Pleiotropic functions of SscA on the asexual spore of the human pathogenic fungus Aspergillus fumigatus

Figures & data

Table 1. Aspergillus fumigatus strains used in this study.

Strain name	Relevant genotype	References
AF293	A. fumigatus wild type	Brookman and Denning (2000)
AF293.1	pyrG1	Xue et al. (2004)
TYE53.1-3	pyrG1; ΔsscA::AnipyrG^+	This study
TYE61.1	pyrG1; ΔsscA::AnipyrG^+; sscA(p)::sscA::FLAG4x::ptrA^R	This study

Table 2. Oligonucleotides used in this study.

Name	Sequence (5′ → 3′)	Purpose
OHS2008	TCATCCATGGTGTCCTCGTC	AnipyrG_Marker_F
OHS2009	CTGGAATCAGTGGAGCGAAC	AnipyrG_Marker_R
OHS1724	CCTGCGACCAGATCTCTCC	5′ sscA DF
OHS1725	TTTGTAGGCTTTGGGCTGTTCACAA CTTCCGCTCTAGGAAGCGAC	3′ sscA with AnipyrG tail
OHS1726	CTGATCTACCCCTTGGAACGCAGCA GCAGTCCACAGCTTTCGTATG	5′ sscA with AnipyrG tail
OHS1727	AGCAACCAAGGTCAGGGT	3′ sscA DR
OHS1728	ATCCTGCAGTCTCGACTCC	5′ sscA NF
OHS1729	CCTATGACGTTGTCGACGAGA	3′ sscA NR
OHS1730	TCCTCACTTAGCTTCGGCAA	5′ sscA RT_F
OHS1731	GATGCAAAGGGATTCGCACT	3′ sscA RT_R
OHS1826	AATT CTGCAG GCTCCATCCGTTCCTTGTAGG	5′ sscA with promoter and PstI
OHS1827	AATT CTGCAG CGTGGCGACTGCAACAAG	3′ sscA with PstI
OHS2512	TCTGGGTGCTTTCTGGTTCT	5′ fksA RT_F
OHS2513	GCGGACCAAATCCTGTAACC	3′ fksA RT_R
OHS2514	CTGACTGTCCCTAGCCTGAC	5′ gelA RT_F
OHS2515	GACGGTGGACAGGAGTGTAA	3′ gelA RT_R
OHS2792	GTCGCGTTACAGAACGACAA	5′ chsA RT_F
OHS2793	CCAGGTACATGTTGGCTGTG	3′ chsA RT_R
OHS2794	GGGTACCAAGGGAGACAACA	5′ chsB RT_F
OHS2795	AGCCACTGTCAATGTCAAGC	3′ chsB RT_R
OHS2796	GGCAACTTCCTCTCCTCCTT	5′ chsC RT_F
OHS2797	TGCCAAGGGTCCATGTAGAG	3′ chsC RT_R
OHS2798	TGGATAGCGCTCGTTTACCT	5′ chsE RT_F
OHS2799	TCGCACATCTTTGCGCTTTA	3′ chsE RT_R
OHS2800	CGCCCTTCTATGAGACCAGT	5′ gliT RT_F
OHS2801	GCCAAAGATCCCATCGACAC	3′ gliT RT_R
OHS2802	GATTCTCGAGGCGTATGTGG	5′ gliA RT_F
OHS2803	AGACAACAAGGTCGCAATCC	3′ gliA RT_R
OHS2806	TCACAACCACCTATGGCAGT	5′ rglT RT_F
OHS2807	CTCGACGGTAATGTGAACGG	3′ rglT RT_R

Tail sequence is in italic.

Figure 1. In silico analysis of SscA in Aspergillus species. a phylogenetic tree of SscA homologues identified in Aspergillus species. Protein sequences were obtained from the NCBI database, and alignment was performed with clustal W. The tree was generated via MEGA X to examine SscA homologues. The tree based on the neighbour-joining method was replicated 1,000 times, and the scale represented the number of substitutions per site.

Figure 2. Functions of SscA in Aspergillus fumigatus development. (a) Photographs of colonies of WT, ΔsscA and Cʹ sscA strains point-inoculated onto solid MMY and grown at 37 °C for 4 days. (b) Quantitative analysis of fungal growth of strains shown in (a); error bars indicate the standard error of the mean in three biological replicates (**p < 0.01). (c) Quantitative analysis of asexual spore formation of the strains shown in (a); error bars indicate the standard error of the mean in three biological replicates (**p < 0.01).

