Purpose: The objective of this study was to determine the invasion pathway of S. aureus using next generation sequencing.
Methods: Among S. aureus strains, two strains were selected for invasion positive (S. aureus KACC10768) and invasion negative (S. aureus KACC10778) according to the results of previous studies. To construct cDNA libraries, 1 μg of total RNA was used. For RNA fragmentation, random hexamer primed reverse transcription and 100 nt paired-end sequencing by Illumina HiSeq2000. The libraries were quantified using qPCR and qualified using Agilent Technologies 2100 Bioanalyzer. To estimate expression levels, the RNA-Seq reads were mapped to the genome of S. aureus using TopHat. The reference genome sequence of S. aureus and annotation data were downloaded from the Pepper Genome Platform (PGP) ftp site. The transcript counts in isoform level were calculated, and the relative transcript abundances were measured in FPKM using Cufflinks. In addition, novel transcripts were found per sample. These results were obtained by Cufflinks Reference Annotation Based Transcript Assembly (RABT) method, allowing discovery of reference transcripts and novel transcripts. All data analysis and visualization of differentially expressed genes was conducted using R.
Results: Relative expression for attachment related genes was higher (P<0.05) in invasion positive strain than in invasion negative strain, and various regulator genes especially for translation were highly expressed in invasion positive strain. However, the relative expression levels for most stress response genes were not different between two strains.
Significance: This result indicates that invasive S. aureus has high relative expression level for attachment-related gene and regulator genes, especially for translation.