Article Figures & Data
Figures
- FIG 1
Experimental infection of the natural host of Arthroderma benhamiae. Cutaneously infected guinea pigs developed skin symptoms that were the most severe at 14 days postinfection (dpi) due to inflammation, while 8 dpi was the time point for the peak of infection.
- FIG 2
Prediction and manual correction of the gene coding for the autophagy protein Atg27 (ARB_01857; a transmembrane protein). (A) Original gene prediction; (B) automatic prediction from Augustus (signal peptide is missing); (C) final (new) gene prediction after manual correction. The reannotation of this particular gene is remarkable, as it produced a new intron, an alternative stop codon, and a manually corrected start codon.
- FIG 3
Characterization of the secretome. (A) Pie chart showing the main functional groups identified within the 457 proteins of the secretome. See detailed description in Data Set S1 in the supplemental material. (B) Pie charts showing the same functional groups as in panel A but within the 100 most expressed genes in Gp8 (in vivo 8 days postinfection), K (in vitro in keratin medium), and S (in vitro in soy medium). (C) Venn diagram of proteases (top) and carbohydrate/cell wall metabolism proteins (bottom) present in the 100 most expressed secreted proteins under the 3 conditions described for panel B. Proteases represent about 20% of the 100 most expressed proteins under the 3 conditions; however, the batch of proteins in Gp8 is clearly different from those in K and S. This trend is not as significant when comparing carbohydrate/cell wall metabolism proteins.
- FIG 4
Hierarchical clustering (A and C) and principal component (PC) analysis (B and D) of RNA sequencing samples considering the genes from the complete genome (A and B) or only the secretome subset (C and D). The sample names reflect the growth conditions: Cb, in vivo in guinea pig; S, in vitro in soy medium; Sa, in vitro in Sabouraud medium; K, in vitro in keratin medium. The in vivo samples cluster together.
- FIG 5
Number of differentially expressed genes versus the enumeration of all possible contrasting conditions in the genome and the secretome, using a cutoff of 1e−3 for FDR and 2 for the fold change.
- FIG 6
(A) The twenty-five most highly expressed genes encoding secreted proteins during infection compared to in vitro expression. (B) The twenty-five most highly expressed genes encoding secreted proteins in vitro (keratin medium) compared to in vivo expression. Abbreviations are as defined in the Fig. 4 legend.
Tables
- TABLE 1
RNA-seq data summarya
Library Total no. of cleaned reads (M) Reads aligned with organism: A. benhamiae Cavia porcellus No. of reads % No. of reads (M) % 8 dpi 34.5 0.5 M 1.5 31.8 92.3 31.7 1 M 3.3 28.9 91.0 26 1 M 4 23.5 90.6 14 dpi 31.8 44.5 K 0.1 30 94.3 30.6 51.2 K 0.2 28.9 94.4 31.4 24.8 K 0.1 29.5 93.8 27 dpi 33.8 623 0 31.6 93.5 39 657 0 36.6 94.0 30.8 452 0 28.8 93.3 44 dpi 35.7 458 0 33.1 92.9 31.9 857 0 29.6 92.7 25.3 808 0 23.5 92.8 Control 26.1 637 0 24.3 93.4 38.9 840 0 36.3 93.3 35.7 3,143 0 33.2 93.0 Keratin 12.4 6.1 M 49.2 13.5 7.9 M 58.3 13.9 8 M 57.6 Soy 11.7 7.3 M 62.6 10.5 6 M 57.1 12.8 7.5 M 58.8 Sabouraud 12.4 7.9 M 63.5 14.8 8.7 M 59.1 11.6 7.2 M 61.6 ↵a M, million; K, thousand; dpi, days postinfection.
- TABLE 2
Comparison of new gene set and original onea
New versus old gene prediction Gene count in complete genome Gene count in secretome only With GPI Without GPI Auto Manual Auto Manual Matched 2,662 47 (13) 2 (2) 155 (55) 0 Alternative 1,246 19 (6) 0 49 (19) 1 Different 2,752 31 (6) 1 83 (19) 10 (4) Merged 286 5 (2) 0 7 (2) 1 Split 76 1 (1) 0 5 (2) 0 New 383 6 0 34 (8) 0 Total 7,405 109 (28) 3 (2) 333 (105) 12 (4) ↵a Matched, identical old and new gene annotations; alternative, conserved start and stop codons but different splicing; different, different start or stop codons, possibly different splicing; merged, more than one old gene merged into a single new one; split, old gene split into several new ones; new, genes found only in the new predictions (708 original genes were lost); auto, gene annotations as produced by Augustus; manual, manual correction of the start codon. The number of genes whose products were confirmed by mass spectrometry in culture supernatants is given in parentheses. GPI, glycosylphosphatidylinositol.
