Fig 1.
Identification of ligand activity and enrichment by ConA.
(A) Silver-stained SDS-PAGE gel of CWE after water wash and sonication. (B) Dectin-2 reporter cells were stimulated with plate-coated CWE treated with or without proteinase K (pro-K), α-Mannosidase (α-M), or β-Mannosidase (β-M). After 18 h, lacZ activity was measured. Data are the mean ± SD of duplicate wells. (C) Flow chart of ligand enrichment and purification. (D) CWE was incubated with ConA resin. Flow-through (FL) and eluate were run on SDS-PAGE gel, silver stained (E) and analyzed for ligand activity. (F) ConA eluate was further separated by size exclusion using a BioLogic LP system (Biorad) and Ultro Gel ACA44 resin (Pall Corporation) at a flow rate of 1 ml/min (blue line represents the trace line of A280 absorption). Fractions were tested by Dectin-2 reporter cells for ligand activity. Fractions 4–6 contained most of the ligand activity and were separated by a second run over the size exclusion column (see S1C Fig).
Fig 2.
Mass spec analysis identified Bl-Eng2 as a Dectin-2 ligand candidate.
(A) The ligand- negative and -positive fractions (#9–13 and #1–7 from S1C Fig, respectively) from the second gel filtration were analyzed by Mass spectrometry. Numbers on the right represent number of peptide specific fragments detected. (B) Domains of native B. dermatitidis Eng2 (Bl-Eng2) and recombinant Bl-Eng2 expressed in Pichia pastoris: SP denotes Signal peptide; GH16 denotes glycosyl hydrolase catalytic domain; Ser/Thr-rich domain harbors 68 potential O-linked glycosylation sites; and Myc and His tags are placed at the C terminus for purification. (C) 0.6 μg Bl-Eng2 and 0.3 μg PDIA1 were run on SDS-PAGE gel under reducing conditions and stained for protein (left) or carbohydrate (right). (D) Monosaccharide composition of Bl-Eng2 measured by gas chromatography (GC). GC chromatogram of the alditol acetate-derivatized monosugars of hydrolyzed Bl-Eng2 (top). Monosaccharides are labeled as follows: Rha—rhamnose, Rib—ribose, Xyl—xylose, Man—mannose, and Glu–glucose. Unlabeled peak at 5.953 min resulted from component degradation during alditol acetate derivatization. Pie diagram shows the relative contribution of monosaccharides (bottom).
Fig 3.
Bl-Eng2 is a bona-fide, superior Dectin-2 ligand.
(A) Pichia-expressed proteins were plate-bound and tested for ligand activity using CLR expressing B3Z reporter cells expressing FcRγ chain, Dectin-2 + FcRγ, MCL + FcRγ, and Mincle + FcRγ, and BWZ cells and a subline expressing Dectin-1-CD3ζ (Dectin-1). (B) Supernatants from murine BMDCs (2 × 105 per well) co-cultured with plate-bound Bl-Eng2 or PDIA1 were analyzed for IL-6 by ELISA. Blastomyces vaccine yeast (4 × 105 per well) was used as positive control. (C) Supernatants from BMDCs (105 per well) co-cultured with 1, 10, or 100 ng and 0.01, 0.1 or 1 pmol plate-bound Bl-Eng2, Man-LAM, Furfurman or MP98 were analyzed for IL-6 by ELISA. Blastomyces vaccine yeast (104, 105 or 106 per well) was used as positive control. Data in A-C represent the mean ± SEM of one representative experiment of 3 independent experiments. (D) Bl-Eng2 induces IL-6 and IL-1β by human PBMCs. Human PBMCs were stimulated with plate-bound Bl-Eng2 for 24h and cytokines in cell culture supernatants were measured by ELISA. Data represent the mean ± SEM of 5 healthy individuals. *, p < 0.05 vs. no Bl-Eng2.
Fig 4.
Bl-Eng2 augments CD4+ T cell development in vivo.
Mice received 106 adoptively transferred naïve 1807 T cells prior to vaccination (A-D) or no transfer (E+F). Mice were subcutaneously vaccinated with 5μg calnexin and 10μg Bl-Eng2 or alum twice, two weeks apart, and then challenged intratracheally with B. dermatitidis 26199 yeast two weeks post-vaccination. At day 4 post-infection, the frequencies of IL-17 and IFN-γ producing 1807 T cells (A) and numbers of activated (CD44+) and cytokine-producing 1807 cells in the lung were enumerated by FACS (B). Almost all of the 1807 T cells recruited to the lung were CD44+. Data represent the average ± SEM of two independent experiments with 8–10 mice/group. *, p < 0.05 vs. control mice vaccinated with calnexin and IFA alone and **, p < 0.05 vs. control mice vaccinated with soluble calnexin alone. Cytokines from lymph node cells stimulated ex vivo with calnexin were measured by ELISA (D). The number indicates the n-fold change of mice vaccinated with calnexin+Bl-Eng2 vs. mice vaccinated with calnexin alone. *, p < vs. all other groups. Lung CFU were counted at day 18 post-infection when naïve mice were moribund, (C+E). *, p < 0.05 vs. all other groups. Numbers reflect the n-fold change in lung CFU of mice vaccinated with calnexin and Bl-Eng2 vs. control mice vaccinated with calnexin or IFA alone. The survival of vaccinated mice was recorded for 30 days post-infection (E). *, p < 0.05 vs. all other groups. At day 4 post-infection, the number of calnexin-specific CD4+ T cells were enumerated by tetramer staining (F). Data represent the average ± SEM of tetramer positive cells from one of two independent experiments with 4–5 mice/group. *, p < 0.05 vs. all other groups. Cnx denotes calnexin.
Fig 5.
Myeloid effector mechanisms by Bl-Eng-2.
Mice received 1807 cells prior to vaccination and were vaccinated and boosted with indicated adjuvants and formulated calnexin. Two weeks after the boost, mice were challenged i.t. with 105 DsRed yeast and lungs were harvested 3 days later. The percentage of dead (DsRed-Uvitex+)(blue) yeast among total neutrophil-associated yeast (all Uvitex+ events)(blue and red together) (see gating strategy in S6A Fig) were analyzed and calculated (dot plots are concatenates from 5 mice/group) to depict the amount of killing by neutrophils (A+B). The percentage of killing by alveolar macrophages is shown in (C). The number of live yeast was depicted by showing the total number of DsRed+ events (D) or plating lung CFU (E). The numbers indicate the n-fold reduction in live yeast (DsRed+ or CFU) vs. the calnexin control groups. *p<0.05 control groups without Bl-Eng2. Cnx denotes calnexin.