Study of the role of iron in the anticryptococcal activity of human serum and fluconazole

Grover, Devina D.; Brummer, Elmer; Stevens, David A.

doi:10.1007/bf00439116

Cited by 11 publications

(2 citation statements)

References 6 publications

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“…There were some indications in the literature that the C. neoformans-inhibitory activity of serum was due to the iron binding capacity of serum (9,10,17). Reversal by FeCl 3 is demonstrable with freshly prepared (3, 5) but not stored (9) reagent, presumably due to absorption, reduction, or complexing of ferric ion in stored material.…”

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Characterization of an Anticryptococcal Protein Isolated from Human Serum

et al. 2000

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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%...

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Characterization of an Anticryptococcal Protein Isolated from Human Serum

et al. 2000

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“…In these studies we found human serum (10%) in RPMI‐1640 inhibited growth of C. albicans and this inhibition is completely reversed by iron overload (10 μ m FeCl 3 ). Grover et al reported previously [6] that inhibition of C. neoformans growth by 10% human serum was not reversed by 10 μ m FeCl 3 , though partial reversal was noted in some present experiments performed concurrently with CSF experiments. There was also at least partial reversal, by iron overload, of inhibition of C. neoformans and C. albicans growth by 50% CSF in preliminary studies, a point currently under more extensive study.…”

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Antifungal activity of cerebrospinal fluid against Cryptococcus neoformans and Candida species

et al. 2008

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The effect of human cerebrospinal fluid (CSF) on the growth of Cryptococcus neoformans and Candida species was tested in RPMI‐1640. CSF alone was highly fungistatic for both yeasts and inhibited growth in a concentration‐dependent manner. Unlike human serum, CSF did not collaborate with fluconazole for killing C. neoformans. Molecular sieve fractionation of CSF on a G‐200 Sephadex column yielded a highly antifungal fraction with a molecular weight around 66 kDa. On SDS‐PAGE this fraction migrated as a major and a minor band corresponding to the mobility of bovine serum albumin. These novel findings suggest that CSF contains a factor(s) that provides resistance to the growth of C. neoformans or Candida species.

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Malassezia Species and Immunity: Host–Pathogen Interactions

Ashbee

Bond²

2010

Malassezia and the Skin

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The Infectious Diseases Data Observatory (IDDO, https://www.iddo.org) has launched a clinical data platform for the collation, curation, standardisation and reuse of individual participant data (IPD) on treatments for two of the most globally important neglected tropical diseases (NTDs), schistosomiasis (SCH) and soiltransmitted helminthiases (STHs). This initiative aims to harness the power of data-sharing by facilitating collaborative joint analyses of pooled datasets to generate robust evidence on the efficacy and safety of anthelminthic treatment regimens. A crucial component of this endeavour has been the development of a Research Agenda to

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Study of the role of iron in the anticryptococcal activity of human serum and fluconazole

Cited by 11 publications

References 6 publications

Characterization of an Anticryptococcal Protein Isolated from Human Serum

Characterization of an Anticryptococcal Protein Isolated from Human Serum

Antifungal activity of cerebrospinal fluid against Cryptococcus neoformans and Candida species

Malassezia Species and Immunity: Host–Pathogen Interactions

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