Tristan Brandhorst

The WI-1 Adhesin Blocks Phagocyte TNF-α Production, Imparting Pathogenicity on Blastomyces dermatitidis

The WI-1 adhesin is indispensable for pathogenicity of Blastomyces dermatitidis and is thought to promote pulmonary infection by fixing yeast to lung tissue and cells. Recent findings suggest that WI-1 confers pathogenicity by mechanisms in addition to adherence. Here, we investigated whether WI-1 modulates host immunity by altering production of pro-inflammatory cytokines. Production of TNF-α in lung alveolar fluids of mice infected with B. dermatitidis was severalfold higher for WI-1 knockout yeast compared with wild-type yeast, and in vitro coculture of unseparated lung cells with these isogenic yeast disclosed similar differences. Upon coculture with purified macrophages and neutrophils, wild-type yeast blocked TNF-α production, yet WI-1 knockout yeast stimulated production. Coating knockout yeast with purified WI-1 converted them from stimulating TNF-α production to inhibiting production. Addition of purified WI-1 into stimulated phagocyte cultures led to concentration-dependent inhibition of TNF-α production. Neutralization of TNF-α in vivo exacerbated experimental pulmonary infection, particularly for the nonpathogenic WI-1 knockout yeast. Inducing increased TNF-α levels in the lung by adenovirus-vectored gene therapy controlled infection with wild-type yeast. Thus, the WI-1 adhesin on yeast modulates host immunity through blocking TNF-α production by phagocytes, which fosters progression of pulmonary infection.

Exploiting Type 3 Complement Receptor for TNF-α Suppression, Immune Evasion, and Progressive Pulmonary Fungal Infection

TNF-α is crucial in defense against intracellular microbes. Host immune cells use type 3 complement receptors (CR3) to regulate excess TNF-α production during physiological clearance of apoptotic cells. BAD1, a virulence factor of Blastomyces dermatitidis, is displayed on yeast and released during infection. BAD1 binds yeast to macrophages (Mφ) via CR3 and CD14 and suppresses TNF-α, which is required for resistance. We investigated whether blastomyces adhesin 1 (BAD1) exploits host receptors for immune deviation and pathogen survival. Soluble BAD1 rapidly entered Mφ, accumulated intracellularly by 10 min after introduction to cells, and trafficked to early and late endosomes. Inhibition of receptor recycling by monodansyl cadaverine blocked association of BAD1 with Mφ and reversed TNF-α suppression in vitro. Inhibition of BAD1 uptake with cytochalasin D and FcR-redirected delivery of soluble BAD1 as Ag-Ab complexes or of wild-type yeast opsonized with IgG similarly reversed TNF-α suppression. Hence, receptor-mediated entry of BAD1 is requisite in TNF-α suppression, and the route of entry is critical. Binding of soluble BAD1 to Mφ of CR3−/− and CD14−/− mice was reduced to 50 and 33%, respectively, of that in wild-type mice. Mφ of CR3−/− and CD14−/− mice resisted soluble BAD1 TNF-α suppression in vitro, but, in contrast to CR3−/− cells, CD14−/− cells were still subject to suppression mediated by surface BAD1 on wild-type yeast. CR3−/− mice resisted both infection and TNF-α suppression in vivo, in contrast to wild-type and CD14−/− mice. BAD1 of B. dermatitidis thus co-opts normal host cell physiology by exploiting CR3 to subdue TNF-α production and foster pathogen survival.

A C-terminal EGF-like domain governs BAD1 localization to the yeast surface and fungal adherence to phagocytes, but is dispensable in immune modulation and pathogenicity of Blastomyces dermatitidis.

BAD1, an adhesin and immune modulator of Blastomyces dermatitidis, is an essential virulence factor that is released extracellularly before association with the yeast surface. Here, deletion of the C‐terminal EGF‐like domain profoundly affected BAD1 function, leading to non‐association with yeast, extracellular accumulation and impaired yeast adherence to macrophages. In equilibrium binding assays, ΔC‐term BAD1, lacking an EGF‐like domain, bound poorly to BAD1 null yeast, yielding a low affinity (Kd, 3 × 10−7 M versus 5 × 10−8 M) and Bmax (1.9 × 105 versus 7.9 × 105) compared with BAD1. Similar protein binding profiles were observed using chitin particles, reinforcing the notion that chitin fibrils are a receptor for BAD1, and that the EGF‐like domain is critical for BAD1 interactions with chitin on yeast. ΔC‐term strains bound poorly to macrophages, compared with parental or BAD1‐reconstituted null strains. However, ΔC‐term strains and the purified protein itself sharply suppressed tumour necrosis factor (TNF)‐α release by phagocytes in vitro and in lung in vivo, and the strains retained pathogenicity in a murine model of blastomycosis. Our results illustrate the previously undefined role of the EGF‐like domain for BAD1 localization to yeast surfaces during cell wall biogenesis. They also demonstrate that the requirements for host cell binding and immune modulation by BAD1 can be dissociated from one another, and that the former is unexpectedly dispensable in the requisite role of BAD1 in pathogenesis.

