Novel role for albumin in innate immunity: serum albumin inhibits the growth of Blastomyces dermatitidis yeast form in vitro

Abstract

In this study we found that serum inhibitory activity against Blastomyces dermatitidis was principally mediated by albumin. This was confirmed in experiments using albumin from several mammalian species. Analbuminemic rat serum did not inhibit B. dermatitidis growth in vivo; however, the addition of albumin restored inhibitory activity. Inhibitory activity does not require albumin domain III and appears to involve binding of a low-molecular-weight yeast-derived growth factor.

FIG. 1.

Fraction V albumin from several animal species inhibits the growth of the B. dermatitidis yeast form. B. dermatitidis yeast cells (10⁵) were suspended in RPMI 1640 medium supplemented with the following: fraction V BSA (1 mg/ml) or whole serum (FBS; 5%, vol/vol) (A), fraction V HSA (2.28 mg/ml) or whole serum (HS; 5%, vol/vol) (B), fraction V murine serum albumin (MSA; 1.5 mg/ml) or whole serum (MS: 5%, vol/vol) (C), or fraction V canine serum albumin (CSA; 1.48 mg/ml) or whole serum (CS; 5%, vol/vol) (D). B. dermatitidis yeast cells (10⁵) suspended in RPMI 1640 medium alone served as a control in all four panels. The concentrations of the various fraction V albumins were chosen because they are equivalent to the estimated albumin concentrations in the respective sera. The data illustrated are the means ± standard errors of the means of results of three separate experiments (asterisks indicate results significantly different from those of the medium control at a P value of <0.001).

FIG. 2.

Analbuminemic Nagase rat serum does not inhibit the growth of the B. dermatitidis yeast form. B. dermatitidis yeast cells (10⁵) were grown in RPMI 1640 medium supplemented with analbuminemic Nagase rat serum (ARS; 5%, vol/vol), fraction V rat serum albumin (RSA; 1.28 mg/ml), normal rat serum (RS; 5%, vol/vol), or analbuminemic Nagase rat serum supplemented with fraction V rat albumin (ARS + RSA). Analbuminemic Nagase rat serum had no inhibitory effect on the growth of the B. dermatitidis yeast form. However, rat serum (5%, vol/vol), rat serum albumin, and analbuminemic Nagase rat serum supplemented with rat serum albumin significantly inhibited the growth of B. dermatitidis yeast cells at 48 h (P < 0.001; indicated by asterisks) relative to the growth of B. dermatitidis in RPMI 1640 medium alone (control). The data illustrated are the means ± standard errors of the means of the results of four separate experiments.

FIG. 3.

Albumin inhibitory activity is mediated by a mechanism that does not require direct contact between the B. dermatitidis yeast form and albumin. (A) B. dermatitidis yeast cells (10⁷) were grown in RPMI 1640 medium (40 ml) in which a dialysis membrane containing immunoaffinity chromatography-purified BSA (BSA in dialysis tubing) was suspended. Controls included B. dermatitidis grown in RPMI 1640 medium containing a control dialysis membrane (no BSA) (dialysis tubing control), RPMI 1640 medium containing immunoaffinity chromatography-purified BSA, or RPMI 1640 medium alone (medium). Immunoaffinity chromatography-purified BSA that was contained within the dialysis membrane inhibited the growth of B. dermatitidis to an extent comparable to that of BSA added directly to the medium (results of both were significantly different from those of the medium control at a P of <0.01; indicated by asterisks). The data illustrated are the means ± standard errors of the means of results of three separate experiments. (B) Nondenaturing PAGE analysis was performed with culture supernatants from flasks that contained medium plus BSA and B. dermatitidis (lanes 1 to 3) or medium plus BSA without B. dermatitidis (lanes 4 to 6). The BSA incubated with B. dermatitidis did not migrate as far on a 10% PAGE gel as did BSA incubated with medium alone. Results are for one representative experiment of three performed.

FIG. 4.

Albumin inhibitory activity does not require the drug binding site 2 on domain III. (A) Diagram of the structure of serum albumin indicating the three structural domains and the two known drug binding sites. (B) B. dermatitidis yeast cells (10⁵) were grown in RPMI 1640 medium supplemented with fraction V HSA (HSA), whole rHSA, rHSA domains I and II (contains drug binding site 1), or rHSA domain III (contains drug binding site 2). rHSA proteins and fraction V HSA were added at a final concentration of 1 mg/ml, which is equivalent to the molar concentrations of albumin in 2.5% human serum. B. dermatitidis yeast cells (10⁵) were grown in RPMI 1640 medium alone as a control (medium). B. dermatitidis growth was significantly inhibited by fraction V HSA, rHSA, and rHSA domains I and II at 48 h (P < 0.01; indicated by asterisks). The data illustrated are the means ± standard deviations of results from one representative experiment of three that were performed.