Background: Pseudomonas aeruginosa (P. aeruginosa ) is the second-most common commensal bacterium in healthy conjunctival sacs. When the corneal epithelial barrier is damaged, P. aeruginosa in a healthy conjunctival sac can cause infectious keratitis, which can result in the loss of vision. This study was designed to investigate the differentially expressed genes (DEGs) of P. aeruginosa isolates from eyes with keratitis and from healthy conjunctival sacs to predict their functions and pathways through Illumina high-throughput RNA sequencing (RNA-seq).
Methods: P. aeruginosa isolates from keratitis and healthy conjunctival sacs were obtained. The transcriptome profile of P. aeruginosa was characterized by a high throughput RNA-seq strategy using the Illumina HiSeq 2500 platform. The DEGs were analyzed with DESeq and validated through quantitative real-time polymerase chain reaction (PCR) and with experimental mice. GO enrichment and the KEGG pathway were also analyzed.
Results: In genome-wide transcriptional analysis, 557 genes (332 upregulated and 225 downregulated) were found to be differentially expressed (fold change ≥ 2, p ≤ 0.05) in the strains from keratitis. GO enrichment analysis suggested that DEGs tended to be associated with cellular and metabolic processes. KEGG pathway analysis revealed the DEGs were typically associated with the pathways of the bacterial secretion system and pyoverdine metabolism. Eleven DEGs were validated using quantitative reverse-transcription PCR and verified with experimental mice. The results were consistent with those obtained in RNA-seq.
Conclusion: The DEGs related to pilin, T2SS, T3SS, and pyoverdine metabolisms were significantly altered in the strains from keratitis. The findings may be helpful for further investigations on genes or pathways related to the pathogenesis of and therapeutic targets for P. aeruginosa keratitis.
Keywords: P. aeruginosa , genotype, keratitis, conjunctival sac, microflora