Transcriptomic analysis of chemotherapy-associated oropharyngeal candidiasis reveals a novel role of indigenous mucosal bacteria in pathogenesis

This work reports a new mechanism of pathogenic synergy between C. albicans and E. faecalis, which may be responsible for increased epithelial barrier damage and mucosal invasion by C. albicans hyphae in cancer chemotherapy. Overall design: Tongue tissues (stored in RNAlater at -80oC) were thawed on ice for 15 min and RNA isolation was carried out using the RNeasy Kit (Qiagen) with few modifications. Briefly, thawed samples were homogenized in 800µl of RLT buffer containing 1% of ßeta-mercaptoethanol (Biorad) using a tissue homogenizer. Subsequently 800µl of Phenol:chloroform:isoamyl-alcohol (Invitrogen) was added to the homogenate in 2ml screw-capped tubes. Following centrifugation, the aqueous phase was collected, and an equal volume of 70% molecular-grade ethanol was added. Kit instructions were followed for subsequent column separation steps. DNAse treatment was performed using the TURBO DNA-free kit (Invitrogen). RNA was analyzed for quality using the Experion automated gel electrophoretic system (Biorad) and only samples with RNA integrity value (RIN) >7 were used for RNA seq. For the RNA sequencing, cDNA libraries were prepared using the TruSeq RNA kit (Illumina) and subjected to poly(A) enrichment according to kit protocol. cDNA fragments (one hundred nucleotides from each end) were sequenced using the Illumina NextSeq platform. To analyze the RNA sequences, we combined the raw reads from each sample and performed a quality check using FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Next, we used Sickle to trim the reads and QC them again with FASTQC (https://github.com/najoshi/sickle). Reads were aligned using HISAT2 (Hierarchical Indexing for Spliced Alignment of Transcripts) against the Mus musculus (GRCm38) index (Kim et al., 2015). The aligned reads were then converted into binary format using samtools and the duplicates were removed using the tool PICARD (Li et al., 2009). The counts were calculated using HTseq-count program and comparisons were done between the antibiotics and non-antibiotics oropharyngeal candidiasis groups (Anders et al., 2015). False discovery rate adjusted p-values = 0.05 were applied to identify significant differentially expressed (DE) genes using the DESeq2 package. Samples within a group were compared using principal component analysis and sample-to-sample distance matrix. Finally, the R software was used to compare the two groups and DE genes were tabulated and subjected to further downstream analysis.