Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and ?Rsp MRSA Transcriptomes

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of gene regulon. The goal of this study is to investigate the genes regulated by Rsp in MRSA BD02-25 Methods: mRNA profiles of wild-type (WT) and Rsp knockout (?Rsp) MRSA at mid-logarithmic growth phase (4h) were generated by deep sequencing, respectively in duplicate samples, using the Hiseq2000 (Illumina, CA) sequencer. The sequence reads that passed quality filters were aligned to S. aureus COL (RefSeq accession number NC_002951.2) using the Burrows-Wheeler Alignment tool (BWA) followed by ANOVA (ANOVA). Only the consistent data between the two WT or mutant samples were reserved for further analysis. qRT–PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, RNA-seq analyses revealed that 328 genes were up-regulated and 176 genes were down-regulated in ?rsp compared with wild type. In order to explain the major relevant changes in ?rsp compare to wild-type, the differential expressed genes obtained in RNA-seq with three biological replicates were used to construct a regulation network. 108 up-regulated genes and 33 down-regulated genes in RNA-seq data had gathered in the protein-protein interaction network. 15 modules of biological processes were responsible, including ribosome, metabolism, biofilm formation, cell wall degradation, cytolysis, two-component system and so on. Biological processes related to biofilm formation, such as cell wall degradation and metabolism were up regulated, indicating that the defense systems were activated. However, the genes that responsible for the two-component system and cytolysis were down regulated, suggesting a possible pathway how Rsp controls the virulence. Conclusions: Our data provide new information to the virulence regulatory network in S. aureus. Overall design: mRNA profiles of wild-type (WT) and Rsp knockout (?Rsp) MRSA were generated by deep sequencing, in duplicate, using the Hiseq2000 (Illumina, CA) sequencer.