female balb c mice  (Jackson Laboratory)

Journal: iScience

Article Title: Systematic infectome–phenome profiling reveals cryptococcal infection-associated proteins driving immune system remodeling and immunization potential

doi: 10.1016/j.isci.2026.116263

Figure Lengend Snippet: Systematic phenome profiling of C. neoformans infection-associated candidates (A) Capsule production of infection-associated mutant strains. Strains grown in iron-limited capsule-inducing media to mid-log phase (∼24 h), visualized by India Ink staining with differential interference (DIC) microscopy. Representative images of mutant strains are shown. Scale bars, 4.6 μm. A minimum of 50 cells were measured for capsule and cell size assays across three biological replicates. (B) Normalized percentage of melanin (pigment) produced by mutant strains. Experiment performed in biological duplicate and technical triplicate. Representative melanized colonies are shown in the figure below figure. (C) Normalized 37°C growth profiles obtained from area under the curve (AUC) measurements of mutant strain growth curves in YNB. Representative spot dilution plates are shown in the figure below. Experiment performed in biological triplicate or quadruplicate and technical duplicate. (D) Normalized percentage of host cell death by the quantification of lactate dehydrogenase (LDH) release from BALB/c macrophages at 27 hpi following co-culture with infection-associated mutant fungal strains. Mutant strains opsonized by monoclonal 18b7 antibody prior to co-culture. Experiment completed in biological triplicate and technical duplicate or triplicate. (E) Phenome heatmap of infection-associated mutant fungal strains. Phenome scores generated by normalization against WT based on quantitative or semi-quantitative measurements. (F) Categorization of candidates as ‘antifungal’ based on defect across evaluated criteria. Red and blue schemes represent enhanced and reduced phenotype scores, respectively. Data are represented as mean ± SEM. Significance determined by Student’s t test: p < 0.05, ∗; p ≤ 0.01, ∗∗; p ≤ 0.005, ∗∗∗; p ≤ 0.0001, ∗∗∗∗. Phenotypic assays performed with two independent mutant strains; figure generated with a representative single independent mutant strain.

Article Snippet: Six female BALB/c elite mice aged 7–9 weeks old (Charles River Laboratories, ON, Canada) were used for each group within the immunization experiment.

Techniques: Infection, Mutagenesis, Staining, Microscopy, Produced, Co-Culture Assay, Generated

Journal: iScience

Article Title: Systematic infectome–phenome profiling reveals cryptococcal infection-associated proteins driving immune system remodeling and immunization potential

doi: 10.1016/j.isci.2026.116263

Figure Lengend Snippet: Evaluation and characterization of antifungal targets (A) Survival curve for the murine model of cryptococcosis performed with WT and two independent mutants of CNAG_05997 Δ. (B) Fungal dissemination profiles assessed by the quantification of fungal burden from lung, brain, and spleen for CNAG_05997 Δ. (C) Survival curve for murine model of cryptococcosis performed with WT and two independent mutants of cipCΔ . (D) Fungal dissemination profiles assessed by the quantification of fungal burden from lung, brain, and spleen for cipC Δ. Experimental groups of mouse survival and fungal dissemination assays following inhalation model of cryptococcosis completed with n = 10 female BALB/c mice. Differences in survival were statistically tested using a log rank (Mantel-Cox) test (∗, p ≤ 0.05; ∗∗∗, p ≤ 0.0001). Differences in fungal burden statistically tested using Student’s t test (∗, p < 0.01; ∗∗∗∗, p ≤ 0.0001). (E) Cell wall and membrane stress susceptibility of cipC Δ growth on YPD supplemented with caffeine (1 mg/mL), SDS 0.01%, amphotericin B (2 μg/mL), and fluconazole (8 μg/mL), compared to YPD. (F) Osmotic and oxidative stress susceptibility of cipC Δ growth on YPD supplemented with H 2 O 2 (5 mM) and KCl (1.5 M) compared to YPD. Serial dilutions of strains were spotted on YPD supplemented with stressor and incubated at 30°C for 2–5 days. Experiment completed in biological duplicates and a technical duplicate. (G) Microdilution assay of amphotericin B sensitivity to strains at concentrations of 0–2.9 μg/mL and fluconazole sensitivity to strains at concentrations of 0–7.6 μg/mL. Cell density was measured at OD 600nm and represented as a percentage of the growth of the untreated control, corresponding to the percentage of growth labeled in each individual heatmap cell. Experiment completed in biological quadruplicate and technical duplicate. (H) CipC localization during C. neoformans infection of macrophages with cipC Δ::CIPC-1x FLAG strain detected during 3 h co-culture (MOI 100). Images captured with DAPI and Alexa Fluor 488 (FLAG). Scale bars, 5.1 μm. (I) Engulfed fungal cells were quantified by co-culturing macrophages with C. neoformans strains for 3 h at an MOI of 10. Phagocytosed cells were enumerated for CFUs following PBS washes of infected macrophages. Intracellular fungal burden was quantified following initial 3 h co-culture, and PBS washes to remove extracellular and non-adhered cells, followed by maintenance in fresh DMEM. At 12 and 24 hpi, lysed macrophages were plated for CFUs. Data are represented as mean ± SEM. Statistical analysis using Student’s t test (∗, p ≤ 0.05; ∗∗, p ≤ 0.01). Experiment completed in biological triplicate. (J) Normalized LFQ intensities (CipC-FLAG vs. WT) of host 20S proteasome and immunoproteasome subunits identified from CipC-1xFLAG interactome. Co-immunoprecipitation experiment completed in biological quadruplicate.

