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, DeltaC-term BAD1, lacking an EGF-like domain, bound poorly to BAD1 null yeast, yielding a low affinity (Kd, 3 x 10(-7) M versus 5 x 10(-8) M) and Bmax (1.9 x 10(5) versus 7.9 x 10(5)) 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. DeltaC-term strains bound poorly to macrophages, compared with parental or BAD1-reconstituted null strains. However, DeltaC-term strains and the purified protein itself sharply suppressed tumour necrosis factor (TNF)-alpha 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.
Brandhorst
Tristan
T
Department of Pediatrics, University of Wisconsin Medical School, 600 Highland Ave., K4/434, Madison, WI 53792, USA.
Wüthrich
Marcel
M
Finkel-Jimenez
Beatriz
B
Klein
Bruce
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001758:Blastomyces / Q000276:immunology* / Q000378:metabolism / Q000472:pathogenicity*; D017931:DNA Primers; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins; D006023:Glycoproteins / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D014774:Virulence
D017931:DNA Primers; D005656:Fungal Proteins; D006023:Glycoproteins; D004815:Epidermal Growth Factor