Wang resin

MB (34 amino acid sequence: NH2-CWLCRALIKRIQAMIPKGGRMLPQLVCRLVLRCSCOOH; see Fig. 2B), S-MB (41 amino acid sequence: NH2-FPIPLPYCWLCRALIKRIQAMIPKGGRMLPQLVCRLVLRCS-COOH; see Fig. 2A) and SP-B(1–8) [8 amino acid sequence: NH2-FPIPLPYC-CONH2] were prepared with either a ABI 431A solid phase peptide synthesizer (Applied Biosystems, Foster City, CA) configured for FastMoc™ chemistry [54] (link), a Symphony Multiple Peptide Synthesizer (Protein Technologies, Tucson, AZ) using standard Fmoc synthesis, or a Liberty Microwave Peptide Synthesizer (CEM Corp., Matthews, NC) configured for standard Fmoc synthesis. A low substitution (0.3 mmole/gm) pre-derivatized Fmoc-serine (tBu) Wang resin (NovaBiochem, San Diego, CA) or H-Ser(OtBu)-HMPB Nova PEG resin (NovaBiochem, San Diego, CA) were used to minimize the formation of truncated sequences with the MB and S-MB peptide, while a Rink Amide MBHA resin (NovaBiochem, San Diego, CA) was employed for synthesis of the SP-B(1–8) peptide. All residues were double-coupled to insure optimal yield [48] (link). After synthesis of the respective linear sequences, peptides were cleaved from the resin and deprotected using a mixture of 0.75 gm phenol, 0.25 ml ethanedithiol, 0.5 ml of thioanisole, 0.5 ml of deionized water and 10 ml trifluoroacetic acid per gram of resin initially chilled to 5°C, and then allowed to come to 25°C with continuous stirring over a period of 2 h to insure complete peptide deprotection [48] (link). Crude peptides were removed from the resin by vacuum-assisted filtration, and by washing on a medium porosity sintered glass filter with trifluoroacetic acid and dichloromethane to maximize yield. Filtered crude peptides were precipitated in ice cold tertiary butyl ether, and separated by centrifugation at 2000×g for 10 min (2–3 cycles of ether-precipitation and centrifugation were used to minimize cleavage-deprotection byproducts). Reduced crude peptides from ether-precipitation were verified for molecular mass by MALDI-TOF spectroscopy, dissolved in trifluoroethanol (TFE):10 mM HCl (1∶1, v∶v), freeze dried, and purified by preparative HPLC [48] (link). Final folding of HPLC-purified peptides was facilitated by air-oxidation for at least 48 h at 25°C in TFE and 10 mM ammonium bicarbonate buffer (4∶6, v∶v) at pH 8.0 [55] (link). Final oxidized MB and S-MB were re-purified by reverse phase HPLC, verified in molecular mass via MALDI-TOF, and disulfide connectivity was confirmed by mass spectroscopy of enzyme-digested fragments (trypsin and chymotrypsin digestion).

Cell suspensions were prepared at a concentration 10⁶ cells/mL in parallel in Sabouraud with 15 μM sodium azide and TCM. Serial 1/10 dilutions were made up to 10³ cells/mL. A volume of 170 μL of these suspensions was incubated in a 96 well plate at 37°C overnight with 5% CO₂ without shaking. The plates were observed with a Leica DMI 3000B microscope. Pictures were taken with a Leica DFC 300FX camera using Leica Mycrosystems software. The cell body diameter of thirty to fifty cells was measured with Adobe Photoshop 7.0.
Cryptococcus neoformans was inoculated at 10⁴ and 10⁶ cells/mL in TCM as described above and incubated at 37°C in a 5% CO₂ enriched atmosphere. After 18 h of incubation, the cultures were centrifuged and the supernatants collected to yield Titan-like Cell Supernatant (TCS) and regular cells supernatant (RCS). To evaluate the influence of these supernatants on the titan-like cell formation, C. neoformans cultures inoculated at 10⁴ cells/mL in 96 wells plates were prepared in different growth conditions: 1) Fresh TCM medium (TCM), 2) Supernatant from cultures of titan-like cells (TCS), 3) Supernatant from cultures of cells of regular size (RCS). These conditioned media were mixed with fresh TCM (1:1 proportion v/v). As control, we carried out a culture in which fresh TCM was diluted with the same volume of distilled sterile H₂O. After the inoculation of the different media and mixtures with C. neoformans at 10⁴ and 10⁶ cells/mL and incubation at 37°C in a CO₂ incubator for 18 h, the cell size was measured by microscopy as described above.
Chemical synthesis of the peptides was done by the proteomic facility of the National Centre for Biotechnology (CSIC, Spain) using an Multipep automatic synthesizer (Intavis, Köln, Germany) and Fmoc-Amino Acid Wang resins (Merck, Darmstadt, Germany). After release from the resin, the peptides were purified by reverse-phase chromatography in a semipreparative HPLC system (Jasco, Tokio, Japan) with a C18 Kromaphase column (Scharlab, Barcelona, Spain). The fractions were analyzed by mass spectrometry and lyophilized until their use. We synthesized peptides described in [32 (link)]: Qsp1 (NFGAPGGAYPW), an inactive version of this peptide (NFGAPGAAYPW) and a scrambled Qsp1 peptide (AWAGYFPGPNG). The peptides were dissolved in sterile PBS at 1 mM, and their effect on titan-like cell formation was tested at 30 μM and 15 μM in TCM. The samples were incubated for 18 hours at 37°C with 5% of CO₂ in 96 wells plates. After the incubation period, the cells were observed by optical microscopy and the body cell sizes were measured.