasmosis. The expression of SID1 (coding for L-ornithine-N(5)-monooxygenase) was silenced by RNA interference (RNAi) in H. capsulatum strain G217B, and abolished by gene targeting in strain G186AR. G217B SID1-silenced yeasts grew normally in rich medium, did not synthesize siderophores, and were unable to grow on apotransferrin-chelated medium. Their intracellular growth in human and murine MΦ was significantly decreased compared to wild type (WT) yeasts, but growth was restored to WT levels by the addition of exogenous iron, or restoration of SID1 expression. Similar results were obtained with G186AR Δsid1 yeasts. Compared to WT yeasts, G217B SID1-silenced yeasts demonstrated in C57BL/6 mice significantly reduced growth in the lungs and spleens seven days after infection, and 40% of the mice given a normally lethal inoculum of G217B SID1-silenced yeasts survived. These experiments demonstrate that: (1) SID1 expression is required for siderophore biosynthesis by H. capsulatum strain G217B, (2) SID1 expression is required for optimum intracellular growth in MΦ, and (3) inhibition of SID1 expression in vivo reduces the virulence of H. capsulatum yeasts.

Dendritic cells restrict the transformation of Histoplasma capsulatum conidia into yeasts.

Infections due to Histoplasma capsulatum occur as a result of the inhalation of airborne microconidia of the mold into the alveoli of the lungs. In this study we quantified the transformation over time of conidia into yeast-like cells within macrophages (MΦ) and dendritic cells (DC). Conidia from strain G217B which had been surface labeled with carboxy-fluorescein succinimidyl ester (CFSE), or conidia from strain G217B that expresses green fluorescent protein (GFP) only in the yeast phase, were used to infect MΦ and DC. At various time points, numbers of intracellular conidia or yeasts were quantified via phase-contrast and fluorescent microscopy. Transformation of conidia from non-GFP-expressing G217B also was quantified by their incorporation of ³H-leucine. In both human and murine MΦ, numerous yeast-like cells appeared by day 3 post-infection. The time course of conidia transformation into yeasts in culture medium was the same as in MΦ. However, transformation of conidia to yeasts was significantly restricted in human DC and murine lung DC. In DC, significant numbers of yeasts did not appear until 5 days post-infection. Further, MΦ monolayers were destroyed by day 6-7 post-infection, whereas DC monolayers remained intact throughout the study period. These data suggest that in vivo, conidia may transform into yeast-like cells efficiently whether or not they are phagocytosed by MΦ, but not when ingested by DC.

Histoplasma capsulatum cyclophilin A mediates attachment to dendritic cell VLA-5.

Histoplasma capsulatum (Hc) is a pathogenic fungus that replicates in macrophages (Mphi). In dendritic cells (DC), Hc is killed and fungal Ags are processed and presented to T cells. DC recognize Hc yeasts via the VLA-5 receptor, whereas Mphi recognize yeasts via CD18. To identify ligand(s) on Hc recognized by DC, VLA-5 was used to probe a Far Western blot of a yeast freeze/thaw extract (F/TE) that inhibited Hc binding to DC. VLA-5 recognized a 20-kDa protein, identified as cyclophilin A (CypA), and CypA was present on the surface of Hc yeasts. rCypA inhibited the attachment of Hc to DC, but not to Mphi. Silencing of Hc CypA by RNA interference reduced yeast binding to DC by 65-85%, but had no effect on binding to Mphi. However, F/TE from CypA-silenced yeasts still inhibited binding of wild-type Hc to DC, and F/TE from wild-type yeasts depleted of CypA also inhibited yeast binding to DC. rCypA did not further inhibit the binding of CypA-silenced yeasts to DC. Polystyrene beads coated with rCypA or fibronectin bound to DC and Mphi and to Chinese hamster ovary cells transfected with VLA-5. Binding of rCypA-coated beads, but not fibronectin-coated beads, was inhibited by rCypA. These data demonstrate that CypA serves as a ligand for DC VLA-5, that binding of CypA to VLA-5 is at a site different from FN, and that there is at least one other ligand on the surface of Hc yeasts that mediates binding of Hc to DC.

The Histoplasma capsulatum vacuolar ATPase is required for iron homeostasis, intracellular replication in macrophages and virulence in a murine model of histoplasmosis.

Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (Mphi). To identify specific genes required for intracellular survival, we utilized Agrobacterium tumefaciens-mediated mutagenesis, and screened for H. capsulatum insertional mutants that were unable to survive in human Mphi. One colony was identified that had an insertion within VMA1, the catalytic subunit A of the vacuolar ATPase (V-ATPase). The vma1 mutant (vma1::HPH) grew normally on iron-replete medium, but not on iron-deficient media. On iron-deficient medium, the growth of the vma1 mutant was restored in the presence of wild-type (WT) H. capsulatum yeasts, or the hydroxamate siderophore, rhodotorulic acid. However, the inability to replicate within Mphi was only partially restored by the addition of exogenous iron. The vma1::HPH mutant also did not grow as a mold at 28 degrees C. Complementation of the mutant (vma/VMA1) restored its ability to replicate in Mphi, grow on iron-poor medium and grow as a mold at 28 degrees C. The vma1::HPH mutant was avirulent in a mouse model of histoplasmosis, whereas the vma1/VMA1 strain was as pathogenic as WT yeasts. These studies demonstrate the importance of V-ATPase function in the pathogenicity of H. capsulatum, in iron homeostasis and in fungal dimorphism.

Interaction of Histoplasma capsulatum with human macrophages, dendritic cells, and neutrophils.

Histoplasma capsulatum (Hc) is a dimorphic fungal pathogen indigenous to the Ohio and Mississippi River Valleys in the United States. Infection is initiated by inhalation of microconidia or small mycelial fragments into the terminal bronchioles of the lung. The conidia are taken up by alveolar macrophages (Mphi), in which they convert to the pathogenic yeast phase. The yeasts replicate in the alveolar Mphi and other Mphi recruited to the lung as part of the inflammatory response. Thus, the yeasts are able to disseminate from the lung to other organs, such as the liver and spleen. As a facultative intracellular parasite, the interaction of Hc yeasts with Mphi is a critical component of the host response to infection. In addition, Hc yeasts have critical interactions with inflammatory neutrophils, and with dendritic cells (DCs) in the lung and other organs. Indeed, recent new evidence suggests that DCs may be the key antigen-presenting cells that initiate cell-mediated immunity. Thus, the methods described in this chapter cover quantitation of the binding, ingestion, and intracellular replication of Hc yeasts in human Mphi, DCs, and neutrophils.

Iron uptake and virulence in Histoplasma capsulatum.

Histoplasma capsulatum (Hc) is the causative organism of a spectrum of disease affecting both the immunocompetent and the immunocompromised host. Hc is a dimporhic fungus that converts from conidia to the pathogenic yeast phase after entry into the mammalian host. Despite rapid ingestion by macrophages, it survives intracellularly within the macrophage. The intracellular survival strategy of Hc yeasts focuses on regulating the phagosomal compartment by modulating the intraphagosomal pH to 6.5. As an intracellular pathogen of MΦ, Hc obtains iron from Fe-transferrin, ferritin, or both, via the production of hydroxamate siderophores, and the production of ferric reductases. A better understanding of the mechanisms by which Hc yeasts acquire iron from the host may lead to novel therapeutics for histoplasmosis.

Human macrophages do not require phagosome acidification to mediate fungistatic/fungicidal activity against Histoplasma capsulatum.

Histoplasma capsulatum (Hc) is a facultative intracellular fungus that modulates the intraphagosomal environment to survive within macrophages (Mphi). In the present study, we sought to quantify the intraphagosomal pH under conditions in which Hc yeasts replicated or were killed. Human Mphi that had ingested both viable and heat-killed or fixed yeasts maintained an intraphagosomal pH of approximately 6.4-6.5 over a period of several hours. These results were obtained using a fluorescent ratio technique and by electron microscopy using the 3-(2,4-dinitroanilo)-3'-amino-N-methyldipropylamine reagent. Mphi that had ingested Saccharomyces cerevisae, a nonpathogenic yeast that is rapidly killed and degraded by Mphi, also maintained an intraphagosomal pH of approximately 6.5 over a period of several hours. Stimulation of human Mphi fungicidal activity by coculture with chloroquine or by adherence to type 1 collagen matrices was not reversed by bafilomycin, an inhibitor of the vacuolar ATPase. Human Mphi cultured in the presence of bafilomycin also completely degraded heat-killed Hc yeasts, whereas mouse peritoneal Mphi digestion of yeasts was completely reversed in the presence of bafilomycin. However, bafilomycin did not inhibit mouse Mphi fungistatic activity induced by IFN-gamma. Thus, human Mphi do not require phagosomal acidification to kill and degrade Hc yeasts, whereas mouse Mphi do require acidification for fungicidal but not fungistatic activity.

Enhanced killing of Candida albicans by human macrophages adherent to type 1 collagen matrices via induction of phagolysosomal fusion.

