The sino-nasal warzone: transcriptomic and genomic studies on sino-nasal aspergillosis in dogs

Abstract

We previously showed that each dog with chronic non-invasive sino-nasal aspergillosis (SNA) was infected with a single genotype of Aspergillus fumigatus. Here, we studied the transcriptome of this fungal pathogen and the canine host within the biofilm resulting from the infection. We describe here transcriptomes resulting from natural infections in animal species with A. fumigatus. The host transcriptome showed high expression of IL-8 and alarmins, uncontrolled inflammatory reaction and dysregulation of the Th17 response. The fungal transcriptome showed in particular expression of genes involved in secondary metabolites and nutrient acquisition. Single-nucleotide polymorphism analysis of fungal isolates from the biofilms showed large genetic variability and changes related with adaptation to host environmental factors. This was accompanied with large phenotypic variability in in vitro stress assays, even between isolates from the same canine patient. Our analysis provides insights in genetic and phenotypic variability of Aspergillus fumigatus in biofilms of naturally infected dogs reflecting in-host adaptation. Absence of a Th17 response and dampening of the Th1 response contributes to the formation of a chronic sino-nasal warzone.

Fig. 1. Transcriptomic profiles.

Transcriptome profiles of four fungal plaques of three different dogs with SNA. Venn diagram a showing number and percentage of shared transcripts (TPM > 1) between all four fungal plaques and heatmaps with hierarchical clustering of the 6536 shared transcripts b and the 660 transcripts mapped to the InnateDB database c. Number of transcripts with TPM > 1 is shown under each patient code in a.

Fig. 2. GO and KEGG term enrichment analysis.

GO and KEGG term enrichment analysis of the 6536 shared expressed transcripts in four fungal plaques from three dogs with SNA. BP indicates biological process, CC cellular component and MF molecular function (for full list see Supplementary data set 1).

Fig. 3. Comparison of RNAseq data.

Comparison between the RNAseq data of this study and the microarray from. Shared transcripts between our RNAseq data and found to be upregulated, downregulated, and not differentially expressed (log2 FC ≥ 2) in the microarray are in green, red, and gray, respectively. Transcripts mentioned in the Results and/or Discussion section are labeled.

Fig. 4. Heatmap of expressed and shared fungal genes.

Heatmap clustering expression of shared genes expressed in the fungal plaques of CP6, CP7, CP8, and CP8.2.

Fig. 5. GO and FunCat analysis.

GO a and FunCat (b; representing the first five terms) enrichment of shared expressed genes with low and high variation in expression. See Supplementary data set 2 for the complete enrichment analysis.

Fig. 6. SNP analysis.

Number of biallelic SNPs per A. fumigatus isolate.

Fig. 7. SNP-based phylogenetic tree.

SNP-based phylogenetic tree of the 26 isolates from eight SNA patients and from eight environmental isolates. The tree was inferred by using the maximum likelihood method based on the general time-reversible model. The tree with the highest log likelihood (−49,077.29) is shown and drawn to scale, with branch lengths measured in the number of substitutions per site. There was a total of 3826 nucleotide positions in the final data set. Evolutionary analyses were conducted in MEGA X. Selected clades (A, B, C, D) and sub clades (B1, D1, D2) are depicted with letters and numbers. The dog and environmental isolates are indicated with a dog and house pictogram, respectively, whereas G, D, and E indicate the number of STRAf genotypes, dog isolates, and environmental isolates, respectively. Two isolates that were not grouped with isolates from the same STRAf genotype are denoted by a star (*). Isolates with a notable high-SNP density are denoted by a plus (+) symbol.

Fig. 8. View of SNPs per strain.

SNPs present in an isolate are indicated by black shading of the cell. The origin of the isolates is depicted as a colored bar to the left.

Fig. 9. Overview of phenotyping.

The color of the cells corresponds to a z score of the test (red = poor growth, blue = good growth, white = average growth). Note that in the case of H₂O₂ the color relates to the size of the inhibition halo with a red color indicating large inhibition. Average measurements per strain are depicted in each cell. The colored bar to the right indicates the origin of the isolate.