SRA

Cryptococcus neoformans is an opportunistic basidiomycete pathogen that is a major etiological agent of fungal meningoencephalitis leading to more than 180,000 deaths worldwide annually. For this pathogen, the polysaccharide capsule is a key virulence factor, which interferes with the phagocytosis by host innate immune cells, but its complex signaling networks remain elusive. In this study, we systematically analyzed capsule biosynthesis and signaling networks by using C. neoformans transcription factor (TF) and kinase mutant libraries under diverse capsule-inducing conditions, such as Dulbecco's Modified Eagle's (DME), Littman's medium (LIT) and fetal bovine serum (FBS) medium. We found that deletion of GAT201, YAP1, BZP4, and ADA2 consistently causes capsule production defects in all tested media, indicating that they are capsule-regulating core TFs. Epistatic and expression analysis showed that Yap1 and Ada2 control Gat201 upstream, whereas Bzp4 and Gat201 regulate capsule production independently. We next searched for potential upstream kinases and found that mutants deleted of PKA1, BUD32, POS5, IRE1 or CDC2801 showed reduced capsule production under all three capsule induction conditions, whereas mutants deleted of HOG1 and IRK5 displayed enhanced capsule production. Notably, Pka1 and Irk5 controls induction of GAT201 and BZP4, respectively, under capsule induction condition. Finally, we monitored transcriptome profiles governed by Bzp4, Gat201, and Ada2 under capsule-inducing condition and demonstrated that these TFs regulate redundant and unique sets of downstream target genes. In conclusion, this study provides further insight into the complex regulatory mechanism of capsule production related signaling pathways in C. neoformans. Overall design: Transcriptome patterns of H99S, bzp4?, ada2? and gat201? strains were comparatively analyzed by RNA-sequencing. Cryptococcus neoformans strains were grown at 30°C for 16hr in a YPD medium and transferred to 50 mL of fresh YPD medium and incubated at 30°C until the OD600 reached 0.8. Half of the cells were further incubated in an LIT liquid medium for 2hr. Then the cells were harvested by centrifugation and lyophilized. Total RNAs were prepared as described above and purified with RNeasy Mini Kit (QIAGEN). The concentration was measured by Quant-IT RiboGreen (Invitrogen). The quality of the RNA was verified by the TapeStation RNA screentape (Agilent). Cultured samples were prepared independently three times, and the cDNA library was constructed with the 1 µg of total RNAs for each sample by Illumina TruSeq mRNA library kit (Illumina) and sequenced by the Illumina platform. The adapter sequences were trimmed from the sequencing reads by using Cutadpat v3.4 with Python 3.7.4. The reference genome sequence of C. neoformans H99 and annotation data were downloaded from the NCBI FTP server, derived from Broad Insitute. The reads were aligned to the C. neoformans H99 genome sequence using Hisat2 v2.2.1 with the HISAT and Bowtie2 algorithm and processed as previously reported. Hisat2 was performed with “-p 30” and “– dta -1” options and other parameters set as default. Aligned reads were converted and then sorted by Samtools v1.9 with the “-Sb -@ 8” option for converting, and “-@ 20 –m 2000000000” option for sorting, and the other parameters were set as default. Transcript assembly and abundance estimation were performed using Stringtie v2.1.1 by using the “-p 12” option, and also the “-B” option to run the Ballgown analysis. Assembled transcripts were merged to a single GTF ?le, and the relative transcript abundances were calculated via Fragments Per Kilobase of exon per Million fragments mapped (FPKM). The FPKM and read count matrix were generated by the R package “isoformswitchanalyzerR” and analyzed by DESeq2. Differentially expressed genes (DEG) analysis was performed using DESeq2 v1.24.0 with default sets with the Ballgown. The volcano plot was illustrated by using R v4.1.0 and R package "EnhancedVolcano", with the cutoff (more than two-fold changes with p< 0.05). PCA plot was generated by R package "DeBrowser" and heatmap was also generated by following R package. KEGG pathway analysis was performed using the R package "Cluster profiler", after changing H99S gene locus ID to JEC21 orthologue locus ID.