Proteogenomic analysis of pathogenic yeast Cryptococcus neoformans using high resolution mass spectrometry

Clinical Proteomics

Thottethodi Subrahmanya Keshava Prasad²  Harsha Gowda⁴  Raju Ravikumar⁶  Akhilesh Pandey¹  Aditi Chatterjee²  Bipin Nair⁷  Anil K Madugundu⁴  Praveen Kumar⁴  Sneha M Pinto²  Sartaj Ahmad²  Dhanashree S Kelkar⁷  Aneesha Radhakrishnan⁵  Nazia Syed⁵  Tejaswini Subbannayya⁷  Vinuth N Puttamallesh⁴  Babylakshmi Muthusamy³  Raja Sekhar Nirujogi³  Jyothi Embekkat Kaviyil⁶  Lakshmi Dhevi Nagarajha Selvan⁷ 

[1] Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;Manipal University, Madhav Nagar, Manipal 576 104, India;Centre of Excellence in Bioinformatics, School of Life Sciences, Pondicherry University, Puducherry 605 014, India;Institute of Bioinformatics, International Technology Park, Bangalore 560 066, India;Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Puducherry 605 014, India;Department of Neuromicrobiology, National Institute of Mental Health and Neuro Sciences, Bangalore 560 029, India;Amrita School of Biotechnology, Amrita University, Kollam 690 525, India

关键词: Genome annotation;    Computational prediction;    Cryptococcal meningitis;    Antifungal drugs;    Fungal genomics;    Fungal infection;   

Others  :  803061
DOI  :  10.1186/1559-0275-11-5

 received in 2013-04-30, accepted in 2013-12-17,  发布年份 2014

【 摘 要 】

Background

Cryptococcus 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.

Results

We 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.

Conclusions

Proteogenomic 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.

