Abstract
Objective: The form of human fungal sinusitis that most closely approximates the disease occurring in the dog is chronic erosive non-invasive fungal sinusitis. This disease is characterized by final destruction of bone in the absence of tissue invasion by the fungus and requires both removal of fungal plaques, necrotic tissue
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and medical therapy with antifungals. Remarkably, these fungal plaques are white indicated that asexual development does not proceed in the patients. Immune response in SNA infections has been studied via biopsy and cytokine profiling as well as transcriptomic analysis of the host tissue. However, a transcriptomic study of this fungal pathogen growing in patients causing a non-invasive infection has never being performed. We obtained fungal plaques directly from canine patients suffering from SNA and characterize the transcriptome of the causative fungus A. fumigatus in order understand gene expression in the context of the host and particularly in the field of in-host adaptation Methods: Four different fungal plaques were isolated from dogs suffering from SNA. After surgical removal using endoscope or trephination part of fungal plaques were immediately frozen in liquid nitrogen and stored at -80°C for RNA isolation and sequencing. RNA isolation was performed using RNeasy Mini Kit® from Qiagen and sequencing was performed by ServiceXS (Leiden, The Netherlands). RNA-seq analysis involved quality check with fastQC. Cleaning and trimming of reads with Fastx-toolkit. Kallisto was used for transcript quantification(TPM) with A. fumigatus Af293 (AspGD) as reference. For functional characterization of the transcriptome, highly variable expressed genes between samples were removed, and 3 subjective levels of expression were established: low (1 to 39.8 TPM), median (39.8 to 1584.8 TPM) and high (1584.8 to 79432.8 TPM), for each group an enrichment analysis was performed. Additionally a more targeted categorization of the transcriptome was done using published lists of genes involved in stress, reproduction and virulence. Results: According to the used criteria 17% of the expressed genes presented a stable expression across all 4 fungal plaques. Careful examination of this group of genes showed genes previously reported to be involved in SskA-Hog/SakA signalling pathway with some of them (bck1 and hdaA) also involved in the regulation of secondary metabolism. Interestingly, central regulators of asexual reproduction like BrlA, WetA and AbaA showed variable or null expression. A similar pattern was observed for catalases and superoxide dismutases. Finally comparison with published biofilm expression data showed that 18% of the stable expressed genes were also differentially expressed in A. fumigatus biofilm, of which approximately half of them were described to be differentially expressed in an “mature” biofilm (48 h). Conclusion: 1-To our knowledge this is the first transcriptomic study of A. fumigatus in the context of a natural non-invasive infection in dogs suffering from SNA. 2-Variability in gene expression in SNA fungal plaques could be caused by several factors like time of infection, host response, and genomic differences. 3-SNA fungal plaques resemble a non-sporulation mature biofilm, explained partially by low expression of central regulators of sporulation and the expression of some genes related to previously reported biofilm formation.
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