Simon L. Newman

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 (Mφ). To identify the ligand(s) on Hc yeasts that is recognized by Mφ, 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 Mφ. 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 Mφ, 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 Mφ. Depletion of hsp60 from F/TE removed the capacity of F/TE to block binding of Hc to Mφ. 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 Mφ CD11/CD18, whereas DC recognize Hc via a different ligand(s).

Candida albicans Is Phagocytosed, Killed, and Processed for Antigen Presentation by Human Dendritic Cells

ABSTRACT Candida albicans is a component of the normal flora of the alimentary tract and also is found on the mucocutaneous membranes of the healthy host. Candida is the leading cause of invasive fungal disease in premature infants, diabetics, and surgical patients, and of oropharyngeal disease in AIDS patients. As the induction of cell-mediated immunity to Candida is of critical importance in host defense, we sought to determine whether human dendritic cells (DC) could phagocytose and degradeCandida and subsequently present Candidaantigens to T cells. Immature DC obtained by culture of human monocytes in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 phagocytosed unopsonized Candida in a time-dependent manner, and phagocytosis was not enhanced by opsonization of Candida in serum. Like macrophages (Mφ), DC recognized Candida by the mannose-fucose receptor. Upon ingestion, DC killed Candida as efficiently as human Mφ, and fungicidal activity was not enhanced by the presence of fresh serum. Although phagocytosis ofCandida by DC stimulated the production of superoxide anion, inhibitors of the respiratory burst (or NO production) did not inhibit killing of Candida, even when phagocytosis was blocked by preincubation of DC with cytochalasin D. Further, although apparently only modest phagolysosomal fusion occurred upon DC phagocytosis of Candida, killing ofCandida under anaerobic conditions was almost equivalent to killing under aerobic conditions. Finally, DC stimulatedCandida-specific lymphocyte proliferation in a concentration-dependent manner after phagocytosis of both viable and heat-killed Candida cells. These data suggest that, in vivo, such interactions between DC and C. albicans may facilitate the induction of cell-mediated immunity.

Macrophages in host defense against Histoplasma capsulatum

Macrophages function in both innate and cell-mediated immunity in host defense against pathogenic fungi. They initially serve as a protected environment in which the primary fungal pathogen Histoplasma capsulatum multiplies and disseminates from the lung to other organs. Upon induction of cell-mediated immunity, cytokines activate macrophages to destroy the yeasts and thus remove them from the host.

Histoplasma capsulatum Yeasts Are Phagocytosed Via Very Late Antigen-5, Killed, and Processed for Antigen Presentation by Human Dendritic Cells

Histoplasma capsulatum (Hc) is a facultative, intracellular parasite of world-wide importance. As the induction of cell-mediated immunity to Hc is of critical importance in host defense, we sought to determine whether dendritic cells (DC) could function as a primary APC for this pathogenic fungus. DC obtained by culture of human monocytes in the presence of GM-CSF and IL-4 phagocytosed Hc yeasts in a time-dependent manner. Upon ingestion, the intracellular growth of yeasts within DC was completely inhibited compared with rapid growth within human macrophages. Electron microscopy of DC with ingested Hc revealed that many of the yeasts were degraded as early as 2 h postingestion. In contrast to macrophages, human DC recognized Hc yeasts via the fibronectin receptor, very late Ag-5, and not via CD18 receptors. DC stimulated Hc-specific lymphocyte proliferation in a concentration-dependent manner after phagocytosis of viable and heat-killed Hc yeasts, but greater proliferation was achieved after ingestion of viable yeasts. These data demonstrate that human DC can phagocytose and degrade a fungal pathogen and subsequently process the appropriate Ags for stimulation of lymphocyte proliferation. In vivo, such interactions between DC and Hc may facilitate the induction of cell-mediated immunity.

Identification of Constituents of Human Neutrophil Azurophil Granules That Mediate Fungistasis against Histoplasma capsulatum

ABSTRACT Previously we demonstrated that human neutrophils mediate potent and long-lasting fungistasis against Histoplasma capsulatumyeasts and that all of the fungistatic activity resides in the azurophil granules. In the present study, specific azurophil granule constituents with fungistatic activity were identified by incubation with H. capsulatum yeasts for 24 h and by quantifying the subsequent growth of yeasts via the incorporation of [3H]leucine. Human neutrophil defensins HNP-1, HNP-2, and HNP-3 inhibited the growth of H. capsulatum yeasts in a concentration-dependent manner with maximum inhibition at 8 μg/ml. At a concentration of 4 μg/ml, all possible paired combinations of defensins exhibited additive fungistatic activity against H. capsulatum yeasts. Cathepsin G and bactericidal-permeability-increasing protein (BPI) also mediated fungistasis against H. capsulatum in a concentration-dependent manner. The fungistatic activities of combinations of cathepsin G and BPI were additive, as were those of combinations of cathepsin G or BPI with HNP-1, HNP-2, and HNP-3. Lysozyme and elastase exhibited modest antifungal activity, and azurocidin and proteinase 3 exhibited no significant fungistasis against H. capsulatum yeasts. Thus, defensins, cathepsin G, and BPI are the major anti-H. capsulatum effector molecules in the azurophil granules of human neutrophils.

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.

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 (Mφ). 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 Mφ. 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 Mφ was only partially restored by the addition of exogenous iron. The vma1::HPH mutant also did not grow as a mold at 28°C. Complementation of the mutant (vma/VMA1) restored its ability to replicate in Mφ, grow on iron‐poor medium and grow as a mold at 28°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.

Enhanced Killing of Candida albicans by Human Macrophages Adherent to Type 1 Collagen Matrices via Induction of Phagolysosomal Fusion

ABSTRACT 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.

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.

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 (Mφ). In the present study, we sought to quantify the intraphagosomal pH under conditions in which Hc yeasts replicated or were killed. Human Mφ that had ingested both viable and heat-killed or fixed yeasts maintained an intraphagosomal pH of ∼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. Mφ that had ingested Saccharomyces cerevisae, a nonpathogenic yeast that is rapidly killed and degraded by Mφ, also maintained an intraphagosomal pH of ∼6.5 over a period of several hours. Stimulation of human Mφ 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 Mφ cultured in the presence of bafilomycin also completely degraded heat-killed Hc yeasts, whereas mouse peritoneal Mφ digestion of yeasts was completely reversed in the presence of bafilomycin. However, bafilomycin did not inhibit mouse Mφ fungistatic activity induced by IFN-γ. Thus, human Mφ do not require phagosomal acidification to kill and degrade Hc yeasts, whereas mouse Mφ do require acidification for fungicidal but not fungistatic activity.