Figure 3. Roles of SscA in Aspergillus fumigatus conidia. (a) The relative survival rates of WT, ΔsscA and Cʹ sscA conidia grown for 2-, 15-, or 30-days (**p < 0.01, *p < 0.05). (b–d) The relative conidial survival rates of the designated strains treated with thermal stress (55 °C) for 30 or 60 min (b), oxidative stress (0.05 mol/L H₂O₂) for 30 min (c), or UV stress (50 J/m²) (d); error bars indicate the standard error of the mean in three biological replicates (**p < 0.01, *p < 0.05).

Figure 4. Transcriptomic analyses of ΔsscA conidia. (a) Heatmap showing DEGs between WT and ΔsscA conidia. (b) GO analyses of upregulated DEGs (left; 1,291 genes) and downregulated (right; 865 genes) DEGs.

Figure 5. Effect of SscA on key conidial wall components in Aspergillus fumigatus. (a) Heatmap showing the DEGs among β-glucan biosynthetic genes in WT and ΔsscA conidia. (b) The relative mRNA expression levels of major β-glucan biosynthetic genes (fksA and gelA) in WT, ΔsscA, and Cʹ sscA conidia as assessed by RT-qPCR (*p < 0.05). (c) Amount of β-glucan in conidia of WT, ΔsscA, and Cʹ sscA strains (**p < 0.01). (d) Heatmap showing the DEGs among chitin biosynthetic genes in WT and ΔsscA conidia. (e) The relative mRNA expression levels of four chitin biosynthetic genes (chsA, chsB, chsC, and chsE) in WT, ΔsscA, and Cʹ sscA conidia as assessed by RT-PCR (*p < 0.05). (f) Amount of chitin in conidia of WT, ΔsscA, and Cʹ sscA strains (***p < 0.001). (g) Sensitivity of the designated strains to cell wall disturbing agents. The error bars indicate the standard error of the mean in three biological replicates.

Figure 6. Functions of SscA in gliotoxin (GT) production. (a) TLC of gliotoxin from the 2-day-grown conidia of WT, ΔsscA, and Cʹ sscA strains. The right bar plot indicates the relative band intensity of gliotoxin shown in TLC plate; error bars indicate the standard error of the mean in three biological replicates (**p < 0.01). (b) The relative mRNA expression levels of gliotoxin biosynthetic genes (gliA, gliT, and rglT) in WT, ΔsscA and Cʹ sscA conidia grown for 2 days (***p < 0.001, **p < 0.01). (c) TLC of gliotoxin from the 7-day-grown conidia of WT, ΔsscA, and Cʹ sscA strains. The right bar plot indicates the relative band intensity of gliotoxin shown in TLC plate; error bars indicate the standard error of the mean in three biological replicates (**p < 0.01). (d) The relative mRNA expression levels of gliotoxin biosynthetic genes (gliA, gliT, and rglT) in WT, ΔsscA, and Cʹ sscA conidia grown for 7 days (**p < 0.01, *p < 0.05).

Figure 7. Functions of SscA in the virulence of Aspergillus fumigatus. (a) Schematic representation of fungal infection in the mice model. (b) Survival rate of A. fumigatus WT, ΔsscA, and Cʹ sscA strains in mice model. Statistical differences were calculated using the Log-rank (mantel-cox) test (WT vs. KO, 0.1328; WT vs. complemented, 0.6767; KO vs. complemented, 0.0483).

Brookman JL, Denning DW. 2000. Molecular genetics in Aspergillus fumigatus. Curr Opin Microbiol. 3(5):468–474. doi: 10.1016/S1369-5274(00)00124-7.

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Xue T, Nguyen CK, Romans A, Kontoyiannis DP, May GS. 2004. Isogenic auxotrophic mutant strains in the Aspergillus fumigatus genome reference strain AF293. Arch Microbiol. 182(5):346–353. doi: 10.1007/s00203-004-0707-z.

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Data availability statement

All RNA-seq data files are available at the NCBI Sequence Read Archive (SRA) database under the accession number PRJNA983063 for WT and ΔsscA of Aspergillus fumigatus conidia RNA-seq.