Molecular Regulation Of GdpS On Spa In Staphylococcus Aureus

Staphylococcus aureus is an important opportunistic pathogen responsible for a broad spectrum of diseases, ranging from minor superficial skin infections to life-threatening systemic infectious diseases The virulence of S. aureus is essentially determined by a wide range of virulence factors, which are precisely controlled in response to various environmental changes. Protein A is a significant surface matrix binding protein in S. aureus, which functions to avoid phagocytosis by binding to the Fc regions of immunoglobulin G (IgG) in several mammalian species. In S. aureus, spa is regulated by a range of regulatory facrors.3’,5’-Cyclic diguanylic acid GMP (c-di-GMP) is a ubiquitous bacterial second messenger that involves in the regulation of biofilm formation, motility, and virulence in several pathogens. C-di-GMP is synthesized from two GTP molecules by the GGDEF domain-containing proteins, namely diguanylate cyclases (DGCs) and is hydrolyzed into pGpG or two GMPs by the EAL or HD-GYP domain-containing proteins, namely phodiesterases (PDEs), respectively. Typically, the numbers of GGDEF, EAL, and HD-GYP domain proteins are various in different bacteria. In S. aureus NCTC8325, only GdpS was identified as the only one conserved GGDEF domain containing protein, while no EAL or HD-GYP domain-containing proteins have been reported.Recent studies have revealed that GdpS cannot synthesize c-di-GMP in both S. epidermidis and S. aureus, while our previous research has found that gdpS can regulate a wide range of virulence factors, especially, the transcription of spa through sarS.Based on our previous studies, the influences of gdpS on spa and sarS depend on its N-terminal domain rather than its C-terminal, suggesting that gdpS functions independently of c-di-GMP signaling. In this study, further experiments were carried out for the details of the regulatory mechanisms of gdpS. We constructed the complementary strains with mutated ATG or deleted ATG by site-directed mutagenesis and proved that the regularion of sarS is independent of the translation product of gdpS. Then, the RNA transcript forms of gdpS were analyzed by Northern blot assay and only whole gdpS mRNA was detected, ruling out the existence of other small RNAs. Besides, we also performed electrophoretic mobility shift assay to determine the interaction of gdpS mRNA and the 5’UTR of sarS mRNA.By sequence alignments, we found an 18-nt region represents the possible binding site of gdpS mRNA with the 5’UTR of sarS mRNA and demonstrated the effect of gdpS on sarS relies on the putative 18-nt region by real-time RT PCR and lacZ reporter assays. In addition, mRNA half-life analysis indicated that gdpS can positively regulate sarS mRNA levels by extending the half-life of sarS mRNA, suggesting that gdpS regulates the expression of spa through sarS by direct RNA-RNA base pairing.In summary, this work can prompt the study of the regulatory network controlling spa transcription in S. aurues. The identification of gdpS as a dual-function RNA might will develop a deep understanding of regulatory RNAs in bacteria. In addition, many species possess GGDEF and EAL domain proteins in which the GGDEF and EAL motifs are not involved in the enzymatic function of DGCs and PDEs, and the function elucidation of gdpS can prompt the study of such genes.