- TABLE 3
Designation of samples and growth conditions
RNA sample Growth condition Code Description Cb1 Gp8 In vivo: guinea pig 8 days postinfection Cb2 Cb3 Cb4 Gp14 In vivo: guinea pig 14 days postinfection Cb5 Cb6 K1 K In vitro: keratin medium K2 K3 S1 S In vitro: soy medium S2 S4 Sa1 Sa In vitro: Sabouraud medium Sa2 Sa3
Table S1
The secretome: predicted cell surface/secreted proteins, putative functions, and expression. 1Open reading frame (ORF) names in this study. 2The status indicates the changes between previous proteome annotation and our prediction. 3ORF names in the previous genome annotation (Burmester et al., 2011). 4Names attributed to some proteases by Burmester et al. (2011) and Sriranganadane et al. (2011). 5UniProt accession numbers corresponding to the previous prediction. When two ORFs have been merged in the new prediction and both are present in UniProtKB, the two corresponding ACs are indicated. 6The presence of a signal peptide is indicated by SIG. SIG+GPI indicates that the gene product is predicted to have a GPI anchor. 7Mass spectrometry data were extracted from the work of Sriranganadane et al. (2011). For each identified gene product, we indicate the medium pH in which it was detected (either pH 4 or 7). 8Function has been assigned based on homology search in well-characterized fungi and/or from InterPro scanning to identify specific domains and families. Green identifies proteins with a potential role in proteolytic activity; red, proteins involved in carbohydrate metabolism; and orange, proteins involved in lipid metabolism. 9Homologous fungal allergens extracted from the Allergome database (http://www.allergome.org/). 10Name of weighted gene correlation network analysis (WGCNA) gene coexpression module. 11Summary of differential gene expression in vivo versus in vitro. Cutoffs: FDR = 1e−3 and 2-fold change. 12Significant expression trends from RNA sequencing data are indicated. The cutoff of −1 for the Z-score of transcripts per million was applied. 13Mean expression values expressed in transcripts per million for every growth condition. The detailed counts per sample are given in Table S3. Download Table S1, XLSX file, 0.1 MB.
Copyright © 2016 Tran et al.
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Data Set S1
Supplemental materials and results. Download Data Set S1, PDF file, 0.2 MB.
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Table S2
Potential allergens based on sequence homology. 1Open reading frame (ORF) names in this study that have homologs acting as allergens in other fungi (and wasp in the case of ARB_02861). 2Allergens were retrieved from the Allergome database (http://www.allergome.org/). 3The (+) indicates A. benhamiae allergen homologs identified as encoding putative cell surface/secreted proteins in our study. 4Function has been assigned based on homology search in well-characterized fungi and/or from InterPro scanning to identify specific domains and families. Download Table S2, XLSX file, 0.1 MB.
Copyright © 2016 Tran et al.
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Figure S1
Manual curation on SUB10 and SED3. Actual and previous open reading frame predictions for SUB10 (A) and SED3 (B). N-terminal signal peptides, propeptides, and peptidase domains are illustrated with boxes of different hues. The corrections of assembly errors in actual open reading frames are indicated in red. Full ORFs were restored by the manual addition of the missing T in the SUB10 DNA sequence and G in the SED3 DNA sequence. These corrections have been confirmed by Sanger resequencing. The actual protein sequences of SUB10 and SED3 are available on the UniProtKB database (http://www.uniprot.org/) with respective accession numbers D4AQG0 and D4AK75. Download Figure S1, PDF file, 0.1 MB.
Copyright © 2016 Tran et al.
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Table S3
Detailed counts per sample, differential expression, and weighted gene correlation network analysis module attribution. Download Table S3, XLSX file, 1.4 MB.
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Figure S2
Module-contrast correlations. Download Figure S2, PDF file, 0.1 MB.
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Figure S3
Heat map of the turquoise module (A), blue module (B), tan module (C), midnight blue module (D), and yellow module (E). Download Figure S3, PDF file, 1.1 MB.
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Table S4
Pathway enrichment analysis of weighted gene correlation network analysis modules. Only the most significant gene ontology terms are reported. Download Table S4, XLSX file, 0.1 MB.
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Figure S4
The twelve most highly expressed genes encoding secreted proteases during infection (left table) and during in vitro growth in keratin medium (right table). Download Figure S4, PDF file, 0.1 MB.
Copyright © 2016 Tran et al.
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Figure S5
Quality of RNA extracted using the Qiagen RNA extraction kit (A) and the protocol described in Materials and Methods (B). The 18S rRNA and 28S rRNA absorbance peaks are shown in purple and blue, respectively. Download Figure S5, PDF file, 0.4 MB.
Copyright © 2016 Tran et al.
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