Candida albicans, a component of the normal flora of the alimentary tract and mucocutaneous membranes, is the leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients and of oropharyngeal disease in AIDS patients. As little is known about the regulation of monocyte/macrophage anti-Candida activity, we sought to determine if fungicidal activity might be regulated by extracellular matrix proteins to which monocytes/macrophages are adherent in vivo. Compared to monocyte/macrophages that adhered to plastic, human monocytes and monocyte-derived macrophages that adhered to type 1 collagen matrices, but not to fibronectin, vitronectin, or laminin, demonstrated a significant increase in candidacidal activity. The enhancement of monocyte fungicidal activity was maintained over a 4-h period, whereas macrophage fungicidal activity was maximum at 1 h. Although adherence of monocytes and macrophages to collagen matrices concomitantly enhanced the production of superoxide anion, only the fungicidal activity of collagen-adherent monocytes was partially blocked by superoxide dismutase and catalase. Remarkably, we found that only 10% of the phagosomes in C. albicans-infected macrophages that adhered to plastic fused with lysosomes. In contrast, 80% of yeast-containing phagosomes of collagen-adherent macrophages fused with lysosomes. These data suggest that nonoxidative mechanisms are critical for human macrophage anti-Candida activity and that C. albicans pathogenicity is mediated, in part, by its ability to inhibit phagolysosomal fusion in macrophages.

Human dendritic cell activity against Histoplasma capsulatum is mediated via phagolysosomal fusion.

Histoplasma capsulatum is a fungal pathogen that requires the induction of cell-mediated immunity (CMI) for host survival. We have demonstrated that human dendritic cells (DC) phagocytose H. capsulatum yeasts and, unlike human macrophages (Mø) that are permissive for intracellular growth, DC killed and degraded the fungus. In the present study, we sought to determine whether the mechanism(s) by which DC kill Histoplasma is via lysosomal hydrolases, via the production of toxic oxygen metabolites, or both. Phagosome-lysosome fusion (PL-fusion) was quantified by using fluorescein isothiocyanate-dextran and phase and fluorescence microscopy and by electron microscopy with horseradish peroxidase colloidal gold to label lysosomes. Unlike Mphi, Histoplasma-infected DC exhibited marked PL-fusion. The addition of suramin to Histoplasma-infected DC inhibited PL-fusion and DC fungicidal activity. Incubation of Histoplasma-infected DC at 18 degrees C also concomitantly reduced PL-fusion and decreased the capacity of DC to kill and degrade H. capsulatum yeasts. Further, culture of Histoplasma-infected DC in the presence of bafilomycin, an inhibitor of the vacuolar ATPase, did not block DC anti-Histoplasma activity, indicating that phagosome acidification was not required for lysosome enzyme activity. In contrast, culture of Histoplasma-infected DC in the presence of inhibitors of the respiratory burst or inhibitors of NO synthase had little to no effect on DC fungicidal activity. These data suggest that the major mechanism by which human DC mediate anti-Histoplasma activity is through the exposure of yeasts to DC lysosomal hydrolases. Thus, DC can override one of the strategies used by H. capsulatum yeasts to survive intracellularly within Mø.

Identification of heat shock protein 60 as the ligand on Histoplasma capsulatum that mediates binding to CD18 receptors on human macrophages.

Histoplasma capsulatum (Hc), is a facultative intracellular fungus that binds to CD11/CD18 receptors on macrophages (Mphi). To identify the ligand(s) on Hc yeasts that is recognized by Mphi, purified human complement receptor type 3 (CR3, CD11b/CD18) was used to probe a Far Western blot of a detergent extract of Hc cell wall and cell membrane. CR3 recognized a single 60-kDa protein, which was identified as heat shock protein 60 (hsp60). Biotinylation of viable yeasts, followed by precipitation with streptavidin-coated beads, and Western blotting with anti-hsp60 demonstrated that hsp60 was on the surface of Hc yeasts. Electron and confocal microscopy revealed that hsp60 resided on the yeast cell wall in discrete clusters. Recombinant hsp60 (rhsp60) inhibited attachment of Hc yeasts to Mphi. Recombinant hsp60 and Abs to CD11b and CD18 inhibited binding of yeasts to Chinese hamster ovary cells transfected with CR3 (CHO3). Polystyrene beads coated with rhsp60 bound to Mphi, and attachment was inhibited by Abs to CD11 and CD18. Freeze/thaw extract (F/TE), a preparation of Hc yeast surface proteins that contained hsp60, inhibited the attachment of Hc yeasts to Mphi. Depletion of hsp60 from F/TE removed the capacity of F/TE to block binding of Hc to Mphi. Interestingly, rhsp60 did not inhibit binding of Hc yeasts to dendritic cells (DC), which recognize Hc via very late Ag 5. Moreover, F/TE inhibited attachment of Hc to DC even when depleted of hsp60. Thus, Hc hsp60 appears to be a major ligand that mediates attachment of Hc to Mphi CD11/CD18, whereas DC recognize Hc via a different ligand(s).