Human serum at low concentrations inhibits the growth of Cryptococcus neoformans in vitro. Fractionation of serum yielded a purified inhibitory protein with a molecular mass of ϳ81.8 kDa, a pI of ϳ6.2, and an amino acid sequence that matched that of human transferrin. The inhibitory activity and that of apotransferrin and 5% human serum were reversed by 10 M freshly prepared FeCl 3 .Human serum inhibits the growth of Cryptococcus neoformans (1, 11). Early attempts to identify an inhibitory component(s) were limited by the technology available (12,18). Here a sequential method was used for characterization of the anticryptococcal activity.C. neoformans isolates CDC 9759 and 46545 were grown on Sabouraud's dextrose agar or blood agar plates at 35°C for 48 h. Yeast cells were washed in saline and suspended in RPMI 1640 (GIBCO, Grand Island, N.Y.) with or without human serum fractions and dispensed, 0.1 ml per microtest plate well, in sets of quadruplicate cultures. In other experiments, C. neoformans was cultured in iron-free human transferrin (siderophilin) (Sigma Chemical Company, St. Louis, Mo.).Cultures were incubated at 37°C in 5% CO 2 -95% air for 24 h, harvested, and washed with sterile distilled water. Dilutions of harvested material were plated on blood agar plates, CFU were enumerated after 48 h at 35°C. Percent inhibition was calculated by the formula [1 Ϫ (experimental CFU/control CFU)] ϫ 100. CFU from RPMI 1640 cultures constituted the control. This definition measures the sum of events during incubation where some cells may be static, some multiply, and others die.Serum from healthy donors and human AB serum (GIBCO) were stored at Ϫ80°C.For anion-exchange gel chromatography, DEAE-Sephacel (Pharmacia LKB, Uppsala, Sweden) columns (2.5 by 6.5 cm and 3 by 12 cm) were prepared. Four milliliters of serum was applied to the smaller column, and 12 ml was applied to the larger. Columns were eluted sequentially with Tris-HCl (0.1 M, pH 8.5) buffer and then 0.1, 0.2, and 0.3 M NaCl in buffer. Five-milliliter fractions were collected, and the protein concentration of fractions was estimated by absorbance at 280 nm. Fractions that formed a protein peak were pooled, lyophilized, dialyzed against distilled water, and filter sterilized.Sephadex G-200 (Pharmacia, Piscataway, N.J.) columns (2.5 by 90 cm) were used for molecular sieve chromatography. Samples were eluted from the column with phosphate-buffered saline diluted 1:10. Fractions were processed as described above.A precast Bis-Tris polyacrylamide minigel electrophoresis system, which runs at neutral pH, 7.0 (NuPAGE; NOVEX, San Diego, Calif.), was used for one-dimensional gel electrophoresis. Electrophoresis was done on 4 to 12% gels using a 3-(N-morpholino)propanesulfonic acid-sodium dodecyl sulfate buffer in an Xcell-II apparatus. Proteins were fixed, stained with Coomassie blue, photographed with a digital camera, and printed with a graphic printer.NOVEX isoelectric focusing (IEF) gels, pH 3 to 7, nondenaturing (no urea), containing 5% polyacrylamide and 2%...

An earlier study reported that human cerebrospinal¯uid (CSF) has fungistatic activity for Cryptococcus neoformans. The present study reports that molecular sieve fractionation of concentrated CSF yielded three protein peaks, one of which (p2) had anticryptococcal activity. On a DEAE-Sephacel anion-exchange column the active molecular sieve peak (p2) gave two peaks that contained anticryptococcal activity. The ®rst (DEAE-1) eluted with 0.1 M NaCl and the second (DEAE-2) eluted with 0.2 M NaCl in buffer. Fungistatic activity of DEAE-1 was reversed by FeCl 3 . Moreover, FeCl 3 reversed inhibition of C. neoformans growth by CSF. In contrast, activity of DEAE-2 was not reversed by FeCl 3 , indicating that inhibition was produced by an ironindependent mechanism. Immunoblot assays showed that transferrin was present in DEAE-1 but not in DEAE-2, whereas albumin was present in DEAE-2 but not in DEAE-1. On NuPAGE, DEAE-1 protein migrated as a single band corresponding to transferrin and DEAE-2 protein gave a single band corresponding to albumin. In control experiments, human serum albumin subjected to the same isolation protocol acquired anticryptococcal activity similar to that of DEAE-2. Therefore, CSF albumin (DEAE-2) activity was associated with the isolation protocol. These data indicate that transferrin, present in or isolated from CSF, sequesters trace amounts of ferric iron, inhibits growth of C. neoformans and acts as an innate defence mechanism.