Purification in Quantity of the Secreted Form of WI-1: A Major Adhesin onBlastomyces dermatitidisYeasts

et al. 2004

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.

“…Secreted BAD1 was purified as previously described (18). Briefly, yeast were grown in liquid Histoplasma M medium in a gyratory shaker at 37°C for 2 wk.…”

Section: Antigensmentioning

confidence: 99%

“…dermatitidis releases sBAD1 in quantity (up to 25 mg/l) during growth in vitro (18,29) and in the host lung alveolus during infection (7). In this study we analyzed the fate of sBAD1 after interaction with M, cells that mediate host defense against the fungus, but are also modulated by B. dermatitidis surface and sBAD1.…”

Section: Internalization Of Sbad1 By Mmentioning

confidence: 99%

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

Brandhorst

et al. 2004

“…Secreted BAD1 was purified as described (9). Briefly, yeast were grown in liquid Histoplasma macrophage medium in a gyratory shaker at 37°C for 2 wk.…”

Section: Antigensmentioning

confidence: 99%

BAD1, an Essential Virulence Factor ofBlastomyces dermatitidis, Suppresses Host TNF-α Production Through TGF-β-Dependent and -Independent Mechanisms

Klein

2002

We investigated how BAD1, an adhesin and virulence factor of Blastomyces dermatitidis, suppresses phagocyte proinflammatory responses. Wild-type yeast cocultured with murine neutrophils or macrophages prompted release of a soluble factor into conditioned supernatant that abolished TNF-α production in response to the fungus; isogenic, attenuated BAD1 knockout yeast did not have this effect. Phagocytes released 4- to 5-fold more TGF-β in vitro in response to wild-type yeast vs BAD1 knockout yeast. Treatment of inhibitory, conditioned supernatant with anti-TGF-β mAb neutralized detectable TGF-β and restored phagocyte TNF-α production. Similarly, addition of anti-TGF-β mAb into cultures of phagocytes and wild-type yeast reversed BAD1 inhibition of TNF-α production. Conversely, TGF-β treatment of phagocytes cultured with knockout yeast suppressed TNF-α production. Hence, TGF-β mediates BAD1 suppression of TNF-α by wild-type B. dermatitidis cultured in vitro with phagocytes. In contrast to these findings, neutralization of elevated TGF-β levels during experimental pulmonary blastomycosis did not restore BAD1-suppressed TNF-α levels in the lung or ameliorate disease. Soluble BAD1 was found to accumulate in the alveoli of infected mice at levels that suppressed TNF-α production by phagocytes. However, in contrast to yeast cell surface BAD1, which induced TGF-β, soluble BAD1 failed to do so and TNF-α suppression mediated by soluble BAD1 was unaffected by neutralization of TGF-β. Thus, BAD1 of B. dermatitidis induces suppression of TNF-α and progressive infection by both TGF-β-dependent and -independent mechanisms.

“…Secreted WI-1 was purified as described (12). Briefly, yeasts were grown in liquid Histoplasma macrophage medium in a gyratory shaker at 37°C for 1 wk.…”

Section: Antigensmentioning

confidence: 99%

“…WI-1 is released into culture supernatant in large amounts during log-phase growth (12). Therefore, we investigated whether soluble, purified WI-1 alone was capable of inhibiting TNF-␣ production by phagocytes.…”

Section: Wi-1 Itself Inhibits Tnf-␣ Production By Phagocytesmentioning

confidence: 99%

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

Brandhorst

et al. 2001

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.