Functional Study Of ArtR And Identification Of RNAIII-binding Proteins In Staphylococcus Aureus

Staphylococcus aureus is a gram-positive and opportunistic human pathogen, which is widely distributed in the environment. The bacterium has become a major clinical pathogen and causes millions of infectious cases in hospital every year. S. aureus can cause a variety of infections ranging from localized skin infections to life-threatening systemic infections. The infection ability of S. aureus mainly depends on the production of many virulence factors, in which a-toxin is a notorious one. All these virulence factors are under control of different regulatory pathways, including two-component systems, transcriptional regulator proteins, and non-coding RNAs. Recent studies have pointed to the importance of small-noncoding RNAs (sRNAs) in bacterial virulence control.Here, we report the identification and functional analyses of a novel sRNA, ArtR, which is overlapped with luxS gene on the opposite strand. The full length of ArtR is345nt, as determined by RACE experiment. Our data showed that the AgrA protein can bind to the artR promoter and repress artR transcription, suggesting that, besides RNAIII, ArtR is the second sRNA regulated by AgrA. Therefore, we designated it as ArtR (AgrA-repressed and toxin regulating sRNA). Furthermore, ArtR is unique in S. aureus and involved in virulence regulation by activating a-toxin expression. ArtR can directly bind to the5’untranslated region of the sarT mRNA, thus promoting the degradation of sarT mRNA by RNase III and arresting the translation of SarT. This suggests that the activation of ArtR on the a-toxin is through SarT. This study reveals another kind of staphylococcal regulatory small RNA that plays a role in virulence control. It also indicates the diversity of small RNA-target mRNA interactions and how these multiple interactions can mediate virulence regulation in S. aureus.RNA regulation may involves proteins such as RNase, RNA chaperones and those proteins targeted by sRNA. Since it is difficult to identify RNA-binding proteins (RBPs), very little was known about the RBPs in S. aureus, especially those associated with sRNAs. Here we adopted a tRNA scaffold streptavidin aptamer based pull-down assay to identify RBPs in S. aureus. The tethered RNA was successfully captured by the streptavidin magnetic beads, and proteins binding to RNAIII were isolated and analyzed by mass spectrometry. We have identified81proteins, and expressed heterologously9of them in Escherichia coli. The binding ability of the recombinant proteins with RNAIII was further analyzed by electrophoresis mobility shift assay. RNase III has an RNA-binding motif, and exhibited RNA degradation activity. CshA, RNase J2, and Era also have RNA-binding motifs and showed binding ability with RNAIII. Hu and WalR have DNA-binding domain, and also showed RNAIII binding ability. Surprisingly, Pyk and FtsZ, without RNA/DNA-binding domains, also had binding activity with RNAIII. PfkA did not bind to RNAIII even at high concentration. PNPase and Enolase were suggested to be the components of RNA degradosome, but they were not found in our pull-down assay and they did not bind to RNAIII as expected. This study suggests that some proteins can bind to RNAIII in S. aureus, and may be involved in RNAIII functions. And tRSA based pull-down assay is an effective method to search for RBPs in bacteria, and this method should facilitate the identification and functional study of RBPs in diverse bacterial species.