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Isolation and characterisation of an anticryptococcal protein in human cerebrospinal fluid

M. AHLUWALIA^*, E. BRUMMER^*,, S. SRIDHAR^*, R SINGH^* and D.A. STEVENS^*,

*Division of Infectious Diseases, Department of Medicine, Santa Clara Valley Medical Center and California Institute for Medical Research, San Jose, CA and Stanford University School of Medicine, Stanford, CA, USA

Corresponding author: Dr D.A. Stevens, Dept. Med., Santa Clara Valley Med Ctr., 751 South Bascom Ave, San Jose, CA 95128-2699, USA.

Received 24 Jan. 2000; revised version received 16 May 2000; accepted 20 May 2000.

Abstract

An earlier study reported that human cerebrospinal fluid (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 first (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 FeCl₃. Moreover, FeCl₃ reversed inhibition of C. neoformans growth by CSF. In contrast, activity of DEAE-2 was not reversed by FeCl₃, indicating that inhibition was produced by an iron-independent 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.

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