Article Snippet: Six female BALB/c elite mice aged 7–9 weeks old (Charles River Laboratories, ON, Canada) were used for each group within the immunization experiment.

Techniques: Membrane, Incubation, Microdilution Assay, Control, Labeling, Infection, Co-Culture Assay, Immunoprecipitation

Journal: iScience

Article Title: Systematic infectome–phenome profiling reveals cryptococcal infection-associated proteins driving immune system remodeling and immunization potential

doi: 10.1016/j.isci.2026.116263

Figure Lengend Snippet: cipC Δ-derived EVs feature altered sterol and proteome profiles (A) EV production from C. neoformans strains on YPD solid agar at 30°C; sterol quantity expressed per 10 9 cells for each strain. Statistical analysis using Student’s t test (∗∗, p ≤ 0.01). (B) Host cell cytotoxicity following treatment with 10 μg of WT and cipC Δ-derived EVs compared to PBS control. Treatment applied to BALB/c immortalized macrophages in serum-free DMEM for 3 h at 37°C and 5% CO 2 , followed by the quantification of LDH release. (C) During co-culture of C. neoformans WT with macrophages (MOI 10), 10 μg EV (WT and cipC Δ-derived) were added at the same time as fungal cells in serum-free DMEM. Phagocytosed fungal cells were quantified by CFUs at the initial 3 h co-culture and PBS washes, followed by maintenance in fresh DMEM. (D) At 24 h.p.i, lysed macrophages were plated for CFU quantification to assess the effect of initial EV exposure on long-term infection dynamics. Statistical analysis using Student’s t test (∗, p ≤ 0.05). (E) Total protein content of purified EVs normalized to sterol content, representative of the amount of EV molecules per purification. Statistical significance tested using Student’s t test, ns = not significant. Experiment completed in biological triplicate and technical duplicate. Values shown are representative of technical replicates from one independent assay. (F) Principal component analysis for WT- and cipC Δ-derived EVs. (G) Volcano plot compares EV-associated proteins between WT and cipC Δ strains; Venn diagram depicts the number of significantly different proteins identified within each condition. Student’s t test, p < 0.05; FDR = 0.05; S 0 = 1. (H) Distribution of GOCC terms between significantly different EV-associated proteins with significant increases or decreases between WT and cipC Δ origin strain is shown. (I) Antigenicity score of significantly different EV-associated proteins from the corresponding origin strain classified with the GOCC term ‘membrane’ or ‘secreted’. Scores calculated using VaxiJen 2.0 Antigen Predictor. Data are represented as mean ± SEM. Statistical analysis of antigenicity scores completed using Student’s t test (∗, p ≤ 0.05). Proteomics experiment performed in biological quadruplicate.

Article Snippet: Six female BALB/c elite mice aged 7–9 weeks old (Charles River Laboratories, ON, Canada) were used for each group within the immunization experiment.

Techniques: Derivative Assay, Control, Co-Culture Assay, Infection, Purification, Membrane

Journal: iScience

Article Title: Systematic infectome–phenome profiling reveals cryptococcal infection-associated proteins driving immune system remodeling and immunization potential

doi: 10.1016/j.isci.2026.116263

Figure Lengend Snippet: Immunization with cipC Δ-derived EVs leads to increased serological reaction (A) Experimental design of vaccination regimen and H99 challenge. Female BALB/c mice ( n = 6 per group) were immunized by intraperitoneal injections of 10 μg EV isolated from WT or cipC Δ strain in 100 μL of PBS. Control mice were injected with 100 μL PBS, followed by the intranasal inoculation of C. neoformans WT. (B) Western blot of WT-derived EVs probed by sera from nonimmunized and immunized mice. (C) Quantification of serological response to EVs across vaccination regimes. Net protein values obtained from the quantification of protein bands in western blot analysis using ImageJ software. Experiment completed in technical triplicate. Statistical analysis using Student’s t test (∗∗, p ≤ 0.005; ∗∗∗, p ≤ 0.0005; ∗∗∗∗, p ≤ 0.0001). (D) Survival rates of vaccinated animals with either WT- or cipC Δ-derived EVs and unvaccinated animals. (E) Lung weights of vaccinated and unvaccinated mice infected with C. neoformans WT. (F) Spleen weights of vaccinated and unvaccinated mice infected with C. neoformans WT. Statistical analysis using Student’s t test (∗, p ≤ 0.05). (G) Fungal burden from lung, bronchoalveolar lavage, brain, spleen, and liver was determined from homogenized tissues and quantification of CFUs. Statistical analysis using Student’s t test (∗∗, p ≤ 0.005). (H) Cross-reactivity of serological reaction of immunized mice to other major human fungal pathogens, including a C. neoformans clinical isolate of strain H99 serotype A, Aspergillus fumigatus ATCC 204305, Candida glabrata BG87, and C. albicans SC3514. Experiment completed in technical duplicate. Data are represented as mean ± SEM.

Article Snippet: Six female BALB/c elite mice aged 7–9 weeks old (Charles River Laboratories, ON, Canada) were used for each group within the immunization experiment.

Techniques: Derivative Assay, Isolation, Control, Injection, Western Blot, Software, Infection