T4 cell counter

Wild-type (CM026), cmp1Δ, and CMP1C. neoformans strains were grown in YPD broth at 30°C in a shaking incubator (220 rpm) for 24 h, centrifuged, and washed twice in phosphate-buffered saline (PBS). The cells were resuspended in PBS and quantified using a T4 cell counter (Nexcelom). Equal numbers of female and male CD-1 mice (Charles River Laboratories) were infected with approximately 5 × 10⁴ CFU per mouse via intranasal aspiration while under isoflurane anesthesia. Mice were monitored daily and observed for acute and chronic adverse symptoms. Mice were sacrificed on day 14. The brain and left lung were homogenized in 1 ml PBS for 25 s using two steel beads and a Mini-Beadbeater 16 apparatus (Biospec Products). The homogenized tissues were serially diluted, and 100 μl from each dilution was plated onto YPD containing 100 μg/ml chloramphenicol. The plates were incubated for 3 days at 30°C. Colonies were counted, and the tissue burden (CFU per gram of tissue) was determined. Fungal burden data were log₁₀ transformed and evaluated using t tests for unpaired means (Prism software, v9.1.0; GraphPad Software). A P value of ≤0.05 was considered statistically significant.

C. neoformans strains were grown in YPD broth at 30°C in a shaking incubator (220 rpm) for 24 h, centrifuged, and washed twice in phosphate-buffered saline (PBS). Yeast cell counts were determined using a T4 cell counter (Nexcelom). For gene deletion strains, five male, 22 to 24 g CD-1 mice (Charles River Laboratories) were infected with approximately 5 × 10⁴ yeast cells per mouse via oropharyngeal aspiration while under isoflurane anesthesia. For the clinical isolates, 15 male, 22 to 24 g CD-1 mice (Charles River Laboratories) were infected with approximately 5 × 10⁴ yeast cells per mouse via oropharyngeal aspiration while under isoflurane anesthesia. The mice were monitored and weighed daily. Mice with a total body weight loss of ≥20% or exhibiting behavioral, neurological, or respiratory symptoms were sacrificed, following the IACUC guidelines, via CO₂ asphyxiation. The Kaplan-Meier survival plots and analyses (the log-rank test) were completed using Prism software v9.1.0., GraphPad Software. A P value of ≤0.05 was considered to be indicative of a statistically significant result. Outliers were excluded based on a ROUT analysis that was performed using Prism software v9.1.0., GraphPad Software.