Skin peptides collection and anti-Bd growth inhibition assays
Skin secretions of two L. nigriventer individuals were collected from two sterile polyethylene bags (Whirl-Pak, Nasco) in which they were kept prior to measurement, by washing the bags with ~50 ml of sterile water. Samples were then frozen until further processing. After defrosting, 2 ml of the skin secretion wash was removed for direct testing against Bd. The remaining volume from each sample was acidified to a final volume of 1% HCL to inactivate potential endogenous peptidases (Resnick et al., 1991; Steinborner et al., 1997) and immediately loaded onto C-18 Sep-Pak cartridges (Waters Corporation, Milford, Massachusetts, USA) which were then stored in vials with 2–5 ml of 0.1% HCL until further processing.
We eluted the peptides bound to the Sep-Paks with 70% acetonitrile, 29.9% water, and 0.1% trifluoracetic acid (v/v/v) and centrifuged them under vacuum to concentrate them to dryness. After Sep-Pak purification, we determined the total concentration of the recovered skin peptides by Micro BCA Assay (Pierce, Rockford, Illinois, USA) following manufacturer’s instructions, except that we used bradykinin (RPPGFSPFR; Sigma) to establish a standard curve (Rollins-Smith et al., 2002). Skin peptides were analysed by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI MS) as described in Pask et al. (2012). We analysed each sample by averaging signals from 250 consecutive laser shots. Mass spectrometry data was acquired in the mass/charge (m/z) range 500 to 7,000, truncated at m/z 4,000 and baseline-corrected with Data Explorer v4.4 (Applied Biosystems). The peak values shown represent the monoisotopic mass, [M+H]+. A few signals may show secondary peaks 22 mass units greater than the primary peak and probably represent a peptide plus sodium adduct [M+Na]+. Spectra may also show peaks at m/z 568.1 and 650.0 because of matrix or background signal.
We conducted in vitro growth inhibition assays of the enriched skin peptide mixtures against two Bd isolates (JEL 197 and ‘Section Line’) (Longcore et al., 1999; Piovia-Scott et al., 2015) as described previously (Rollins-Smith et al., 2006; Ramsey et al., 2010; Holden et al., 2015). Briefly, B. dendrobatidis zoospores were grown on 1% tryptone agar for one week at 23°C. Freshly isolated zoospores were added (5 × 104/50 µl, 5 replicates) in tryptone broth to a 96-well flat-bottom microtiter plates with 50 µl of a serially diluted mixture of skin peptides dissolved in HPLC-grade water. Positive control wells contained zoospores and 50 µl HPLC water. Negative control wells contained heat-killed zoospores (60°C for 10 minutes) and 50 µl of HPLC water. Plates were incubated at 23°C for one week, and growth was measured as an increased optical density at 490 nm (OD490 ) using an MRX Microplate Reader (Dynex Technologies, Inc., Chantilly, VA, USA). Percent growth was calculated as follows: OD at Day 7 – OD at Day 0 for test sample / OD at Day 7 – OD at Day 0 for positive control.
We performed Bd growth inhibition assays with the direct skin secretion solution following the methods explained above, with 50 µl of the direct skin secretion solution being added to the microtiter plate instead of the peptide dilutions. The assays were completed with the isolate Bd VMV 813.