Biofilm Formation And Fibrinogen Binding Ability Of Staphylococcus Aureus

Staphylococcus aureus is an important human pathogen that causes a variety of diseases, ranging from localized skin infections to life-threatening systemic infections. S. aureus NCTC8325(RN1and derivatives) was used widely as a model strain for which an incomparable wealth of knowledge has accumulated in the almost last50years since its isolation in1960from a sepsis patient. This thesis focuses on the regulatory mechanism of S. aureus NCTC8325(RN1and derivatives), and the studies fall into two aspects as listed below:1. GbaAB proteins influence biofilm formation in a PIA-dependent mannerThe Gram-positive bacteria Staphylococcus aureus and Staphylococcus epidermidis are capable of attaching to a biomaterial surface and forming resistant biofilms. The identification of biomolecular and regulatory factors involved in staphylococcal adhesion and biofilm formation is needed to understand biofilm-associated infection in humans. Here, we have identified a new operon, gbaAB (glucose induced biofilm accessory gene), that affects biofilm formation in S. aureus NCTC8325. Real-time reverse transcription PCR (RT-PCR) and Gel shift assays showed that GbaA and GbaB are transcribed from the same transcript, and GbaA directly inhibits the transcription of the gbaAB operon through self-repression. Our results indicated that the gbaA mutant displays enhanced biofilm formation compared with the wild type. However, the gbaB and the gbaAB double mutant displayed reduced biofilm formation, suggesting that the gbaAB operon is involved in biofilm formation and that gbaB might be the key gene in biofilm regulation. Phenotypic analysis suggested that the gbaAB operon mediates biofilm formation of S. aureus at the multicellular aggregation stage rather than during initial attachment. In addition, real-time RT-PCR analysis showed that icaA was upregulated in the gbaA mutant and downregulated in the gbaB and gbaAB mutants compared with the wild type. In addition, the gbaA and the gbaB mutants affected the induction of biofilm formation by glucose. Our results suggest that the gbaAB operon is involved in the regulation of the multicellular aggregation step of S. aureus biofilm formation in response to glucose and that this regulation may be mediated through the ica operon.2. Rot and Agr system modulate fibrinogen-binding ability mainly via ClfBThe success of S. aureus as a pathogen is partly due to its ability to adhere to a wide range of host tissues, including host extracellular matrix proteins such as fibrinogen, fibronectin, and collagen. S. aureus expresses two proteins that can bind specifically to fibrinogen, clumping factors A and B (ClfA and ClfB). Repressor of toxins (Rot) is known to be a global regulator of virulence gene expression in S. aureus. The translation of Rot is regulated by the staphylococcal accessory gene regulator (Agr) quorum-sensing system. In this study, we demonstrated that Rot and the Agr system can affect the bacterial binding ability to human fibrinogen (Fg) under different bacterial growth phases. According to our real-time RT-PCR results, we confirmed that both Rot and the Agr system have no significant effect on clf A expression. However, Rot is an activator of clfB and Agr/RNAⅢ can regulate clfB expression via Rot. Our gel shift data further suggested that Rot might regulate clfB expression by directly binding to the promoter region of clfB. Moreover, Rot and the Agr system exhibited consistent regulatory effects on clfB transcription and bacterial Fg-binding ability, suggesting that Rot and the Agr system might affect bacterial Fg-binding ability mainly through regulating clfB transcription.