Were also sensitive to 1-10-phenanthroline. (B) Screen with the effect of pH on proteolytic activity in YNB and DMEM supernatants. Three effectively cleaved IQ substrates have been selected for this evaluation. The activity scale is differs for this experiment due to the fact this assay was conducted on a SpectraMax Gemini plate reader (Molecular Devices) despite the fact that conditions had been otherwise equivalent. Averages and S.D. are shown for triplicates. (PDF) S2 Fig. Reproducibility of MSP-MS assay. (A) YNB media conditioned by wild sort C. neoformans was incubated with the 228-member MSP-MS peptide library for 15, 60, 240, and 1200 minutes. The amount of cleavage web-sites was assessed at each time point, in triplicate. Error bars represent S.D. (B) Overlap of MSP-MS cleavage web sites at the 1200 minute time point, amongst 3 replicates. (C-E) Substrate specificity profile of YNB media conditioned by wild sort C. neoformans, as assessed in 3 technical replicates. (PDF) S3 Fig. Functional categorization and evaluation of secretion mechanism for proteomics benefits. (A) Functional categorization of all 24 proteins predicted to have a secretion signal. Functions have been determined for unannotated proteins by the closest annotated protein following conducting a Blastp search. (B) Evaluation of predicted secretion strategy for all proteins detected in YNB or DMEM conditioned media by proteomics. (PDF) S4 Fig. MSP-MS analysis of secreted peptidase activity in prb1, CNAG_00150, scx1 and cxd1 strains cultured in DMEM. (A) Substrate specificity profiles with the serine peptidase deletion strains prb1 and CNAG_00150 and the carboxypeptidase deletion strains scx1 and cxd1 grown in DMEM, p 0.05. (B) Positional analysis from the bonds cleaved within the 4 deletion strains. (C) Representative example of a peptide cleaved by peptidases in media conditioned by every single of the 4 deletion strains.Ethyl 2-bromothiophene-3-carboxylate site (PDF) S5 Fig.BrettPhos Pd G4 Chemical name MSP-MS evaluation of secreted peptidase activity in cxd2, cxd3 and pep4 strains cultured in YNB media.PMID:23847952 (A) Substrate specificity profiles of the carboxypeptidase deletion strains cxd2 and cxd3 also as the aspartyl peptidase deletion strain pep4 grown in YNB, p 0.05. (B) Positional evaluation from the bonds cleaved in the 4 deletion strains. (C) An example of a representative peptide cleaved by conditioned media from every single deletion strain. (PDF) S6 Fig. IQ-2 is cleaved by May1. (A) Proteolysis of IQ-2 was measured in a fluorogenic assay of YNB supernatants from all peptidase deletion strains. Deletion of MAY1 led to finish loss of cleavage of IQ-2. Columns represent mean S.D. (B) May1 was diluted to 14.six nM in 100 mM MES pH four.five, 100 mM NaCl and incubated with IQ-2. At the get started of your reaction and after 24 hours of incubation at room temperature, samples have been collected and analyzed by Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF). Based on evaluation of its substrate specificity, it was hypothesized that May1 would cleave involving the phenylalanine and leucine in IQ-2. The sodium adduct was observed for the N-terminal fragment of the expected cleavage product, confirming the website of cleavage. (PDF)PLOS Pathogens | DOI:ten.1371/journal.ppat.1006051 December 15,23 /Secreted Peptidases Influence Virulence of C. neoformansS7 Fig. Development curves for all peptidase deletion strains. OD600 measurements have been recorded for cultures grown in triplicate. Averages and S.D. of triplicates are shown. (PDF) S8 Fig. Temperature and pH tolerance of peptidase deletion strains. (A) Two independen.