Microorganisms and culture conditions
R. delemar (99-880)9, R. arrhizus (557969)48 and Cunninghamella bertholletiae (506313)48 have been previously described. Mucor circinelloides (JMRC:NRZ:0774), Rhizomucor pusillus (JMRC:NRZ:0496), Rhizopus microsporus (JMRC:NRZ:0680), A. fumigatus (ATCC, 46645), Aspergillus flavus (JMRC:NRZ:0756), Aspergillus terreus (JMRC:NRZ:0442), C. albicans (JMRC:NRZ:1000), C. glabrata (JMRC:NRZ:1006), C. albicans SC5314 and Fusarium proliferatum (JMRC:NRZ:0657) were obtained from the Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute. All Mucorales isolates were cultured on potato dextrose agar (Becton Dickinson) plates for 7 days at 37 °C. R. delemar M16 is a previously described pyrF-null mutant derived from R. delemar 99-880 that is unable to synthetize uracil49. R. delemar transformed with RNAi targeting mucoricin expression and R. delemar transformed with empty plasmid (control) were derived from strain M16, as previously described9. For the experiments involving these RNAi strains, a synthetic medium containing yeast nitrogen base (YNB, BD) supplemented with a complete supplemental mixture without uracil (CSM−URA, MP Biochemicals) (that is, YNB + CSM−URA) was used.
All of the bacterial isolates used (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus) were clinical isolates obtained from the University Hospital of Heraklion, Crete. All bacteria were streaked onto plates containing LB agar plates from a freshly prepared frozen glycerol stock. After growth on LB agar plates, single colonies were used to inoculate overnight cultures of LB (50 ml at 37 °C). The next day, a volume of 1 ml of each of these cultures was diluted 1:10 to a final volume of 10 ml in LB medium and incubated in conical flasks for 2 h, at 37 °C, with constant shaking, to reach mid-log phase of growth. A volume of 200 μl was taken from each culture and the optical density was measured at 600 nm (OD 600 ). The desired volume from each culture was used and centrifuged at 2,000 rpm for 2–3 min. Bacteria pellets were washed three times with LB medium. Bacteria pellets were diluted in RPMI medium or RPMI plus 4.5 g dl−1 of albumin to the desired OD 600 of 0.2. A volume of 200 μl from each test condition was transferred to a flat-bottom 96-well plate. The plates were incubated at 37 °C, with constant shaking. At regular time intervals (45 min), 10 μl from each culture condition was diluted in RPMI medium to a final volume of 100 μl and the OD 600 was measured spectrophotometrically. The mean value of triplicate measurements of bacterial growth in regular medium at t = 340 min was defined as 100% growth.
For filamentous fungi, the effect of albumin on fungal growth was assessed by counting spore germination rates in RPMI medium compared with RPMI supplemented with 4.5 g dl−1 albumin at 6 h using light microscopy. At least 300 spores were counted per condition in biological triplicates. For the effect of human IgG (i.v. human IgG, Gaminex, 100 mg ml−1, Grifols) fungal spores were incubated in medium containing increasing concentrations of human IgG (from 1.5 g dl−1 to 4.5 g dl−1). At 6 h, spores were assessed by light microscopy and photos were taken.
For Candida isolates, the impact of albumin on growth was evaluated spectrophotometrically. C. albicans strain SC5314 and Candida glabrata (JMRC:NRZ:1006) were streaked from freshly prepared glycerol stocks onto Sabouraud dextrose agar plates and incubated overnight at 37 °C. Single colonies were used to inoculate Sabouraud dextrose broth (50 ml) and liquid cultures were grown overnight at 37 °C with agitation at 150 rpm. The OD 600 of the overnight cultures was determined, and aliquots were diluted with phosphate-buffered saline (PBS) to an OD 600 of 0.1. Cells were collected by centrifugation at 2,000 rpm for 10 min, washed three times with PBS, counted and resuspended in either RPMI medium alone or RPMI supplemented with 4.5 g dl−1 albumin to an OD 600 of 0.1. For each condition, 200 μl of the cell suspension was transferred into wells of a flat-bottom 96-well plate and incubated at 37 °C with continuous shaking. After 8 h, 10 μl from each well was diluted in RPMI to a final volume of 100 μl, and the OD 600 was measured spectrophotometrically. Growth in standard RPMI medium was assessed in triplicate, and the mean OD 600 value at 8 h was defined as 100% growth for normalization.
Human albumin depletion and purification
Albumin was depleted from human serum using an Albumin Depletion Kit (Abcam) according to the manufacturer’s instructions with a modification in the rehydration step of Cibacron Blue 3G-A beads, which was performed using albumin-free serum filtrate (generated through serum filtration using Amicon 50 kDa molecular weight cut-off (MWCO) ultracentrifugal filters; Merck).
For HSA purification, Blue Sepharose 6 Fast Flow (GE Healthcare) was first rehydrated with an albumin-free serum filtrate, incubated with fresh human serum (3 ml) at 4 °C overnight with rotation and then packed back in a column. The first volume (3 ml) of the flow-through contained albumin-depleted human serum. The column was subsequently washed with 7 ml of wash buffer (20 mM Na 2 HPO 4, 20 mM NaH 2 PO 4, pH 8). Albumin was isolated from the column in six consecutive elutions using 7 ml of elution buffer (2 M NaCl, 20 mM Na 2 HPO 4 , pH 8) each time. Elution fractions 2–6 were pooled and further processed for in vitro experiments.
Owing to the increased amount of NaCl in the eluted fractions, dialysis was performed using a CelluSep T2 membrane (Orange Scientific, Cellusep T2 Tubings, 6,000–8,000 MWCO), which was embedded in the appropriate buffer (20 mM Na 2 HPO 4, 20 mM NaH 2 PO 4, pH 8) for 4 h at 4 °C to achieve a physiologically relevant NaCl concentration (150 mM). Elutions were then condensed using Amicon 3 kDa MWCO ultracentrifugal filters (Merck), to a final volume of 2 ml and filter-sterilized through 0.22 μm Spin-X centrifuge tube filters (Costar). The flow-through generated during the condensation procedure was pooled, measured for any remnants of albumin (no traces of albumin were found) and also filter-sterilized using the same 0.22-μm filters.
Chemical modifications of albumin
... continue reading