A Proteomic Approach to Identify Virulence Factors Regulated by Polysaccharide Intercellular Adhesin (Pia) in Staphylococcus Aureus

Staphylococcus aureus is a major cause of nosocomial 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 extracellular factors, such as the 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 role of PIA in the gene regulation in S. aureus, particularly the regulatory network between PIA and Agr, as well as the pathogenesis of PIA, and host immune response to PIA. A comparative proteomic approach is employed to identify and characterize PIA-regulated proteins and expression of genes of interest will be confirmed using qRT-PCR. Transcription regulators belonged to the SarA family control 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. Purified PIA, different S. aureus strains and their ΔicaA (PIA-) counterparts will be employed to infect different cell lines to evaluate the cytotoxicity of PIA. Expression level of pro-inflammatory cytokines is determined to study the immune response to PIA in macrophages. The potential receptors and signaling pathways activated by PIA in macrophages will be investigated through a PCR array assay. A murine systemic infection model is used for evaluating the pathogenesis of PIA in vivo. BALB/c mice will be challenged with purified PIA, wild-type S. aureus strain and its PIA-deficient counterpart. The effect of PIA on the in vivo immune response is evaluated through the determination of the expression level of pro-inflammatory cytokines in mice. 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:PC10707-0165
External Project ID:MOST107-2320-B182-010