Role of glucan and surface protein BAD1 in complement activation by Blastomyces dermatitidis yeast.

ABSTRACT Our previous studies showed that Blastomyces dermatitidis yeast activates the human complement system, leading to deposition of opsonic complement fragments onto the yeast surface. This report examines the influence of altered surface expression of glucan or BAD1 protein (formerly WI-1) on the yeasts ability to activate and bind C3. Compared to the wild type, a glucan-deficient mutant yeast delayed initiation of C3 deposition and reduced C3-binding capacity by 50%. Linkage of bakers-yeast β-glucan to the glucan-deficient yeast restored initial C3 deposition kinetics to the wild-type level and partially restored C3-binding capacity, suggesting that β-glucan is an initiator of complement activation and a C3 acceptor. The role of BAD1 in B. dermatitidis yeast-complement interaction was also assessed.BAD1 knockout yeast initiated faster C3 deposition and increased C3-binding capacity compared to the wild-type yeast or aBAD1-reconstituted yeast, suggesting either a lack of an intrinsic ability in BAD1 or an inhibitory role of BAD1 in complement activation and binding. However, both complement activation and the capacity for C3 binding by the wild-type yeast were enhanced in normal human serum supplemented with an anti-BAD1 monoclonal antibody (MAb) or in immune sera from blastomycosis patients. Microscopic analysis revealed that more initial C3-binding sites were formed on yeast in the presence of both naturally occurring complement initiators and exogenous anti-BAD1 MAb, suggesting that anti-BAD1 antibody enhanced the ability of B. dermatitidis yeast to interact with the host complement system. Thus, glucan and BAD1 have distinctly different regulatory effects on complement activation by B. dermatitidis.

Development of a Highly Sensitive and Specific Blastomycosis Antibody Enzyme Immunoassay Using Blastomyces dermatitidis Surface Protein BAD-1

ABSTRACT Serologic tests for antibodies to Blastomyces dermatitidis are not thought to be useful for the diagnosis of blastomycosis, in part due to the low sensitivity of immunodiffusion and complement fixation. Earlier studies have shown that the enzyme immunoassay improves the sensitivity of antibody detection for the diagnosis of blastomycosis. Microplates coated with the B. dermatitidis surface protein BAD-1 were used for testing sera from patients with proven blastomycosis or histoplasmosis and controls. Semiquantification was accomplished by using standards containing human anti-B. dermatitidis antibodies. The antibodies were detected in 87.8% of the patients with blastomycosis by the enzyme immunoassay compared to 15.0% by immunodiffusion. The specificities were 99.2% for patients with nonfungal infections and healthy subjects and 94.0% for patients with histoplasmosis. The results were highly reproducible on repeat testing. When combined with antigen testing, antibody testing improved the sensitivity from 87.8% to 97.6%. Enzyme immunoassay detection of antibodies against BAD-1 is highly specific, has greatly improved sensitivity over immunodiffusion, and may identify cases with negative results by antigen testing. This assay has the potential to aid in the diagnosis of blastomycosis.

Calnexin induces expansion of antigen-specific CD4+ T cells that confer immunity to fungal ascomycetes via conserved epitopes

Fungal infections remain a threat due to the lack of broad-spectrum fungal vaccines and protective antigens. Recent studies showed that attenuated Blastomyces dermatitidis confers protection via T cell recognition of an unknown but conserved antigen. Using transgenic CD4(+) T cells recognizing this antigen, we identify an amino acid determinant within the chaperone calnexin that is conserved across diverse fungal ascomycetes. Calnexin, typically an ER protein, also localizes to the surface of yeast, hyphae, and spores. T cell epitope mapping unveiled a 13-residue sequence conserved across Ascomycota. Infection with divergent ascomycetes, including dimorphic fungi, opportunistic molds, and the agent causing white nose syndrome in bats, induces expansion of calnexin-specific CD4(+) T cells. Vaccine delivery of calnexin in glucan particles induces fungal antigen-specific CD4(+) T cell expansion and resistance to lethal challenge with multiple fungal pathogens. Thus, the immunogenicity and conservation of calnexin make this fungal protein a promising vaccine target.

