To Study the Role of Polysaccharide Intercellular Adhesion on the Regulation of Agr Quorum Sensing System in Staphylococcus Aureus

Staphylococcus aureus is a major cause of human infections and the development of biofilm can provide an extracellular barrier to antimicrobial agents or host immune defenses. Maturation of biofilms is mediated by the production of polysaccharide intercellular adhesin (PIA) regulated by icaRADBC locus. PIA plays a fundamental role in intercellular adhesion of staphylococci within a biofilm and also an important structural component of the biofilm matrix. We recently found that PIA constitutively expressed under planktonic growth whereas an environment is not suitable for the biofilm formation. We propose that PIA may act as a virulence factor or a regulator controlling gene expression in S. aureus. Results from our pilot study show that a significant difference of the expression profiles of both intracellular and extracellular proteins was observed between PIA wild-type and deficient strains. A decreased expression of protein A (encoded by spa), an important virulence factor for immune evasion, was observed in PIA-deficient strain. PIA-contained broth also showed a higher cytotoxicity to epithelial cells than PIA-free broth. Strikingly, hemolytic activity was significantly enhanced in PIA-deficient strains and an up-regulation of hla and hlb (α and β-hemolysin) was observed either in an Agr+ (accessory gene regulator) or Agr- strain. Expression of hld (δ-hemolysin, gene in agr P3 operon) was blocked in PIA-/Agr+ strain suggesting that PIA positively regulates Agr quorum sensing system, one of the most important global regulators in S. aureus. We aim to study the regulatory network between PIA and Agr and its effect on the pathogenesis. Regulation of the expression of protein A and hemolysins by PIA-Agr network will particularly be addressed. A comparative proteomic approach is employed to identify and characterize PIA-regulated proteins, particularly their association with Agr system. SarA family, a group of transcription regulators, controls the expression of agr, spa, and hemolysin-associated genes. Whether SarA family connects the crosstalk between PIA and Agr or hemolysis will be investigated through the deletion of different SarA family genes. Expression level of varies hemolysin-associated genes will be determined in wild-type and their ΔsarA, Δsae, ΔsarA/ΔicaA, Δsae/ΔicaA mutant strains to characterize the interaction between PIA and those regulators. Phagocytosis and cytokines expression of macrophages upon infection with PIA wild-type or deficient strains will be evaluated. A murine systemic infection model is used for evaluating the pathogenesis of PIA-Agr system in vivo. BALB/c mice will be challenged with purified PIA, wild-type S. aureus strain and its PIA-deficient counterpart. Pathogenesis in vivo will be investigated through the evaluation of the survival of mice, bacterial loads in blood and different organs, and expression levels of pro-inflammatory cytokines. Less has been known about the PIA behind its fundamental role in biofilm matrix architecture. Our study will get more insight into the role of PIA in the global gene regulation in S. aureus and the clinical significance of PIA to hosts.

Project ID:PC10901-1715
External Project ID:MOST108-2320-B182-031-MY3