T isolates of each and every peptidase deletion strain were spotted in a 10-fold dilution series on YNB agar plates and grown for 48 hours before imaging. (B) pH tolerance of may1 strains after 72 hours of growth. (PDF) S9 Fig. Tolerance to solute, peroxide and cell wall pressure and production of melanin of peptidase deletion strains. (A) 10-fold dilution series of all peptidase deletion strains were spotted on YNB agar plates containing the indicated pressure and grown for 48 hours, except for H2O2 plates, which were grown for four days just before imaging. (B) 10-fold dilution series of peptidase deletion strains grown on wealthy media plates (YPAD) containing 0.02 SDS and imaged soon after four days of development. (C) Melanin production within the presence of L-DOPA. Strains had been spotted in triplicate and photos have been taken following 72 hours of development. (PDF) S10 Fig. Screen of aspartyl peptidase inhibitors. Panels (A), (B) and (C) show the results of every inhibitor compound tested in triplicate at 100M, 10M and 1M.885588-14-7 web The May1 activity against IQ-2 was measured. The average worth and S.D. of triplicates are shown. (D) IC50 values had been calculated for Brecanavir, pepstatin A and compounds 4, 16, 18 and 21. Values are averaged from triplicates and S.D. is shown by error bars. (PDF) S11 Fig. May1 activity in cultures treated with aspartyl peptidase inhibitors. (A) Activity was recorded against the substrate IQ-2. Typical values and S.D. of triplicate measurements are shown. (B) Density at saturation (following 48 hours of development) is shown for YNB cultures of wild kind or may1 C. neoformans treated with May1 inhibitors.89284-85-5 Order Typical values and S.D. of triplicates are shown. (PDF) S12 Fig. Expression of genes neighboring may1. (A-B) Transcript levels for the duration of situations of low (A) or higher (B) cell density in YNB medium, as assessed by RT-qPCR and normalized to 18S rRNA levels. Low density samples have been harvested at a concentration of OD600 = 1.0, and high density samples were harvested soon after 32 hr of development, as in conditioned media experiments. Typical values and S.D. of duplicate samples are shown. (C) Map of MAY1 locus, with indication of region deleted in may1 strains. (PDF) S13 Fig. May1 is essential for C. neoformans accumulation in macrophages. (A) Phagocytic index of opsonized C. neoformans. Error bars represent S.D. (B) Intracellular accumulation of C.PMID:24202965 neoformans in macrophages. p 0.05 versus wild sort handle. Error bars represent 95 self-confidence intervals. (PDF) S1 Table. Sequences of internally quenched fluorogenic substrates. All peptides contain an N-terminal fluorophore: aminomethylcoumarin bound for the side chain of lysine or straight for the N-terminus as indicated, as well as a C-terminal quencher: di-nitrophenol bound to the side chain of lysine or straight towards the C-terminus as indicated. “t” represents tert-butyl glycine andPLOS Pathogens | DOI:10.1371/journal.ppat.1006051 December 15,24 /Secreted Peptidases Impact Virulence of C. neoformans”n” represents norleucine. (XLSX) S2 Table. Pearson correlations amongst of MSP-MS assay outcomes and technical replicates. YNB media conditioned by wild kind C. neoformans was incubated with all the 228-member MSP-MS peptide library in 3 technical replicates. In every single replicate, the frequency of just about every amino acid identified at each and every on the 8 positions surrounding the cleaved bond was assessed. P4-P4′ substrate specificity profiles have been then made and compared using Pearson correlation. Correlation among the substrate specificity profiles of YNB media.