Abstract
BackgroundCryptococcus neoformans, a basidiomycetous fungus of universal occurrence, is a significant opportunistic human pathogen causing meningitis. Owing to an increase in the number of immunosuppressed individuals along with emergence of drug-resistant strains, C. neoformans is gaining importance as a pathogen. Although, whole genome sequencing of three varieties of C. neoformans has been completed recently, no global proteomic studies have yet been reported.ResultsWe performed a comprehensive proteomic analysis of C. neoformans var. grubii (Serotype A), which is the most virulent variety, in order to provide protein-level evidence for computationally predicted gene models and to refine the existing annotations. We confirmed the protein-coding potential of 3,674 genes from a total of 6,980 predicted protein-coding genes. We also identified 4 novel genes and corrected 104 predicted gene models. In addition, our studies led to the correction of translational start site, splice junctions and reading frame used for translation in a number of proteins. Finally, we validated a subset of our novel findings by RT-PCR and sequencing.ConclusionsProteogenomic investigation described here facilitated the validation and refinement of computationally derived gene models in the intron-rich genome of C. neoformans, an important fungal pathogen in humans.
Highlights
Cryptococcus neoformans, a basidiomycetous fungus of universal occurrence, is a significant opportunistic human pathogen causing meningitis
485,714 Tandem mass spectrometry (MS/MS) spectra obtained were searched against three databases, i) protein database containing sequences of 6,980 known/predicted proteins of C. neoformans var. grubii as reported in the genome database hosted by the Broad Institute; ii) six-frame translated genome database; and iii) Nacetylated peptide database
Proteomic evidence for predicted protein-coding genes in C. neoformans var. grubii A search of the MS/MS data against the C. neoformans var. grubii protein database resulted in the identification of 184,030 peptide-spectrum matches (PSMs) that were filtered for first rank assignments and passed a 1% FDR cut-off
Summary
Cryptococcus neoformans, a basidiomycetous fungus of universal occurrence, is a significant opportunistic human pathogen causing meningitis. We used high accuracy mass spectrometry-derived data as a complementary approach to validate and improve the annotation of C. neoformans var. Grubii to identify novel protein-coding regions in its genome in addition to the validation of predicted genes from this genome. We searched MS/MS data against protein database of C. neoformans var. By searching MS/MS data against a six-frame translated genome database, we identified 286 novel peptides with good spectral assignments that were unique to C. neoformans var. By corroborating these peptide sequences with ESTs and comparative genomics data, we identified 4 novel genes and revised the annotation of 104 gene structures in 111 instances. We confirmed the annotated translational start sites of 524 proteins and identified 65 novel splice junctions
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