【 授权许可】

   
2014 Nagarajha Selvan et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Mitchell TG, Perfect JR: Cryptococcosis in the era of AIDS–100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev 1995, 8:515-548.
• [2]Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG, Chiller TM: Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 2009, 23:525-530.
• [3]Franzot SP, Salkin IF, Casadevall A: Cryptococcus neoformans var. grubii: separate varietal status for Cryptococcus neoformans serotype A isolates. J Clin Microbiol 1999, 37:838-840.
• [4]Lin X, Heitman J: The biology of the Cryptococcus neoformans species complex. Annu Rev Microbiol 2006, 60:69-105.
• [5]Loftus BJ, Fung E, Roncaglia P, Rowley D, Amedeo P, Bruno D, Vamathevan J, Miranda M, Anderson IJ, Fraser JA, et al.: The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans. Science 2005, 307:1321-1324.
• [6]Broad Institute of Harvard and MIT: Cryptococcus neoformans var. grubii H99 Sequencing Project. http://www.broadinstitute.org/ webcite
• [7]D’Souza CA, Kronstad JW, Taylor G, Warren R, Yuen M, Hu G, Jung WH, Sham A, Kidd SE, Tangen K, et al.: Genome variation in Cryptococcus gattii, an emerging pathogen of immunocompetent hosts. MBio 2011, 2:e00342-00310.
• [8]Steenbergen JN, Casadevall A: Prevalence of Cryptococcus neoformans var. neoformans (Serotype D) and Cryptococcus neoformans var. grubii (Serotype A) isolates in New York City. J Clin Microbiol 2000, 38:1974-1976.
• [9]Banerjee U, Datta K, Casadevall A: Serotype distribution of Cryptococcus neoformans in patients in a tertiary care center in India. Med Mycol 2004, 42:181-186.
• [10]Kupfer DM, Drabenstot SD, Buchanan KL, Lai H, Zhu H, Dyer DW, Roe BA, Murphy JW: Introns and splicing elements of five diverse fungi. Eukaryot Cell 2004, 3:1088-1100.
• [11]Steen BR, Zuyderduyn S, Toffaletti DL, Marra M, Jones SJ, Perfect JR, Kronstad J: Cryptococcus neoformans gene expression during experimental cryptococcal meningitis. Eukaryot Cell 2003, 2:1336-1349.
• [12]Cryptococcus neoformans cDNA sequencing. [http://www.genome.ou.edu/cneo.html webcite]
• [13]Castellana N, Bafna V: Proteogenomics to discover the full coding content of genomes: a computational perspective. J Proteomics 2010, 73:2124-2135.
• [14]Pandey A, Lewitter F: Nucleotide sequence databases: a gold mine for biologists. Trends Biochem Sci 1999, 24:276-280.
• [15]Renuse S, Chaerkady R, Pandey A: Proteogenomics. Proteomics 2011, 11:620-630.
• [16]Khatun J, Yu Y, Wrobel JA, Risk BA, Gunawardena HP, Secrest A, Spitzer WJ, Xie L, Wang L, Chen X, Giddings MC: Whole human genome proteogenomic mapping for ENCODE cell line data: identifying protein-coding regions. BMC Genomics 2013, 14:141. BioMed Central Full Text
• [17]Sevinsky JR, Cargile BJ, Bunger MK, Meng F, Yates NA, Hendrickson RC, Stephenson JL Jr: Whole genome searching with shotgun proteomic data: applications for genome annotation. J Proteome Res 2008, 7:80-88.
• [18]Brunner E, Ahrens CH, Mohanty S, Baetschmann H, Loevenich S, Potthast F, Deutsch EW, Panse C, de Lichtenberg U, Rinner O, et al.: A high-quality catalog of the Drosophila melanogaster proteome. Nat Biotechnol 2007, 25:576-583.
• [19]Merrihew GE, Davis C, Ewing B, Williams G, Kall L, Frewen BE, Noble WS, Green P, Thomas JH, MacCoss MJ: Use of shotgun proteomics for the identification, confirmation, and correction of C. elegans gene annotations. Genome Res 2008, 18:1660-1669.
• [20]Schrimpf SP, Weiss M, Reiter L, Ahrens CH, Jovanovic M, Malmstrom J, Brunner E, Mohanty S, Lercher MJ, Hunziker PE, et al.: Comparative functional analysis of the Caenorhabditis elegans and Drosophila melanogaster proteomes. PLoS Biol 2009, 7:e48.
• [21]Washburn MP, Wolters D, Yates JR 3rd: Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 2001, 19:242-247.
• [22]Oshiro G, Wodicka LM, Washburn MP, Yates JR 3rd, Lockhart DJ, Winzeler EA: Parallel identification of new genes in Saccharomyces cerevisiae. Genome Res 2002, 12:1210-1220.
• [23]Wright JC, Sugden D, Francis-McIntyre S, Riba-Garcia I, Gaskell SJ, Grigoriev IV, Baker SE, Beynon RJ, Hubbard SJ: Exploiting proteomic data for genome annotation and gene model validation in Aspergillus niger. BMC Genomics 2009, 10:61. BioMed Central Full Text
• [24]Kelkar DS, Kumar D, Kumar P, Balakrishnan L, Muthusamy B, Yadav AK, Shrivastava P, Marimuthu A, Anand S, Sundaram H, et al.: Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry. Mol Cell Proteomics 2011, 10:M111 011627.
• [25]de Souza GA, Malen H, Softeland T, Saelensminde G, Prasad S, Jonassen I, Wiker HG: High accuracy mass spectrometry analysis as a tool to verify and improve gene annotation using Mycobacterium tuberculosis as an example. BMC Genomics 2008, 9:316. BioMed Central Full Text
• [26]Prasad TS, Harsha HC, Keerthikumar S, Sekhar NR, Selvan LD, Kumar P, Pinto SM, Muthusamy B, Subbannayya Y, Renuse S, et al.: Proteogenomic analysis of Candida glabrata using high resolution mass spectrometry. J Proteome Res 2012, 11:247-260.