Performance comparison of immunodiffusion, enzyme-linked immunosorbent assay, immunochromatography and hemagglutination for serodiagnosis of human pythiosis

Pythiosis is a life-threatening infectious disease caused by the fungus-like organism Pythium insidiosum. Morbidity and mortality rates of pythiosis are high. The treatment of choice for pythiosis is surgical debridement of infected tissue. Early and accurate diagnosis is critical for effective treatment. In-house serodiagnostic tests, including immunodiffusion (ID), enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICT) and hemagglutination (HA) have been developed to detect antibodies against P. insidiosum in sera. This study compares the diagnostic performance of ID, ELISA, ICT, and HA, using sera from 37 pythiosis patients and 248 control subjects. ICT and ELISA showed optimal diagnostic performance (100% sensitivity, specificity, positive predictive value and negative predictive value). ICT was both rapid and user-friendly. ELISA results were readily quantitated. ID is relatively insensitive. HA was rapid, but diagnostic performance was poor. Understanding the advantages offered by each assay facilitates selection of an assay that is circumstance-appropriate. This will promote earlier diagnoses and improved outcomes for patients with pythiosis.

The ribosome-inactivating protein restrictocin deters insect feeding on Aspergillus restrictus

The fungus-feeding beetle, Carpophilus freemani, consumed equal quantities of young mycelia, fewer phialides bearing mature spores and much fewer phialides bearing developing spores of Aspergillus restrictus compared to those of Aspergillus nidulans when tested in diet choice assays. The degree to which specific fungal structures were consumed was inversely related to the localization of high levels of restrictocin, a ribosome-inactivating protein, to those structures. Pure restrictocin added to the insect diet at 1000 p.p.m. killed 38.5% of C. freemani larvae and 62.5% of Spodoptera frugiperda larvae in 48 h, but did not affect C. freemani adults or Helicoverpa zea larvae over the same interval. In diet choice assays, 1000 p.p.m. of restrictocin deterred feeding by adult C. freemani and Sitophilus zeamais compared to control diets. Thus, restrictocin production and localization may have a natural defensive role against insect feeding at times critical to spore formation by A. restrictus, and may have potential as an insect control agent.

Production and localization of restrictocin in Aspergillus restrictus

The production and secretion of restrictocin (a cytotoxin that cleaves ribosomal RNA) by cultures of the fungus Aspergillus restrictus was investigated. Previous studies have indicated that restrictocin production in liquid culture coincides with the appearance of differentiated cell structures. A study of the correlation between the appearance of differentiated structures and restrictocin production was conducted with A. restrictus grown on agar medium. Restrictocin was found to be associated with the cell mass of the agar-grown culture (in contrast to liquid cultures), and was first observed when aerial hyphae emerged. Restrictocin levels increased until the time of conidiation, after which they fell off sharply. No restrictocin could be found in the agar medium. The presence of restrictocin upon and within various cell structures was determined by immunofluorescent laser microscopy. This study showed that restrictocin became localized to the conidiophores and phialides during the process of conidiation. Prior to this, restrictocin was found within the hyphae in localized concentrations that may correspond to secretory vesicles.

Fungal Mimicry of a Mammalian Aminopeptidase Disables Innate Immunity and Promotes Pathogenicity

Systemic fungal infections trigger marked immune-regulatory disturbances, but the mechanisms are poorly understood. We report that the pathogenic yeast of Blastomyces dermatitidis elaborates dipeptidyl-peptidase IVA (DppIVA), a close mimic of the mammalian ectopeptidase CD26, which modulates critical aspects of hematopoiesis. We show that, like the mammalian enzyme, fungal DppIVA cleaved C-C chemokines and GM-CSF. Yeast producing DppIVA crippled the recruitment and differentiation of monocytes and prevented phagocyte activation and ROS production. Silencing fungal DppIVA gene expression curtailed virulence and restored recruitment of CCR2(+) monocytes, generation of TipDC, and phagocyte killing of yeast. Pharmacological blockade of DppIVA restored leukocyte effector functions and stemmed infection, while addition of recombinant DppIVA to gene-silenced yeast enabled them to evade leukocyte defense. Thus, fungal DppIVA mediates immune-regulatory disturbances that underlie invasive fungal disease. These findings reveal a form of molecular piracy by a broadly conserved aminopeptidase during disease pathogenesis.