• [27]Maillet I, Berndt P, Malo C, Rodriguez S, Brunisholz RA, Pragai Z, Arnold S, Langen H, Wyss M: From the genome sequence to the proteome and back: evaluation of E. coli genome annotation with a 2-D gel-based proteomics approach. Proteomics 2007, 7:1097-1106.
• [28]Rodrigues ML, Nakayasu ES, Oliveira DL, Nimrichter L, Nosanchuk JD, Almeida IC, Casadevall A: Extracellular vesicles produced by Cryptococcus neoformans contain protein components associated with virulence. Eukaryot Cell 2008, 7:58-67.
• [29]Missall TA, Pusateri ME, Donlin MJ, Chambers KT, Corbett JA, Lodge JK: Posttranslational, translational, and transcriptional responses to nitric oxide stress in Cryptococcus neoformans: implications for virulence. Eukaryot Cell 2006, 5:518-529.
• [30]Li J, Su Z, Ma ZQ, Slebos RJ, Halvey P, Tabb DL, Liebler DC, Pao W, Zhang B: A bioinformatics workflow for variant peptide detection in shotgun proteomics. Mol Cell Proteomics 2011, 10:M110 006536.
• [31]Ansong C, Purvine SO, Adkins JN, Lipton MS, Smith RD: Proteogenomics: needs and roles to be filled by proteomics in genome annotation. Brief Funct Genomic Proteomic 2008, 7:50-62.
• [32]Peri S, Pandey A: A reassessment of the translation initiation codon in vertebrates. Trends Genet 2001, 17:685-687.
• [33]Bonissone S, Gupta N, Romine M, Bradshaw RA, Pevzner PA: N-terminal protein processing: a comparative proteogenomic analysis. Mol Cell Proteomics 2012, 12:14-28.
• [34]Rison SC, Mattow J, Jungblut PR, Stoker NG: Experimental determination of translational starts using peptide mass mapping and tandem mass spectrometry within the proteome of Mycobacterium tuberculosis. Microbiology 2007, 153:521-528.
• [35]Goetze S, Qeli E, Mosimann C, Staes A, Gerrits B, Roschitzki B, Mohanty S, Niederer EM, Laczko E, Timmerman E, et al.: Identification and functional characterization of N-terminally acetylated proteins in Drosophila melanogaster. PLoS Biol 2009, 7:e1000236.
• [36]Helbig AO, Gauci S, Raijmakers R, van Breukelen B, Slijper M, Mohammed S, Heck AJ: Profiling of N-acetylated protein termini provides in-depth insights into the N-terminal nature of the proteome. Mol Cell Proteomics 2010, 9:928-939.
• [37]Saag MS, Graybill RJ, Larsen RA, Pappas PG, Perfect JR, Powderly WG, Sobel JD, Dismukes WE: Practice guidelines for the management of cryptococcal disease. Infectious Diseases Society of America. Clin Infect Dis 2000, 30:710-718.
• [38]Stajich JE, Dietrich FS, Roy SW: Comparative genomic analysis of fungal genomes reveals intron-rich ancestors. Genome Biol 2007, 8:R223. BioMed Central Full Text
• [39]Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 1951, 193:265-275.
• [40]Crestani J, Carvalho PC, Han X, Seixas A, Broetto L, de Saldanha da Gama Fischer J, Staats CC, Schrank A, Yates JR III, Vainstein MH: Proteomic profiling of the influence of iron availability on Cryptococcus gattii. J Proteome Res 2012, 11:189-205.
• [41]Harsha HC, Molina H, Pandey A: Quantitative proteomics using stable isotope labeling with amino acids in cell culture. Nat Protoc 2008, 3:505-516.
• [42]Chaerkady R, Harsha HC, Nalli A, Gucek M, Vivekanandan P, Akhtar J, Cole RN, Simmers J, Schulick RD, Singh S, et al.: A quantitative proteomic approach for identification of potential biomarkers in hepatocellular carcinoma. J Proteome Res 2008, 7:4289-4298.
• [43]Wang Y, Yang F, Gritsenko MA, Clauss T, Liu T, Shen Y, Monroe ME, Lopez-Ferrer D, Reno T, Moore RJ, et al.: Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells. Proteomics 2011, 11:2019-2026.
• [44]Olsen JV, de Godoy LM, Li G, Macek B, Mortensen P, Pesch R, Makarov A, Lange O, Horning S, Mann M: Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 2005, 4:2010-2021.
• [45]Paoletti AC, Parmely TJ, Tomomori-Sato C, Sato S, Zhu D, Conaway RC, Conaway JW, Florens L, Washburn MP: Quantitative proteomic analysis of distinct mammalian mediator complexes using normalized spectral abundance factors. Proc Natl Acad Sci USA 2006, 103:18928-18933.
• [46]Stanke M, Morgenstern B: AUGUSTUS: a web server for gene prediction in eukaryotes that allows user-defined constraints. Nucleic Acids Res 2005, 33:W465-W467.
• [47]Blanco E, Parra G, Guigo R: Using geneid to identify genes. Curr Protoc Bioinformatics 2007, Chapter 4:Unit 4 3.
• [48]Elsik CG, Mackey AJ, Reese JT, Milshina NV, Roos DS, Weinstock GM: Creating a honey bee consensus gene set. Genome Biol 2007, 8:R13. BioMed Central Full Text
• [49]Lukashin AV, Borodovsky M: GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res 1998, 26:1107-1115.
• [50]van Baren MJ, Koebbe BC, Brent MR: Using N-SCAN or TWINSCAN to predict gene structures in genomic DNA sequences. Curr Protoc Bioinformatics 2007, Chapter 4:Unit 4 8.
• [51]Polevoda B, Brown S, Cardillo TS, Rigby S, Sherman F: Yeast N(alpha)-terminal acetyltransferases are associated with ribosomes. J Cell Biochem 2008, 103:492-508.
• [52]Rozen S, Skaletsky H: Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 2000, 132:365-386.
• [53]Yang Z, Pascon RC, Alspaugh A, Cox GM, McCusker JH: Molecular and genetic analysis of the Cryptococcus neoformans MET3 gene and a met3 mutant. Microbiology 2002, 148:2617-2625.